Most recent papers:

• Hydroxychloroquine affects bone resorption both in vitro and in vivo.
  Tim Both, M. Carola Zillikens, Marijke Koedam, Marijn Vis, Wai‐Kwan Lam, Angelique E.A. M. Weel, Johannes P.T. M. van Leeuwen, P. Martin van Hagen, Bram C.J. van der Eerden, Paul L.A. van Daele.
  Journal of Cellular Physiology. May 30, 2017
  We recently showed that patients with primary Sjögren syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favorable effects on BMD. The aim of the study was to evaluate whether HCQ modulates osteoclast function. Osteoclasts were cultured from PBMC‐sorted monocytes for 14 days and treated with different HCQ doses (control, 1 and 5 µg/ml). TRAP staining and resorption assays were performed to evaluate osteoclast differentiation and activity, respectively. Staining with an acidification marker (acridine orange) was performed to evaluate intracellular pH at multiple timepoints. Additionally, a fluorescent cholesterol uptake assay was performed to evaluate cholesterol trafficking. Serum bone resorption marker β‐CTx was evaluated in rheumatoid arthritis patients. HCQ inhibits the formation of multinuclear osteoclasts and leads to decreased bone resorption. Continuous HCQ treatment significantly decreases intracellular pH and significantly enhanced cholesterol uptake in mature osteoclasts along with increased expression of the lowdensity lipoprotein receptor. Serum β‐CTx was significantly decreased after six months of HCQ treatment. In agreement with our clinical data, we demonstrate that HCQ suppresses bone resorption in vitro and decreases the resorption marker β‐CTx in vivo. We also showed that HCQ decreases the intracellular pH in mature osteoclasts and stimulates cholesterol uptake, suggesting that HCQ induces osteoclastic lysosomal membrane permeabilization (LMP) leading to decreased resorption without changes in apoptosis. We hypothesize that skeletal health of patients with increased risk of osteoporosis and fractures may benefit from HCQ by preventing BMD loss. This article is protected by copyright. All rights reserved

  May 30, 2017   doi: 10.1002/jcp.26028   open full text
• Microtubules Regulate Brush Border Formation.
  Facundo M. Tonucci, Anabela Ferretti, Evangelina Almada, Pamela Cribb, Rodrigo Vena, Florencia Hidalgo, Cristián Favre, Matt J. Tyska, Irina Kaverina, M. Cecilia Larocca.
  Journal of Cellular Physiology. May 26, 2017
  Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation towards the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity. This article is protected by copyright. All rights reserved

  May 26, 2017   doi: 10.1002/jcp.26033   open full text
• The nucleus is irreversibly shaped by motion of cell boundaries in cancer and non‐cancer cells.
  Vincent J. Tocco, Yuan Li, Keith G. Christopher, James H. Matthews, Varun Aggarwal, Lauren Paschall, Hendrik Luesch, Jonathan D Licht, Richard B. Dickinson, Tanmay P. Lele.
  Journal of Cellular Physiology. May 25, 2017
  Actomyosin stress fibers impinge on the nucleus and can exert compressive forces on it. These compressive forces have been proposed to elongate nuclei in fibroblasts, and lead to abnormally shaped nuclei in cancer cells. In these models, the elongated or flattened nuclear shape is proposed to store elastic energy. However, we found that deformed shapes of nuclei are unchanged even after removal of the cell with micro‐dissection, both for smooth, elongated nuclei in fibroblasts and abnormally shaped nuclei in breast cancer cells. The lack of shape relaxation implies that the nuclear shape in spread cells does not store any elastic energy, and the cellular stresses that deform the nucleus are dissipative, not static. During cell spreading, the deviation of the nucleus from a convex shape increased in MDA‐MB‐231 cancer cells, but decreased in MCF‐10A cells. Tracking changes of nuclear and cellular shape on micropatterned substrata revealed that fibroblast nuclei deform only during deformations in cell shape and only in the direction of nearby moving cell boundaries. We propose that motion of cell boundaries exert a stress on the nucleus, which allows the nucleus to mimic cell shape. The lack of elastic energy in the nuclear shape suggests that nuclear shape changes in cells occur at constant surface area and volume. This article is protected by copyright. All rights reserved

  May 25, 2017   doi: 10.1002/jcp.26031   open full text
• Zebrafish Tmem230a cooperates with the Delta/Notch signaling pathway to modulate endothelial cell number in angiogenic vessels.
  Silvia Carra, Lorenzo Sangiorgio, Paride Pelucchi, Solei Cermenati, Alessandra Mezzelani, Valentina Martino, Mira Palizban, Alberto Albertini, Martin Götte, James Kehler, Gianluca Deflorian, Monica Beltrame, Antonio Giordano, Rolland Reinbold, Franco Cotelli, Gianfranco Bellipanni, Ileana Zucchi.
  Journal of Cellular Physiology. May 25, 2017
  During embryonic development, new arteries and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre‐existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co‐operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel‐network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis. This article is protected by copyright. All rights reserved

  May 25, 2017   doi: 10.1002/jcp.26032   open full text
• Integrated transcriptomic and metabolomic analysis reveals adaptive changes of hibernating retinas.
  Yizhao Luan, Jingxing Ou, Vincent P Kunze, Fengyu Qiao, Yan Wang, Lai Wei, Wei Li, Zhi Xie.
  Journal of Cellular Physiology. May 25, 2017
  Hibernation is a seasonally adaptive strategy that allows hibernators to live through extremely cold conditions. Despite the profound reduction of blood flow to the retinas, hibernation causes no lasting retinal injury. Instead, hibernators show an increased tolerance to ischemic insults during the hibernation period. To understand the molecular changes of the retinas in response to hibernation, we applied an integrative transcriptome and metabolome analysis to explore changes in gene expression and metabolites of 13‐lined ground squirrel retinas during hibernation. Metabolomic analysis showed a global decrease of ATP synthesis in hibernating retinas. Decreased glucose and galactose, increased beta‐oxidation of carnitine and decreased storage of some amino acids in hibernating retinas indicated a shift of fuel use from carbohydrates to lipids and alternative usage of amino acids. Transcriptomic analysis revealed that the down‐regulated genes were enriched in DNA‐templated transcription and immune‐related functions, while the up‐regulated genes were enriched in mitochondrial inner membrane and DNA packaging‐related functions. We further showed that a subset of genes underwent active alternative splicing events in response to hibernation. Finally, integrative analysis of the transcriptome and metabolome confirmed the shift of fuel use in the hibernating retina by the regulation of catabolism of amino acids and lipids. Through transcriptomic and metabolomic data, our analysis revealed the altered state of mitochondrial oxidative phosphorylation and the shift of energy source in the hibernating retina, advancing our understanding of the molecular mechanisms employed by hibernators. The data will also serve as a useful resource for the ocular and hibernation research communities. This article is protected by copyright. All rights reserved

  May 25, 2017   doi: 10.1002/jcp.26030   open full text
• Nanoparticles as new tools for inhibition of cancer angiogenesis.
  Nasser Hashemi Goradel, Farshid Ghiyami Hoor, Samira Jahangiri, Babak Negahdari, Amirhossein Sahebkar, Aria Masoudifar, Hamed Mirzaei.
  Journal of Cellular Physiology. May 25, 2017
  Angiogenesis is known as one of the hallmarks of cancer. Multiple lines evidence indicated that vascular endothelium growth factor (VEGF) is a key player in the progression of angiogenesis and exerts its functions via interaction with tyrosine kinase receptors (TKRs). These receptors could trigger a variety of cascades that lead to the supply of oxygen and nutrients to tumor cells and survival of these cells. With respect to pivotal role of angiogenesis in the tumor growth and survival, finding new therapeutic approaches via targeting angiogenesis could open a new horizon in cancer therapy. Among various types of therapeutic strategies, nanotechnology has emerged as new approach for the treatment of various cancers. Nanoparticles (NPs) could be used as effective tools for targeting a variety of therapeutic agents. According to in vitro and in vivo studies, NPs are efficient in depriving tumor cells from nutrients and oxygen by inhibiting angiogenesis. However, the utilization of NPs are associated with a variety of limitations. It seems that new approaches such as NPs conjugated with hydrogels could overcome to some limitations. In the present review, we summarize various mechanisms involved in angiogenesis, common anti‐angiogenesis strategies and application of NPs for targeting angiogenesis in various cancers. This article is protected by copyright. All rights reserved

  May 25, 2017   doi: 10.1002/jcp.26029   open full text
• Chick derived induced pluripotent stem cells by the poly‐cistronic transposon with enhanced transcriptional activity.
  Masafumi Katayama, Takashi Hirayama, Tetsuya Tani, Katsuhiko Nishimori, Manabu Onuma, Tomokazu Fukuda.
  Journal of Cellular Physiology. May 24, 2017
  Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly‐cistronic reprogramming vector (PB‐R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c‐Myc, Lin28, and Nanog. The PB‐R6F derived iPS cells were alkaline‐phosphatase and SSEA‐1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three‐germ‐layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly‐cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species. This is the first report of establishment of avian derived iPS cells with a single poly‐cistronic transposon based expression system. Our established iPS cells depend on the activation of FGF signaling pathway, but does not to the LIF signaling.

  May 24, 2017   doi: 10.1002/jcp.25947   open full text
• DJ‐1/Park7 Sensitive Na+/H+ Exchanger 1 (NHE1) in CD4+ T Cells.
  Yuetao Zhou, Xiaolong Shi, Hong Chen, Shaqiu Zhang, Madhuri S. Salker, Andreas F. Mack, Michael Föller, Tak W. Mak, Yogesh Singh, Florian Lang.
  Journal of Cellular Physiology. May 24, 2017
  DJ‐1/Park7 is a redox‐sensitive chaperone protein counteracting oxidation and presumably contributing to the control of oxidative stress responses and thus inflammation. DJ‐1 gene deletion exacerbates the progression of Parkinson's disease presumably by augmenting oxidative stress. Formation of reactive oxygen species (ROS) is paralleled by activation of the Na+/H+ exchanger 1 (NHE1). ROS formation in CD4+ T cells plays a decisive role in regulating inflammatory responses. In the present study, we explored whether DJ‐1 is expressed in CD4+ T cells, and affects ROS production as well as NHE1 in those cells. To this end, DJ‐1 and NHE1 transcript, and protein levels were quantified by qRT‐PCR and Western blotting, respectively, intracellular pH (pHi) utilizing bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF) fluorescence, NHE activity from realkalinization after an ammonium pulse, and ROS production utilizing 2′,7′ −dichlorofluorescin diacetate (DCFDA) fluorescence. As a result DJ‐1 was expressed in CD4+ T cells. ROS formation, NHE1 transcript levels, NHE1 protein, and NHE activity were higher in CD4+ T cells from DJ‐1 deficient mice than in CD4+ T cells from wild type mice. Antioxidant N‐acetyl‐cysteine (NAC) and protein tyrosine kinase (PTK) inhibitor staurosporine decreased the NHE activity in DJ‐1 deficient CD4+ T cells, and blunted the difference between DJ‐1−/− and DJ‐1+/+ CD4+ T cells, an observation pointing to a role of ROS in the up‐regulation of NHE1 in DJ‐1−/− CD4+ T cells. In conclusion, DJ‐1 is a powerful regulator of ROS production as well as NHE1 expression and activity in CD4+ T cells. J. Cell. Physiol. 9999: 1–10, 2017. © 2017 Wiley Periodicals, Inc. Our study described that DJ‐1 deficient CD4+ T cells leads to upregulation of NHE1 and enhanced ROS production, therefore results in increase in NHE1 activity. NHE1 regulation in DJ‐1 deficient CD4+ T cells is dependent on ROS and PTK signaling. Thus, our data first time described that link between DJ‐1 proteins with responses on ROS production and NHE1 functions.

  May 24, 2017   doi: 10.1002/jcp.25516   open full text
• Characterization of Runx2 phosphorylation sites required for TGF‐β1‐mediated stimulation of matrix metalloproteinase‐13 expression in osteoblastic cells.
  Balasubramanian Arumugam, Mariappanadar Vairamani, Nicola C. Partridge, Nagarajan Selvamurugan.
  Journal of Cellular Physiology. May 24, 2017
  Transforming growth factor‐beta1 (TGF‐β1), a highly abundant growth factor in skeletal tissues, stimulates matrix metalloproteinase‐13 (MMP‐13) expression in osteoblastic cells. MMP‐13 plays a critical role in bone remodeling. Runx2, a bone transcription factor, is required for TGF‐β1‐mediated stimulation of MMP‐13 expression in osteoblastic cells. In this study, the molecular mechanism responsible for TGF‐β1‐stimulation of MMP‐13 expression via Runx2 in osteoblastic cells was elucidated. TGF‐β1 stimulated the phosphorylation of Runx2 at serine amino acids, and ERK inhibition blocked this effect in rat (UMR106‐01) and human (MG‐63) osteoblastic cells. Pretreatment with okadaic acid, a serine‐threonine phosphatase inhibitor, increased Runx2 serine phosphorylation in osteoblastic cells. When cells were pretreated with an ERK inhibitor, TGF‐β1‐mediated stimulation of MMP‐13 mRNA expression decreased. Nano‐ESI/LC/MS analysis identified that TGF‐β1 stimulates Runx2 phosphorylation at three serine amino acids. Transient transfection of mouse mesenchymal stem cells (C3H10T1/2) with Runx2 serine mutant constructs decreased TGF‐β1‐mediated Runx2 serine phosphorylation. A luciferase reporter assay identified that TGF‐β1 stimulated MMP‐13 promoter activity in these cells only in the presence of the wild Runx2 construct, and not with mutant Runx2. Thus, TGF‐β1 stimulates the phosphorylation of Runx2 at three serine amino acids, and this event is required for MMP‐13 expression in osteoblastic cells. Hence, this study contributes to the knowledge of events governing bone remodeling and bone‐related diseases. A schematic diagram representing the TGF‐β1‐induced stimulation of ERK‐dependent Runx2 phosphorylation, with the corresponding sites, required for MMP‐13 promoter activation in human osteoblast cells.

  May 24, 2017   doi: 10.1002/jcp.25964   open full text
• Isorhapontigenin induced cell growth inhibition and apoptosis by targeting EGFR‐related pathways in prostate cancer.
  Cuicui Zhu, Qingyi Zhu, Zhaomeng Wu, Yingying Yin, Dan Kang, Shan Lu, Ping Liu.
  Journal of Cellular Physiology. May 24, 2017
  Isorhapontigenin (ISO), a naturally phytopolyphenol compound existing in Chinese herb, apples, and various vegetables, has attracted extensive interest in recent years for its diverse pharmacological characteristics. Increasing evidences reveal that ISO can inhibit cancer cell growth by induced apoptosis, however, the molecular mechanisms is not fully understood. In this study, we found for the first time that ISO apparently induced cell growth inhibition and apoptosis by targeting EGFR and its downstream signal pathways in prostate cancer (PCa) cells both in vitro and in vivo, whereas no obviously effect on normal prostate cells. From the results, we found that ISO competitively targeted EGFR with EGF and inhibited EGFR auto‐phosphorylation, and then decreased the levels of p‐Erk1/2, p‐PI3 K, and p‐AKT, and further induced down‐regulation of p‐FOXO1 and promoted FOXO1 nuclear translocation; and finally resulted in a significantly up‐regulation of Bim/p21/27/Bax/cleaved Caspase‐3/cleaved PARP‐1 and a markedly down‐regulation of Sp1/Bcl‐2/XIAP/Cyclin D1. Moreover, our experimental data demonstrated that treatment of ISO decreased protein level of AR via both inhibiting the expression of AR gene and promoting the ubiquitination/degradation of AR proteins in proteasome. In vivo, we also found that ISO inhibited the growth of subcutaneous xenotransplanted tumor in nude mice by inducing PCa cell growth inhibition and apoptosis. Taken together, all findings here clearly implicated that EGFR‐related signal pathways, including EGFR‐PI3K‐Akt and EGFR‐Erk1/2 pathways, were involved in ISO‐induced cell growth inhibition and apoptosis in PCa cells, providing a more solid theoretical basis for the application of ISO to treat patients with prostate cancer in clinic. In this study, we find for the first time that ISO competitively targets EGFR with EGF and inhibits the auto‐phosphorylation of EGFR at Tyr1173, resulting in inactivation of EGFR and its downstream signal pathways, including EGFR/PI3K/Akt pathway, and EGFR/Erk pathway; and finally induce cell growth inhibition and apoptosis by further activating FOXO1 and inactivating androgen receptor (AR) in prostate cancer cells.

  May 24, 2017   doi: 10.1002/jcp.25968   open full text
• Hematopoietic stem/progenitor cell differentiation towards myeloid lineage is modulated by LIGHT/LIGHT receptor signaling.
  Weikai Chen, Xin Lv, Changlong Liu, Ruoping Chen, Jianhe Liu, Haiyan Dai, Gang‐Ming Zou.
  Journal of Cellular Physiology. May 24, 2017
  The cytokine LT‐related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) is a member of the tumor necrosis factor (TNF) superfamily. It is expressed primarily on activated T lymphocytes, and detectable on monocytes, granulocytes, and immune dendritic cells. It mainly plays a role in immune regulation including T cell activation and dendritic cell maturation. We recently reported its role as an inducer in embryonic stem cell differentiation, but its role in regulation of adult stem cell has not been defined. In the present study, we examined the expression of LIGHT receptor in Lin−c‐kit+Sca‐1+ hematopoietic stem/progenitor cells (HSC/HPCs). We found that HSC express HVEM, a LIGHT receptor, on its surface. We further identified the role of LIGHT in promoting myeloid differentiation of HSCs driven by granulocyte‐monocyte colony stimulating factor (GM‐CSF). Further studies showed that LIGHT enhances both GM‐CSF and GM‐CSF receptor (GM‐CSFR) expression in HSCs. LIGHT stimulation increases PU.1 expression in HSC/HPCs. In vivo administration of LIGHT increases the colony‐forming unit‐granulocyte/monocyte (CFU‐GM) colony formation and plasma GM‐CSF level. Altogether, the data suggest LIGHT promote myeloid differentiation of HSC/HPCs. In the present study, we examined the expression of LIGHT receptor in Lin‐c‐kit+Sca‐1+ hematopoietic stem/progenitor cells (HSC/HPCs). We found that HSC express HVEM, a LIGHT receptor, on its surface. We further identified the role of LIGHT in promoting myeloid differentiation of HSCs driven by granulocyte‐monocyte colony stimulating factor (GM‐CSF).

  May 24, 2017   doi: 10.1002/jcp.25967   open full text
• Acyl‐CoA synthetase short‐chain family member 2 (ACSS2) is regulated by SREBP‐1 and plays a role in fatty acid synthesis in caprine mammary epithelial cells.
  Huifen Xu, Jun Luo, Gongzhen Ma, Xueying Zhang, Dawei Yao, Ming Li, Juan J. Loor.
  Journal of Cellular Physiology. May 24, 2017
  Sterol regulatory element binding protein 1 (SREBP‐1) is well‐known as the master regulator of lipogenesis in rodents. Acyl‐CoA synthetase short‐chain family member 2 (ACSS2) plays a key role in lipogenesis by synthesizing acetyl‐CoA from acetate for lipogenesis. ATP citrate lyase (ACLY) catalyzes the conversion of citrate and coenzyme A to acetyl‐CoA, hence, it is also important for lipogenesis. Although ACSS2 function in cancer cells has been elucidated, its essentiality in ruminant mammary lipogenesis is unknown. Furthermore, ACSS2 gene promoter and its regulatory mechanisms have not known. Expression of ACSS2 was high in lipid synthesizing tissues, and its expression increased during lactation compared with non‐lactating period. Simultaneous knockdown of both ACSS2 and ACLY by siRNA in primary goat mammary epithelial cells decreased (p < 0.05) the mRNA abundance of genes associated with de novo fatty acid synthesis (FASN, ACACA, SCD1) and triacylglycerol (TAG) synthesis (DGAT1, DGAT2, GPAM, and AGPAT6). Genes responsible for lipid droplet formation and secretion (PLIN2 and PLIN3) and fatty acid oxidation (ATGL, HSL, ACOX, and CPT1A) all decreased (p < 0.05) after ACSS2 and ACLY knockdown. Total cellular TAG content and lipid droplet formation also decreased. Use of a luciferase reporter assay revealed a direct regulation of ACSS2 by SREBP‐1. Furthermore, SREBP‐1 interacted with an SRE (SREBP response element) spanning at −475 to −483 bp on the ACSS2 promoter. Taken together, our results revealed a novel pathway that SREBP‐1 may regulate fatty acid and TAG synthesis by regulating the expression of ACSS2. The expression of ACSS2 is crucial for triacylglycerol synthesis in goat mammary epithelial cells. Its expression is controlled by the transcription factor SREBP‐1 through an SRE element on the promoter region.

  May 24, 2017   doi: 10.1002/jcp.25954   open full text
• End stage renal disease‐induced hypercalcemia may promote aortic valve calcification via Annexin VI enrichment of valve interstitial cell derived‐matrix vesicles.
  Lin Cui, Nabil A. Rashdan, Dongxing Zhu, Elspeth M. Milne, Paul Ajuh, Gillian Milne, Miep H. Helfrich, Kelvin Lim, Sai Prasad, Daniel A. Lerman, Alex T. Vesey, Marc R. Dweck, William S. Jenkins, David E. Newby, Colin Farquharson, Vicky E. Macrae.
  Journal of Cellular Physiology. May 24, 2017
  Patients with end‐stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4‐fold; p < 0.05) whereas Pi treatment alone had no effect. Ca (2.7 mM) and Pi (2.5 mM) synergistically induced calcium deposition (10.8‐fold; p < 0.001) in VICs. Ca treatment increased the mRNA of the osteogenic markers Msx2, Runx2, and Alpl (p < 0.01). MVs were harvested by ultracentrifugation from VICs cultured with control or calcification media (containing 2.7 mM Ca and 2.5 mM Pi) for 16 hr. Proteomics analysis revealed the marked enrichment of exosomal proteins, including CD9, CD63, LAMP‐1, and LAMP‐2 and a concomitant up‐regulation of the Annexin family of calcium‐binding proteins. Of particular note Annexin VI was shown to be enriched in calcifying VIC‐derived MVs (51.9‐fold; p < 0.05). Through bioinformatic analysis using Ingenuity Pathway Analysis (IPA), the up‐regulation of canonical signaling pathways relevant to cardiovascular function were identified in calcifying VIC‐derived MVs, including aldosterone, Rho kinase, and metal binding. Further studies using human calcified valve tissue revealed the co‐localization of Annexin VI with areas of MVs in the extracellular matrix by transmission electron microscopy (TEM). Together these findings highlight a critical role for VIC‐derived MVs in CAVD. Furthermore, we identify calcium as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of ESRD patients to accelerated development of CAVD. Matrix vesicle (MV) deposition in the extracellular matrix of human calcified aortic valve tissue. Aortic valve calcification was confirmed by Alizarin red and Von Kossa staining. Arrows indicate positive areas of staining. Transmission electron microscopy (TEM) shows MVs with spindle‐like projections resembling hydroxyapatite crystal needles in calcified tissue, and “empty” vesicle structures in control tissue (arrows). Scale bars = 500 µm (Alizarin red/Von Kossa); 500 nm (TEM).

  May 24, 2017   doi: 10.1002/jcp.25935   open full text
• Stimulation of oral fibroblast chemokine receptors identifies CCR3 and CCR4 as potential wound healing targets.
  Jeroen K. Buskermolen, Sanne Roffel, Susan Gibbs.
  Journal of Cellular Physiology. May 23, 2017
  The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL‐6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP‐1 secretion was reduced by CCL15/CCR3. In conclusion, this in‐vitro study identifies chemokine receptor‐ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL‐6 and HGF and reducing the secretion of TIMP‐1. Gingiva fibroblasts express 12 chemokine receptors. Fourteen chemokines were used to study proliferation (top), migration (middle), and secretion of wound healing mediators by gingiva fibroblasts (bottom). Predominantly CCR3 and CCR4 stimulation influenced these oral wound healing parameters.

  May 23, 2017   doi: 10.1002/jcp.25946   open full text
• APN/CD13 is over‐expressed by Psoriatic fibroblasts and is modulated by CGRP and IL‐4 but not by retinoic acid treatment.
  Pascale Gerbaud, Jean Guibourdenche, Rafika Jarray, Marc Conti, Patricia Palmic, Stéphanie Leclerc‐Mercier, Julie Bruneau, Olivier Hermine, Yves Lepelletier, Françoise Raynaud.
  Journal of Cellular Physiology. May 23, 2017
  Psoriasis vulgaris is a common skin inflammatory disease characterized by recurrent flare episodes associated with scaly well‐demarcated skin plaques. Skin biopsies from psoriatic patients with high PASI score (22.67 ± 8.67) and from HD were used to study APN/CD13. APN/CD13 is over‐expressed in LP and nLP compare to HD skins and fibroblasts. This over‐expression is positively correlated with specific enzymatic activity enhancement. However, discrepancies between APN/CD13 expression in LP and nLP prompt us to focus our study on APN/CD13 modulation. Calcitonin Gene Related Peptide (CGRP), a neuropeptide, positively modulated expression and activity of APN/CD13. CGRP consistently induced IL4 secretion, which is also involved in the increase of APN/CD13 expression and activity, which is significantly reversed using IL‐4 blocking antibody. Surprisingly, retinoic acid altered the APN/CD13 enzymatic activity only in nLP fibroblasts without modification of APN/CD13 expression. APN/CD13 is over‐expressed on psoriatic fibroblasts and exerted high level of activity compare to HD fibroblasts. Taken together, several factors such as CGRP and IL‐4 acted on positive regulation of APN/CD13 expression and activity. This study highlighted the interest of APN/CD13 as a new potential target, which should be investigated in psoriasis. APN/CD13 aminopeptidase‐N is upregalated in sporiatic skins compare to healthy donors. APN/CD13 is mostly increased in nLP (non lesional psoriatic) compare to LP (lesional psoriatic) skins. APN/CD13 is positively regulated by IL‐4 and CGRP.

  May 23, 2017   doi: 10.1002/jcp.25941   open full text
• Therapeutic potential of novel formulated forms of curcumin in the treatment of breast cancer by the targeting of cellular and physiological dysregulated pathways.
  Amir Tajbakhsh, Malihe Hasanzadeh, Mehdi Rezaee, Mostafa Khedri, Majid Khazaei, Soodabeh ShahidSales, Gordon A. Ferns, Seyed Mahdi Hassanian, Amir Avan.
  Journal of Cellular Physiology. May 23, 2017
  Breast cancer is among the most important causes of cancer related death in women. There is a need for novel agents for targeting key signaling pathways to either improve the efficacy of the current therapy, or reduce toxicity. There is some evidence that curcumin may have antitumor activity in breast cancer. Several clinical trials have investigated its activity in patients with breast cancer, including a recent trial in breast cancer patients receiving radiotherapy, in whom it was shown that curcumin reduced the severity of radiation dermatitis, although it is associated with low bioavailability. Several approaches have been developed to increase its absorption rate (e.g., nano crystals, liposomes, polymers, and micelles) and co‐delivery of curcumin with adjuvants as well as different conjugation to enhance its bioavailability. In particular, micro‐emulsions is an option for transdermal curcumin delivery, which has been reported to increase its absorption. Lipid‐based nano‐micelles is another approach to enhance curcumin absorption via gastrointestinal tract, while polymer‐based nano‐formulations (e.g., poly D, L‐lactic‐co‐glycolic [PLGA]) allows the release of curcumin at a sustained level. This review summarizes the current data of the therapeutic potential of novel formulations of curcumin with particular emphasis on recent preclinical and clinical studies in the treatment of breast cancer.

  May 23, 2017   doi: 10.1002/jcp.25961   open full text
• The effect of substrate stiffness on cancer cell volume homeostasis.
  Meng Wang, Na Chai, Baoyong Sha, Manyun Guo, Jian Zhuang, Feng Xu, Fei Li.
  Journal of Cellular Physiology. May 23, 2017
  Existing studies on the mechanism of cell volume regulation are mainly relevant to ion channels and osmosis in extracellular fluid. Recently, accumulating evidence has shown that cellular mechanical microenvironment also influences the cell volume. Herein, we investigated the regulation of substrate stiffness on the cell volume homeostasis of MCF‐7 cells and their following migration behaviors. We found that cell volume increases with increasing substrate stiffness, which could be affected by blocking the cell membrane anion permeability and dopamine receptor. In addition, the cell migration is significantly inhibited by decreasing the cell volume using tamoxifen and such inhibition effect on migration is enhanced by increasing substrate stiffness. The cell membrane anion permeability might be the linker between cellular mechanical microenvironment and cellular volume homeostasis regulation. This work revealed the regulation of substrate stiffness on cell volume homeostasis for the first time, which would provide a new perspective into the understanding of cancer metastasis and a promising anti‐cancer therapy through regulation of cell volume homeostasis. This article is protected by copyright. All rights reserved

  May 23, 2017   doi: 10.1002/jcp.26026   open full text
• Melatonin‐induced increase of lipid droplets accumulation and in vitro maturation in porcine oocytes is mediated by mitochondrial quiescence.
  Bin He, Chao Yin, Yabin Gong, Jie Liu, Huiduo Guo, Ruqian Zhao.
  Journal of Cellular Physiology. May 23, 2017
  Melatonin, the major pineal secretory product, has a significant impact on the female reproductive system. Recently, the beneficial effects of melatonin on mammalian oocyte maturation and embryonic development have drawn increased attention. However, the exact underlying mechanisms remain to be fully elucidated. This study demonstrates that supplementing melatonin to in vitro maturation (IVM) medium enhances IVM rate, lipid droplets (LDs) accumulation as well as triglyceride content in porcine oocytes. Decrease of mitochondrial membrane potential, mitochondrial respiratory chain complex IV activity as well as mitochondrial reactive oxygen species (mROS) content indicated that melatonin induced a decrease of mitochondrial activity. The copy number of mitochondrial DNA (mtDNA) which encodes essential subunits of oxidative phosphorylation (OXPHOS), was not affected by melatonin. However, the expression of mtDNA‐encoded genes was significantly down‐regulated after melatonin treatment. The DNA methyltransferase DNMT1, which regulates methylation and expression of mtDNA, was increased and translocated into the mitochondria in melatonin‐treated oocytes. The inhibitory effect of melatonin on the expression of mtDNA was significantly prevented by simultaneous addition of DNMT1 inhibitor, which suggests that melatonin regulates the transcription of mtDNA through up‐regulation of DNMT1 and mtDNA methylation. Increase of triglyceride contents after inhibition of OXPHOS indicated that mitochondrial quiescence is crucial for LDs accumulation in oocytes. Taken together, our results suggest that melatonin‐induced reduction in mROS production and increase in IVM, and LDs accumulation in porcine oocytes is mediated by mitochondrial quiescence.

  May 23, 2017   doi: 10.1002/jcp.25876   open full text
• NLRP3 inflammasome: Its regulation and involvement in atherosclerosis.
  Zahra Hoseini, Fatemeh Sepahvand, Bahman Rashidi, Amirhossein Sahebkar, Aria Masoudifar, Hamed Mirzaei.
  Journal of Cellular Physiology. May 23, 2017
  Inflammasomes are intracellular complexes involved in the innate immunity that convert proIL‐1β and proIL‐18 to mature forms and initiate pyroptosis via cleaving procaspase‐1. The most well‐known inflammasome is NLRP3. Several studies have indicated a decisive and important role of NLRP3 inflammasome, IL‐1β, IL‐18, and pyroptosis in atherosclerosis. Modern hypotheses introduce atherosclerosis as an inflammatory/lipid‐based disease and NLRP3 inflammasome has been considered as a link between lipid metabolism and inflammation because crystalline cholesterol and oxidized low‐density lipoprotein (oxLDL) (two abundant components in atherosclerotic plaques) activate NLRP3 inflammasome. In addition, oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and lysosome rupture, which are implicated in inflammasome activation, have been discussed as important events in atherosclerosis. In spite of these clues, some studies have reported that NLRP3 inflammasome has no significant effect in atherogenesis. Our review reveals that some molecules such as JNK‐1 and ASK‐1 (upstream regulators of inflammasome activation) can reduce atherosclerosis through inducing apoptosis in macrophages. Notably, NLRP3 inflammasome can also cause apoptosis in macrophages, suggesting that NLRP3 inflammasome may mediate JNK‐induced apoptosis, and the apoptotic function of NLRP3 inflammasome may be a reason for the conflicting results reported. The present review shows that the role of NLRP3 in atherogenesis can be significant. Here, the molecular pathways of NLRP3 inflammasome activation and the implications of this activation in atherosclerosis are explained.

  May 23, 2017   doi: 10.1002/jcp.25930   open full text
• Targeting RAS signaling pathway as a potential therapeutic target in the treatment of colorectal cancer.
  Afsane Bahrami, Seyed Mahdi Hassanian, Soodabeh ShahidSales, Zahra Farjami, Malihe Hasanzadeh, Kazem Anvari, Amir Aledavood, Mina Maftouh, Gordon A. Ferns, Majid Khazaei, Amir Avan.
  Journal of Cellular Physiology. May 23, 2017
  The V‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) is frequently dysregulated in colorectal cancer (CRC). It is involved in the modulation of several downstream effectors, that include: Raf/Mek/Erk, PI3K/Akt, RalGDS/p38MAPK, and Rac/Rho, and thereby influences tumorigenesis, the invasive behaviors of tumor cell, and resistance to therapy. There is growing evidence exploring the use of drugs that target these pathways in the treatment of CRC. Cetuximab has been approved for CRC patients without a KRAS mutation, or for EGFR‐expressing metastatic CRC, although some of the patients have a mutation of KRAS and NRAS. This review summarizes the recent knowledge about the therapeutic potential of targeting RAS with particular emphasis on recent preclinical and clinical studies in treatment of CRC.

  May 23, 2017   doi: 10.1002/jcp.25890   open full text
• Fibroblast dynamics as an in vitro screening platform for anti‐fibrotic drugs in primary myelofibrosis.
  Ciprian Tomuleasa, Sonia Selicean, Grigore Gafencu, Bobe Petrushev, Laura Pop, Cristian Berce, Anca Jurj, Adrian Trifa, Ana‐Maria Rosu, Sergiu Pasca, Lorand Magdo, Mihnea Zdrenghea, Delia Dima, Alina Tanase, Ioana Frinc, Anca Bojan, Ioana Berindan‐Neagoe, Gabriel Ghiaur, Stefan O. Ciurea.
  Journal of Cellular Physiology. May 23, 2017
  Although the cause for bone marrow fibrosis in patients with myelofibrosis remains controversial, it has been hypothesized that it is caused by extensive fibroblast proliferation under the influence of cytokines generated by the malignant megakaryocytes. Moreover, there is no known drug therapy which could reverse the process. We studied the fibroblasts in a novel system using the hanging drop method, evaluated whether the fibroblasts obtain from patients are part of the malignant clone of not and, using this system, we screen a large library of FDA‐approved drugs to identify potential drugs candidates that might be useful in the treatment of this disease, specifically which would inhibit fibroblast proliferation and the development of bone marrow fibrosis. We have found that the BM fibroblasts are not part of the malignant clone, as previously suspected and two immunosuppressive medications—cyclosporine and mycophenolate mophetil, as most potent suppressors of the fibroblast collagen production thus potentially inhibitors of bone marrow fibrosis production in myelofibrosis. Although the cause for bone marrow fibrosis in patients with myelofibrosis remains controversial, it has been hypothesized that it is caused by extensive fibroblast proliferation under the influence of cytokines generated by the malignant megakaryocytes. Moreover, there is no known drug therapy which could reverse the process. We studied the fibroblasts in a novel system using the hanging drop method, evaluated whether the fibroblasts obtain from patients are part of the malignant clone of not and, using this system, we screen a large library of FDA‐approved drugs to identify potential drugs candidates that might be useful in the treatment of this disease, specifically which would inhibit fibroblast proliferation and the development of bone marrow fibrosis. We have found that the BM fibroblasts are not part of the malignant clone, as previously suspected and two immunosuppressive medications—cyclosporine and mycophenolate mophetil, as most potent suppressors of the fibroblast collagen production thus potentially inhibitors of bone marrow fibrosis production in myelofibrosis.

  May 23, 2017   doi: 10.1002/jcp.25902   open full text
• Genetic variants as potential predictive biomarkers in advanced colorectal cancer patients treated with oxaliplatin‐based chemotherapy.
  Afsane Bahrami, Forouzan Amerizadeh, Seyed Mahdi Hassanian, Soodabeh ShahidSales, Majid Khazaei, Mina Maftouh, Majid Ghayour‐Mobarhan, Gordon A. Ferns, Amir Avan.
  Journal of Cellular Physiology. May 23, 2017
  Chemotherapy regimen containing oxaliplatin is often the first‐line treatment for patient with advanced colorectal cancer. Oxaliplatin binds to DNA, leading to the formation of crosslinks and bulky adducts. Approximately 50% of patients with CRC benefit from treatment with oxaliplatin. It is possible that genetic variants in biological pathways involved in drug transportation, drug metabolism, DNA damage repair, and cell cycle modulation might affect the activity, or efficacy of oxaliplatin. Because oxaliplatin resistance may be related to these genetic variants and may therefore be an important reason for treatment failure, we have summarized the genetic variations that have been reported to be predictive markers of the response to oxaliplatin based therapy in patients with advanced CRC.

  May 23, 2017   doi: 10.1002/jcp.25966   open full text
• GD2‐targeted immunotherapy and potential value of circulating microRNAs in neuroblastoma.
  Sharareh Gholamin, Hamed Mirzaei, Seyed‐Mostafa Razavi, Seyed Mahdi Hassanian, Leila Saadatpour, Aria Masoudifar, Soodabeh ShahidSales, Amir Avan.
  Journal of Cellular Physiology. May 23, 2017
  Neuroblastoma (NB) with various clinical presentation is a known childhood malignancy. Despite significant progress in treatment of NB afflicted patients, high risk disease is usually associated with poor outcome, resulting in long‐term survival of less that 50%. Known as a disease most commonly originated form the nerve roots, the variants involved in NB imitation and progression remain to be elucidated. The outcome of low to intermediate risk disease is favorable whereas the high risk NB disease with dismal prognosis, positing the necessity of novel approaches for early detection and prognostication of advanced disease. Tailored immunotherapy approaches have shown significant improvement in high‐risk NB patients. It has found a link between Gangliosides and progression of NB. The vast majority of neuroblastoma tumors express elevated levels of GD2, opening new insight into using anti‐GD2 drugs as potential treatments for NBs. Implication of anti‐GD2 monoclonal antibodies for treatment of high risk NBs triggers further investigation to unearth novel biomarkers as prognostic and response biomarker to guide additional multimodal tailored treatment approaches. A growing body of evidence supports the usefulness of miRNAs to evaluate high risk NBs response to anti‐GD2 drugs and further prevent drug‐related toxicities in refractory or recurrent NBs. miRNAs and circulating proteins in body fluids (plasma and serum) present as potential biomarkers in early detection of NBs. Here, we summarize various biomarkers involved in diagnosis, prognosis and response to treatment in patients with NB. We further attempted to overview prognostic biomarkers in response to treatment with anti‐GD2 drugs.

  May 23, 2017   doi: 10.1002/jcp.25793   open full text
• The therapeutic potential of targeting the BRAF mutation in patients with colorectal cancer.
  Afsane Bahrami, AmirReza Hesari, Majid Khazaei, Seyed Mahdi Hassanian, Gordon A. Ferns, Amir Avan.
  Journal of Cellular Physiology. May 23, 2017
  Colorectal cancer is among the most lethal malignancies globally. BRAF is a member of the RAS/RAF/MEK/ERK signaling pathway. Its constitutive activation can result in increased cellular growth, development, invasion, and resistance to therapy. A mutation of the BRAF gene is present in 5–10% of metastatic colorectal cancers. BRAF mutations have been found to predict a lack of benefit to anti‐EGFR therapy in metastatic CRC. Furthermore, CRC containing the BRAF V600E mutation display an innate resistance to BRAF inhibitors. The mechanisms of cell resistance can be explained at least in part by ERK dependent and ERK in‐dependent pathway. Clinical trials evaluating the combinations of BRAF, PI3K, EGFR, and/or MEK inhibitors have revealed promising activity in BRAF mutant containing CRCs. There may be some benefit from future studies that focus on improving the efficacy of combined therapy in CRC with respect to the sustained effects. The aim of current review is to give an overview about the current status and prospective regarding the therapeutic potential of targeting BRAF mutant colorectal cancer.

  May 23, 2017   doi: 10.1002/jcp.25952   open full text
• Roles of neurotrophins in skeletal tissue formation and healing.
  Yu‐Wen Su, Xin‐Fu Zhou, Bruce K. Foster, Brian L. Grills, Jiake Xu, Cory J. Xian.
  Journal of Cellular Physiology. May 23, 2017
  Neurotrophins and their receptors are key molecules that are known to be critical in regulating nervous system development and maintenance and have been recognized to be also involved in regulating tissue formation and healing in skeletal tissues. Studies have shown that neurotrophins and their receptors are widely expressed in skeletal tissues, implicated in chondrogenesis, osteoblastogenesis, and osteoclastogenesis, and are also involved in regulating tissue formation and healing events in skeletal tissue. Increased mRNA expression for neurotrophins NGF, BDNF, NT‐3, and NT‐4, and their Trk receptors has been observed in injured bone tissues, and NT‐3 and its receptor, TrkC, have been identified to have the highest induction at the injury site in a drill‐hole injury repair model in both bone and the growth plate. In addition, NT‐3 has also recently been shown to be both an osteogenic and angiogenic factor, and this neurotrophin can also enhance expression of the key osteogenic factor, BMP‐2, as well as the major angiogenic factor, VEGF, to promote bone formation, vascularization, and healing of the injury site. Further studies, however, are needed to investigate if different neurotrophins have differential roles in skeletal repair, and if NT‐3 can be a potential target of intervention for promoting bone fracture healing. Increasing evidence now suggests that neurotrophins also have important roles in bone. Among the neurotrophins and receptors, the NT‐3/TrkC signaling (induced far more prominently compared to other NTs and other Trk receptors) may potentially play a prominent role in the bone healing.

  May 23, 2017   doi: 10.1002/jcp.25936   open full text
• Connexin43 intercellular communication drives the early differentiation of human bone marrow stromal cells into osteoblasts.
  Julie Talbot, Régis Brion, Audrey Lamora, Mathilde Mullard, Sarah Morice, Dominique Heymann, Franck Verrecchia.
  Journal of Cellular Physiology. May 23, 2017
  Although it has been demonstrated that human bone marrow stromal cells (hBMSCs) express the ubiquitous connexin43 (Cx43) and form functional gap junctions, their role in the early differentiation of hBMSCs into osteoblasts remains poorly documented. Using in vitro assays, we show that Cx43 expression and gap junctional intercellular communication (GJIC) are increased during the differentiation of hBMSCs into osteoblasts, both at the protein and mRNA levels. Two independent procedures to reduce GJIC, a pharmacological approach with GJIC inhibitors (18α‐glycyrrhetinic acid and Gap27 peptide) and a molecular approach using small interfering RNA against Cx43, demonstrated that the presence of Cx43 and functional junctional channels are essential to the ability of hBMSCs to differentiate into osteoblasts in vitro. In addition, a reduced GJIC decreases the expression of Runx2, the major transcription factor implicated in the control of osteoblast commitment and early differentiation of hBMSCs into osteoblasts, suggesting that GJIC mediated by Cx43 is implicated in this process. Together our results demonstrate that GJIC mediated by the Cx43 channels plays a central role throughout the differentiation of hBMSC into osteoblasts, from the early stages to the process of mineralization. Our results demonstrate that GJIC mediated by the Cx43 channels plays a central role throughout the differentiation of hBMSC into osteoblasts, from the early stages to the process of mineralization.

  May 23, 2017   doi: 10.1002/jcp.25938   open full text
• TWEAK promotes migration and invasion in MEFs through a mechanism dependent on ERKs activation and Fibulin 3 down‐regulation.
  Celia Sequera, Ana Vázquez‐Carballo, María Arechederra, Sonia Fernández‐Veledo, Almudena Porras.
  Journal of Cellular Physiology. May 23, 2017
  TWEAK regulates multiple physio‐pathological processes in fibroblasts such as fibrosis. It also induces migration and invasion in tumors and it can activate p38 MAPK in various cell types. Moreover, p38α MAPK promotes migration and invasion in several cancer cells types and in mouse embryonic fibroblasts (MEFs). However, it remains unknown if TWEAK could promote migration in fibroblasts and whether p38α MAPK might play a role. Our results reveal that TWEAK activates ERKs, Akt, and p38α/β MAPKs and reduces secreted Fibulin 3 in MEFs. TWEAK also increases migration and invasion in wt and p38α deficient MEFs, which indicates that p38α MAPK is not required to mediate these effects. In contrast, ERKs inhibition significantly decreases TWEAK‐induced migration and Fibulin 3 knock‐down mimics TWEAK effect. These results indicate that both ERKs activation and Fibulin 3 down‐regulation would contribute to mediate TWEAK pro‐migratory effect. In fact, the additional regulation of ERKs and/or p38β as a consequence of Fibulin 3 decrease might be also involved in the pro‐migratory effect of TWEAK in MEFs. In conclusion, our studies uncover novel mechanisms by which TWEAK would favor tissue repair by promoting fibroblasts migration. TWEAK promotes migration of mouse embryonic fibroblasts through ERKs activation and Fibulin 3 down‐regulation. The additional regulation of ERKs and/or p38beta as a consequence of Fibulin 3 decrease might be also involved in this pro‐migratory effect of TWEAK.

  May 23, 2017   doi: 10.1002/jcp.25942   open full text
• The genetic factors contributing to the development of Wilm's tumor and their clinical utility in its diagnosis and prognosis.
  Afsane Bahrami, Marjan Joodi, Mina Maftooh, Gordon A. Ferns, Mehrdad Ahmadi, Seyed Mahdi Hassanian, Amir Avan.
  Journal of Cellular Physiology. May 20, 2017
  Mutations in the Wilm's tumor 1 (WT1) gene are associated with a wide spectrum of renal manifestations, ultimately leading to end‐stage kidney failure. There is an inadequate understanding of the molecular functions of WT1 in renal development, and this has limited the potential for therapeutic interventions in WT1‐related diseases. In this review we discuss the existing data on the genetic and epigenetic abnormalities that have been described in WTs and their potential utility as biomarkers for risk stratification, prediction and prognosis in patients withWTs. This article is protected by copyright. All rights reserved

  May 20, 2017   doi: 10.1002/jcp.26021   open full text
• Humanized Mice: A Brief Overview On Their Diverse Applications In Biomedical Research.
  Shigeyoshi Fujiwara.
  Journal of Cellular Physiology. May 20, 2017
  Model animals naturally differ from humans in various respects and results from the former are not directly translatable to the latter. One approach to address this issue is humanized mice that are defined as mice engrafted with functional human cells or tissues. In humanized mice, we can investigate the development and function of human cells or tissues (including their products encoded by human genes) in the in vivo context of a small animal. As such, humanized mouse models have played important roles that cannot be substituted by other animal models in various areas of biomedical research. Although there are obvious limitations in humanized mice and we may need some caution in interpreting the results obtained from them, it is reasonably expected that they will be utilized in increasingly diverse areas of biomedical research, as the technology for preparing humanized mice are rapidly improved. In this review, I will describe the methodology for generating humanized mice and overview their recent applications in various disciplines including immunology, infectious diseases, drug metabolism, and neuroscience. This article is protected by copyright. All rights reserved

  May 20, 2017   doi: 10.1002/jcp.26022   open full text
• GLUT 1 receptor expression and circulating levels of fasting glucose in high grade serous ovarian cancer.
  L Pizzuti, D Sergi, Chiara Mandoj, B Antoniani, Francesca Sperati, Andrea Chirico, Luigi Di Lauro, Mario Valle, Alfredo Garofalo, Enrico Vizza, Gabriele Corrado, Federica Tomao, Massimo Rinaldi, Silvia Carpano, M Maugeri‐Saccà, Laura Conti, Giovanna Digiesi, Paolo Marchetti, Ruggero De Maria, A Giordano, Maddalena Barba, Maria Antonietta Carosi, Patrizia Vici.
  Journal of Cellular Physiology. May 20, 2017
  In recent years, the poorly remarkable goals achieved in terms of patients' important outcomes for ovarian cancer have fueled our interest towards the study of its metabolic roots. Within this research pipeline, we assessed the association between the expression of the glucose transporter GLUT1, as expressed at the tumor tissue level, and circulating pre‐surgical levels of fasting glucose in a case series including data from 40 patients with high FIGO stage serous ovarian cancer. Patients who provided data to the current analysis were randomly selected from a larger cohort. To our purposes, the procedures related to serum and tissue collection, storage and biomarker assessment were highly standardized and centralized at the institutional laboratories. The GLUT1 antibody SPM498 SPRING (REF. E13810) was used at a 1:500 dilution in 2 µm slides. Staining for GLUT1 was observed at the cell membrane level in all the cases assessed, but strong staining was described in 29 (72.5) of them. The agreement between the two independent reviewers was 100%. Strong GLUT1 staining was inversely associated with the circulating levels of fasting glucose, with a particularly striking difference in the distribution for patients in the lowest fasting glucose tertile (p = 0.044). These results support the biological plausibility of the association of interest. If confirmed in larger studies, our findings may help clarify the potentials of biomarkers related to energy metabolism in terms of prognosis definition, treatment assignment and outcome interpretation for patients with high FIGO stage serous ovarian cancer. This article is protected by copyright. All rights reserved

  May 20, 2017   doi: 10.1002/jcp.26023   open full text
• TNF‐α has both stimulatory and inhibitory effects on mouse monocyte‐derived osteoclastogenesis.
  Yixuan Cao, Ineke D.C. Jansen, Sara Sprangers, Teun J. de Vries, Vincent Everts.
  Journal of Cellular Physiology. May 20, 2017
  Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31hiLy‐6C‐), myeloid blasts (CD31+Ly‐6C+) and monocytes (CD31‐Ly‐6Chi) were sorted from mouse bone marrow using flow cytometry and cultured with M‐CSF and RANKL, with or without TNF‐α. Surprisingly, TNF‐α prevented the differentiation of TRAcP+ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes' osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1 and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes' osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone. This article is protected by copyright. All rights reserved

  May 20, 2017   doi: 10.1002/jcp.26024   open full text
• Estrogen receptor β regulates the tumoral suppressor PTEN to modulate pituitary cell growth.
  Pablo Anibal Perez, Juan Pablo Petiti, Florencia Picech, Carolina Beatriz Guido, Liliana Valle Sosa, Ezequiel Grondona, Jorge Humberto Mukdsi, Ana Lucia De Paul, Alicia Ines Torres, Silvina Gutierrez.
  Journal of Cellular Physiology. May 20, 2017
  In this study, we focused on ERβ regulation in the adenohypophysis under different estrogenic milieu, by analyzing whether ER modulates the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression and its subcellular localization on anterior pituitary glands from Wistar rats and GH3 lactosomatotroph cells that over‐expressed ERβ. ERβ was regulated in a cyclic manner, and underwent dynamic changes throughout the estrous cycle, with decreased ERβ+ cells in estrus and under E2 treatment, but increased in ovariectomized rats. In addition, the ERα/β ratio increased in estrus and under E2 stimulation, but decreased in ovariectomized rats. Double immunofluorescence revealed that lactotroph and somatotroph ERβ+ were significantly decreased in estrus. Also, variations in the PTEN expression was observed, which was diminished with high E2 conditions but augmented with low E2 milieu. The subcellular localization of this phosphatase was cell cycle–dependent, with remarkable changes in the immunostaining pattern: nuclear in arrested pituitary cells but cytoplasmic in stimulated cells, and responding differently to ER agonists, with only DPN being able to increase PTEN expression and retaining it in the nucleus. Finally, ERβ over‐expression increased PTEN with a noticeable subcellular redistribution, and with a significant nuclear signal increase in correlation with an increase of cells in G0/G1 phase. These results showed that E2 is able to inhibit ERβ expression and suggests that the tumoral suppressor PTEN might be one of the signaling proteins by which E2, through ERβ, acts to modulate pituitary cell proliferation, thereby adapting endocrine populations in relation with hormonal necessities. This article is protected by copyright. All rights reserved

  May 20, 2017   doi: 10.1002/jcp.26025   open full text
• Identification and characterization of site‐specific N‐glycosylation in the potassium channel Kv3.1b.
  Paul Christian Vicente, Jin Young Kim, Jeong‐Ju Ha, Min‐Young Song, Hyun‐Kyung Lee, Dong‐Hyun Kim, Jin‐Sung Choi, Kang‐Sik Park.
  Journal of Cellular Physiology. May 19, 2017
  The potassium ion channel Kv3.1b is a member of a family of voltage‐gated ion channels that are glycosylated in their mature form. In the present study, we demonstrate the impact of N‐glycosylation at specific asparagine residues on the trafficking of the Kv3.1b protein. Large quantities of asparagine 229 (N229)‐glycosylated Kv3.1b reached the plasma membrane, whereas N220‐glycosylated and unglycosylated Kv3.1b were mainly retained in the endoplasmic reticulum (ER). These ER‐retained Kv3.1b proteins were susceptible to degradation, when co‐expressed with calnexin, whereas Kv3.1b pools located at the plasma membrane were resistant. Mass spectrometry analysis revealed a complex type Hex3HexNAc4Fuc1 glycan as the major glycan component of the N229‐glycosylated Kv3.1b protein, as opposed to a high‐mannose type Man8GlcNAc2 glycan for N220‐glycosylated Kv3.1b. Taken together, these results suggest that trafficking‐dependent roles of the Kv3.1b potassium channel are dependent on N229 site‐specific glycosylation and N‐glycan structure, and operate through a mechanism whereby specific N‐glycan structures regulate cell surface expression. Site‐specific N‐glycosylation and N‐glycan structure regulate cell surface trafficking and expression of the Kv3.1b potassium channel. Mass spectrometry analysis identified Man8GlcNAc2 (high‐mannose type) and Hex3HexNAc4Fuc1 (complex type) as the glycan moieties on N220 and N229.

  May 19, 2017   doi: 10.1002/jcp.25915   open full text
• Transplantation of mesenchymal stem cells overexpressing IL10 attenuates cardiac impairments in rats with myocardial infarction.
  Xin Meng, Jianping Li, Ming Yu, Jian Yang, Minjuan Zheng, Jinzhou Zhang, Chao Sun, Hongliang Liang, Liwen Liu.
  Journal of Cellular Physiology. May 19, 2017
  Mesenchymal stem cell (MSC) has been well known to exert therapeutic potential for patients with myocardial infarction (MI). In addition, interleukin‐10 (IL10) could attenuate MI through suppressing inflammation. Thus, the combination of MSC implantation with IL10 delivery may extend health benefits to ameliorate cardiac injury after MI. Here we established overexpression of IL10 in bone marrow‐derived MSC through adenoviral transduction. Cell viability, apoptosis, and IL10 secretion under ischemic challenge in vitro were examined. In addition, MSC was transplanted into the injured hearts in a rat model of MI. Four weeks after the MI induction, MI, cardiac functions, apoptotic cells, and inflammation cytokines were assessed. In response to in vitro oxygen‐glucose deprivation (OGD), IL10 overexpression in MSC (Ad.IL10‐MSC) enhanced cell viability, decreased apoptosis, and increased IL10 secretion. Consistently, the implantation of Ad.IL10‐MSCs into MI animals resulted in more reductions in myocardial infarct size, cardiac impairment, and cell apoptosis, compared to the individual treatments of either MSC or IL10 administration. Moreover, the attenuation of both systemic and local inflammations was most prominent for Ad.IL10‐MSC treatment. IL10 overexpression and MSC may exert a synergistic anti‐inflammatory effect to alleviate cardiac injury after MI. IL10 overexpression in mesenchymal stem cells enhances cell viability and decreases apoptosis. Implantation of IL10‐overexpressed mesenchymal stem cells into myocardial infarct animals results in more reductions in myocardial infarct size, cardiac impairment, and cell apoptosis.

  May 19, 2017   doi: 10.1002/jcp.25919   open full text
• All‐trans retinoic acid ameliorates hepatic stellate cell activation via suppression of thioredoxin interacting protein expression.
  Hiroki Shimizu, Toshiaki Tsubota, Keita Kanki, Goshi Shiota.
  Journal of Cellular Physiology. May 19, 2017
  Activation of hepatic stellate cells (HSCs) is the effector factor of hepatic fibrosis and hepatocellular carcinoma (HCC) development. Accumulating evidence suggests that retinoic acids (RAs), derivatives of vitamin A, contribute to prevention of liver fibrosis and carcinogenesis, however, regulatory mechanisms of RAs still remain exclusive. To elucidate RA signaling pathway, we previously performed a genome‐wide screening of RA‐responsive genes by in silico analysis of RA‐response elements, and identified 26 RA‐responsive genes. We found that thioredoxin interacting protein (TXNIP), which inhibits antioxidant activity of thioredoxin (TRX), was downregulated by all‐trans retinoic acid (ATRA). In the present study, we demonstrate that ATRA ameliorates activation of HSCs through TXNIP suppression. HSC activation was attenuated by TXNIP downregulation, whereas potentiated by TXNIP upregulation, indicating that TXNIP plays a crucial role in activation of HSCs. Notably, we showed that TXNIP‐mediated HSC activation was suppressed by antioxidant N‐acetylcysteine. In addition, ATRA treatment or downregulation of TXNIP clearly declined oxidative stress levels in activated HSCs. These data suggest that ATRA plays a key role in inhibition of HSC activation via suppressing TXNIP expression, which reduces oxidative stress levels. ATRA plays a key role in inhibition of HSC activation via suppressing TXNIP expression, which reduces oxidative stress levels.

  May 19, 2017   doi: 10.1002/jcp.25921   open full text
• The role of Runx2 in facilitating autophagy in metastatic breast cancer cells.
  Manish Tandon, Ahmad H. Othman, Vivek Ashok, Gary S. Stein, Jitesh Pratap.
  Journal of Cellular Physiology. May 19, 2017
  Breast cancer metastases cause significant patient mortality. During metastases, cancer cells use autophagy, a catabolic process to recycle nutrients via lysosomal degradation, to overcome nutritional stress for their survival. The Runt‐related transcription factor, Runx2, promotes cell survival under metabolic stress, and regulates breast cancer progression and bone metastases. Here, we identify that Runx2 enhances autophagy in metastatic breast cancer cells. We defined Runx2 function in cellular autophagy by monitoring microtubule‐associated protein light chain (LC3B‐II) levels, an autophagy‐specific marker. The electron and confocal microscopic analyses were utilized to identify alterations in autophagic vesicles. The Runx2 knockdown cells accumulate LC3B‐II protein and autophagic vesicles due to reduced turnover. Interestingly, Runx2 promotes autophagy by enhancing trafficking of LC3B vesicles. Our mechanistic studies revealed that Runx2 promotes autophagy by increasing acetylation of α‐tubulin sub‐units of microtubules. Inhibiting autophagy decreased cell adhesion and survival of Runx2 knockdown cells. Furthermore, analysis of LC3B protein in clinical breast cancer specimens and tumor xenografts revealed significant association between high Runx2 and low LC3B protein levels. Our studies reveal a novel regulatory mechanism of autophagy via Runx2 and provide molecular insights into the role of autophagy in metastatic cancer cells. We demonstrate a novel function of Runx2 in facilitating autophagy by altering cytoskeletal elements during cellular stress conditions such as nutrient deprivation. Our results in bone metastatic MDA‐MB‐231 breast cancer cells show that Runx2 promotes autophagy via α‐tubulin acetylation and autophagosome trafficking. Since autophagy promotes tumor cell adhesion, the Runx2‐mediated autophagy supporting cell adhesion highlights its function in metastatic breast cancer cell survival.

  May 19, 2017   doi: 10.1002/jcp.25916   open full text
• Gax suppresses chemerin/CMKLR1‐induced preadipocyte biofunctions through the inhibition of Akt/mTOR and ERK signaling pathways.
  Yunqi Jiang, Ping Liu, Wenlin Jiao, Juan Meng, Jinbo Feng.
  Journal of Cellular Physiology. May 19, 2017
  Adipose tissue is closely associated with angiogenesis and vascular remodeling. Chemerin is involved in inflammatory reaction and vascular dysfunction. However, the mechanisms of chemerin participating in vascular remodeling and whether Growth arrest‐specific homeobox (Gax) can effectively intervene it remain obscured. Here, 3T3‐F442A preadipocytes were cultured, injected into athymic mice to model fat pads, and treated respectively with Ad‐chemerin, Ad‐Gax, or specific inhibitors in vitro and in vivo. MTT, flow cytometry, Western blotting, and imunohisto(cyto)‐chemistry analyses showed that chemerin enhanced the expression of FABP4 and VEGF, activated Akt/mTOR and ERK pathways, increased the cell percent of S phase, decreased the percent of G0‐G1 phase and apoptotic cells, and augmented neovascular density in fat pads. Inversely, Gax suppressed the expression of these adipogenic and vasifactive markers and these signaling proteins, decreased the percent of S phase cells, and increased those of G0‐G1 phase and apoptotic cells, and reduced the neovascular density. Our results indicate that chemerin‐CMKLR1 activates Akt/mTOR and ERK pathways and facilitates preadipocyte proliferation, adipogenesis, and angiogenesis. Contrarily, Gax weakens the effect of chemerin on preadipocyte biofunctions. Chemerin/CMKLR1 activates Akt/mTOR/p70SK1/4EBP1 and ERK1/2 pathways and enhances preadipocyte proliferation, adipogenesis, and angiogenesis. Contrarily, Gax weakens the effect of chemerin on the preadipocyte biofunctions.

  May 19, 2017   doi: 10.1002/jcp.25918   open full text
• Global chondrocyte gene expression after a single anabolic loading period: Time evolution and re‐inducibility of mechano‐responses.
  Simone Scholtes, Elisabeth Krämer, Melanie Weisser, Wolfgang Roth, Reto Luginbühl, Tobias Grossner, Wiltrud Richter.
  Journal of Cellular Physiology. May 19, 2017
  Aim of this study was a genome‐wide identification of mechano‐regulated genes and candidate pathways in human chondrocytes subjected to a single anabolic loading episode and characterization of time evolution and re‐inducibility of the response. Osteochondral constructs consisting of a chondrocyte‐seeded collagen‐scaffold connected to β‐tricalcium‐phosphate were pre‐cultured for 35 days and subjected to dynamic compression (25% strain, 1 Hz, 9 × 10 min over 3 hr) before microarray‐profiling was performed. Proteoglycan synthesis was determined by 35S‐sulfate‐incorporation over 24 hr. Cell viability and hardness of constructs were unaltered by dynamic compression while proteoglycan synthesis was significantly stimulated (1.45‐fold, p = 0.016). Among 115 significantly regulated genes, 114 were up‐regulated, 48 of them ≥ twofold. AP‐1‐relevant transcription factors FOSB and FOS strongly increased in line with elevated ERK1/2‐phosphorylation and rising MAP3K4 expression. Expression of proteoglycan‐synthesizing enzymes CHSY1 and GALNT4 was load‐responsive as were factors associated with the MAPK‐, TGF‐β‐, calcium‐, retinoic‐acid‐, Wnt‐, and Notch‐signaling pathway which were significantly upregulated SOX9, and BMP6 levels rose significantly also after multiple loading episodes at daily intervals even at the 14th cycle with no indication for desensitation. Canonical pSmad2/3 and pSmad1/5/9‐signaling showed no consistent regulation. This study associates novel genes with mechanoregulation in chondrocytes, raising SOX9 protein levels with anabolic loading and suggests that more pathways than so far anticipated apparently work together in a complex network of stimulators and feedback‐regulators. Upregulation of mechanosensitive indicators extending differentially into the resting time provides crucial knowledge to maximize cartilage matrix deposition for the generation of high‐level cartilage replacement tissue. We here identified mechano‐regulation of SOX9 in chondrocytes in the center of a complex signaling network of an anabolic loading context and describe its robust re‐inducibility in daily intervals without desensitization. We suggest to use SOX9 protein levels and a promising selection of critical mechanosensitive genes as diagnostic markers to optimize loading regimes for future refinement of anabolic loading protocols to enhance cartilage tissue functionality in vitro and of tissue formation after surgical therapy in patients in vivo.

  May 19, 2017   doi: 10.1002/jcp.25933   open full text
• Acute ethanol exposure‐induced autophagy‐mediated cardiac injury via activation of the ROS‐JNK‐Bcl‐2 pathway.
  Zhongxin Zhu, Yewei Huang, Lingchun Lv, Youli Tao, Minglong Shao, Congcong Zhao, Mei Xue, Jia Sun, Chao Niu, Yang Wang, Sunam Kim, Weitao Cong, Wei Mao, Litai Jin.
  Journal of Cellular Physiology. May 19, 2017
  Binge drinking is associated with increased cardiac autophagy, and often triggers heart injury. Given the essential role of autophagy in various cardiac diseases, this study was designed to investigate the role of autophagy in ethanol‐induced cardiac injury and the underlying mechanism. Our study showed that ethanol exposure enhanced the levels of LC3‐II and LC3‐II positive puncta and promoted cardiomyocyte apoptosis in vivo and in vitro. In addition, we found that ethanol induced autophagy and cardiac injury largely via the sequential triggering of reactive oxygen species (ROS) accumulation, activation of c‐Jun NH2‐terminal kinase (JNK), phosphorylation of Bcl‐2, and dissociation of the Beclin 1/Bcl‐2 complex. By contrast, inhibition of ethanol‐induced autophagic flux with pharmacologic agents in the hearts of mice and cultured cells significantly alleviated ethanol‐induced cardiomyocyte apoptosis and heart injury. Elimination of ROS with the antioxidant N‐acetyl cysteine (NAC) or inhibition of JNK with the JNK inhibitor SP600125 reduced ethanol‐induced autophagy and subsequent autophagy‐mediated apoptosis. Moreover, metallothionein (MT), which can scavenge reactive oxygen and nitrogen species, also attenuated ethanol‐induced autophagy and cell apoptosis in MT‐TG mice. In conclusion, our findings suggest that acute ethanol exposure induced autophagy‐mediated heart toxicity and injury mainly through the ROS‐JNK‐Bcl‐2 signaling pathway. Acute ethanol‐induces cardiac dysfunction and cardiomyocyte apoptosis through upregulated autophagy. Acute ethanol modulation of autophagic activity may be mediated by the ROS‐JNK‐Bcl‐2 signaling pathway.

  May 19, 2017   doi: 10.1002/jcp.25934   open full text
• Autophagy maintains the integrity of endothelial barrier in LPS‐induced lung injury.
  Dan Zhang, Jian Zhou, Le Chi Ye, Jing Li, Zhenzhou Wu, Yuping Li, Chichi Li.
  Journal of Cellular Physiology. May 19, 2017
  Understanding the role and underlying regulation mechanism of autophagy in lipopolysaccharide‐induced lung injury (LPS‐LI) may provide potentially new pharmacological targets for treatment of acute lung injury. The aim of this study was to investigate the functional significance of autophagy in LPS‐LI. The autophagy of human pulmonary microvascular endothelial cells (HPMVECs) and mice was inhibited before they were challenged with LPS. In vitro, permeability, vitality, and the LDH release rate of the cells were detected, the zonula occluden‐1 (ZO‐1) expression and the stress fiber formation were determined. In vivo, the lung injury was assessed. We found LPS caused high permeability and increased lactate dehydrogenase (LDH) release rate, lowered viability of the cells, inhibited the ZO‐1 expression and induced stress fiber formation, these effects were further aggravated by prohibiting the level of autophagy. Consistently, in in vivo experiments, LPS‐induced serious lung injury, which was reflected as edema, leukocyte infiltration and hemorrhage in lung tissue, and the high concentration of pro‐inflammation cytokines tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β in bronchoalveolar lavage fluid (BALF). Inhibiting autophagy further exacerbated LPS‐LI. It appears that autophagy played a protective role in LPS‐LI in part through restricting the injury of lung microvascular barrier.

  May 19, 2017   doi: 10.1002/jcp.25928   open full text
• Mechanism of prolactin inhibition of miR‐135b via methylation in goat mammary epithelial cells.
  Zhi Chen, Jun Luo, ChangHui Zhang, Yue Ma, Shuang Sun, Tianyin Zhang, Juan J. Loor.
  Journal of Cellular Physiology. May 19, 2017
  Prolactin is an important endocrine activator of lactogenesis. This study investigated the function and mechanism of miR‐135b in the enhancement of lactation by prolactin in goat mammary epithelial tissue. We utilized S‐Poly (T) sequencing to evaluate changes in gene regulation in the goat mammary gland after incubation with 2.5 μg/ml prolactin and 2.5 μg/ml IGF‐1 by examining highly expressed miRNAs during early lactation and late‐lactation. The results illustrated that miR‐135b is highly expressed in the goat mammary gland during early lactation and late‐lactation, and also after treatment with 2.5 μg/ml prolactin and 2.5 μg/ml IGF‐1. We used Q‐RT PCR, Western Blot, immunofluorescence, and luciferase reporter assay analysis, and found that PRL was significantly down‐regulated in response to the expression of miR‐135b in a manner that was functionally related to TAG synthesis via the large tumor suppressor 2 gene (Lats2), an important regulator of adipocyte proliferation via Hippo Signaling. Furthermore, using bisulfite‐sequencing PCR (BSP), Q‐PCR, and Western Blot we discovered an increase in expression of DNMT I (DNA methyl transferase I) in goat mammary epithelial cells with the 2.5 μg/ml PRL incubation, which led to DNA methylation of the CpG island upstream of miR‐135b and inhibited the transcription and expression of miR‐135b. This study clarified the mechanism by which PRL induces milk formation and identified a role of miR‐135b in this process. With PRL stimulation, the expression of DNMT I in mammary epithelial cells increased, resulting in DNA methylation of the CpG island upstream of miR‐135b's, thus inhibiting transcription of miR‐135b. At the same time, down‐regulation of miR‐135b resulted in increased expression of the target gene LATS2 thereby causing proliferation and metabolic changes in the mammary gland, eventually leading to lactation.

  May 19, 2017   doi: 10.1002/jcp.25925   open full text
• Primary cilium alterations and expression changes of Patched1 proteins in niemann‐pick type C disease.
  Patrizia Formichi, Carla Battisti, Maria M. De Santi, Raffaella Guazzo, Sergio A. Tripodi, Elena Radi, Benedetta Rossi, Ermelinda Tarquini, Antonio Federico.
  Journal of Cellular Physiology. May 19, 2017
  Niemann‐Pick type C disease (NPC) is a disorder characterized by abnormal intracellular accumulation of unesterified cholesterol and glycolipids. Two distinct disease‐causing genes have been isolated, NPC1 and NPC2. The NPC1 protein is involved in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. It has extensive homology with the Patched1 (Ptc1) receptor, a transmembrane protein localized in the primary cilium, and involved in the Hedgehog signaling (Shh) pathway. We assessed the presence of NPC1 and Ptc1 proteins and evaluated the relative distribution and morphology of primary cilia in fibroblasts from five NPC1 patients and controls, and in normal fibroblasts treated with 3‐ß‐[2‐(diethylamino)ethoxy]androst‐5‐en‐17‐one (U18666A), a cholesterol transport‐inhibiting drug that is widely used to mimic NPC. Immunofluorescence and western blot analyses showed a significant decrease in expression of NPC1 and Ptc1 in NPC1 fibroblasts, while they were normally expressed in U18666A‐treated fibroblasts. Moreover, fibroblasts from NPC1 patients and U18666A‐treated cells showed a lower percentage distribution of primary cilia and a significant reduction in median cilia length with respect to controls. These are the first results demonstrating altered cytoplasmic expression of Ptc1 and reduced number and length of primary cilia, where Ptc1 is located, in fibroblasts from NPC1 patients. We suggest that the alterations in Ptc1 expression in cells from NPC1 patients are closely related to NPC1 expression deficit, while the primary cilia alterations observed in NPC1 and U18666A‐treated fibroblasts may represent a secondary event derived from a defective metabolic pathway. We assessed the presence of NPC1 and Ptc1 proteins and evaluated the relative distribution and morphology of primary cilia in fibroblasts from five NPC1 patients. We demonstrated altered cytoplasmic expression of Ptc1 and reduced number and length of primary cilia in fibroblasts from NPC1 patients.

  May 19, 2017   doi: 10.1002/jcp.25926   open full text
• Retinoic acid receptor‐related orphan receptor RORα regulates differentiation and survival of keratinocytes during hypoxia.
  Hongyu Li, Longjian Zhou, Jun Dai.
  Journal of Cellular Physiology. May 19, 2017
  Low O2 pressures present in the microenvironment of epidermis control keratinocyte differentiation and epidermal barrier function through hypoxia inducible factors (HIFs) dependent gene expression. This study focuses on investigating relations of the retinoic acid receptor‐related orphan receptor alpha (RORα) to HIF‐1α in keratinocytes under hypoxic conditions. The expression level of RORα is significantly elevated under hypoxia in both human and murine keratinocytes. Gene silencing of RORA attenuates hypoxia‐stimulated expression of genes related to late differentiation and epidermal barrier function, and leads to an enhanced apoptotic response. While the hypoxic induction of RORα is dependent on HIF‐1α, RORα is in turn critical for nuclear accumulation of HIF‐1α and activation of HIF transcriptional activity. These results collectively suggest that RORα functions as an important mediator of HIF‐1α activities in regulating keratinocyte differentiation/survival and epidermal barrier function during the oxygen sensing stage. We have identified RORα as a regulator imperative to HIF‐1α activities in promoting terminal differentiation, epidermal barrier function, and survival of keratinocytes under low O2 tension. Given highly diversified functions of HIF targets in maintaining skin homeostasis, manipulations of HIF activities through RORα ligands can represent a novel strategy for therapeutic treatment of pathophysiological conditions such as cutaneous diseases related to defective epidermal barrier functions and wound healing.

  May 19, 2017   doi: 10.1002/jcp.25924   open full text
• Involvement of testicular DAAM1 expression in zinc protection against cadmium‐induced male rat reproductive toxicity.
  Marouane Chemek, Massimo Venditti, Sana Boughamoura, Safa B. Mimouna, Imed Messaoudi, Sergio Minucci.
  Journal of Cellular Physiology. May 19, 2017
  In order to verify the effects of exposure to Cd and Zn on testicular DAAM1 gene and protein expression and also to ascertain their involvement in the protective role of Zn in prevent the testicular toxicity Cd‐induced in male offspring rats at adult age after gestational and lactational exposure, male offspring rats, from mothers receiving either tap water, Cd, Zn, or Cd + Zn during gestation and lactation periods, were scarified on postnatal days (PND) 70. The reproductive organ (testis, epididymis, and vesicle seminal) were collected, weighed, and analyzed. The results showed that exposure to Cd in utero and through lactation decreased the relative reproductive organ weight, altered the testicular histology at the interstitial and tubular levels, and causing a significant reduction in the daily sperm production (DSP) per testis and per gram of testis, and other then altering the epididymal sperm quality. Furthermore, both mRNA and protein expression of rat testicular DAAM1 were also inhibited in Cd‐treated group. Zn supply has completely corrected the most of these toxic effects. Our results imply that Zn could prevent Cd‐induced testicular toxicity and sperm quality alteration in adult male rat after gestational and lactational exposure, probably via the restoration of the testicular DAAM1 expression inhibited by Cd. The effects of exposure to Cd and Zn on testicular DAAM1 gene and protein expression and heir involvement in the protective role of Zn in prevent the testicular toxicity Cd‐induced in male offspring rats have been studied. The reproductive organ were collected, weighed and analyzed. The results showed that Cd exposure decreased the relative reproductive organ weight, altered the testicular histology, and causing a significant reduction in the daily sperm production other then altering the epididymal sperm quality. In addition, both mRNA and protein expression of rat testicular DAAM1 were also inhibited. Zn supply has completely corrected the most of these toxic effects. Our results imply that Zn could prevent Cd‐induced testicular toxicity probably via the restoration of the testicular DAAM1 expression inhibited by Cd.

  May 19, 2017   doi: 10.1002/jcp.25923   open full text
• Dual‐specificity tyrosine phosphorylation‐regulated kinase 2 regulates osteoclast fusion in a cell heterotypic manner.
  Gali Guterman‐Ram, Milena Pesic, Ayelet Orenbuch, Tal Czeiger, Anastasia Aflalo, Noam Levaot.
  Journal of Cellular Physiology. May 19, 2017
  Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign‐body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion‐founder cells that fuse to fusion‐follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells. This study investigate the molecular mechanisms regulating osteoclast fusion. Osteoclast fusion involve a fusion initiation cell termed fusion founder which fuse to cells lacking fusion initiation abilities termed fusion followers. This study show for the fist time that the protein Dyrk2 inhibit fusion by limiting the distribution of fusion follower cells.

  May 19, 2017   doi: 10.1002/jcp.25922   open full text
• miR‐124 and miR‐9 mediated downregulation of HDAC5 promotes neurite development through activating MEF2C‐GPM6A pathway.
  Xi Gu, Congcong Fu, Lifang Lin, Shuhu Liu, Xiaohong Su, Aili Li, Qiaoqi Wu, Chunhong Jia, Peidong Zhang, Lu Chen, Xinhong Zhu, Xuemin Wang.
  Journal of Cellular Physiology. May 19, 2017
  The class IIa histone deacetylases (HDACs) play important roles in the central nervous system during diverse biological processes such as synaptic plasticity, axon regeneration, cell apoptosis, and neural differentiation. Although it is known that HDAC5 regulates neuronal differentiation, neither the physiological function nor the regulation of HDAC5 in neuronal differentiation is clear. Here, we identify HDAC5 as an inhibitor of neurite elongation and show that HDAC5 is regulated by the brain enriched microRNA miR‐124 and miR‐9. We discover that HDAC5 inhibits neurite extension both in differentiated P19 cells and primary neurons. We also show that the neuronal membrane glycoprotein GPM6A (M6a) is a direct target gene of HDAC5 regulated transcriptional factor MEF2C. HDAC5 inhibits neurite elongation, acting at least partially via a MEF2C/M6a signaling pathway. We also confirmed the miR‐124/miR‐9 regulated HDAC5‐MEF2C‐M6a pathway regulates neurite development in primary neurons. Thus, HDAC5 emerges as a cellular conductor of MEF2C and M6a activity and is regulated by miR‐124 and miR‐9 to control neurite development. We provide evidence that through the direct repression of HDAC5, miR‐124, and miR‐9 intrinsically regulate MEF2C transcription activity and M6a expression, and this ensures proper neurite elongation. Our findings further suggest that by acting in neurons, brain‐enriched miRNAs can play key roles in coordinating vertebrate central nervous system development.

  May 19, 2017   doi: 10.1002/jcp.25927   open full text
• Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling.
  Tamara Kukolj, Drenka Trivanović, Ivana Okić Djordjević, Slavko Mojsilović, Jelena Krstić, Hristina Obradović, Srdja Janković, Juan Francisco Santibanez, Aleksandra Jauković, Diana Bugarski.
  Journal of Cellular Physiology. May 19, 2017
  Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast‐like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF‐β, fibronectin (FN), α‐SMA, and NG2. LPS also increased protein and gene expression levels of anti‐inflammatory COX‐2 and pro‐inflammatory IL‐6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS‐treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen‐stimulated proliferation of CD4+ and the ratio of CD4+CD25high/CD4+CD25low lymphocytes. LPS‐treated PDLSCs did not change the frequency of CD34+ and CD45+ cells, but decreased the frequency of CD33+ and CD14+ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU‐GM number. The results indicated that LPS‐activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features. In this study, we examined functional properties of human periodontal ligament stem cells (PDLSCs) in presence of LPS (E. coli). Our results indicated that LPS modified PDLSCs’ mesodermal lineage commitment, favoring myofibroblastic‐like phenotype, without affecting their growth and immunophenotype. LPS‐treated PDLSCs strongly inhibited transendothelial migration (TEM) of human peripheral blood mononuclear cells (MNCs). LPS‐treated PDLSCs decreased the frequency of CD33+ and CD14+ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity, predominantly the CFU‐GM number, of MNCs. Our results indicate that changes in differentiation and immunomodulatory properties of PDLSCs may be in partly governed by LPS‐activated ERK1,2 signaling cascade.

  May 19, 2017   doi: 10.1002/jcp.25904   open full text
• Comparative proteomic profiling of human osteoblast‐derived extracellular matrices identifies proteins involved in mesenchymal stromal cell osteogenic differentiation and mineralization.
  Marta Baroncelli, Bram C. van der Eerden, Yik‐Yang Kan, Rodrigo D. Alves, Jeroen A. Demmers, Jeroen van de Peppel, Johannes P. van Leeuwen.
  Journal of Cellular Physiology. May 19, 2017
  The extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cell‐secreted ECMs have become of interest as they reproduce tissue‐architecture and modulate the promising properties of mesenchymal stem cells (MSCs). We have previously created an in vitro model of human osteoblast‐derived devitalized ECM that was osteopromotive for MSCs. The aim of this study was to identify ECM regulatory proteins able to modulate MSC differentiation to broaden the spectrum of MSC clinical applications. To this end, we created two additional models of devitalized ECMs with different mineralization phenotypes. Our results showed that the ECM derived from osteoblast‐differentiated MSCs had increased osteogenic potential compared to ECM derived from undifferentiated MSCs and non‐ECM cultures. Proteomic analysis revealed that structural ECM proteins and ribosomal proteins were upregulated in the ECM from undifferentiated MSCs. A similar response profile was obtained by treating osteoblast‐differentiating MSCs with Activin‐A. Extracellular proteins were upregulated in Activin‐A ECM, whereas mitochondrial and membrane proteins were downregulated. In summary, this study illustrates that the composition of different MSC‐secreted ECMs is important to regulate the osteogenic differentiation of MSCs. These models of devitalized ECMs could be used to modulate MSC properties to regulate bone quality. In vitro cell‐derived extracellular matrices (ECM) mimic the ECM role in tissue architecture and in actively modulating cell behavior, and they have been proposed for tissue engineering applications. We created three models of human osteoblast‐derived ECM with extremely different mineralization phenotypes, to identify regulatory proteins to modulate mesenchymal stromal cell (MSC) differentiation. Comparative proteomic profiles of these ECMs reveal that the protein composition is important to regulate MSC behavior, and could be used to modulate MSC properties and bone quality for bone tissue engineering applications.

  May 19, 2017   doi: 10.1002/jcp.25898   open full text
• Nanotubes impregnated human olfactory bulb neural stem cells promote neuronal differentiation in trimethyltin‐induced neurodegeneration rat model.
  Hany E. Marei, Ahmed A. Elnegiry, Adel Zaghloul, Asma Althani, Nahla Afifi, Ahmed Abd‐Elmaksoud, Amany Farag, Samah Lashen, Shymaa Rezk, Zeinab Shouman, Carlo Cenciarelli, Anwarul Hasan.
  Journal of Cellular Physiology. May 18, 2017
  Neural stem cells (NSCs) are multipotent self‐renewing cells that could be used in cellular‐based therapy for a wide variety of neurodegenerative diseases including Alzheimer's diseases (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Being multipotent in nature, they are practically capable of giving rise to major cell types of the nervous tissue including neurons, astrocytes, and oligodendrocytes. This is in marked contrast to neural progenitor cells which are committed to a specific lineage fate. In previous studies, we have demonstrated the ability of NSCs isolated from human olfactory bulb (OB) to survive, proliferate, differentiate, and restore cognitive and motor deficits associated with AD, and PD rat models, respectively. The use of carbon nanotubes (CNTs) to enhance the survivability and differentiation potential of NSCs following their in vivo engraftment have been recently suggested. Here, in order to assess the ability of CNTs to enhance the therapeutic potential of human OBNSCs for restoring cognitive deficits and neurodegenerative lesions, we co‐engrafted CNTs and human OBNSCs in TMT‐neurodegeneration rat model. The present study revealed that engrafted human OBNSCS‐CNTs restored cognitive deficits, and neurodegenerative changes associated with TMT‐induced rat neurodegeneration model. Moreover, the CNTs seemed to provide a support for engrafted OBNSCs, with increasing their tendency to differentiate into neurons rather than into glia cells. The present study indicate the marked ability of CNTs to enhance the therapeutic potential of human OBNSCs which qualify this novel therapeutic paradigm as a promising candidate for cell‐based therapy of different neurodegenerative diseases. In order to assess the ability of CNTs to enhance the therapeutic potential of human OBNSCs for restoring cognitive deficits and neurodegenerative lesions, we co‐engrafted CNTs and human OBNSCs in TMT‐neurodegeneration rat model. The present study revealed that engrafted human OBNSCS‐CNTs restored cognitive deficits, and neurodegenerative changes associated with TMT‐induced rat neurodegeneration model. The present study indicate the marked ability of CNTs to enhance the therapeutic potential of human OBNSCs which qualify this novel therapeutic paradigm as a promising candidate for cell‐based therapy of different neurodegenerative diseases.

  May 18, 2017   doi: 10.1002/jcp.25826   open full text
• PP2A deactivation is a common event in oral cancer and reactivation by FTY720 shows promising therapeutic potential.
  Bharath Kumar Velmurugan, Chien‐Hung Lee, Shang‐Lun Chiang, Chun‐Hung Hua, Mei‐Chung Chen, Shu‐Hui Lin, Kun‐Tu Yeh, Ying‐Chin Ko.
  Journal of Cellular Physiology. May 18, 2017
  Protein phosphatase 2A (PP2A) is a tumor suppressor gene, that has been frequently deactivated in many types of cancer. However its molecular and clinical relevance in oral squamous cell carcinoma (OSCC) remain unclear. Here we show that, PP2A deactivation is a common event in oral cancer cells and hyperphosphorylation in its tyrosine‐307 (Y307) residue contributes to PP2A deactivation. PP2A restoration by FTY720 treatment reduced cell growth and decreased GSK‐3β phosphorylation without significantly altering other PP2A targets. We further detected PP2A phosphorylation in 262 OSCC tissues. Increased expression of p‐PP2A in the tumor tissues was significantly correlated with higher N2/N3‐ stage (aOR =2.1, 95% CI: 1.2‐3.8). Patients with high p‐PP2A expression had lower overall survival rates than those with low expression. Hazard ratio analysis showed that, high p‐PP2A expression was significantly associated with mortality density (aOR= 2.2, 95% CI: 1.2‐4.0) and lower 10‐year overall survival (P = 0.027) in lymph node metastasis. However, no interaction was observed between p‐PP2A expression and lymph node metastasis. All our results suggest that PP2A is frequently deactivated is oral cancer and determines poor outcome, restoring its expression by FTY720 can be an alternative therapeutic approach in OSCC. This article is protected by copyright. All rights reserved

  May 18, 2017   doi: 10.1002/jcp.26001   open full text
• S‐Adenosylmethionine‐Mediated Apoptosis Is Potentiated By Autophagy Inhibition Induced By Chloroquine In Human Breast Cancer Cells.
  Donatella Delle Cave, Vincenzo Desiderio, Laura Mosca, Concetta Paola Ilisso, Luigi Mele, Michele Caraglia, Giovanna Cacciapuoti, Marina Porcelli.
  Journal of Cellular Physiology. May 18, 2017
  The naturally‐occurring sulfonium compound S‐adenosyl‐L‐methionine (AdoMet) is an ubiquitous sulfur‐nucleoside that represents the main methyl donor in numerous methylation reactions. In recent years, it has been shown that AdoMet possesses antiproliferative properties in various cancer cells, but the molecular mechanisms at the basis of the effect induced by AdoMet have been only in part investigated. In the present study, we found that AdoMet strongly inhibited the proliferation of breast cancer cells MCF‐7 by inducing both autophagy and apoptosis. AdoMet consistently enhanced the levels of the autophagy markers beclin‐1 and LC3B‐II, and caused a significant increase of pro‐apoptotic Bax/Bcl‐2 ratio paralleled by poly (ADP ribose) polymerase (PARP) and caspase 9, and 6 cleavage. Notably, AdoMet, already at low doses, raised the percentage of cells in G2/M phase of cell cycle by down‐regulating the expression of cell cycle‐regulatory proteins cyclin B and cyclin E with a remarkable increase of p53, p27 and p21. We also evaluated the combination of AdoMet and the autophagy inhibitor chloroquine (CLC) showing that autophagy block is synergistic in inducing both growth inhibition and apoptosis. These effects were paralleled by a strong inhibition of the activity of AKT and of the downstream effector mTOR and by an increased cleavage of caspase‐6 and PARP. These data suggest, for the first time, that autophagy can act as an escape mechanism from the apoptotic activity of AdoMet, and that AdoMet could be used in combination with CLC or its analogs in the treatment of breast cancer. This article is protected by copyright. All rights reserved

  May 18, 2017   doi: 10.1002/jcp.26015   open full text
• Low shear stress induces endothelial reactive oxygen species via the AT1R/eNOS/NO pathway.
  Yuelin Chao, Peng Ye, Linlin Zhu, Xiangquan Kong, Xinliang Qu, Junxia Zhang, Jie luo, Hongfeng Yang, Shaoliang Chen.
  Journal of Cellular Physiology. May 18, 2017
  Reactive oxygen species (ROS) contribute to many aspects of physiological and pathological cardiovascular processes. However, the underlying mechanism of ROS induction by low shear stress (LSS) remains unclear. Accumulating evidence has shown that the angiotensin II type 1 receptor (AT1R) is involved in inflammation, apoptosis, and ROS production. Our aim was to explore the role of AT1R in LSS‐mediated ROS induction. We exposed human umbilical vein endothelial cells (HUVECs) to LSS (3 dyne/cm2) for different periods of time. Western blotting and immunofluorescence showed that LSS significantly induced AT1R expression in a time‐dependent manner. Using immunohistochemistry, we also noted a similar increase in AT1R expression in the inner curvature of the aortic arch compared to the descending aorta in C57BL/6 mice. Additionally, HUVECs were cultured with a fluorescent probe, either DCFH, DHE or DAF, after being subjected to LSS. Cell chemiluminescence and flow cytometry results revealed that LSS stimulated ROS levels and suppressed nitric oxide (NO) generation in a time‐dependent manner, which was reversed by the AT1R antagonist Losartan. We also found that Losartan markedly increased endothelial NO synthase (eNOS) phosphorylation at Ser(633,1177) and dephosphorylation at Thr(495), which involved AKT and ERK. Moreover, the ROS level was significantly reduced by endogenous and exogenous NO donors (L‐arginine, SNP) and increased by the eNOS inhibitor L‐NAME. Overall, we conclude that LSS induces ROS via AT1R/eNOS/NO. This article is protected by copyright. All rights reserved

  May 18, 2017   doi: 10.1002/jcp.26016   open full text
• Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL‐1 cardiac cell line.
  Federica Campolo, Alessandra Zevini, Silvia Cardarelli, Lucia Monaco, Federica Barbagallo, Manuela Pellegrini, Marisa Cornacchione, Antonio Di Grazia, Valeria De Arcangelis, Daniele Gianfrilli, Mauro Giorgi, Andrea Lenzi, Andrea M. Isidori, Fabio Naro.
  Journal of Cellular Physiology. May 18, 2017
  Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N‐terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT‐PCR analysis showed that mPde5a1, mPde5a2, and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL‐1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL‐1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). Overexpression of mPDE5As induced cell cycle progression of HL‐1 cells. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL‐1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms.

  May 18, 2017   doi: 10.1002/jcp.25880   open full text
• Transcription factor EGR1 promotes differentiation of bovine skeletal muscle satellite cells by regulating MyoG gene expression.
  WeiWei Zhang, HuiLi Tong, ZiHeng Zhang, ShuLi Shao, Dan Liu, ShuFeng Li, YunQin Yan.
  Journal of Cellular Physiology. May 18, 2017
  The transcription factor, early growth response 1 (EGR1), has important roles in various cell types in response to different stimuli. EGR1 is thought to be involved in differentiation of bovine skeletal muscle‐derived satellite cells (MDSCs); however, the precise effects of EGR1 on differentiation of MDSCs and its mechanism of action remain unknown. In the present study, a time course of EGR1 expression and the effects of EGR1 on MDSC differentiation were determined. The results demonstrated that the expression of EGR1 mRNA and protein increased significantly in differentiating MDSCs relative to that in proliferating cells. Over‐expression of the EGR1 gene in MDSCs promoted their differentiation and inhibited proliferation. Conversely, knock‐down of EGR1 inhibited differentiation of MDSCs and promoted their proliferation, indicating that EGR1 promotes MDSC differentiation. Moreover, over‐expression of EGR1 in MDSCs increased the expression of MyoG mRNA and protein, whereas its knock‐down had the opposite effect. Furthermore, ChIP‐PCR analyses demonstrated that EGR1 could bind directly to its putative binding site within the promoter region of MyoG, and determination of ERG1 subcellular localization in MDSCs demonstrated that it could relocate to the nucleus, indicating MyoG is likely an EGR1 target gene whose expression is positively regulated by this transcription factor. In conclusion, EGR1 can promote MDSC differentiation through positive regulation of MyoG gene expression. The transcription factor, EGR1, can promote differentiation of bovine skeletal muscle‐derived satellite cells. In addition, EGR1 can relocate to the nucleus and bind directly to the MyoG gene promoter, hence positively regulating MyoG gene expression. These data prove that EGR1 promotes MDSC differentiation by regulation of MyoG gene expression.

  May 18, 2017   doi: 10.1002/jcp.25883   open full text
• Histomorphology and Innate Immunity during the Progression of Osteoarthritis: Does Synovitis Affect Cartilage Degradation?
  Wang Huan, Wang Qingguo, Yang Meijuan, Yang Lili, Wang Weili, Ding Haobin, Zhang Dong, Xu Jing, Tang Xuezhang, Ding Haitao, Wang Qingfu.
  Journal of Cellular Physiology. May 17, 2017
  Osteoarthritis (OA) is a common chronic degenerative disease that affects all joints. At present, the pathological processes and mechanisms of OA are still unclear. Innate immunity, a key player in damage to the structure of the joint and the mechanism by which the host attempts to repair OA, affects all pathological stages of the disease. In the present study, our aim was to assess changes in innate immunity during the pathological processes of OA in articular cartilage (AC) and the synovial membrane (SM), which are the major structures in joints, and to systematically examine the histological changes in AC and SM in mild, moderate and severe cases of OA, in order to further speculate about the manner in which the interactions of AC and SM are facilitated by innate immunity. Histological methods (including HE and Safranin O‐fast green staining) were used to assess the morphological changes within AC and SM tissues in healthy and mild, moderate or severe OA rats. Mankin's and Wakitani scorings were conducted on the AC to assess the degradation of the cartilage and the extent of synovial inflammation. Localization and intensity of expression of the three key proteins: TLR4 (upstream), MyD88 (midstream) and NF‐κB (downstream) of the inflammatory pathway believed to be involved in innate immune system–induced AC and SM structural degeneration, were examined using immunofluorescent staining. This technique allows a more stereoscopic visualization of the cellular structure by labeling the relative positions of the proteins of interest using fluorescent markers. In addition, significantly enhanced fluorescence signals could be detected by fluorescence microscopy in areas with more severe tissue damage. Combined with the terminal‐deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, the phenomenon of apoptosis in chondrocytes and synoviocytes was observed. We found that TLR4, MyD88 and NF‐κB were significantly expressed in the AC and SM of the OA rats, while very little expression was detected in healthy rats. We speculate that innate immunity is programmed to play a role in mediating the repair of tissue damage. Considering the differences in the expression levels of these proteins in AC and SM during different stages of the disease, the SM might play a driving role in the process of creating lesions. Western blots (WBs) were combined to confirm the trend of expression of TLR4, MyD88 and NF‐κB in SM tissues during different stages of OA. The expression of an important specificity protein TRAF6, in addition to IL‐1β and TNF‐α, representative downstream inflammatory mediators, were also measured in order to verify the key role of the signaling pathway in OA. In addition, we systematically describe the principal cell types in the SM which participate in innate immunity. Our results showed that the damage to AC and SM within the joints progressively worsened during the course of the disease, and that the innate immune system was closely involved in the AC and SM during each stage of OA. These findings also confirmed that SM may affect the pathological changes in AC through the innate immune system, and therefore affect the progress of OA. This article is protected by copyright. All rights reserved

  May 17, 2017   doi: 10.1002/jcp.26011   open full text
• Loss of TET1 facilitates DLD1 colon cancer cell migration via H3K27me3‐mediated down‐regulation of E‐cadherin.
  Zhen Zhou, Hong‐Sheng Zhang, Yang Liu, Zhong‐Guo Zhang, Guang‐Yuan Du, Hu Li, Xiao‐Ying Yu, Ying‐Hui Huang.
  Journal of Cellular Physiology. May 17, 2017
  Epigenetic modifications such as histone modifications and cytosine hydroxymethylation are linked to tumorigenesis. Loss of 5‐hydroxymethylcytosine (5hmC) by ten‐eleven translocation 1 (TET1) down‐regulation facilitates tumor initiation and development. However, the mechanisms by which loss of TET1 knockdown promotes malignancy development remains unclear. Here, we report that TET1 knockdown induced epithelial‐mesenchymal transition (EMT) and increased cancer cell growth, migration and invasion in DLD1 cells. Loss of TET1 increased EZH2 expression and reduced UTX‐1 expression, thus increasing histone H3K27 tri‐methylation causing repression of the target gene E‐cadherin. Ectopic expression of the H3K27 demethylase UTX‐1 or EZH2 depletion both impeded EZH2 binding caused a loss of H3K27 methylation at epithelial gene E‐cadherin promoter, thereby suppressing EMT and tumor invasion in shTET1 cells. Conversely, UTX‐1 depletion and ectopic expression of EZH2 enhanced EMT and tumor metastasis in DLD1 cells. These findings provide insight into the regulation of TET1 and E‐cadherin and identify EZH2 as a critical mediator of E‐cadherin repression and tumor progression.This article is protected by copyright. All rights reserved

  May 17, 2017   doi: 10.1002/jcp.26012   open full text
• A systems approach to physiologic evolution: From micelles to consciousness.
  John S. Torday, William B. Miller.
  Journal of Cellular Physiology. May 16, 2017
  A systems approach to evolutionary biology offers the promise of an improved understanding of the fundamental principles of life through the effective integration of many biologic disciplines. It is presented that any critical integrative approach to evolutionary development involves a paradigmatic shift in perspective, more than just the engagement of a large number of disciplines. Critical to this differing viewpoint is the recognition that all biological processes originate from the unicellular state and remain permanently anchored to that phase throughout evolutionary development despite their macroscopic appearances. Multicellular eukaryotic development can, therefore, be viewed as a series of connected responses to epiphenomena that proceeds from that base in continuous iterative maintenance of collective cellular homeostatic equipoise juxtaposed against an ever‐changing and challenging environment. By following this trajectory of multicellular eukaryotic evolution from within unicellular First Principles of Physiology forward, the mechanistic nature of complex physiology can be identified through a step‐wise analysis of a continuous arc of vertebrate evolution based upon serial exaptations. There is no systematic way of understanding physiology at the present time. By reducing the process of physiologic evolution to an integrated series of cellular‐molecular steps from the unicellular state to complex traits, this goal has now been accomplished.

  May 16, 2017   doi: 10.1002/jcp.25820   open full text
• Promising anti‐tumor properties of bisdemethoxycurcumin: A naturally occurring curcumin analogue.
  Mahin Ramezani, Mahdi Hatamipour, Amirhosein Sahebkar.
  Journal of Cellular Physiology. May 16, 2017
  Curcuminoids are turmeric‐extracted phytochemicals with documented chemopreventive and anti‐tumor activities against several types of malignancies. Curcuminoids can modulate several molecular pathways and cellular targets involved in different stages of tumor initiation, growth, and metastasis. Bisdemethoxycurcumin (BDMC) is a minor constituent (approximately 3%) of curcuminoids that has been shown to be more stable than the other two main curcuminoids, that is, curcumin and demthoxycurcumin. Recent studies have revealed that BDMC has anti‐tumor effects exerted through a multimechanistic mode of action involving inhibition of cell proliferation, invasion and migration, metastasis and tumour growth, and induction of apoptotic death in cancer cells. The present review discusses the findings on the anti‐tumor effects of BDMC, underlying mechanisms, and the relevance of finding for translational studies in human. This review discusses the findings on the anti‐tumor effects of bisdemethoxycurcumin, underlying mechanisms, and the relevance of finding for translational studies in human.

  May 16, 2017   doi: 10.1002/jcp.25795   open full text
• The prognostic value of MGMT promoter methylation in glioblastoma: A meta‐analysis of clinical trials.
  Maryam Moradi Binabaj, Afsane Bahrami, Soodabeh ShahidSales, Marjan Joodi, Mona Joudi Mashhad, Seyed Mahdi Hassanian, Kazem Anvari, Amir Avan.
  Journal of Cellular Physiology. May 16, 2017
  The DNA repair protein O6‐Methylguanine‐DNA methyltransferase (MGMT) is suggested to be associated with resistance to alkylating agents such as Temozolomide which is being used in treatment of patients with glioblastoma (GBM). Therefore, we evaluated the associations between MGMT promoter methylation and prognosis of patients with glioblastoma (GBM). Data were extracted from publications in Embase, PubMed, and the Cochrane Library. Data on overall survival (OS), progression‐free survival (PFS), and MGMT methylation status were obtained and 4,097 subjects were enrolled. Data from 34 studies showed that MGMT methylated patients had better OS, compared to GBM unmethylated patients (pooled HRs, 0.494; 95%CI 0.412–0.591; p = 0.001). Meta‐analysis of 10 eligible studies reporting on PFS, demonstrated that MGMT promoter methylation was not significantly associated with better PFS (pooled HRs, 0.653; 95%CI 0.414–1.030; p = 0.067). GBM patients with MGMT methylation were associated with longer overall survival, although this effect was not detected for PFS. Moreover, we performed further analysis in patients underwent a comprehensive imaging evaluation. This data showed a significant association with better OS and PFS, although further studies are warranted to assess the value of emerging marker in prospective setting in patients with glioblastoma as a risk stratification biomarker in clinical management of the patients.

  May 16, 2017   doi: 10.1002/jcp.25896   open full text
• Myocardial proteases and cardiac remodeling.
  Sadaf Riaz, Asad Zeidan, Fatima Mraiche.
  Journal of Cellular Physiology. May 16, 2017
  Cardiac hypertrophy (CH), characterized by the enlargement of cardiomyocytes, fibrosis and apoptosis, is one of the leading causes of death worldwide. Despite the advances in cardiovascular research, there remains a need to further investigate the signaling pathways that mediate CH in order to identify novel therapeutic targets. One of the hallmarks of CH is the remodeling of the extracellular matrix (ECM). Multiple studies have shown an important role of cysteine proteases and matrix metalloproteinases (MMPs) in the remodeled heart. This review focuses on the role of cysteine cathepins and MMPs in cardiac remodeling.

  May 16, 2017   doi: 10.1002/jcp.25884   open full text
• Intracellular transcytosis of albumin in glomerular endothelial cells after endocytosis through caveolae.
  Takahito Moriyama, Kayo Sasaki, Kazunori Karasawa, Keiko Uchida, Kosaku Nitta.
  Journal of Cellular Physiology. May 15, 2017
  We previously described albumin endocytosis through caveolae in human renal glomerular endothelial cells (HRGECs). This suggested a new albumin transcytosis pathway, in addition to the fenestral pathway. As a next step, we investigated albumin transcytosis in HRGECs after caveolar endocytosis. HRGECs were incubated with Alexa Fluor 488‐labeled bovine serum albumin from 0 to 360 min. Next, markers for endosomes, endoplasmic reticulum (ER), golgi apparatus (GA), lysosomes, and proteasomes and Fc receptors, microtubules, and actin were monitored by immunofluorescence. Labeled albumin co‐localization with endosomes was gradually and significantly increased and it was significantly higher than with the other markers at any timepoint. Albumin, placed on inside of the Transwell membrane, diffused through HRGEC monolayers during a 360 min incubation period. This transportation of albumin through HRGECs was inhibited by methyl beta cyclodextrin (MBCD), a caveolae disrupting agent. MBCD also decreased albuminuria, causing decreased caveolin‐1 (Cav‐1) expression on glomerular capillaries, in puromycin aminonucleoside induced nephrotic mice. Albumin transcytosis depends on early endosomes, but not on other organelles, Fc receptors, or cytoskeletal components. Caveolae disruption prevented albumin transportation through HRGECs and decreased albuminuria in nephrotic mice. This newly described caveolae‐dependent albumin pathway through glomerular endothelial cells is a potential pathogenetic mechanism for albuminuria, independent of the fenestrae. After endocytosis through caveolae into glomerular endothelial cells, albumin was transported to early endosome to sort to bypass lysosomes, proteasomes, the golgi apparatus, and endoplasmic reticulum, and is then transported to the other side of the cells. This newly described caveolae‐dependent albumin pathway through glomerular endothelial cells is a potential pathogenetic mechanism for albuminuria, independent of the fenestrae.

  May 15, 2017   doi: 10.1002/jcp.25817   open full text
• Individual‐specific variation in the respiratory activities of HMECs and their bioenergetic response to IGF1 and TNFα.
  Sallie S. Schneider, Elizabeth M. Henchey, Nazneen Sultana, Stephanie M. Morin, D. Joseph Jerry, Grace Makari‐Judson, Giovanna M. Crisi, Richard B. Arenas, Melissa Johnson, Holly S. Mason, Nagendra Yadava.
  Journal of Cellular Physiology. May 15, 2017
  Metabolic reprograming is a hallmark of cancer cells. However, the roles of pre‐existing differences in normal cells metabolism toward cancer risk is not known. In order to assess pre‐existing variations in normal cell metabolism, we have quantified the inter‐individual variation in oxidative metabolism of normal primary human mammary epithelial cells (HMECs). We then assessed their response to selected cytokines such as insulin growth factor 1 (IGF1) and tumor necrosis factor alpha (TNFα), which are associated with breast cancer risk. Specifically, we compared the oxidative metabolism of HMECs obtained from women with breast cancer and without cancer. Our data show considerable inter‐individual variation in respiratory activities of HMECs from different women. A bioenergetic parameter called pyruvate‐stimulated respiration (PySR) was identified as a key distinguishing feature of HMECs from women with breast cancer and without cancer. Samples showing PySR over 20% of basal respiration rate were considered PySR+ve and the rest as PySR−ve. By this criterion, HMECs from tumor‐affected breasts (AB) and non‐tumor affected breasts (NAB) of cancer patients were mostly PySR−ve (88% and 89%, respectively), while HMECs from non‐cancer patients were mostly PySR+ve (57%). This suggests that PySR−ve/+ve phenotypes are individual‐specific and are not caused by field effects due to the presence of tumor. The effects of IGF1 and TNFα treatments on HMECs revealed that both suppressed respiration and extracellular acidification. In addition, IGF1 altered PySR−ve/+ve phenotypes. These results reveal individual‐specific differences in pyruvate metabolism of normal breast epithelial cells and its association with breast cancer risk. We describe individual‐specific variation in respiratory activity and pyruvate metabolism of normal HMECs from tumor affected (AB) and non‐affected breasts (NAB). Our analyses identified a bioenergetic parameter, the pyruvate‐stimulated respiration (PySR), which distinguishes cells from cancer patients versus mammoplasty patients that did not have any cancer history.

  May 15, 2017   doi: 10.1002/jcp.25932   open full text
• Pathogenic Role of Associated Adherent‐Invasive Escherichia coli in Crohn's Disease.
  Giuseppe Mazzarella, Angelica Perna, Angela Marano, Angela Lucariello, Vera Rotondi Aufiero, Alida Sorrentino, Raffaele Melina, Germano Guerra, Fabio Silvio Taccone, Gaetano Iaquinto, Antonio De Luca.
  Journal of Cellular Physiology. May 15, 2017
  Several lines of evidence suggest that adherent‐invasive Escherichia coli (AIEC) strains play an important role in Crohn's disease (CD). The objective of this study was to investigate the pathogenic role of two AIEC strains, LF82 and O83:H1, in CD patients. Organ cultures of colonic biopsies from patients were set up to assess the effects of LF82 and O83:H1 on the expression of CEACAM6, LAMP1, HLA‐DR, ICAM1 by immunohistochemistry and of IL‐8, IFNʏ, and TNF‐α genes by RT‐PCR. Moreover, on Caco2 cells, we analyzed the cell cycle, the expression of MGMT and DNMT1 genes, and DNA damage induced by LF82 and O83:H1, by FACS, RT‐PCR, and DAPI staining, respectively. Epithelial and lamina propria mononuclear cells (LPMNC) expression of CEACAM6 and LAMP1 were higher in biopsies cultured in the presence of both O83:H1 and LF82 than in biopsies cultured with non‐pathogenic E. coli. Both AIEC strains induced increased expression of ICAM‐1 on blood vessels and HLA‐DR on LPMNC. We observed higher levels of TNF‐α, IFN‐γ, and IL‐8 transcripts in biopsies cultured with both AIEC strains than in those cultured with NP. Both LF82 and O83:H1, block the cell cycle into S phase, inducing DNA damage, and modulate the expression of DNMT1 and MGMT genes. Our data suggest that LF82 and 083:H1 strains of E. coli are able to increase in CD colonic biopsies the expression of all the pro‐inflammatory cytokines and all the mucosal immune markers investigated. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc. Adherent‐invasive Escherichia coli (AIEC) strains play an important role in Crohn's disease (CD). The objective of this study was to investigate the pathogenic role of two AIEC strains, LF82 and O83:H1, in CD patients. LF82 and 083:H1 strains of E. coli are able to increase in CD colonic biopsies the expression of all the pro‐inflammatory cytokines and all the mucosal immune markers investigated.

  May 15, 2017   doi: 10.1002/jcp.25717   open full text
• Intranuclear and higher‐order chromatin organization of the major histone gene cluster in breast cancer.
  Andrew J. Fritz, Prachi N. Ghule, Joseph R. Boyd, Coralee E. Tye, Natalie A. Page, Deli Hong, Adam S. Weinheimer, A Rasim Barutcu, Diana L. Gerrard, Seth Frietze, Sayyed K. Zaidi, Anthony Imbalzano, Jane B. Lian, Janet L. Stein, Gary S. Stein.
  Journal of Cellular Physiology. May 15, 2017
  Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher‐order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA‐seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal‐like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub‐clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub‐clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in‐between them, suggesting that the histone sub‐clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub‐nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher‐order chromatin organization of the major HLB and its regulation during breast cancer progression. This article is protected by copyright. All rights reserved

  May 15, 2017   doi: 10.1002/jcp.25996   open full text
• Activating transcription factor 3 (ATF3) protects against lipopolysaccharide‐induced acute lung injury via inhibiting the expression of TL1A.
  Lanlan Qian, Yunfeng Zhao, Liang Guo, Shaoying Li, Xueling Wu.
  Journal of Cellular Physiology. May 15, 2017
  Excessive inflammatory responses are critical in the pathogenesis of acute lung injury (ALI). Activating transcription factor 3 (ATF3) is a stress‐induced transcriptional regulator that is a negative regulator of inflammatory responses. Therefore, we investigated the role and signaling pathways of ATF3 in lipopolysaccharide (LPS)‐induced ALI in mice. The mouse macrophage RAW264.7 cells were cultured on HTS 24‐Transwell filter plates in presence of ATF3 siRNA before exposure to LPS. ATF3 knock‐out (KO) and wild type (WT) mice were challenged by intra‐peritoneal injection of LPS (15 mg/kg). Gene analysis was used to analyze differential gene expression between ATF3 KO and WT mice. LPS increased the expression of ATF3 in RAW264.7 cells and in lung tissues of mice, The concentration of TNFα and IL‐6 was significantly increased in ATF3 siRNA‐treated RAW264.7 cells compared to control cells after LPS stimulation. The concentration of TNFα, IL‐6 and IL‐1β in serum and lung tissue of ATF3 KO mice was significantly increased compared to ATF3 WT mice. In addition, the lung wet/dry weight and BALF protein were significantly increased in ATF3 KO mice after LPS injection at 6, 24, and 48 hr. The survival of ATF3 KO mice significantly decreased. Differential gene analysis showed that TL1A was highly expressed in LPS‐induced lung tissues of ATF3 KO mice.Moreover, ATF3 down‐regulated the expression of TL1A in RAW264.7 cells and in lung tissues. These findings suggest that ATF3 protects against LPS‐induced ALI via inhibiting TL1A expression. Excessive inflammatory responses are critical in the pathogenesis of acute lung injury (ALI). Activating transcription factor 3 (ATF3) is a stress‐induced transcriptional regulator that is a negative regulator of inflammatory responses.The survival of ATF3 KO mice significantly decreased. Differential gene analysis showed that TL1A was highly expressed in LPS‐induced lung tissues of ATF3 KO mice.Moreover, ATF3 down‐regulated the expression of TL1A in RAW264.7 cells and in lung tissues.These findings suggest that ATF3 protects against LPS‐induced ALI via inhibiting TL1A expression.

  May 15, 2017   doi: 10.1002/jcp.25849   open full text
• MicroRNAs: Potential candidates for diagnosis and treatment of colorectal cancer.
  Abdullah Moridikia, Hamed Mirzaei, Amirhossein Sahebkar, Jafar Salimian.
  Journal of Cellular Physiology. May 15, 2017
  Colorectal cancer (CRC) is known as the third common cancer worldwide and an important public health problem in different populations. Several genetics and environmental risk factors are involved in the development and progression of CRC including chromosomal abnormalities, epigenetic alterations, and unhealthy lifestyle. Identification of risk factors and biomarkers could lead to a better understanding of molecular pathways involved in CRC pathogenesis. MicroRNAs (miRNAs) are important regulatory molecules which could affect a variety of cellular and molecular targets in CRC. A large number of studies have indicated deregulations of some known tissue‐specific miRNAs, for example, miR‐21, miR‐9, miR‐155, miR‐17, miR‐19, let‐7, and miR‐24 as well as circulating miRNAs, for example, miR‐181b, miR‐21, miR‐183, let‐7g, miR‐17, and miR‐126, in patients with CRC. In the current review, we focus on the findings of preclinical and clinical studies performed on tissue‐specific and circulating miRNAs as diagnostic biomarkers and therapeutic targets for the detection of patients at various stages of CRC.

  May 15, 2017   doi: 10.1002/jcp.25801   open full text
• E2F is Involved in Radioresistance of Carbon Ion Induced Apoptosis via Bax/caspase 3 Signal Pathway in Human Hepatoma Cell.
  XIE Yi, SI Jing, WANG Yu‐Pei, LI Hong‐Yan, DI Cui‐Xia, YAN Jun‐Fang, YE Yan‐Cheng, ZHANG Yan‐Shan, ZHANG Hong.
  Journal of Cellular Physiology. May 13, 2017
  Deletion of p53, most common genetic alteration, is observed in human tumors and reported to lead to improve in cell radioresistance. Heavy‐ion irradiation (IR) could induce p53‐/‐ cancer cells apoptosis. However, little is known regarding the molecular mechanism in this type of cell apoptosis. The present studies have focused on mechanisms state of signaling pathways as an activator of the cell fate decisions induced by heavy ion IR without p53. Carbon ion IR could induce up‐regulation of E2F1 expression in cancer cells. This phenomenon was not observed in X‐ray IR group. Up‐regulation of E2F1 could cause a higher reduction in clonogenic survival, low level of cellular activity, G2/M phase arrest, promotion of apoptosis rate, up‐regulation of phosphor‐Rb, Bax, and cleaved‐caspase 3 proteins expressions without p53. Changes of E2F1 expressions could partly alter radioresistance in cancer cells. The results were suggested that heavy ion IR could induce p53‐/‐ cancer cells apoptosis via E2F1 signal pathway. Our study provides a scientific rationale for the clinical use of heavy ion as radiotherapy in patients with p53‐deficient tumors, which are often resistant to radiotherapy. This article is protected by copyright. All rights reserved

  May 13, 2017   doi: 10.1002/jcp.26005   open full text
• The novel role of pyrvinium in cancer therapy.
  Amir Abbas Momtazi‐borojeni, Elham Abdollahi, Faezeh Ghasemi, Michele Caraglia, Amirhossein Sahebkar.
  Journal of Cellular Physiology. May 13, 2017
  Pyrvinium pamoate (PP) is a quinoline‐derived cyanine dye which was officially approved by FDA for its anthelmintic properties and therapeutic function against animal‐like protists such as Cryp‐tosporidium parvum and Plasmodium falciparum in the 1950 s. In the last 10 years, several studies have shown the novel activity of pyrvinium in tumor therapy. Some investigations have indicated that pyrvinium could delay or inhibit tumor cell proliferation in cancer models including colon, breast, lung and prostate cancer and some hematological malignancies. In this review, we discuss multiple critical signaling pathways and mechanisms underlying the anticancer effects of PP. In details, pyrvinium acts through the following main mechanisms: i) energy and autophagy depletion, and ii) inhibition of Akt and Wnt‐β‐catenin‐dependent pathways. Interestingly, pyrvinium has also shown potent anti‐cancer stem cell activity. The overwhelming insights into the mechanism of anticancer properties of PP can help establishing novel and future anti‐tumor treatment strategies. This article is protected by copyright. All rights reserved

  May 13, 2017   doi: 10.1002/jcp.26006   open full text
• Differentiation of human olfactory bulb‐derived neural stem cell towards oligodendrocyte development.
  Marei HE, Shouman Z, Althani A, Afifi N, Abd‐Elmaksoud A, Lashen S, Hasan A, Caceci T, Rizzi R, Cenciarelli C, Casalbore P.
  Journal of Cellular Physiology. May 13, 2017
  In the central nervous system, oligodendrocytes are the glial element in charge of myelin formation. Obtaining an overall presence of oligodendrocyte precursor cells/oligodendrocytes. (OPCs/OLs) in culture from different sources of NSCs is an important research area, because OPCs/OLs may provide a promising therapeutic strategy for diseases affecting myelination of axons. The present study was designed to differentiate human olfactory bulb NSCs (OBNSCs) into OPCs/OLs and using expression profiling (RT‐qPCR) gene, immunocytochemistry and specific protein expression to highlight molecular mechanism(s) underlying differentiation of human OBNSCs into OPCs/OLs. The differentiation of OBNSCs was characterized by a simultaneous appearance of neurons and glial cells. The differentiation medium, containing cAMP, PDGFA, T3, and all‐trans‐retinoic acid (RA), promotes OBNSCs to generate mostly oligodendrocytes displaying morphological changes and appearance of long cytoplasmic processes. OBNSCs showed, after five days in oligodendrocytes differentiation medium, a considerable decrease in the number of nestin positive cells, which was associated with a concomitant increase of NG2 immunoreactive cells and few O4(+)‐OPCs. In addition, a significant up regulation in gene and protein expression profile of stage specific cell markers for OPCs/OLs (CNPase, Galc, NG2, MOG, OLIG1, OLIG2, MBP), neurons and astrocytes (MAP2, β‐TubulinIII, GFAP) and concomitant decrease of OBNSCs pluripotency markers (Oct4, Sox2, Nestin), was demonstrated following induction of OBNSCs differentiation. Taken together, the present study demonstrate the marked ability of a cocktail of factors containing PDGFA, T3, cAMP and ATRA, to induce OBNSCs differentiation into OPCs/OLs and shed light on the key genes and pathological pathways involved in this process. This article is protected by copyright. All rights reserved

  May 13, 2017   doi: 10.1002/jcp.26008   open full text
• High density lipoprotein (HDL) reverses palmitic acid induced energy metabolism imbalance by switching CD36 and GLUT4 signaling pathways in cardiomyocyte.
  Su‐Ying Wen, Bharath Kumar Velmurugan, Cecilia Hsuan Day, Chia‐Yao Shen, Li‐Chin Chun, Yi‐Chieh Tsai, Yueh‐Min Lin, Ray‐Jade Chen, Chia‐Hua Kuo, Chih‐Yang Huang.
  Journal of Cellular Physiology. May 13, 2017
  In our previous study palmitic acid (PA) induced lipotoxicity and switches energy metabolism from CD36 to GLUT4 in H9c2 cells. Low level of high density lipoprotein (HDL) is an independent risk factor for cardiac hypertrophy. Therefore we in the present study investigated whether HDL can reverse PA induced lipotoxicity in H9c2 cardiomyoblast cells. In this study, we treated H9c2 cells with PA to create a hyperlipidemia model in vitro and analyzed for CD36 and GLUT4 metabolic pathway proteins. CD36 metabolic pathway proteins (phospho‐AMPK, SIRT1, PGC1α, PPARα, CPT1β and CD36) were decreased by high PA (150 and 200 µg/µL) concentration. Interestingly, expression of GLUT4 metabolic pathway proteins (p‐PI3K and pAKT) were increased at low concentration (50 µg/µL) and decreased at high PA concentration. Whereas, phospho‐PKCζ, GLUT4 and PDH proteins expression was increased in a dose dependent manner. PA treated H9c2 cells were treated with HDL and analyzed for cell viability. Results showed that HDL treatment induced cell proliferation efficiency in PA treated cells. In addition, HDL reversed the metabolic effects of PA: CD36 translocation was increased and reduced GLUT4 translocation, but HDL treatment significantly increased CD36 metabolic pathway proteins and reduced GLUT4 pathway proteins. Rat neonatal cardiomyocytes showed similar results. In conclusion, HDL reversed palmatic acid‐induced lipotoxicity and energy metabolism imbalance in H9c2 cardiomyoblast cells and in neonatal rat cardiomyocyte cells. This article is protected by copyright. All rights reserved

  May 13, 2017   doi: 10.1002/jcp.26007   open full text
• MSCs Ameliorates DPN induced Cellular Pathology via [Ca2+]i Homeostasis and Scavenging the Pro‐inflammatory cytokines.
  Harish C Chandramoorthy, Ismaeel Bin‐Jaliah, Hussian Karari, Prasanna Rajagopalan, Mohammed Eajaz Ahmed Shariff, Ahmed Al‐Hakami, Suliman M Al‐Humayad, Fawzi A Baptain, Humeda Suekit Ahmed, Hanaa Z Yassin, Mohamed A Haidara.
  Journal of Cellular Physiology. May 13, 2017
  The MSCs of various origins are known to ameliorate or modulate cell survival strategies. We investigated, whether UCB MSCs could improve the survival of the human neuronal cells and/or fibroblast assaulted with DPN sera. The results showed, the co‐culture of UCB MSCs with human neuronal cells and/or fibroblasts could effectively scavenge the pro‐inflammatory cytokines TNF‐α, IL‐1β, IFN‐γ and IL − 12 and control the pro‐apoptotic expression of p53/Bax. Further co‐culture of UCB MSCs have shown to induce anti‐inflammatory cytokines like IL‐4, IL‐10 and TGF‐β and anti‐apoptotic Bclxl/Bcl2 expression in the DPN sera stressed cells. Amelioration of elevated [Ca2+]i and cROS, the portent behind the NFκB/Caspase‐3 mediated inflammation in DPN rescued the cells from apoptosis. The results of systemic administration of BM MSCs improved DPN pathology in rat as extrapolated from human cell model. The BM MSCs ameliorated prolonged distal motor latency (control: 0.70 ± 0.06, DPN: 1.29 ± 0.13 m/s DPN + BM MSCs: 0.89 ± 0.02 m/s, p < 0.05) and lowered high amplitude of compound muscle action potentials (CMAPs) (control: 12.36 ± 0.41, DPN: 7.52 ± 0.61 mV, DPN + MSCs: 8.79 ± 0.53 mV, p < 0.05), while slowly restoring the plasma glucose levels. Together, all these results showed that administration of BM or UCB MSCs improved the DPN via ameliorating pro‐inflammatory cytokine signaling and [Ca2+]i homeostasis. This article is protected by copyright. All rights reserved

  May 13, 2017   doi: 10.1002/jcp.26009   open full text
• What role does the stress response have in congestive heart failure?
  Ahmed Badreddin, Youssef Fady, Hamdy Attia, Mohamed Hafez, Ahmed Khairallah, Dina Johar, Larry Bernstein.
  Journal of Cellular Physiology. May 11, 2017
  This review is concerned with cardiac malfunction as a result of an imbalance in protein proteostasis, the homeostatic balance between protein removal and regeneration in a long remodeling process involving the endoplasmic reticulum (ER) and the unfolded protein response (UPR). The importance of this is of special significance with regard to cardiac function as a high energy requiring muscular organ that has a high oxygen requirement and is highly dependent on mitochondria. The importance of mitochondria is not only concerned with high energy dependence on mitochondrial electron transport, but it also has a role in the signaling between the mitochondria and the ER under stress. Proteins made in the ER are folded as a result of sulfhydryl groups (–SH) and attractive and repulsive reactions in the tertiary structure. We discuss how this matters with respect to an imbalance between muscle breakdown and repair in a stressful environment, especially as a result of oxidative and nitrosative byproducts of mitochondrial activity. The normal repair is a remodeling, but under this circumstance, the cell undergoes or even lysosomal “self eating” autophagy, or even necrosis instead of apoptosis. We shall discuss the relationship of the UPR pathway to chronic congestive heart failure (CHF). This article is protected by copyright. All rights reserved

  May 11, 2017   doi: 10.1002/jcp.26003   open full text
• NMDA receptor subunit composition controls dendritogenesis of hippocampal neurons through CAMKII, CREB‐P, and H3K27ac.
  Fernando J. Bustos, Nur Jury, Pablo Martinez, Estibaliz Ampuero, Matias Campos, Sebastian Abarzúa, Karen Jaramillo, Susanne Ibing, Muriel D. Mardones, Henny Haensgen, Julia Kzhyshkowska, Maria Florencia Tevy, Rachael Neve, Magdalena Sanhueza, Lorena Varela‐Nallar, Martín Montecino, Brigitte van Zundert.
  Journal of Cellular Physiology. May 11, 2017
  Dendrite arbor growth, or dendritogenesis, is choreographed by a diverse set of cues, including the NMDA receptor (NMDAR) subunits NR2A and NR2B. While NR1NR2B receptors are predominantly expressed in immature neurons and promote plasticity, NR1NR2A receptors are mainly expressed in mature neurons and induce circuit stability. How the different subunits regulate these processes is unclear, but this is likely related to the presence of their distinct C‐terminal sequences that couple different signaling proteins. Calcium‐calmodulin‐dependent protein kinase II (CaMKII) is an interesting candidate as this protein can be activated by calcium influx through NMDARs. CaMKII triggers a series of biochemical signaling cascades, involving the phosphorylation of diverse targets. Among them, the activation of cAMP response element‐binding protein (CREB‐P) pathway triggers a plasticity‐specific transcriptional program through unknown epigenetic mechanisms. Here, we found that dendritogenesis in hippocampal neurons is impaired by several well‐characterized constructs (i.e., NR2B‐RS/QD) and peptides (i.e., tatCN21) that specifically interfere with the recruitment and interaction of CaMKII with the NR2B C‐terminal domain. Interestingly, we found that transduction of NR2AΔIN, a mutant NR2A construct with increased interaction to CaMKII, reactivates dendritogenesis in mature hippocampal neurons in vitro and in vivo. To gain insights into the signaling and epigenetic mechanisms underlying NMDAR‐mediated dendritogenesis, we used immunofluorescence staining to detect CREB‐P and acetylated lysine 27 of histone H3 (H3K27ac), an activation‐associated histone tail mark. In contrast to control mature neurons, our data shows that activation of the NMDAR/CaMKII/ERK‐P/CREB‐P signaling axis in neurons expressing NR2AΔIN is not correlated with increased nuclear H3K27ac levels. Dendrite arbor growth, or dendritogenesis, is choreographed by a diverse set of cues, including the NMDA receptor (NMDAR) subunits NR2A and NR2B. Here we found that dendritogenesis in hippocampal neurons is controlled by the interaction between NMDAR subunits and CaMKII in a developmental dependent manner.

  May 11, 2017   doi: 10.1002/jcp.25843   open full text
• Estrogens and androgens inhibit association of RANKL with the pre‐osteoblast membrane through post‐translational mechanisms.
  Anthony Martin, Jiali Yu, Jian Xiong, Aysha B. Khalid, Benita Katzenellenbogen, Sung Hoon Kim, John A. Katzenellenbogen, Suchinda Malaivijitnond, Yankel Gabet, Susan A. Krum, Baruch Frenkel.
  Journal of Cellular Physiology. May 11, 2017
  We have recently demonstrated that RUNX2 promoted, and 17β‐Estradiol (E2) diminished, association of RANKL with the cell membrane in pre‐osteoblast cultures. Here we show that, similar to E2, dihydrotestosterone (DHT) diminishes association of RANKL, and transiently transfected GFP‐RANKL with the pre‐osteoblast membrane without decreasing total RANKL mRNA or protein levels. Diminution of membrane‐associated RANKL was accompanied with marked suppression of osteoclast differentiation from co‐cultured pre‐osteoclasts, even though DHT increased, not decreased, RANKL concentrations in pre‐osteoblast conditioned media. A marked decrease in membrane‐associated RANKL was observed after 30 min of either E2 or DHT treatment, and near‐complete inhibition was observed by 1 hr, suggesting that the diminution of RANKL membrane association was mediated through non‐genomic mechanisms. Further indicating dispensability of nuclear action of estrogen receptor, E2‐mediated inhibition of RANKL membrane association was mimicked by an estrogen dendrimer conjugate (EDC) that cannot enter the cell nucleus. Finally, the inhibitory effect of E2 and DHT on RANKL membrane association was counteracted by the MMP inhibitor NNGH, and the effect of E2 (and not DHT) was antagonized by the Src inhibitor SU6656. Taken together, these results suggest that estrogens and androgens inhibit osteoblast‐driven osteoclastogenesis through non‐genomic mechanism(s) that entail, MMP‐mediated RANKL dissociation from the cell membrane. Association of RANKL with the pre‐osteoblast membrane is dramatically reduced within minutes of treatment with either estradiol or dihydrotestosterone. The effect of estradiol is MMP‐ and Src‐dependent, suggesting RANKL ectodomain shedding, and is mimicked by an estrogen dendrimer conjugate that cannot enter the cell nucleus.

  May 11, 2017   doi: 10.1002/jcp.25862   open full text
• Mild mitochondrial uncoupling induces HSL/ATGL‐independent lipolysis relying on a form of autophagy in 3T3‐L1 adipocytes.
  Stéphane Demine, Silvia Tejerina, Benoît Bihin, Marc Thiry, Nagabushana Reddy, Patricia Renard, Martine Raes, Michel Jadot, Thierry Arnould.
  Journal of Cellular Physiology. May 10, 2017
  Obesity is characterized by an excessive triacylglycerol accumulation in white adipocytes. Various mechanisms allowing the tight regulation of triacylglycerol storage and mobilization by lipid droplet‐associated proteins as well as lipolytic enzymes have been identified. Increasing energy expenditure by inducing a mild uncoupling of mitochondria in adipocytes might represent a putative interesting anti‐obesity strategy as it reduces the adipose tissue triacylglycerol content (limiting alterations caused by cell hypertrophy) by stimulating lipolysis through yet unknown mechanisms, limiting the adverse effects of adipocyte hypertrophy. Herein, the molecular mechanisms involved in lipolysis induced by a mild uncoupling of mitochondria in white 3T3‐L1 adipocytes were characterized. Mitochondrial uncoupling‐induced lipolysis was found to be independent from canonical pathways that involve lipolytic enzymes such as HSL and ATGL. Finally, enhanced lipolysis in response to mitochondrial uncoupling relies on a form of autophagy as lipid droplets are captured by endolysosomal vesicles. This new mechanism of triacylglycerol breakdown in adipocytes exposed to mild uncoupling provides new insights on the biology of adipocytes dealing with mitochondria forced to dissipate energy. This article is protected by copyright. All rights reserved

  May 10, 2017   doi: 10.1002/jcp.25994   open full text
• MicroRNA‐224‐5p regulates adipocyte apoptosis induced by TNFα via controlling NF‐κB activation.
  Renli Qi, Jinxiu Huang, Qi Wang, Hong Liu, Ruisheng Wang, Jing Wang, Feiyun Yang.
  Journal of Cellular Physiology. May 10, 2017
  Tumor necrosis factor (TNF) α can induce cell apoptosis and activate nuclear transcription (NF)‐κB in different cell types. Activated NF‐κB further promotes or suppresses cellular apoptosis in different cases. The present study explored the effect of activated NF‐κB on adipocyte apoptosis induced by TNFα and which microRNAs (miRNAs) were involved in the process. Our findings demonstrated that treatment of differentiated 3T3‐L1 adipocytes with TNFα (20ng/mL) rapidly activated NF‐κB and induced moderate apoptosis. Pyrrolidinedithiocarbamic acid (PDTC, 60µM), a specific NF‐κB inhibitor, abated NF‐κB activation that rendered the adipocytes vulnerable to TNFα‐induced apoptosis. Dozens of miRNAs exhibited significant expression changes following TNFα treatment and the addition of PDTC. In which, miRNA‐224‐5p (miR‐224) was up‐regulated by TNFα exposure but down‐regulated by PDTC addition. Furthermore, over‐expression of miR‐224 promoted NF‐κB activation and prevented the adipocyte apoptosis induced by TNFα, while miR‐224 deficiency showed the opposite effects. The TRAF‐associated NF‐κB activator (TANK) gene was identified as a direct target of miR‐224 by computational and luciferase reporter assays. Additionally, silencing the TANK gene by the small interfering RNA similarly promoted NF‐κB activation and attenuated the cellular apoptosis. In conclusion, these findings demonstrate that miR‐224 plays an essential role in adipocyte apoptosis caused by TNFα through control of NF‐κB activation via targeting the TANK gene. This article is protected by copyright. All rights reserved

  May 10, 2017   doi: 10.1002/jcp.25992   open full text
• Antitumoral Potential, Antioxidant Activity and Carotenoid Content of Two Southern Italy Tomato Cultivars Extracts: San Marzano and Corbarino.
  Daniela Barone, Letizia Cito, Giuseppina Tommonaro, Agnese Anna Abate, Danila Penon, Rocco De Prisco, Antonella Penon, Iris Maria Forte, Elisabetta Benedetti, Annamaria Cimini, Paola Indovina, Barbara Nicolaus, Francesca Pentimalli, Antonio Giordano.
  Journal of Cellular Physiology. May 10, 2017
  Gastric cancer represents a diffuse and aggressive neoplasm, whose mortality index is among the highest in the world. Predisposing factors are E‐cadherin mutations, Helicobacter pylori infection and a diet rich in salted and smoked food, with a low intake of fresh fruits and vegetables. Here we analyzed the effect of total lipophilic extracts of two Southern Italy tomato varieties, San Marzano and Corbarino, on an in vitro model of gastric cancer, YCC‐1, YCC‐2 and YCC‐3 cell lines, characterized by different aggressiveness. Our results showed a possible role of these two varieties of tomatoes against typical neoplastic features. The treatment with tomato extracts affected cancer cell ability to grow both in adherence and in semisolid medium, reducing also cell migration ability. No toxic effects were observed on non‐tumoral cells. We found, on gastric cancer cell lines, effects on both cell cycle progression and apoptosis modulation. The extent of antineoplastic effects, however, did not seem to correlate with the carotenoid content and antioxidant activity of the two tomato varieties. Our data indicate that San Marzano and Corbarino intake might be further considered as nutritional support not only in cancer prevention, but also for cancer patient diet. This article is protected by copyright. All rights reserved

  May 10, 2017   doi: 10.1002/jcp.25995   open full text
• Selective expression of long non‐coding RNAs in a breast cancer cell progression model.
  Kirsten M Tracy, Coralee E Tye, Natalie A Page, Andrew J Fritz, Janet L Stein, Jane B Lian, Gary S Stein.
  Journal of Cellular Physiology. May 10, 2017
  Long non‐coding RNAs are acknowledged as regulators of cancer biology and pathology. Our goal was to perform a stringent profiling of breast cancer cell lines that represent disease progression. We used the MCF‐10 series, which includes the normal‐like MCF‐10A, HRAS‐transformed MCF‐10AT1 (pre‐malignant), and MCF‐10CA1a (malignant) cells, to perform transcriptome wide sequencing. From these data, we have identified 346 lncRNAs with dysregulated expression across the progression series. By comparing lncRNAs from these datasets to those from an additional set of cell lines that represent different disease stages and subtypes, MCF‐7 (early stage, luminal), and MDA‐MB‐231 (late stage, basal), 61 lncRNAs that are associated with breast cancer progression were identified. Querying breast cancer patient data from The Cancer Genome Atlas, we selected a lncRNA, IGFL2‐AS1, of potential clinical relevance for functional characterization. Among the 61 lncRNAs, IGFL2‐AS1 was the most significantly decreased. Our results indicate that this lncRNA plays a role in downregulating its nearest neighbor, IGFL1, and affects migration of breast cancer cells. Furthermore, the lncRNAs we identified provide a valuable resource to mechanistically and clinically understand the contribution of lncRNAs in breast cancer progression. This article is protected by copyright. All rights reserved

  May 10, 2017   doi: 10.1002/jcp.25997   open full text
• Hypomorphic conditional deletion of E11/Podoplanin reveals a role in osteocyte dendrite elongation.
  Katherine A. Staines, Behzad Javaheri, Peter Hohenstein, Robert Fleming, Ekele Ikpegbu, Erin Unger, Mark Hopkinson, David J. Buttle, Andrew A. Pitsillides, Colin Farquharson.
  Journal of Cellular Physiology. May 10, 2017
  The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone specific Pdpn knockdown on osteocyte form and function in the post‐natal mouse. Extensive skeletal phenotyping of male and female 6‐week‐old Oc‐cre;Pdpnflox/flox (cKO) mice and their Pdpnflox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (P < 0.05). Consistent with this, we observed an increase in stiffness in female cKO mice in comparison to fl/fl mice (P < 0.01). Moreover, analysis of the osteocyte phenotype by phalloidin staining revealed a significant decrease in the dendrite volume (P < 0.001) and length (P < 0.001) in cKO mice in which deletion of Pdpn also modifies the bone anabolic loading response (P < 0.05) in comparison to age‐matched fl/fl mice. Together, these data confirm a regulatory role for Pdpn in osteocyte dendrite formation and as such, in the control of osteocyte function. As the osteocyte dendritic network is known to play vital roles in regulating bone modeling/remodelling, this highlights an essential role for Pdpn in bone homeostasis.This article is protected by copyright. All rights reserved

  May 10, 2017   doi: 10.1002/jcp.25999   open full text
• Involvement of the Warburg effect in non‐tumor diseases processes.
  Zhe Chen, Meiqing Liu, Lanfang Li, Linxi Chen.
  Journal of Cellular Physiology. May 10, 2017
  Warburg effect, as an energy shift from mitochondrial oxidative phosphorylation to aerobic glycolysis, is extensively found in various cancers. Interestingly, increasing researchers show that Warburg effect plays a crucial role in non‐tumor diseases. For instance, inhibition of Warburg effect can alleviate pulmonary vascular remodeling in the process of pulmonary hypertension. Interference of Warburg effect improves mitochondrial function and cardiac function in the process of cardiac hypertrophy and heart failure. Additionally, the Warburg effect induces vascular smooth muscle cell proliferation and contributes to atherosclerosis. Warburg effect may also involve in axonal damage and neuronal death, which are related with multiple sclerosis. Furthermore, Warburg effect significantly promotes cell proliferation and cyst expansion in polycystic kidney disease. Besides, Warburg effect relieves amyloid β‐mediated cell death in Alzheimer's disease. And Warburg effect also improves the mycobacterium tuberculosis infection. Finally, we also introduce some glycolytic agonists. This review focuses on the newest researches about the role of Warburg effect in non‐tumor diseases, including pulmonary hypertension, tuberculosis, idiopathic pulmonary fibrosis, failing heart, cardiac hypertrophy, atherosclerosis, Alzheimer's diseases, multiple sclerosis and polycystic kidney disease. Obviously, Warburg effect may be a potential therapeutic target for those non‐tumor diseases. This article is protected by copyright. All rights reserved

  May 10, 2017   doi: 10.1002/jcp.25998   open full text
• Nociceptor plasticity: a closer look.
  Maria Caterina Pace, Maria Beatrice Passavanti, Lorenzo Nardis, Fabio Bosco, Pasquale Sansone, Vincenzo Pota, Manlio Barbarisi, Antonio Palagiano, Fabio Arturo Iannotti, Elisabetta Panza, Caterina Aurilio.
  Journal of Cellular Physiology. May 10, 2017
  Nociceptors are receptors specifically involved in detecting a tissue damage and transducing it in an electrical signal. Nociceptor activation provoked by any kind of acute lesion is related to the release of several mediators of inflammation, within the framework of a process defined as “peripheral sensitization”. This results in an exaggerated response to the painful stimulus, clinically defined as “primary hyperalgesia”. The concept of “neuroplasticity” may explain the adaptive mechanisms carried out by the Nervous System in relation to a “harmful” damage; also, neuroplasticity mechanisms are also fundamental for rehabilitative intervention protocols. Here we review several studies that addressed the role of different receptors and ionic channels discovered on nociceptor surface and their role in pain perception. The changes in expression, distribution and functioning of receptors and ionic channels are thought to be a part of the neuroplasticity property, through which the Nervous System constantly adapts to external stimuli. Moreover, some of the reviewed mediators are also been associated to “central sensitization”, a process that results in pain chronicization when the painful stimulation is particularly prolonged or intense, and lastly leads to the memorization of the uncomfortable painful perception. This article is protected by copyright. All rights reserved

  May 10, 2017   doi: 10.1002/jcp.25993   open full text
• The influence of rAAV2‐mediated SOX2 delivery into neonatal and adult human RPE cells; a comparative study.
  Razie Ezati, Azadeh Etemadzadeh, Zahra‐Soheila Soheili, Shahram Samiei, Ehsan Ranaei Pirmardan, Malihe Davari, Hoda Shams Najafabadi.
  Journal of Cellular Physiology. May 08, 2017
  Cell replacement is a promising therapy for degenerative diseases like age‐related macular degeneration (AMD). Since the human retina lacks regeneration capacity, much attention has been directed towards persuading for cells that can differentiate into retinal neurons. In this report we have investigated reprogramming of the human RPE cells and concerned the effect of donor age on the cellular fate as a critical determinant in reprograming competence. We evaluated the effect of SOX2 over‐expression in human neonatal and adult RPE cells in cultures. The coding region of human SOX2 gene was cloned into Adeno‐Associated virus (AAV2) and primary culture of human neonatal/adult RPE cells were infected by recombinant virus. De‐differentiation of RPE to neural/retinal progenitor cells was investigated by quantitative real time PCR and ICC for neural/ retinal progenitor cells' markers. Gene expression analysis showed 80 fold and 12 fold over‐expression for SOX2 gene in infected neonatal and adult hRPE cells respectively. The fold of increase for Nestin in neonatal and adult hRPE cells was 3.8 fold and 2.5 fold respectively. PAX6 expression was increased 3 fold and 2.5 fold in neonatal/adult treated cultures. Howbeit we could not detect rhodopsin, and CHX10 expression in neonatal hRPE cultures and expression of rhodopsin in adult hRPE cells. Results showed SOX2 induced human neonatal/adult RPE cells to de‐differentiate toward retinal progenitor cells. However, the increased number of PAX6, CHX10, Thy1 and rhodopsin positive cells in adult hRPE treated cultures clearly indicated the considerable generation of neuro‐retinal terminally differentiated cells. This article is protected by copyright. All rights reserved

  May 08, 2017   doi: 10.1002/jcp.25991   open full text
• Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro.
  Neil R.W. Martin, Mark C. Turner, Robert Farrington, Darren J. Player, Mark P. Lewis.
  Journal of Cellular Physiology. May 08, 2017
  The amino acid leucine is thought to be important for skeletal muscle growth by virtue of its ability to acutely activate mTORC1 and enhance muscle protein synthesis, yet little data exist regarding its impact on skeletal muscle size and its ability to produce force. We utilized a tissue engineering approach in order to test whether supplementing culture medium with leucine could enhance mTORC1 signaling, myotube growth, and muscle function. Phosphorylation of the mTORC1 target proteins 4EBP‐1 and rpS6 and myotube hypertrophy appeared to occur in a dose dependent manner, with 5 and 20 mM of leucine inducing similar effects, which were greater than those seen with 1 mM. Maximal contractile force was also elevated with leucine supplementation; however, although this did not appear to be enhanced with increasing leucine doses, this effect was completely ablated by co‐incubation with the mTOR inhibitor rapamycin, showing that the augmented force production in the presence of leucine was mTOR sensitive. Finally, by using electrical stimulation to induce chronic (24 hr) contraction of engineered skeletal muscle constructs, we were able to show that the effects of leucine and muscle contraction are additive, since the two stimuli had cumulative effects on maximal contractile force production. These results extend our current knowledge of the efficacy of leucine as an anabolic nutritional aid showing for the first time that leucine supplementation may augment skeletal muscle functional capacity, and furthermore validates the use of engineered skeletal muscle for highly‐controlled investigations into nutritional regulation of muscle physiology. The effect of the amino acid leucine on myotube size and muscle force production were tested in engineered skeletal muscle in vitro. Leucine activated mTORC1 signaling and augmented contractile force, effects which were completely blunted by co‐incubation with rapamycin. Chronic contractions (24 hr) alongside leucine treatment further increased muscle force, but not myotube size. This data provides further evidence of the efficacy of leucine in regulating muscle size and function, and may prove a useful strategy to improve the therapeutic potential of engineered skeletal muscle.

  May 08, 2017   doi: 10.1002/jcp.25960   open full text
• E2F1interactive with BRCA1 pathway induces HCC two different small molecule metabolism or cell cycle regulation via mitochondrion or CD4+T to cytosol.
  Qingchun Chen, Lin Wang, Minghu Jiang, Juxiang Huang, Zhenfu Jiang, Haitao Feng, Zhili Ji.
  Journal of Cellular Physiology. May 05, 2017
  Breast cancer 1 (BRCA1) and E2F transcription factor 1 (E2F1) are related to metabolism and cell cycle regulation. However, the corresponding mechanism is not clear in HCC. High BRCA1 direct pathway was constructed with 11 molecules from E2F1 feedback‐interactive network in HCC by GRNInfer based on 39 Pearson mutual positive corelation CC >0.25 molecules with E2F1. Integration of GRNInfer with GO, KEGG, BioCarta, GNF_U133A, UNIGENE_EST, Disease, GenMAPP databases by DAVID and MAS 3.0, E2F1 feedback‐interactive BRCA1 indirect mitochondrion to cytosol pathway was identified as upstream LAPTM4B activation, feedback UNG, downstream BCAT1‐HIST1H2AD‐TK1 reflecting protein and DNA binding with enrichment of small molecule metabolism; The corresponding BRCA1 indirect membrane to cytosol pathway as upstream CCNB2‐NUSAP1 activation, feedback TTK‐HIST1H2BJ‐CENPF, downstream MCM4‐TK1 reflecting ATP and microtubule binding with enrichment of CD4+T‐related cell cycle regulation in HCC. Therefore, we propose that E2F1 interactive with BRCA1 pathway induces HCC two different small molecule metabolism or cell cycle regulation via mitochondrion or CD4+T to cytosol. Knowledge analysis demonstrates our E2F1 feedback‐interactive BRCA1 pathway wide disease distribution and reflects a novel common one of tumor and cancer. This article is protected by copyright. All rights reserved

  May 05, 2017   doi: 10.1002/jcp.25988   open full text
• Therapeutic Application of Multipotent Stem Cells.
  Hamed Mirzaei, Amirhossein Sahebkar, Laleh Shiri, Abdullah Moridikia, Sara Nazari, Javid Sadri Nahand, Hossein salehi, Jan Stenvang, Aria Masoudifar, Hamid Reza Mirzaei, Mahmoud Reza Jaafari.
  Journal of Cellular Physiology. May 05, 2017
  Cell therapy is an emerging fields in the treatment of various diseases such as cardiovascular, pulmonary, hepatic and neoplastic diseases. Stem cells are an integral tool for cell therapy. Multipotent stem cells are an important class of stem cells which have the ability to self‐renew through dividing and developing into multiple specific cell types in a specific tissue or organ. These cells are capable to activate or inhibit a sequence of cellular and molecular pathways leading to anti‐inflammatory and anti‐apoptotic effects which might contribute to the treatment of various diseases. It has been showed that multipotent stem cells exert their therapeutic effects via inhibition/activation of a sequence of cellular and molecular pathways. Although the advantages of multipotent stem cells are numerous, further investigation is still necessary to clarify the biology and safety of these cells before they could be considered as a potential treatment for different types of diseases. This review summarizes different features of multipotent stem cells including isolation, differentiation and therapeutic applications. This article is protected by copyright. All rights reserved

  May 05, 2017   doi: 10.1002/jcp.25990   open full text
• Direct Delivery of Recombinant Pin1 Protein Rescued Osteoblast Differentiation of Pin1‐Deficient Cells.
  Woo‐Jin Kim, Rabia Islam, Bong‐Soo Kim, Young‐Dan Cho, Won‐Joon Yoon, Jeong‐Hwa Baek, Kyung‐Mi Woo, Hyun‐Mo Ryoo.
  Journal of Cellular Physiology. May 05, 2017
  Pin1 is a peptidyl prolyl cis‐trans isomerase that specifically binds to the phosphoserine‐proline or phosphothreonine‐proline motifs of several proteins. We reported that Pin1 plays a critical role in the fate determination of Smad1/5, Runx2, and β‐catenin that are indispensable nuclear proteins for osteoblast differentiation. Though several chemical inhibitors has been discovered for Pin1, no activator has been reported as of yet. In this study, we directly introduced recombinant Pin1 protein successfully into the cytoplasm via fibroin nanoparticle encapsulated in cationic lipid. This nanoparticle‐lipid complex delivered its cargo with a high efficiency and a low cytotoxicity. Direct delivery of Pin1 leads to increased Runx2 and Smad signaling and resulted in recovery of the osteogenic marker genes expression and the deposition of mineral in Pin1‐deficient cells. These result indicated that a direct Pin1 protein delivery method could be a potential therapeutics for the osteopenic diseases. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc. Direct delivery of Pin1 leads to increased Runx2 and Smad signaling and resulted in recovery of the osteogenic marker genes expression and the deposition of mineral in Pin1‐deficient cells.

  May 05, 2017   doi: 10.1002/jcp.25673   open full text
• The diversified function and potential therapy of ectopic olfactory receptors in non‐olfactory tissues.
  Zhe Chen, Hong Zhao, Nian Fu, Linxi Chen.
  Journal of Cellular Physiology. May 05, 2017
  Olfactory receptors (ORs) are mainly distributed in olfactory neurons and play a key role in detecting volatile odorants, eventually resulting in the production of smell perception. Recently, it is also reported that ORs are expressed in non‐olfactory tissues including heart, lung, sperm, skin, and cancerous tissues. Interestingly, ectopic ORs are associated with the development of diseases in non‐olfactory tissues. For instance, ectopic ORs initiate the hypoxic ventilatory responses and maintain the oxygen homeostasis of breathing in the carotid body when oxygen levels decline. Ectopic ORs induce glucose homeostasis in diabetes. Ectopic ORs regulate systemic blood pressure by increasing renin secretion and vasodilation. Ectopic ORs participate in the process of tumor cell proliferation, apoptosis, metastasis, and invasiveness. Ectopic ORs accelerate the occurrence of obesity, angiogenesis and wound‐healing processes. Ectopic ORs affect fetal hemoglobin levels in sickle cell anemia and thalassemia. Finally, we also elaborate some ligands targeting for ORs. Obviously, the diversified function and related signal pathway of ectopic ORs may play a potential therapeutic target in non‐olfactory tissues. Thus, this review focuses on the latest research results about the diversified function and therapeutic potential of ectopic ORs in non‐olfactory tissues. This review focuses on the latest research results about the diversified function and therapeutic potential of ectopic ORs in non‐olfactory tissues.

  May 05, 2017   doi: 10.1002/jcp.25929   open full text
• Fasting inhibits hepatic stellate cells activation and potentiates anti‐cancer activity of Sorafenib in hepatocellular cancer cells.
  Oriana Lo Re, Concetta Panebianco, Stefania Porto, Carlo Cervi, Francesca Rappa, Stefano Di Biase, Michele Caraglia, Valerio Pazienza, Manlio Vinciguerra.
  Journal of Cellular Physiology. May 04, 2017
  BACKGROUND: Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short‐term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi‐tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short‐lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown. METHODS: 24 hour fasting (2% serum, 0.1% glucose)‐induced changes on human hepatic stellate cells (HSC) LX‐2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)‐induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. 24 hours fasting‐induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib. RESULTS: Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short‐term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells. CONCLUSIONS: Fasting or fasting‐mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use. This article is protected by copyright. All rights reserved

  May 04, 2017   doi: 10.1002/jcp.25987   open full text
• Role of follistatin in muscle and bone alterations induced by gravity change in mice.
  Naoyuki Kawao, Hironobu Morita, Koji Obata, Kohei Tatsumi, Hiroshi Kaji.
  Journal of Cellular Physiology. May 04, 2017
  Interactions between muscle and bone have been recently noted. We reported that the vestibular system plays crucial roles in the changes in muscle and bone induced by hypergravity in mice. However, the details of the mechanisms by which gravity change affects muscle and bone through the vestibular system still remain unknown. Here, we investigated the roles of humoral factors linking muscle to bone and myostatin‐related factors in the hypergravity‐induced changes in muscle and bone in mice with vestibular lesions (VL). Hypergravity elevated serum and mRNA levels of follistatin, an endogenous inhibitor of myostatin, in the soleus muscle of mice. VL blunted the hypergravity‐enhanced levels of follistatin in the soleus muscle of mice. Simulated microgravity decreased follistatin mRNA level in mouse myoblastic C2C12 cells. Follistatin elevated the mRNA levels of myogenic genes as well as the phosphorylation of Akt and p70S6 kinase in C2C12 cells. As for bone metabolism, follistatin antagonized the mRNA levels of osteogenic genes suppressed by activin A during the differentiation of mesenchymal cells into osteoblastic cells. Moreover, follistatin attenuated osteoclast formation enhanced by myostatin in the presence of receptor activator of nuclear factor‐κB ligand in RAW 264.7 cells. Serum follistatin levels were positively related to bone mass in mouse tibia. In conclusion, the present study provides novel evidence that hypergravity affects follistatin levels in muscle through the vestibular system in mice. Follistatin may play some roles in the interactions between muscle and bone metabolism in response to gravity change. This article is protected by copyright. All rights reserved

  May 04, 2017   doi: 10.1002/jcp.25986   open full text
• Irisin plays a pivotal role to protect the heart against ischemia and reperfusion injury.
  Hao Wang, Yu Tina Zhao, Shouyan Zhang, Patrycja M Dubielecka, Jianfeng Du, Naohiro Yano, Y. Eugene Chin, Shougang Zhuang, Gangjian Qin, Ting C Zhao.
  Journal of Cellular Physiology. May 03, 2017
  Irisin, a newly identified hormone, is critical to modulating body metabolism, thermogenesis and reducing oxidative stresses. However, whether irisin protects the heart against myocardial ischemia and reperfusion (I/R) injury remains unknown. In this study, we determine the effect of irisin on myocardial I/R injury in the Langendorff perfused heart and cultured myocytes. Adult C57/BL6 mice were treated with irisin (100 mg/kg) or vehicle for 30 min to elicit preconditioning. The isolated hearts were subjected to 30 min ischemia followed by 30 min reperfusion. Left ventricular function was measured and infarction size were determined using by tetrazolium staining. Western blot was employed to determine myocardial SOD‐1, active‐caspase 3, annexin V, p38, and phospho‐p38. H9c2 cardiomyoblasts were exposed to hypoxia and reoxygenation for assessment of the effects of irisin on mitochondrial respiration and mitochondrial permeability transition pore (mPTP). Irisin treatment produced remarkable improvements in ventricular functional recovery, as evident by the increase in RPP and attenuation in LVEDP. As compared to the vehicle treatment, irisin resulted in a marked reduction of myocardial infarct size. Notably, irisin treatment increased SOD‐1 and p38 phosphorylation, but suppressed levels of active‐caspase 3, cleaved PARP, and annexin V. In cardiomyoblasts exposed to hypoxia/reoxygenation, irisin treatment significantly attenuated hypoxia/reoxygenation (H/R), as indicated by the reduction of lactate dehydrogenase (LDH) leakage and apoptotic cardiomyocytes. Furthermore, irisin treatments suppressed the opening of mPTP, mitochondrial swelling, and protected mitochondria function. Our results indicate that irisin serves as a novel approach to eliciting cardioprotection, which is associated with the improvement of mitochondrial function. Irisin treatment produced remarkable improvements in ventricular functional recovery and resulted in a marked reduction of myocardial infarct size. Furthermore, irisin treatments suppressed the opening of mPTP, mitochondrial swelling, and protected mitochondria function. Our results indicate that irisin serves as a novel approach to eliciting cardioprotection, which is associated with the improvement of mitochondrial function.

  May 03, 2017   doi: 10.1002/jcp.25857   open full text
• MicroRNA‐195‐5p Regulates Osteogenic Differentiation of Periodontal Ligament Cells Under Mechanical Loading.
  Maolin Chang, Heng Lin, Haidi Fu, Beike Wang, Guangli Han, Mingwen Fan.
  Journal of Cellular Physiology. May 03, 2017
  Osteogenic differentiation and bone formation are tightly regulated by several factors, including microRNAs (miRNAs). However, miRNA expression patterns and function during mechanical loading‐induced osteogenic differentiation of human periodontal ligament cells (PDLCs) remain unclear. Here, we investigated the differential expression of miRNA‐195‐5p in the periodontal tissues of mice under orthodontic mechanical loading and in primary human PDLCs exposed to a simulated tension strain. The miR‐195‐5p was observed to be down‐regulated and negatively correlated with osteogenic differentiation. Overexpression of miR‐195‐5p significantly inhibited PDLC differentiation under cyclic tension strain (CTS), whereas the functional inhibition of miR‐195‐5p yielded an opposite effect. Further experiments confirmed that WNT family member 3A (WNT3A), fibroblast growth factor 2 (FGF2), and bone morphogenetic protein receptor‐1A (BMPR1A), proteins important for osteogenic activity and stability, were direct targets of miR‐195‐5p. Mechanical loading increased the WNT3A, FGF2, and BMPR1A protein levels, while miR‐195‐5p inhibited WNT3A, FGF2, and BMPR1A protein expression. WNT, FGF, and BMP signaling were involved in osteogenic differentiation of PDLCs under CTS. Further study confirmed that reintroduction of WNT3A and BMPR1A can rescue the inhibition of miR‐195‐5p on osteogenic differentiation of PDLCs. Our findings are the first to demonstrate that miR‐195‐5p is a mechanosensitive gene that plays an important role in mechanical loading‐induced osteogenic differentiation and bone formation. During orthodontic treatment, periodontal ligament cells can differentiate into osteoblasts under tension strain. The miR‐195‐5p was down‐regulated under tension strain, leading to the disinhibition of WNT3A, FGF2, and BMPR1A, which associated with othodonitc force induced bone formation.

  May 03, 2017   doi: 10.1002/jcp.25856   open full text
• Deubiquitinating enzyme USP22 positively regulates c‐Myc stability and tumorigenic activity in mammalian and breast cancer cells.
  Dongyeon Kim, Ahyoung Hong, Hye In Park, Woo Hyun Shin, Lang Yoo, Seo Jeong Jeon, Kwang Chul Chung.
  Journal of Cellular Physiology. May 03, 2017
  The proto‐oncogene c‐Myc has a pivotal function in growth control, differentiation, and apoptosis and is frequently affected in human cancer, including breast cancer. Ubiquitin‐specific protease 22 (USP22), a member of the USP family of deubiquitinating enzymes (DUBs), mediates deubiquitination of target proteins, including histone H2B and H2A, telomeric repeat binding factor 1, and cyclin B1. USP22 is also a component of the mammalian SAGA transcriptional co‐activating complex. In this study, we explored the functional role of USP22 in modulating c‐Myc stability and its physiological relevance in breast cancer progression. We found that USP22 promotes deubiquitination of c‐Myc in several breast cancer cell lines, resulting in increased levels of c‐Myc. Consistent with this, USP22 knockdown reduces c‐Myc levels. Furthermore, overexpression of USP22 stimulates breast cancer cell growth and colony formation, and increases c‐Myc tumorigenic activity. In conclusion, the present study reveals that USP22 in breast cancer cell lines increases c‐Myc stability through c‐Myc deubiquitination, which is closely correlated with breast cancer progression. The present study reveals that deubiquitinating enzyme USP22 in mammalia and breast cancer cell lines increases c‐Myc stability through c‐Myc deubiquitination, which is closely correlated with breast cancer progression.

  May 03, 2017   doi: 10.1002/jcp.25841   open full text
• An advanced tri‐culture model to evaluate the dynamic interplay among osteoblasts, osteoclasts, and endothelial cells.
  Stefania Pagani, Paola Torricelli, Francesca Veronesi, Francesca Salamanna, Simona Cepollaro, Milena Fini.
  Journal of Cellular Physiology. May 03, 2017
  The dynamic metabolism and the numerous roles of bone tissue necessitate a suitable in vitro model to represent them. In order to investigate the interaction among the several cell types composing bone microenvironment, we studied a tri‐culture model including human osteoblasts (OBs), osteoclasts (OCs), and endothelial cells (HUVEC). While OBs are essential for bone deposition and OCs for bone resorption, the vasculature is necessary to provide growth factors, nutrients, and oxygen in the mature tissue. The results of this study showed a strong mutual influence between OBs, OCs, and HUVEC in term of proliferation, viability, and activity (release of ALP, Coll I, OPG, RANKL, VEGF, CTSK, TGFβ, and IL‐6). The behavior of the single cultures demonstrated to be different compared to the bi‐ or tri‐cultures and depending on the cell types involved: the coexistence of OBs and OCs stimulated the synthetic activity of both cell types, while the presence of HUVEC induced a stimulating role for OBs but mainly an inhibitory effect for OC. In addition, evidence of the effects of OBs and OCs on HUVEC is highlighted by their morphology: regular and able to “sketch” little vessels in presence of OBs, more disorganized and heterogeneous in presence of OCs. Taken together, these observations well characterize an advanced cellular model to be used as starting point for mimicking bone microenvironment in vivo, thus reducing the use of animals in the preclinical phase and offering a more reliable tool to test new and innovative biomaterials. In vitro model of direct co‐colture showing the mutual influence among osteoblast for bone deposition, osteoclast for bone resorption and endothelial cells for angiogenesis, as starting point in mimicking in vivo microenvironment.

  May 03, 2017   doi: 10.1002/jcp.25875   open full text
• Blocking fibrotic signaling in fibroblasts from patients with carpal tunnel syndrome.
  Yoshiaki Yamanaka, Anne Gingery, Gosuke Oki, Tai‐Hua Yang, Chunfeng Zhao, Peter C. Amadio.
  Journal of Cellular Physiology. May 03, 2017
  Fibrosis of the subsynovial connective tissue (SSCT) in carpal tunnel syndrome (CTS) patients is increasingly recognized as an important aspect of CTS pathophysiology. In this study, we evaluated the effect of blocking profibrotic pathways in fibroblasts from the SSCT in CTS patients. Fibroblasts were stimulated with transforming growth factor β1 (TGF‐β1), and then treated either with a specific fibrosis pathway inhibitor targeting TGF‐β receptor type 1 (TβRI), platelet‐derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), or vascular endothelial growth factor receptor (VEGFR). Fibrosis array and quantitative real‐time polymerase chain reaction of fibrotic genes were evaluated. Array gene expression analysis revealed significant down‐regulation of multiple fibrotic genes after treatment with TβRI, PDGFR, and VEGFR inhibitors. No array fibrotic genes were significantly down‐regulated with EGFR inhibition. Further gene expression analysis of known CTS fibrosis markers collagen type I A2 (Col1), collagen type III A1 (Col3), connective tissue growth factor (CTGF), and SERPINE1 showed significantly down‐regulation after TβRI inhibition. In contrast, VEGFR inhibition significantly down‐regulated CTGF and SERPINE1, whereas, PDGFR and EGFR inhibition significantly down‐regulated Col3. Taken together the inhibition of TβRI appears to be the primary mediator of fibrotic gene expression in fibroblasts from CTS patients. TGF‐β/Smad activity was further evaluated, and as expected inhibition of Smad activity was significantly down‐regulated after inhibition of TβRI, but not with PDGFR, VEGFR, or EGFR inhibition. These results indicate that local therapies specifically targeting TGF‐β signaling alone or in combination offer the potential of a novel local antifibrosis therapy for patients with CTS. In this study, we evaluated the effect of blocking profibrotic pathways by receptor blocking agents in fibroblasts from patients with carpal tunnel syndrome (CTS). There was significant down‐regulation of multiple fibrotic genes after treatment with a TFFβ1R (SD208) inhibitor, with smaller effects from PDGFR (AG1296) and VEGFR (Axitinib) inhibitors. These results indicate that local therapies specifically targeting TGF‐β signaling, alone or in combination with other pathways, offer the potential of a novel local anti‐fibrosis therapy for patients with CTS.

  May 03, 2017   doi: 10.1002/jcp.25901   open full text
• Rapamycin attenuates BAFF‐extended proliferation and survival via disruption of mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells.
  Qingyu Zeng, Shanshan Qin, Hai Zhang, Beibei Liu, Jiamin Qin, Xiaoxue Wang, Ruijie Zhang, Chunxiao Liu, Xiaoqing Dong, Shuangquan Zhang, Shile Huang, Long Chen.
  Journal of Cellular Physiology. May 03, 2017
  B cell activating factor from the TNF family (BAFF) stimulates B‐cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)‐stimulated B‐cell proliferation/survival by suppressing mTOR‐mediated PP2A‐Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF‐promoted B cell proliferation/survival is also related to blocking hsBAFF‐stimulated phosphorylation of Akt, S6K1, and 4E‐BP1, as well as expression of survivin in normal and B‐lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF‐induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr‐Akt) or constitutively active S6K1 (S6K1‐ca), or downregulation of 4E‐BP1 conferred resistance to rapamycin's attenuation of hsBAFF‐induced survivin expression and B‐cell proliferation/viability, whereas overexpression of dominant negative Akt (dn‐Akt) or constitutively hypophosphorylated 4E‐BP1 (4EBP1‐5A), or downregulation of S6K1, or co‐treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF‐induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF‐evoked aggressive B‐cell malignancies and autoimmune diseases. Rapamycin, a macrocyclic lactone, prevented B cells from hsBAFF‐promoted proliferation/survival, not only by targeting mTORC1‐mediated S6K1/4E‐BP1 pathways, but also via targeting mTORC2‐mediated Akt pathway. This study highlights that rapamycin may be a potential agent for preventing excessive BAFF‐evoked aggressive B‐cell malignancies and autoimmune diseases.

  May 03, 2017   doi: 10.1002/jcp.25913   open full text
• HDAC1 and HDAC3 underlie dynamic H3K9 acetylation during embryonic neurogenesis and in schizophrenia‐like animals.
  Josef Večeřa, Eva Bártová, Jana Krejčí, Soňa Legartová, Denisa Komůrková, Jana Rudá‐Kučerová, Tibor Štark, Eva Dražanová, Tomáš Kašpárek, Alexandra Šulcová, Frank J. Dekker, Wiktor Szymanski, Christian Seiser, Georg Weitzer, Raphael Mechoulam, Vincenzo Micale, Stanislav Kozubek.
  Journal of Cellular Physiology. May 03, 2017
  Although histone acetylation is one of the most widely studied epigenetic modifications, there is still a lack of information regarding how the acetylome is regulated during brain development and pathophysiological processes. We demonstrate that the embryonic brain (E15) is characterized by an increase in H3K9 acetylation as well as decreases in the levels of HDAC1 and HDAC3. Moreover, experimental induction of H3K9 hyperacetylation led to the overexpression of NCAM in the embryonic cortex and depletion of Sox2 in the subventricular ependyma, which mimicked the differentiation processes. Inducing differentiation in HDAC1‐deficient mouse ESCs resulted in early H3K9 deacetylation, Sox2 downregulation, and enhanced astrogliogenesis, whereas neuro‐differentiation was almost suppressed. Neuro‐differentiation of (wt) ESCs was characterized by H3K9 hyperacetylation that was associated with HDAC1 and HDAC3 depletion. Conversely, the hippocampi of schizophrenia‐like animals showed H3K9 deacetylation that was regulated by an increase in both HDAC1 and HDAC3. The hippocampi of schizophrenia‐like brains that were treated with the cannabinoid receptor‐1 inverse antagonist AM251 expressed H3K9ac at the level observed in normal brains. Together, the results indicate that co‐regulation of H3K9ac by HDAC1 and HDAC3 is important to both embryonic brain development and neuro‐differentiation as well as the pathophysiology of a schizophrenia‐like phenotype. We showed that co‐regulation of H3K9 acetylation by histone deacetylases HDAC1 and HDAC3 is important to both embryonic brain development and neuro‐differentiation as well as the pathophysiology of a schizophrenia‐like phenotype. This knowledge contributes to a basic understanding of epigenetics of the brain.

  May 03, 2017   doi: 10.1002/jcp.25914   open full text
• Chrysin induces death of prostate cancer cells by inducing ROS and ER stress.
  Soomin Ryu, Whasun Lim, Fuller W. Bazer, Gwonhwa Song.
  Journal of Cellular Physiology. May 03, 2017
  Chrysin is a natural flavone found in numerous plant extracts, honey, and propolis that has multiple biological activities including anti‐cancer effects. Understanding of biological mechanisms mediated in response to chrysin in cancerous cells may provide novel insight into chemotherapeutic approaches with reduced side effects in cancers. In the present study, we investigated functional roles of chrysin in progression of prostate cancer cells using DU145 and PC‐3 cell lines. The results showed that chrysin induced apoptosis of cells evidenced by DNA fragmentation and increasing the population of both DU145 and PC‐3 cells in the sub‐G1 phase of the cell cycle. In addition, chrysin reduced expression of proliferating cell nuclear antigen in the prostate cancer cell lines compared to untreated prostate cancer cells. Moreover, chrysin induced loss of mitochondria membrane potential (MMP), while increasing production of reactive oxygen species (ROS) and lipid peroxidation in a dose‐dependent manner. Also, it induced endoplasmic reticulum (ER) stress through activation of unfolded protein response (UPR) proteins including PRKR‐like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), and 78 kDa glucose‐regulated protein (GRP78) in DU145 and PC‐3 cells. The chrysin‐mediated intracellular signaling pathways suppressed phosphoinositide 3‐kinase (PI3K) and the abundance of AKT, P70S6K, S6, and P90RSK proteins, but stimulated mitogen‐activated protein kinases (MAPK) and activation of ERK1/2 and P38 proteins in the prostate cancer cells. Collectively, these results indicate that chrysin initiates cell death through induction of mitochondrial‐mediated apoptosis and ER stress, and regulation of signaling pathways responsible for proliferation of prostate cancer cells. Chrysin initiates cell death through induction of ROS production and ER stress, and regulation of MAPK signaling pathways responsible for proliferation of prostate cancer cells.

  May 03, 2017   doi: 10.1002/jcp.25861   open full text
• Transforming growth factor β induces bone marrow mesenchymal stem cell migration via noncanonical signals and N‐cadherin.
  Maria Jose Dubon, Jinyeong Yu, Sanghyuk Choi, Ki‐Sook Park.
  Journal of Cellular Physiology. May 03, 2017
  Transforming growth factor‐beta (TGF‐β) induces the migration and mobilization of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) to maintain bone homeostasis during bone remodeling and facilitate the repair of peripheral tissues. Although many studies have reported the mechanisms through which TGF‐β mediates the migration of various types of cells, including cancer cells, the intrinsic cellular mechanisms underlying cellular migration, and mobilization of BM‐MSCs mediated by TGF‐β are unclear. In this study, we showed that TGF‐β activated noncanonical signaling molecules, such as Akt, extracellular signal‐regulated kinase 1/2 (ERK1/2), focal adhesion kinase (FAK), and p38, via TGF‐β type I receptor in human BM‐MSCs and murine BM‐MSC‐like ST2 cells. Inhibition of Rac1 by NSC23766 and Src by PP2 resulted in impaired TGF‐β‐mediated migration. These results suggested that the Smad‐independent, noncanonical signals activated by TGF‐β were necessary for migration. We also showed that N‐cadherin‐dependent intercellular interactions were required for TGF‐β‐mediated migration using functional inhibition of N‐cadherin with EDTA treatment and a neutralizing antibody (GC‐4 antibody) or siRNA‐mediated knockdown of N‐cadherin. However, N‐cadherin knockdown did not affect the global activation of noncanonical signals in response to TGF‐β. Therefore, these results suggested that the migration of BM‐MSCs in response to TGF‐β was mediated through N‐cadherin and noncanonical TGF‐β signals. N‐cadherin‐dependent intercellular interaction is required for TGF‐β‐mediated migration of both BM‐MSCs and the BM‐MSC like cell line, ST2. It is likely that in addition to N‐cadherin, non‐canonical TGF‐β signals including Akt, ERKs, p38, FAK, and Rac1 are also required for the cell migration.

  May 03, 2017   doi: 10.1002/jcp.25863   open full text
• Mitochondria‐targeted esculetin inhibits PAI‐1 levels by modulating STAT3 activation and miR‐19b via SIRT3: Role in acute coronary artery syndrome.
  Sujana Katta, Santosh Karnewar, Devayani Panuganti, Mahesh Kumar Jerald, B. K. S. Sastry, Srigiridhar Kotamraju.
  Journal of Cellular Physiology. May 03, 2017
  In this study we explored the microRNAs responsible for the regulation of PAI‐1 during LPS‐stimulated inflammation in human aortic endothelial cells and subsequently studied the effect of a newly synthesized mitochondria‐targeted esculetin (Mito‐Esc) that was shown for its anti‐atherosclerotic potential, in modulating PAI‐1 levels and its targeted miRs during angiotensin‐II‐induced atherosclerosis in ApoE−/− mice. LPS‐stimulated PAI‐1 was accompanied with an upregulation of miR‐19b and down‐regulation of miR‐30c. These effects of LPS on PAI‐1 were reversed in the presence of both parent esculetin and Mito‐Esc. However, the effect of Mito‐Esc was more pronounced in the regulation of PAI‐1. In addition, LPS‐stimulated PAI‐1 expression was significantly decreased in cells treated with Anti‐miR‐19b, thereby suggesting that miR‐19b co‐expression plays a key role in PAI‐1 regulation. The results also show that incubation of cells with Stattic, an inhibitor of STAT‐3, inhibited LPS‐stimulated PAI‐1 expression. Interestingly, knockdown of SIRT3, a mitochondrial biogenetic marker, enhanced PAI‐1 levels via modulation of miR‐19b and ‐30c. Mito‐Esc treatment significantly inhibited Ang‐II‐induced PAI‐1, possibly via altering miR‐19b and 30c in ApoE−/− mice. The association between PAI‐1, miR‐19b and ‐30c were further confirmed in plasma and microparticles isolated from patients suffering from acute coronary syndrome of various degrees. Taken together, LPS‐induced PAI‐1 involves co‐expression of miR‐19b and down regulation of miR‐30c, and Mito‐Esc treatment by modulating miR‐19b and miR‐30c through SIRT3 activation, inhibits PAI‐1 levels that, in part, contribute to its anti‐atherosclerotic effects. Moreover, there exists a strong positive correlation between miR‐19b and PAI‐1 in patients suffering from ST‐elevated myocardial infarction. LPS‐induced PAI‐1 involves co‐expression of miR‐19b and down regulation of miR‐30c. Mito‐Esc treatment by modulating miR‐19b and miR‐30c through SIRT3 activation, inhibits PAI‐1 levels. An association between PAI‐1, miR‐19b, and ‐30c was observed in plasma and microparticles isolated from patients suffering from acute coronary syndrome of various degrees.

  May 03, 2017   doi: 10.1002/jcp.25865   open full text
• Adenosine and adenosine receptors in the immunopathogenesis and treatment of cancer.
  Mohammad H. Kazemi, Sahar Raoofi Mohseni, Mohammad Hojjat‐Farsangi, Enayat Anvari, Ghasem Ghalamfarsa, Hamed Mohammadi, Farhad Jadidi‐Niaragh.
  Journal of Cellular Physiology. May 03, 2017
  Tumor cells overcome anti‐tumor responses in part through immunosuppressive mechanisms. There are several immune modulatory mechanisms. Among them, adenosine is an important factor which is generated by both cancer and immune cells in tumor microenvironment to suppress anti‐tumor responses. Two cell surface expressed molecules including CD73 and CD39 catalyze the generation of adenosine from adenosine triphosphate (ATP). The generation of adenosine can be enhanced under metabolic stress like tumor hypoxic conditions. Adenosine exerts its immune regulatory functions through four different adenosine receptors (ARs) including A1, A2A, A2B, and A3 which are expressed on various immune cells. Several studies have indicated the overexpression of adenosine generating enzymes and ARs in various cancers which was correlated with tumor progression. Since the signaling of ARs enhances tumor progression, their manipulation can be promising therapeutic approach in cancer therapy. Accordingly, several agonists and antagonists against ARs have been designed for cancer therapy. In this review, we will try to clarify the role of different ARs in the immunopathogenesis, as well as their role in the treatment of cancer. Adenosine is one of the important immunosuppressive factors in the tumor microenvironment. Therefore, modulation of adenosine receptors can be considered as the potent cancer therapeutic method.

  May 03, 2017   doi: 10.1002/jcp.25873   open full text
• MicroRNA106a regulates matrix metalloprotease 9 in a sirtuin‐1 dependent mechanism.
  Lincy Edatt, Ashutosh K. Maurya, Grace Raji, Haritha Kunhiraman, Sameer V. B. Kumar.
  Journal of Cellular Physiology. May 03, 2017
  Cellular migration is important during many physiological as well as pathological conditions and is regulated very tightly by an intricate network of signaling and effector molecules. One of the important players during cellular migration are matrix metalloproteases and their levels have been reported to be important in determining the cellular migratory properties during metastasis. MMPs and regulators of MMPs therefore, present themselves as potent candidates for manipulation, to control conditions where they get dysregulated. Micro RNAs are a group of micro regulators that can modulate expression of a gene through transcriptional and post transcriptional regulations. Owing to the fact that many microRNAs have already been reported to regulate MMPs and that miR106a, a member of oncomir17 family has been implicated in metastatic conditions, the present study intended to analyze if miR106a can regulate levels of MMP9, an important inducible matrix metalloproteinase. The results of the in vitro experiments demonstrated that under conditions of migration cells showed elevated levels of miR106a, which could regulate the expression of major MMP9 regulator, SIRT‐1. Decreased levels of SIRT1thus resulted in an increase in the expression and activity of MMP9. Over expression and mRNA stability studies carried out also suggested regulatory role of miR106a. The overall results thus suggested that the levels of miR106a gets modulated during cellular migration, causing a change in the levels of SIRT‐1 mRNA by affecting its stability and the levels of SIRT‐1 in turn can regulate the levels of MMP9. During cellular migration, the level of miRNA 106a is upregulated, which in turn regulates the levels of SIRT‐1 and further, SIRT‐1 modulates the levels of MMP‐9.

  May 03, 2017   doi: 10.1002/jcp.25870   open full text
• Soft tissue engineering with micronized‐gingival connective tissues.
  Sawako Noda, Yoshinori Sumita, Seigo Ohba, Hideyuki Yamamoto, Izumi Asahina.
  Journal of Cellular Physiology. May 03, 2017
  The free gingival graft (FGG) and connective tissue graft (CTG) are currently considered to be the gold standards for keratinized gingival tissue reconstruction and augmentation. However, these procedures have some disadvantages in harvesting large grafts, such as donor‐site morbidity as well as insufficient gingival width and thickness at the recipient site post‐treatment. To solve these problems, we focused on an alternative strategy using micronized tissue transplantation (micro‐graft). In this study, we first investigated whether transplantation of micronized gingival connective tissues (MGCTs) promotes skin wound healing. MGCTs (≤100 µm) were obtained by mincing a small piece (8 mm3) of porcine keratinized gingiva using the RIGENERA system. The MGCTs were then transplanted to a full skin defect (5 mm in diameter) on the dorsal surface of immunodeficient mice after seeding to an atelocollagen matrix. Transplantations of atelocollagen matrixes with and without micronized dermis were employed as experimental controls. The results indicated that MGCTs markedly promote the vascularization and epithelialization of the defect area 14 days after transplantation compared to the experimental controls. After 21 days, complete wound closure with low contraction was obtained only in the MGCT grafts. Tracking analysis of transplanted MGCTs revealed that some mesenchymal cells derived from MGCTs can survive during healing and may function to assist in wound healing. We propose here that micro‐grafting with MGCTs represents an alternative strategy for keratinized tissue reconstruction that is characterized by low morbidity and ready availability. Micronized‐gingival connective tissues promote the skin wound healing.

  May 03, 2017   doi: 10.1002/jcp.25871   open full text
• Profilin1 is expressed in osteocytes and regulates cell shape and migration.
  Wanting Lin, Yayoi Izu, Arayal Smriti, Makiri Kawasaki, Chantida Pawaputanon, Ralph T. Böttcher, Mercedes Costell, Keiji Moriyama, Masaki Noda, Yoichi Ezura.
  Journal of Cellular Physiology. May 03, 2017
  Osteocytes are the most abundant cells in bone and regulate bone metabolism in coordination with osteoblasts and osteoclasts. However, the molecules that control osteocytes are still incompletely understood. Profilin1 is an actin‐binding protein that is involved in actin polymerization. Osteocytes possess characteristic dendritic process formed based on actin cytoskeleton. Here, we examined the expression of profilin1 and its function in osteocytes. Profilin1 mRNA was expressed in osteocytic MLO‐Y4 cells and its levels were gradually increased along with the time in culture. With regard to functional aspect, knockdown of profilin1 by siRNA enhanced BMP‐induced increase in alkaline phosphatase expression levels in MLO‐Y4 cells. Profilin1 knockdown suppressed the levels of dendritic processes and migration of MLO‐Y4 cells. Since aging causes an increase in ROS in the body, we further examined the effects of hydrogen peroxide on the expression of profilin1. Hydrogen peroxide treatment increased the levels of profilin1 mRNA in MLO‐Y4 cells in contrast to the decline in alkaline phosphatase. Profilin1 was expressed not only in MLO‐Y4cells but also in the primary cultures of osteocytes. Importantly, profilin1 mRNA levels in primary cultures of osteocytes were higher than those in primary cultures of osteoblasts. To examine in vivo role of profilin1 in osteocytes, profilin1 was conditionally knocked out by using DMP1‐cre and profilin1 floxed mice. This conditional deletion of profilin1 specifically in osteocytes resulted in reduction in the levels of bone volume and bone mineral density. These data indicate that profilin1 is expressed in osteocytes and regulates cell shape, migration and bone mass. Profilin1 knockdown reduces dendritic process formation in osteocytic MLO‐Y4 cells.

  May 03, 2017   doi: 10.1002/jcp.25872   open full text
• Glucose adsorption to chitosan membranes increases proliferation of human chondrocyte via mammalian target of rapamycin complex 1 and sterol regulatory element‐binding protein‐1 signaling.
  Shun‐Fu Chang, Kuo‐Chin Huang, Chin‐Chang Cheng, Yu‐Ping Su, Ko‐Chao Lee, Cheng‐Nan Chen, Hsin‐I Chang.
  Journal of Cellular Physiology. May 03, 2017
  Osteoarthritis (OA) is currently still an irreversible degenerative disease of the articular cartilage. Recent, dextrose (d‐glucose) intraarticular injection prolotherapy for OA patients has been reported to benefit the chondrogenic stimulation of damaged cartilage. However, the detailed mechanism of glucose's effect on cartilage repair remains unclear. Chitosan, a naturally derived polysaccharide, has recently been investigated as a surgical or dental dressing to control breeding. Therefore, in this study, glucose was adsorbed to chitosan membranes (CTS‐Glc), and the study aimed to investigate whether CTS‐Glc complex membranes could regulate the proliferation of human OA chondrocytes and to explore the underlying mechanism. Human OA and SW1353 chondrocytes were used in this study. The experiments involving the transfection of cells used SW1353 chondrocytes. A specific inhibitor and siRNAs were used to investigate the mechanism underlying the CTS‐Glc‐regulated proliferation of human chondrocytes. We found that CTS‐Glc significantly increased the proliferation of both human OA and SW1353 chondrocytes comparable to glucose‐ or chitosan‐only stimulation. The role of mammalian target of rapamycin complex 1 (mTORC1) signaling, including mTOR, raptor, and S6k proteins, has been demonstrated in the regulation of CTS‐Glc‐increased human chondrocyte proliferation. mTORC1 signaling increased the expression levels of maturated SREBP‐1 and FASN and then induced the expressions of cell cycle regulators, that is, cyclin D, cyclin‐dependent kinase‐4 and ‐6 in human chondrocytes. This study elucidates the detailed mechanism behind the effect of CTS‐Glc complex membranes in promoting chondrocyte proliferation and proposes a possible clinical application of the CTS‐Glc complex in the dextrose intraarticular injection of OA prolotherapy in the future to attenuate the pain and discomfort of OA patients. The adsorption of glucose onto the chitosan membranes increased human chondrocyte proliferation by activating mTORC1 signaling and then induced the expression of mSREBP‐1/FASN/cell cycle regulators (cyclin D and CDK‐4/6).

  May 03, 2017   doi: 10.1002/jcp.25869   open full text
• Mitochondrial ROS‐induced ERK1/2 activation and HSF2‐mediated AT1R upregulation are required for doxorubicin‐induced cardiotoxicity.
  Chih‐Yang Huang, Jia‐Yi Chen, Chia‐Hua Kuo, Pei‐Ying Pai, Tsung‐Jung Ho, Tung‐Sheng Chen, Fu‐Jen Tsai, Vijaya V. Padma, Wei‐Wen Kuo, Chih‐Yang Huang.
  Journal of Cellular Physiology. May 03, 2017
  Doxorubicin (DOX), one useful chemotherapeutic agent, is limited in clinical use because of its serious cardiotoxicity. Growing evidence suggests that angiotensin receptor blockers (ARBs) have cardioprotective effects in DOX‐induced cardiomyopathy. However, the detailed mechanisms underlying the action of ARBs on the prevention of DOX‐induced cardiomyocyte cell death have yet to be investigated. Our results showed that angiotensin II receptor type I (AT1R) plays a critical role in DOX‐induced cardiomyocyte apoptosis. We found that MAPK signaling pathways, especially ERK1/2, participated in modulating AT1R gene expression through DOX‐induced mitochondrial ROS release. These results showed that several potential heat shock binding elements (HSE), which can be recognized by heat shock factors (HSFs), located at the AT1R promoter region. HSF2 markedly translocated from the cytoplasm to the nucleus when cardiomyocytes were damaged by DOX. Furthermore, the DNA binding activity of HSF2 was enhanced by DOX via deSUMOylation. Overexpression of HSF2 enhanced DOX‐induced cardiomyocyte cell death as well. Taken together, we found that DOX induced mitochondrial ROS release to activate ERK‐mediated HSF2 nuclear translocation and AT1R upregulation causing DOX‐damaged heart failure in vitro and in vivo. DOX induces mitochondrial ROS accumulation to activate ERK activation for HSF2‐mediated AT1R upregulation.

  May 03, 2017   doi: 10.1002/jcp.25905   open full text
• Environmental Temperature affect Physiology and Survival of Nanosecond Pulsed Electric Field Treated Cells.
  Shengyong Yin, Xudong Miao, Xueming Zhang, Xinhua Chen, Hao Wen.
  Journal of Cellular Physiology. May 03, 2017
  Nanosecond pulsed electric field (nsPEF) is a novel non‐thermal tumor ablation technique. However, how nsPEF affect cell physiology at different environmental temperature is still kept unknown. But this issue is of critical clinical practice relevance. This work aim to investigate how nsPEF treated cancer cells react to different environmental temperatures (0°C, 4°C, 25°C and 37°C). Their cell viability, apoptosis, mitochondrial membrane potential, and reactive oxygen species (ROS) were examined. Lower temperature resulted in higher apoptosis rate, decreased mitochondria membrane potential and increased ROS levels. Sucrose and N‐acetylcysteine (NAC) pre‐incubation inhibit ROS generation and increase cell survival, protecting nsPEF‐treated cells from low temperature‐caused cell death. This work provides an experimental basis for hypothermia and fluid transfusion during nsPEF ablation with anesthesia. This article is protected by copyright. All rights reserved

  May 03, 2017   doi: 10.1002/jcp.25984   open full text
• Pannexin hemichannels: A novel promising therapy target for oxidative stress related diseases.
  Jin Xu, Linxi Chen, Lanfang Li.
  Journal of Cellular Physiology. May 03, 2017
  Pannexins, which contain three subtypes: pannexin‐1, ‐2, and ‐3, are vertebrate glycoproteins that form non‐junctional plasma membrane intracellular hemichannels via oligomerization. Oxidative stress refers to an imbalance of the generation and elimination of reactive oxygen species (ROS). Studies have shown that elevated ROS levels are pivotal in the development of a variety of diseases. Recent studies indicate that the occurrence of these oxidative stress related diseases is associated with pannexin hemichannels. It is also reported that pannexins regulate the production of ROS which in turn may increase the opening of pannexin hemichannels. In this paper, we review recent researches about the important role of pannexin hemichannels in oxidative stress related diseases. Thus, pannexin hemichannels, novel therapeutic targets, hold promise in managing oxidative stress related diseases such as the tumor, inflammatory bowel diseases (IBD), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), cardiovascular disease, insulin resistance (IR), and neural degeneration diseases. In this review, we find that there is intimate relationship between pannexin hemichannels and oxidative stress, thus we discuss possible mechanism of pannexin hemichannels on oxidative stress related diseases.

  May 03, 2017   doi: 10.1002/jcp.25906   open full text
• Roles of platelet‐derived growth factor in vascular calcification.
  Liu Ouyang, Kun Zhang, Jie Chen, Jingfeng Wang, Hui Huang.
  Journal of Cellular Physiology. May 03, 2017
  Vascular calcification (VC) is prevalent in aging, and patients with hypertension, chronic kidney disease (CKD) or diabetes. VC is regarded as an active and complex process that involves multiple mechanisms responsible for calcium deposition in vessel wall. In light of the complicated pathogenesis of VC, effective therapy for ameliorating VC is limited. Thus, it is urgent to explore the potential mechanisms and find new targets for the therapy of VC. Platelet‐derived growth factor (PDGF), a potent mitogen and chemoattractant, has been found to disturb the vascular homeostasis by inducing inflammation, oxidative stress and phenotype transition, all of which accelerate the process of VC. The aim of current review is to present a review about the roles of PDGF in affecting VC and to establish a potential target for treating VC. This article is protected by copyright. All rights reserved

  May 03, 2017   doi: 10.1002/jcp.25985   open full text
• Reduced glucose‐induced insulin secretion in low‐protein‐fed rats is associated with altered pancreatic islets redox status.
  Ana Paula G. Cappelli, Claudio C. Zoppi, Leonardo R. Silveira, Thiago M. Batista, Flávia M. Paula, Priscilla M. R. da Silva, Alex Rafacho, Helena C. Barbosa‐Sampaio, Antonio C. Boschero, Everardo M. Carneiro.
  Journal of Cellular Physiology. May 03, 2017
  In the present study, we investigated the relationship between early life protein malnutrition‐induced redox imbalance, and reduced glucose‐stimulated insulin secretion. After weaning, male Wistar rats were submitted to a normal‐protein‐diet (17%‐protein, NP) or to a low‐protein‐diet (6%‐protein, LP) for 60 days. Pancreatic islets were isolated and hydrogen peroxide (H2O2), oxidized (GSSG) and reduced (GSH) glutathione content, CuZn‐superoxide dismutase (SOD1), glutathione peroxidase (GPx1) and catalase (CAT) gene expression, as well as enzymatic antioxidant activities were quantified. Islets that were pre‐incubated with H2O2 and/or N‐acetylcysteine, were subsequently incubated with glucose for insulin secretion measurement. Protein malnutrition increased CAT mRNA content by 100%. LP group SOD1 and CAT activities were 50% increased and reduced, respectively. H2O2 production was more than 50% increased whereas GSH/GSSG ratio was near 60% lower in LP group. Insulin secretion was, in most conditions, approximately 50% lower in LP rat islets. When islets were pre‐incubated with H2O2 (100 μM), and incubated with glucose (33 mM), LP rats showed significant decrease of insulin secretion. This effect was attenuated when LP islets were exposed to N‐acetylcysteine. Our data suggest that short‐term early life protein malnourishment induces reduction in redox GIIS signaling only at extreme oxidant challenges, elicited here by high H2O2 pre‐incubation followed by elevated glucose concentration incubation, probably due to lower antioxidant scavenging capacity.

  May 03, 2017   doi: 10.1002/jcp.25908   open full text
• Probiotics are a good choice in remission of inflammatory bowel diseases: A meta analysis and systematic review.
  Mahboube Ganji‐Arjenaki, Mahmoud Rafieian‐Kopaei.
  Journal of Cellular Physiology. May 03, 2017
  Altered gut bacteria and bacterial metabolic pathways are two important factors in initiation and progression of inflammatory bowel disease (IBD). However, efficacy of probiotics in remission of patients with IBD has not been characterized. This study was performed on the studies that specifically assessed the efficacy of probiotics in attaining clinical response on patients with various types of IBD. The efficacy of variant species of probiotics in different conditions and the influence of study quality in outcomes of randomized controlled trials (RCTs) were also assessed. The RCTs were collected by searching in MEDLINE Web of Science and Google scholar. Then all studies were abstracted in abstraction form and the outcomes were analyzed with fixed‐effect and mixed‐effect models for assessment of efficacy of variant species of probiotics in subgroups of IBDs. Analysis of 9 trials showed that probiotics had not significant effect on Crohn's disease (CD) (p = 0.07) but analysis of 3 trials in children with IBD revealed a significant advantage (p < 0.01). Analysis of 18 trials revealed that probiotics in patients with Ulcerative colitis (UC) in different conditions have significant effects (p = 0.007). VSL#3 probiotics in patients with UC had significant effect (p < 0.01). Combination of Lactobacillus probiotic, prebiotics had significant effect (p = 0.03) only in patients with UC. Combination of Saccharomyces boulardii, Lactobacillus, and VSL#3 probiotics in CD had also a trend for efficiency (p = 0.057). In children with IBD, the combination of Lactobacillus with VSL#3 probiotics had significant effect (p < 0.01). Probiotics are beneficial in IBD, especially the combination ones in UC.

  May 03, 2017   doi: 10.1002/jcp.25911   open full text
• Autophagy mediates cytotoxicity of human colorectal cancer cells treated with garcinielliptone FC.
  Shen‐Jeu Won, Cheng‐Hsin Yen, Ting‐Yu Lin, Ya‐Fen Jiang‐Shieh, Chun‐Nan Lin, Jyun‐Ti Chen, Chun‐Li Su.
  Journal of Cellular Physiology. May 03, 2017
  The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT‐29 cells. In the present study, we observed that many autophagy‐related genes in GFC‐treated HT‐29 cells were up‐ and down‐regulated using a cDNA microarray containing oncogenes and kinase genes. GFC‐induced autophagy of HT‐29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double‐membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5‐Atg12 and PI3K/Beclin‐1 complexes were observed using Western blot. Administration of autophagy inhibitor (3‐methyladenine and shRNA Atg5) and apoptosis inhibitor Z‐VAD showed that the GFC‐induced autophagy was cytotoxic form and GFC‐induced apoptosis enhanced GFC‐induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC‐induced anticancer mechanisms of human colorectal cancer. The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. GFC‐induced autophagy was cytotoxic form and GFC‐induced apoptosis enhanced GFC‐induced autophagy.

  May 03, 2017   doi: 10.1002/jcp.25910   open full text
• Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2.
  Sung‐Hee Hwang, Byeal‐I Han, Michael Lee.
  Journal of Cellular Physiology. May 03, 2017
  Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/Cas9 system. In ATG5 KO cells, RT‐PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild‐type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto‐ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G2/M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G2/M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2. ATG5 knockout cells show morphological transformation and anchorage‐independent growth‐ ATG5 KO cells have a lower proportion of the apoptotic cell population as a way to escape cell death in response to PP2.

  May 03, 2017   doi: 10.1002/jcp.25912   open full text
• Modulating tumor hypoxia by nanomedicine for effective cancer therapy.
  Rana Jahanban‐Esfahlan, Miguel de la Guardia, Delshad Ahmadi, Bahman Yousefi.
  Journal of Cellular Physiology. May 03, 2017
  Hypoxia, a characteristic feature of tumors, is indispensable to tumor angiogenesis, metastasis, and multi drug resistance. Hypoxic avascular regions, deeply embedded inside the tumors significantly hinder delivery of therapeutic agents. The low oxygen tension results in resistance to the current applied anti‐cancer therapeutics including radiotherapy, chemotherapy, and photodynamic therapy, the efficacy of which is firmly tied to the level of tumor oxygen supply. However, emerging data indicate that nanocarriers/nanodrugs can offer substantial benefits to improve the efficacy of current therapeutics, through modulation of tumor hypoxia. This review aims to introduce the most recent advances made in nanocarrier mediated targeting of tumor hypoxia. The first part is dedicated to the approaches by which nanocarriers could be designed to target/leverage hypoxia. These approaches include i) inhibiting Hypoxia Inducer Factor (HIF‐1α); ii) hypoxia activated prodrugs/linkers; and iii) obligate anaerobe mediated targeting of tumor hypoxia. The second part, details novel nanosystems proposed to modulate tumor hypoxia through tumor oxygenation. These methods seek to lessen tumor hypoxia through vascular normalization, or reoxygenation therapy. The reoxygenation of tumor could be accomplished by: i) generation of oxygen filled nanocarriers; ii) natural/artificial oxygen nanocarriers; and iii) oxygen generators. The efficacy of each approach and their potential in cancer therapy is further discussed. Hypoxic avascular regions, deeply embedded inside the tumors significantly hinder delivery of therapeutic agents. Hypoxia could be accomplished by developing HIF‐1a and VEGF inhibitors that are now successfully integrated into the oncology practice. Incorporation of nanomedicine in targeting tumor hypoxia could dramatically enhance potential of anti‐cancer therapeutics.

  May 03, 2017   doi: 10.1002/jcp.25859   open full text
• Surface Functionalized Magnetic Nanoparticles Shift Cell Behavior with On/Off Magnetic Fields.
  Seongbeom Jeon, Ramesh Subbiah, Taufik Bonaedy, Seyoung Van, Kwideok Park, Kyusik Yun.
  Journal of Cellular Physiology. May 02, 2017
  Magnetic nanoparticles (MNPs) are used as contrast agents and targeted drug delivery systems (TDDS) due to their favorable size, surface charge, and magnetic properties. Unfortunately, the toxicity associated with MNPs limits their biological applications. Surface functionalization of MNPs with selective polymers alters the surface chemistry to impart better biocompatibility. We report the preparation of surface functionalized MNPs using iron oxide NPs (MNPs), poly (lactic‐co‐glycolic acid) (PLGA) and sodium alginate via co‐precipitation, emulsification, and electro‐spraying, respectively. The NPs are in the nanosize range and negatively charged. Morphological and structural analyses affirm the surface functionalized nanostructure of the NPs. The surface functionalized MNPs are biocompatible, and demonstrate enhanced intracellular delivery under an applied magnetic field (H), which evinces the targeting ability of MNPs. After NP treatment, the physico‐mechanical properties of fibroblasts are decided by the selective MNP uptake under “on” or “off” magnetic field conditions. We envision potential use of biocompatible surface functionalized MNP for intracellular‐, targeted‐DDS, imaging, and for investigating cellular mechanics. This article is protected by copyright. All rights reserved

  May 02, 2017   doi: 10.1002/jcp.25980   open full text
• ABC of multifaceted Dystrophin glycoprotein complex (DGC).
  Hina F Bhat, Saima S Mir, Khalid B Dar, Zuhaib F Bhat, Riaz A Shah, Nazir A Ganai.
  Journal of Cellular Physiology. May 02, 2017
  Dystrophin protein in association with several other cellular proteins and glycoproteins leads to the formation of a large multifaceted protein complex at the cell membrane referred to as dystrophin glycoprotein complex (DGC), that serves distinct functions in cell signalling and maintaining the membrane stability as well as integrity. In accordance with this, several findings suggest exquisite role of DGC in signalling pathways associated with cell development and/or maintenance of homeostasis. In the present review, we summarize the established facts about the various components of this complex with emphasis on recent insights into specific contribution of the DGC in cell signalling at the membrane. We have also discussed the recent advances made in exploring the molecular associations of DGC components within the cells and the functional implications of these interactions. Our review would help to comprehend the composition, role and functioning of DGC and may lead to a deeper understanding of its role in several human diseases. This article is protected by copyright. All rights reserved

  May 02, 2017   doi: 10.1002/jcp.25982   open full text
• The Skeletal Cell‐Derived Molecule Sclerostin Drives Bone Marrow Adipogenesis.
  Heather Fairfield, Carolyne Falank, Elizabeth Harris, Victoria Demambro, Michelle McDonald, Jessica A Pettit, Sindhu T Mohanty, Peter Croucher, Ina Kramer, Michaela Kneissel, Clifford J. Rosen, Michaela R. Reagan.
  Journal of Cellular Physiology. April 29, 2017
  The bone marrow niche is a dynamic and complex microenvironment that can both regulate, and be regulated by the bone matrix. Within the bone marrow (BM), mesenchymal stromal cell (MSC) precursors reside in a multi‐potent state and retain the capacity to differentiate down osteoblastic, adipogenic, or chondrogenic lineages in response to numerous biochemical cues. These signals can be altered in various pathological states including, but not limited to, osteoporotic‐induced fracture, systemic adiposity, and the presence of bone‐homing cancers. Herein we provide evidence that signals from the bone matrix (osteocytes) determine marrow adiposity by regulating adipogenesis in the bone marrow. Specifically, we found that physiologically relevant levels of Sclerostin (SOST), which is a Wnt‐inhibitory molecule secreted from bone matrix‐embedded osteocytes, can induce adipogenesis in 3T3‐L1 cells, mouse ear‐ and BM‐derived MSCs, and human BM‐derived MSCs. We demonstrate that the mechanism of SOST induction of adipogenesis is through inhibition of Wnt signaling in pre‐adipocytes. We also demonstrate that a decrease of sclerostin in vivo, via both genetic and pharmaceutical methods, significantly decreases bone marrow adipose tissue (BMAT) formation. Overall, this work demonstrates a direct role for SOST in regulating fate determination of BM‐adipocyte progenitors. This provides a novel mechanism for which BMAT is governed by the local bone microenvironment, which may prove relevant in the pathogenesis of certain diseases involving marrow adipose. Importantly, with anti‐sclerostin therapy at the forefront of osteoporosis treatment and a greater recognition of the role of BMAT in disease, these data are likely to have important clinical implications.This article is protected by copyright. All rights reserved

  April 29, 2017   doi: 10.1002/jcp.25976   open full text
• Interplay between miRNAs and human diseases.
  Prosenjit Paul, Anindya Chakraborty, Debasree Sarkar, Malobika Langthasa, Musfhia Rahman, Minakshi Bari, RK. Sanamacha Singha, Arup Kumar Malakar, Supriyo Chakraborty.
  Journal of Cellular Physiology. April 27, 2017
  MicroRNAs (miRNAs) are endogenous, non‐coding RNAs, which have evoked a great deal of interest due to their importance in many aspects of homeostasis and diseases. MicroRNAs are stable and are essential components of gene regulatory networks. They play a crucial role in healthy individuals and their dysregulations have also been implicated in a wide range of diseases, including diabetes, cardiovascular disease, kidney disease, and cancer. This review summarized the current understanding of interactions between miRNAs and different diseases and their role in disease diagnosis and therapy. This review summarized the current understanding of interactions between miRNAs and different diseases and their role in disease diagnosis and therapy.

  April 27, 2017   doi: 10.1002/jcp.25854   open full text
• Effect of stress on human biology: Epigenetics, adaptation, inheritance, and social significance.
  David T. Denhardt.
  Journal of Cellular Physiology. April 27, 2017
  I present a brief introduction to epigenetics, focused primarily on methylation of the genome and various regulatory RNAs, modifications of associated histones, and their importance in enabling us to adapt to real and changing environmental, developmental, and social circumstances. Following this is a more extensive overview of how it impacts our inheritance, our entire life (which changes as we age), and how we interact with others. Throughout, I emphasize the critical influence that stress, of many varieties exerts, via epigenetic means, on much of how we live and survive, mostly in the brain. I end with a short section on multigenerational transmission, drugs, and the importance of both social life and early life experiences in the development of adult diseases. There will be nothing about cancer. Although epigenetics is critical in that field, it is a whole different cobweb of complications (some involving stress). This review focuses on the impact of stress of many kinds on human physiology. It discusses DNA and histone modifications and regulation by various forms of RNA. It ends with a critical analysis of the impact of drugs and social interactions on the individual and transgenerational inheritance.

  April 27, 2017   doi: 10.1002/jcp.25837   open full text
• IL‐10 expression in macrophages from neonates born from obese mothers is suppressed by IL‐4 and LPS/INFγ.
  Francisca Cifuentes‐Zúñiga, Viviana Arroyo‐Jousse, Gustavo Soto‐Carrasco, Paola Casanello, Ricardo Uauy, Bernardo J. Krause, José A. Castro‐Rodríguez.
  Journal of Cellular Physiology. April 27, 2017
  Obese women offspring have a higher risk of developing chronic diseases associated with an altered immune function. We aim to determine, in neonatal monocyte‐derived macrophages, whether maternal obesity is associated with an altered expression and DNA methylation of pro‐ and anti‐inflammatory genes, along with a higher pro‐inflammatory response. Cord blood from newborns of obese (Ob) and lean (control) women were obtained at delivery. Monocytes were isolated and differentiated into macrophages, in which M1 (LPS/IFNγ) and M2 (IL‐4) polarization were assayed. The mRNA levels for TNFα, IL‐1β, IL‐12A, IL‐12B, IL‐10, and IL‐4R were quantified by qPCR and the DNA methylation of candidate genes determined by pyrosequencing. Results: Ob‐monocytes had decreased levels of mRNA for pro‐inflammatory cytokines IL‐1β, IL‐10, and IL‐12B compared with controls. Conversely, Ob‐macrophages showed increased levels of mRNA for TNFα, IL‐4R, and IL‐10 compared with controls. M1 response was comparable between both groups, characterized by an important induction of TNFα and IL‐1β. In response to an M2 stimulus, control macrophages showed a decreased expression of inflammatory mediators while Ob‐macrophages had an additional suppression of the anti‐inflammatory mediator IL‐10. Changes in IL‐1β (monocytes) and IL‐10 (macrophages) in Ob‐monocytes were paralleled by changes in their promoter DNA methylation in fetal monocytes. These results suggest that monocyte‐derived macrophages from obese newborns show a basal anti‐inflammatory phenotype with an unbalanced response to M1 and M2 polarization stimuli. The presence of changes in DNA methylation of key inflammatory genes in neonatal monocytes suggests an intrauterine programing of immune function by maternal obesity.

  April 27, 2017   doi: 10.1002/jcp.25845   open full text
• A prognostic 4‐gene expression signature for squamous cell lung carcinoma.
  Jun Li, Jing Wang, Yanbin Chen, Lijie Yang, Sheng Chen.
  Journal of Cellular Physiology. April 27, 2017
  Squamous cell lung carcinoma (SQCLC), a common and fatal subtype of lung cancer, caused lots of mortalities and showed different outcomes in prognosis. This study was to screen key genes and to figure a prognostic signature to cluster the patients with SQCLC. RNA‐Seq data from 550 patients with SQCLC were downloaded from The Cancer Genome Atlas (TCGA). Genetically changed genes were identified and analyzed in univariate survival analysis. Genes significantly influencing prognosis were selected with frequency higher than 100 in lasso regression. Meanwhile, area under the curve (AUC) values and hazard ratios (HR) for seed genes were obtained with R Language. Functional enrichment analysis was performed and clustering effectiveness of the selected common gene set was analyzed with Kaplan–Meier. Finally, the stability and validity of the optimal clustering model were verified. A total of 7,222 genetically changed genes were screened, including 1,045 ones with p < 0.05, 1,746, p < 0.1, and 2,758, p < 0.2. The common gene sets with more than 100 frequencies were 14‐Genes, 10‐Genes and 6‐Genes. Genes with p < 0.05 participated in positive regulation of ERK1 and ERK2 cascade, angiogenesis, platelet degranulation, cell–matrix adhesion, extracellular matrix organization, macrophage activation, and so on. A four‐gene clustering model in 14‐Genes (DPPA, TTTY16, TRIM58, HKDC1, ZNF589, ALDH7A1, LINC01426, IL19, LOC101928358, TMEM92, HRASLS, JPH1, LOC100288778, GCGR) was verified as the optimal. The discovery of four‐gene clustering model in 14‐Genes can cluster the patient samples effectively. This model would help predict the outcomes of patients with SQCLC then improve the treatment strategies. A four‐gene clustering model in 14‐Genes (DPPA, TTTY16, TRIM58, HKDC1, ZNF589, ALDH7A1, LINC01426, IL19, LOC101928358, TMEM92, HRASLS, JPH1, LOC100288778, GCGR) was verified as the optimal. The discovery of four‐gene clustering model in 14‐Genes can cluster the patient samples effectively. This model would help predict the outcomes of patients with SQCLC then improve the treatment strategies.

  April 27, 2017   doi: 10.1002/jcp.25846   open full text
• Molecular pathogenesis in chronic obstructive pulmonary disease and therapeutic potential by targeting AMP‐activated protein kinase.
  Zhihui Zhang, Xiaoyu Cheng, Li Yue, Wenhui Cui, Wencheng Zhou, Jian Gao, Hongwei Yao.
  Journal of Cellular Physiology. April 27, 2017
  Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide, which is characterized by a persistent airflow limitation caused by chronic inflammatory responses to noxious particles or gases. Cigarette smoke and environmental pollutions are major etiological factors for causing COPD. It has been shown that cigarette smoking causes abnormal inflammatory responses, cellular senescence, mitochondrial dysfunction and metabolic dysregulation, suggesting their involvement in the development of COPD. Although the medical care and treatment have advanced, there are no effective therapies to stop or reverse lung destruction in COPD/emphysema. AMP‐activated protein kinase (AMPK) is a serine threonine kinase with α, β, and γ subunits that are highly conserved through evolution. AMPK has been shown to regulate bioenergetics, inflammatory responses, senescence, and metabolism. This review focused on the updated understanding of molecular pathogenesis of COPD, and highlighted the crucial roles of AMPK in lung abnormalities as well as discussed the potential therapeutics of AMPK activators in preventing and halting the progression of COPD. Cigarette smoke as well as other gases and particles cause inflammatory responses, senescence, mitochondrial dysfunction, and metabolic dysregulation, leading to lung destruction and subsequently COPD/emphysema. AMPK is reduced by cigarette smoke and in lungs of COPD patients, and AMPK activators protect against aforementioned cellular processes and pulmonary emphysema.

  April 27, 2017   doi: 10.1002/jcp.25844   open full text
• Downregulation of methyltransferase Dnmt2 results in condition‐dependent telomere shortening and senescence or apoptosis in mouse fibroblasts.
  Anna Lewinska, Jagoda Adamczyk‐Grochala, Ewa Kwasniewicz, Maciej Wnuk.
  Journal of Cellular Physiology. April 27, 2017
  Dnmt2 is a highly conserved methyltransferase of uncertain biological function(s). As Dnmt2 was considered as a driver of fruit fly longevity and a modulator of stress response, we decided to evaluate the role of Dnmt2 during stress‐induced premature senescence in NIH3T3 mouse fibroblasts. Stable knockdown of Dnmt2 resulted in hydrogen peroxide‐mediated sensitivity and apoptosis, whereas in the control conditions, senescence was induced. Cellular senescence was accompanied by elevated levels of p53 and p21, decreased telomere length and telomerase activity, increased production of reactive oxygen species and protein carbonylation, and DNA damage. Dnmt2 silencing also promoted global DNA and RNA hypermethylation, and upregulation of methyltransferases, namely Dnmt1, Dnmt3a, and Dnmt3b. Taken together, we show for the first time that Dnmt2 may promote lifespan in the control conditions and survival during stress conditions in mouse fibroblasts. Dnmt2 silencing results in cellular senescence and apoptosis in the control conditions and after HP treatment, respectively. Senescence was accompanied by mild oxidative stress, DNA damage and telomere shortening, whereas Dnmt2‐silenced fibroblasts were more prone to ROS‐induced apoptotic cell death after HP stimulation.

  April 27, 2017   doi: 10.1002/jcp.25848   open full text
• Sphingolipids metabolism in the salivary glands of rats with obesity and streptozotocin induced diabetes.
  Marta Garbowska, Bartłomiej Łukaszuk, Agnieszka Mikłosz, Igor Wróblewski, Krzysztof Kurek, Lucyna Ostrowska, Adrian Chabowski, Małgorzata Żendzian‐Piotrowska, Anna Zalewska.
  Journal of Cellular Physiology. April 27, 2017
  Diabetes is considered a major public health problem affecting millions of individuals worldwide. Remarkably, scientific reports regarding salivary glands sphingolipid metabolism in diabetes are virtually non‐existent. This is odd given the well‐established link between the both in other tissues (e.g., skeletal muscles, liver) and the key role of these glands in oral health preservation. The aim of this paper is to examine sphingolipids metabolism in the salivary glands in (pre)diabetes (evoked by high fat diet feeding or streptozotocin). Wistar rats were allocated into three groups: control, HFD‐, or STZ‐diabetes. The content of major sphingolipid classes in the parotid (PSG) and submandibular (SMSG) glands was assessed via chromatography. Additionally, Western blot analyses were employed for the evaluation of key sphingolipid signaling pathway enzyme levels. No changes in ceramide content in the PSG were found, whereas an increase in ceramide concentration for SMSG of the STZ group was observed. This was accompanied by an elevation in SPT1 level. Probably also sphingomyelin hydrolysis was increased in the SMSG of the STZ‐diabetic rats, since we observed a significant drop in the amount of SM. PSG and SMSG respond differently to (pre)diabetes, with clearer pattern presented by the later gland. An activation of sphingomyelin signaling pathway was observed in the course of STZ‐diabetes, that is, metabolic condition with rapid onset/progression. Whereas, chronic HFD lead to an inhibition of sphingomyelin signaling pathway in the salivary glands (manifested in an inhibition of ceramide de novo synthesis and accumulation of S1P). In this study we explored sphingolipid metabolism in the rats salivary glands in both diet induced insulin resistance and type 1 diabetic condition. Based on our results we conclude that the SMSG are characterized by somewhat greater metabolic activity in comparison to PSG. Another interesting and novel finding of our study is an activation of sphingomyelin signaling pathway observed only in the course of type 1 diabetes, which is a metabolic condition with rapid onset and progression.

  April 27, 2017   doi: 10.1002/jcp.25939   open full text
• The SGK1 inhibitor SI113 induces autophagy, apoptosis, and endoplasmic reticulum stress in endometrial cancer cells.
  Domenico Conza, Paola Mirra, Gaetano Calì, Teresa Tortora, Luigi Insabato, Francesca Fiory, Silvia Schenone, Rosario Amato, Francesco Beguinot, Nicola Perrotti, Luca Ulianich.
  Journal of Cellular Physiology. April 27, 2017
  Endometrial cancer is often characterized by PI3K/AKT pathway deregulation. Recently it has been suggested that SGK1, a serine/threonine protein kinase that shares structural and functional similarities with the AKT family, might play a role in cancer, since its expression and/or activity has been found to be deregulated in different human tumors. However, the role of SGK1 in endometrial cancer has been poorly investigated. Here, we show that SGK1 expression is increased in tissue specimens from neoplastic endometrium. The SGK1 inhibitor SI113 induced a significant reduction of endometrial cancer cells viability, measured by the (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay. This effect was associated to the increase of autophagy, as revealed by the increase of the markers LC3B‐II and beclin I, detected by both immunofluorescence and western blot analysis. SI113 treatment caused also apoptosis of endometrial cancer cells, evidenced by the cleavage of the apoptotic markers PARP and Caspase‐9. Intriguingly, these effects were associated to the induction of endoplasmic reticulum stress markers GRP78 and CHOP evaluated by both Real‐Time RT‐PCR and Western Blot analysis. Increased expression of SGK1 in endometrial cancer tissues suggest a role for SGK1 in this type of cancer, as reported for other malignancies. Moreover, the efficacy of SI113 in affecting endometrial cancer cells viability, possibly via endoplasmic reticulum stress activation, identifies SGK1 as an attractive molecular target for new tailored therapeutic intervention for the treatment of endometrial cancer. SGK1 expression is increased in endometrial cancer SI113, an inhibitor of SGK1, induces endoplasmic reticulum stress, autophagy, and apoptosis in endometrial cancer cells.

  April 27, 2017   doi: 10.1002/jcp.25850   open full text
• Role of PARP activity in lung cancer‐induced cachexia: Effects on muscle oxidative stress, proteolysis, anabolic markers, and phenotype.
  Alba Chacon‐Cabrera, Mercè Mateu‐Jimenez, Klaus Langohr, Clara Fermoselle, Elena García‐Arumí, Antoni L. Andreu, Jose Yelamos, Esther Barreiro.
  Journal of Cellular Physiology. April 27, 2017
  Strategies to treat cachexia are still at its infancy. Enhanced muscle protein breakdown and ubiquitin‐proteasome system are common features of cachexia associated with chronic conditions including lung cancer (LC). Poly(ADP‐ribose) polymerases (PARP), which play a major role in chromatin structure regulation, also underlie maintenance of muscle metabolism and body composition. We hypothesized that protein catabolism, proteolytic markers, muscle fiber phenotype, and muscle anabolism may improve in respiratory and limb muscles of LC‐cachectic Parp‐1‐deficient (Parp‐1−/−) and Parp‐2−/− mice. In diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing mice (wild type, Parp‐1−/−, and Parp‐2−/−), PARP activity (ADP‐ribose polymers, pADPr), redox balance, muscle fiber phenotype, apoptotic nuclei, tyrosine release, protein ubiquitination, muscle‐specific E3 ligases, NF‐κB signaling pathway, markers of muscle anabolism (Akt, mTOR, p70S6K, and mitochondrial DNA) were evaluated along with body and muscle weights, and limb muscle force. Compared to wild type cachectic animals, in both respiratory and limb muscles of Parp‐1−/− and Parp‐2−/− cachectic mice: cancer induced‐muscle wasting characterized by increased PARP activity, protein oxidation, tyrosine release, and ubiquitin‐proteasome system (total protein ubiquitination, atrogin‐1, and 20S proteasome C8 subunit) were blunted, the reduction in contractile myosin and atrophy of the fibers was attenuated, while no effects were seen in other structural features (inflammatory cells, internal or apoptotic nuclei), and markers of muscle anabolism partly improved. Activation of either PARP‐1 or ‐2 is likely to play a role in muscle protein catabolism via oxidative stress, NF‐κB signaling, and enhanced proteasomal degradation in cancer‐induced cachexia. Therapeutic potential of PARP activity inhibition deserves attention. The study findings imply that activation of either PARP‐1 or ‐2 is likely to play a relevant role in muscle protein catabolism via oxidative stress, NF‐κB signaling, and enhanced proteasomal degradation in cancer‐induced cachexia. Despite the potential limitations of PARP activity inhibition, since it plays a key role in DNA maintenance, exploration of its reliability, and likely applicability as a possible target for future therapeutic interventions deserves attention in models of cancer‐induced cachexia.

  April 27, 2017   doi: 10.1002/jcp.25851   open full text
• Polydatin impairs mitochondria fitness and ameliorates podocyte injury by suppressing Drp1 expression.
  Zheng Ni, Liang Tao, Xu Xiaohui, Zhao Zelin, Liu Jiangang, Song Zhao, Huo Weikang, Xu Hongchao, Wang Qiujing, Li Xin.
  Journal of Cellular Physiology. April 27, 2017
  Polydatin (PD), a resveratrol glycoside, has been shown to protect renal function in diabetic nephropathy (DN), but the underlying molecular mechanism remains unclear. This study demonstrates that PD stabilize the mitochondrial morphology and attenuate mitochondrial malfunction in both KKAy mice and in hyperglycemia (HG)‐induced MPC5 cells. We use Western blot analysis to demonstrate that PD reversed podocyte apoptosis induced by HG via suppressing dynamin‐related protein 1 (Drp1). This effect may depend on the ability of PD to inhibit the generation of cellular reactive oxygen species (ROS). In conclusion, we demonstrate that PD may be therapeutically useful in DN, and that, podocyte apoptosis induced by HG can be reversed by PD through suppressing Drp1 expression. Our findings provide new insights into the pathogenic process of HG‐induced podocyte injury and also identify a new therapeutic target of ROS/Drp1/mitochondrial fission/apoptosis pathway for diabetic nephropathy.

  April 27, 2017   doi: 10.1002/jcp.25943   open full text
• Hypoxia‐induced apoptosis of mouse spermatocytes is mediated by HIF‐1α through a death receptor pathway and a mitochondrial pathway.
  Jun Yin, Bing Ni, Wei‐gong Liao, Yu‐qi Gao.
  Journal of Cellular Physiology. April 26, 2017
  Hypoxia in vivo induces oligozoospermia, azoospermia and degeneration of the germinal epithelium, but the underlying molecular mechanism of this induction is not fully clarified. The aim of this study was to investigate the role of the death receptor pathway and the mitochondrial pathway in hypoxia‐induced apoptosis of mouse GC‐2spd (GC‐2) cells and the relationship between HIF‐1α and apoptosis of GC‐2 cells induced by hypoxia. GC‐2 cells were subjected to 1% oxygen for 48 h. Apoptosis was detected by flow cytometry, TUNEL staining, LDH, caspase‐3/8/9 in the absence and presence of HIF‐1α siRNA. The protein levels of apoptosis‐related markers were determined by western blot in the presence and absence of HIF‐1α siRNA. Mitochondrial transmembrane potential change was observed by in situ JC‐1 staining. Cell viability was assessed upon treatment of caspase‐8 and caspase‐9 inhibitors. The results indicated that hypoxia at 1% oxygen for 48 h induced apoptosis of GC‐2 cells. A prolonged exposure of GC‐2 cells to hypoxic conditions caused downregulation of c‐FLIP, DcR2 and Bcl‐2 and upregulation of DR5, TRAIL, Fas, p53 and Bax, with an overproduction of caspase‐3/8/9. Moreover, hypoxia at this level had an effect on mitochondrial depolarization. In addition, specific inhibitors of caspase‐8/9 partially suppressed hypoxia‐induced GC‐2 cell apoptosis, and the anti‐apoptotic effects of the caspase inhibitors were additive. Of note, HIF‐1α knockdown attenuated hypoxia and induced apoptosis of GC‐2 cells. In conclusion, our data suggest that the death receptor pathway and mitochondrial pathway, which are likely mediated by HIF‐1α, contribute to hypoxia‐induced GC‐2 cell apoptosis. This article is protected by copyright. All rights reserved

  April 26, 2017   doi: 10.1002/jcp.25974   open full text
• VSL#3 probiotic differently influences IEC‐6 intestinal epithelial cell status and function.
  Benedetta Cinque, Cristina La Torre, Francesca Lombardi, Paola Palumbo, Zoran Evtoski, Silvano Jr Santini, Stefano Falone, Annamaria Cimini, Fernanda Amicarelli, Maria Grazia Cifone.
  Journal of Cellular Physiology. April 25, 2017
  The data here reported introduce the wound‐healing assay as a tool for testing probiotics aimed at protecting gastrointestinal mucosal surfaces and to verify the consistency of their manufacturing. At the scope, we compared the in vitro effects of two multi‐strain high concentration formulations both commercialized under the same brand VSL#3 but sourced from different production sites (USA and Italy) on a non‐transformed small‐intestinal epithelial cell line, IEC‐6. The effects on cellular morphology, viability, migration, and H2O2‐induced damage, were assessed before and after the treatment with both VSL#3 formulations. While the USA‐sourced product (“USA‐made”) VSL#3 did not affect monolayer morphology and cellular density, the addition of bacteria from the Italy‐derived product (“Italy‐made”) VSL#3 caused clear morphological cell damage and strongly reduced cellularity. The treatment with “USA‐made” lysate led to a higher rate of wounded monolayer healing, while the addition of “Italy‐made” bacterial lysate did not influence the closure rate as compared to untreated cells. While lysates from “USA‐made” VSL#3 clearly enhanced the formation of elongated and aligned stress fibers, “Italy‐made” lysates had not similar effect. “USA‐made” lysate was able to cause a total inhibition of H2O2‐induced cytotoxic effect whereas “Italy‐made” VSL#3 lysate was unable to protect IEC‐6 cells from H2O2‐induced damage. ROS generation was also differently influenced, thus supporting the hypotesis of a protective action of “USA‐made” VSL#3 lysates, as well as the idea that “Italy‐made” formulation was unable to prevent significantly the H2O2‐induced oxidative stress. The data here reported introduce the wound‐healing assay as a tool for testing probiotics aimed at protecting gastrointestinal mucosal surfaces and to verify the consistency of their manufacturing. At the scope, we compared the in vitro effects of two multi‐strain high concentration formulations both commercialized under the same brand VSL#3 but sourced from different production sites (USA and Italy) on a non‐transformed small‐intestinal epithelial cell line, IEC‐6. Our results suggest that all the cellular parameters analyzed on a rat intestinal epithelial cell line (IEC‐6)—cell viability and death, wounded monolayer healing ability and protection against H2O2‐induced cellular injury—appear to be differently influenced by the “Italy‐made” VSL#3 when compared to the “USA‐made” VSL#3, thus reconfirming previous reports supporting that sourcing is a key element for probiotic safety and or efficacy.

  April 25, 2017   doi: 10.1002/jcp.25814   open full text
• SUMO‐modified insulin‐like growth factor 1 receptor (IGF‐1R) increases cell cycle progression and cell proliferation.
  Yingbo Lin, Hongyu Liu, Ahmed Waraky, Felix Haglund, Prasoon Agarwal, Helena Jernberg‐Wiklund, Dudi Warsito, Olle Larsson.
  Journal of Cellular Physiology. April 25, 2017
  Increasing number of studies have shown nuclear localization of the insulin‐like growth factor 1 receptor (nIGF‐1R) in tumor cells and its links to adverse clinical outcome in various cancers. Any obvious cell physiological roles of nIGF‐1R have, however, still not been disclosed. Previously, we reported that IGF‐1R translocates to cell nucleus and modulates gene expression by binding to enhancers, provided that the receptor is SUMOylated. In this study, we constructed stable transfectants of wild type IGF1R (WT) and triple‐SUMO‐site‐mutated IGF1R (TSM) using igf1r knockout mouse fibroblasts (R‐). Cell clones (R‐WT and R‐TSM) expressing equal amounts of IGF‐1R were selected for experiments. Phosphorylation of IGF‐1R, Akt, and Erk upon IGF‐1 stimulation was equal in R‐WT and R‐TSM. WT was confirmed to enter nuclei. TSM did also undergo nuclear translocation, although to a lesser extent. This may be explained by that TSM heterodimerizes with insulin receptor, which is known to translocate to cell nuclei. R‐WT proliferated substantially faster than R‐TSM, which did not differ significantly from the empty vector control. Upon IGF‐1 stimulation G1‐S‐phase progression of R‐WT increased from 12 to 38%, compared to 13 to 20% of R‐TSM. The G1‐S progression of R‐WT correlated with increased expression of cyclin D1, A, and CDK2, as well as downregulation of p27. This suggests that SUMO‐IGF‐1R affects upstream mechanisms that control and coordinate expression of cell cycle regulators. Further studies to identify such SUMO‐IGF‐1R dependent mechanisms seem important. We constructed stable transfectants of wild type IGF1R (R‐WT) and triple‐SUMO‐site‐mutated IGF1R (R‐TSM) using igf1r knockout mouse fibroblasts (R‐). R‐WT proliferated substantially faster than R‐TSM, which did not differ significantly from the empty vector control. Upon IGF‐1 stimulation G1‐S‐phase progression of R‐WT increased significantly stronger comparing to R‐TSM, which was correlated with expression changes of cell cycle regulatory proteins.

  April 25, 2017   doi: 10.1002/jcp.25818   open full text
• Understanding the role of structural integrity and differential expression of integrin profiling to identify potential therapeutic targets in breast cancer.
  Vishal Das, Gazal Kalyan, Saugata Hazra, Mintu Pal.
  Journal of Cellular Physiology. April 25, 2017
  Breast cancer is found to be the most prevalent neoplasm in women worldwide. Despite the function of physically tethering cells to the matrix, transmembrane protein integrins are crucially involved in diverse cellular functions such as cell differentiation, proliferation, invasion, migration, and metastasis. Dysregulation of integrins and their interactions with the cells and their microenvironment can trigger several signaling cues that determine the cell fate decision. In this review, we spotlight all pre‐existing integrin molecules, their structure, molecular interactions motifs, and function through several cross talks with kinase receptors. We also discuss the role of these integrins as potential prognostic and therapeutic targets and also in the regulation of breast cancer cells differentiation. Understanding of integrin structure and their motifs for ligand interactions in this context will enable the development of new therapeutic approaches to sensitize the tumors and their microenvironment to conventional therapy and overall suppress their metastatic phenotype. In this review, we spotlight all pre‐existing integrin molecules, their structure, molecular interactions motifs, and function through several cross talks with kinase receptors. We also discuss the role of these integrins as potential prognostic and therapeutic targets and also in the regulation of breast cancer cells differentiation. Understanding of integrin structure and their motifs for ligand interactions in this context will enable the development of new therapeutic approaches to sensitize the tumors and their microenvironment to conventional therapy and overall suppress their metastatic phenotype.

  April 25, 2017   doi: 10.1002/jcp.25821   open full text
• NSI‐189, a small molecule with neurogenic properties, exerts behavioral, and neurostructural benefits in stroke rats.
  Naoki Tajiri, David M. Quach, Yuji Kaneko, Stephanie Wu, David Lee, Tina Lam, Ken L. Hayama, Thomas G. Hazel, Karl Johe, Michael C. Wu, Cesar V. Borlongan.
  Journal of Cellular Physiology. April 25, 2017
  Enhancing neurogenesis may be a powerful stroke therapy. Here, we tested in a rat model of ischemic stroke the beneficial effects of NSI‐189, an orally active, new molecular entity (mol. wt. 366) with enhanced neurogenic activity, and indicated as an anti‐depressant drug in a clinical trial (Fava et al., , Molecular Psychiatry, DOI: 10.1038/mp.2015.178) and being tested in a Phase 2 efficacy trial (ClinicalTrials.gov, , ClinicalTrials.gov Identifier: NCT02695472) for treatment of major depression. Oral administration of NSI‐189 in adult Sprague–Dawley rats starting at 6 hr after middle cerebral artery occlusion, and daily thereafter over the next 12 weeks resulted in significant amelioration of stroke‐induced motor and neurological deficits, which was maintained up to 24 weeks post‐stroke. Histopathological assessment of stroke brains from NSI‐189‐treated animals revealed significant increments in neurite outgrowth as evidenced by MAP2 immunoreactivity that was prominently detected in the hippocampus and partially in the cortex. These results suggest NSI‐189 actively stimulated remodeling of the stroke brain. Parallel in vitro studies further probed this remodeling process and demonstrated that oxygen glucose deprivation and reperfusion (OGD/R) initiated typical cell death processes, which were reversed by NSI‐189 treatment characterized by significant attenuation of OGD/R‐mediated hippocampal cell death and increased Ki67 and MAP2 expression, coupled with upregulation of neurogenic factors such as BDNF and SCF. These findings support the use of oral NSI‐189 as a therapeutic agent well beyond the initial 6‐hr time window to accelerate and enhance the overall functional improvement in the initial 6 months post stroke. Here, we show that enhancing neurogenesis may be a powerful stroke therapy. The present study tested in a rat model of ischemic stroke the beneficial effects of NSI‐189, an orally active, new molecular entity (mol. wt. 366) with enhanced neurogenic activity, and indicated as an anti‐depressant drug in a clinical trial (Fava et al., , Molecular Psychiatry, DOI: 10.1038/mp.2015.178) and being tested in a Phase 2 efficacy trial (ClinicalTrials.gov, , ClinicalTrials.gov Identifier: NCT02695472) for treatment of major depression. Behavioral recovery in NSI‐189‐treated stroke animals was accompanied by significant increments in neurite outgrowth as evidenced by MAP2 immunoreactivity that was prominently detected in the hippocampus and partially in the cortex, indicating that NSI‐189 actively stimulated remodeling of the stroke brain.

  April 25, 2017   doi: 10.1002/jcp.25847   open full text
• Osteoactivin regulates head and neck squamous cell carcinoma invasion by modulating matrix metalloproteases.
  Oneida A. Arosarena, Eric W. Barr, Ryan Thorpe, Hilary Yankey, Joseph T. Tarr, Fayez F. Safadi.
  Journal of Cellular Physiology. April 25, 2017
  Nearly 60% of patients with head and neck squamous cell carcinoma (HNSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell migration and invasion, which are in part dependent on extracellular matrix degradation by matrix metalloproteinases. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies, and has been shown to upregulate matrix metalloproteinase (MMP) expression and activity. To determine how OA modulates MMP expression and activity in HNSCC, and to investigate OA effects on cell invasion, we assessed effects of OA treatment on MMP mRNA and protein expression, as well as gelatinase and caseinolytic activity in HNSCC cell lines. We assessed the effects of OA gene silencing on MMP expression, gelatinase and caseinolytic activity, and cell invasion. OA treatment had differential effects on MMP mRNA expression. OA treatment upregulated MMP‐10 expression in UMSCC14a (p = 0.0431) and SCC15 (p < 0.0001) cells, but decreased MMP‐9 expression in UMSCC14a cells (p = 0.0002). OA gene silencing decreased MMP‐10 expression in UMSCC12 cells (p = 0.0001), and MMP‐3 (p = 0.0005) and ‐9 (p = 0.0036) expression in SCC25 cells. In SCC15 and SCC25 cells, OA treatment increased MMP‐2 (p = 0.0408) and MMP‐9 gelatinase activity (p < 0.0001), respectively. OA depletion decreased MMP‐2 (p = 0.0023) and ‐9 (p < 0.0001) activity in SCC25 cells. OA treatment increased 70 kDa caseinolytic activity in UMSCC12 cells consistent with tissue type plasminogen activator (p = 0.0078). OA depletion decreased invasive capacity of UMSCC12 cells (p < 0.0001). OA's effects on MMP expression in HNSCC are variable, and may promote cancer cell invasion. Osteoactivin/gpnmb has been implicated in metastases, particularly bony metastases, in several malignancies. In this study we demonstrated that osteoactivin differentially upregulates matrix metalloprotease expression and activity in head and neck cancer cell lines.

  April 25, 2017   doi: 10.1002/jcp.25900   open full text
• High glucose alters the secretome of mechanically stimulated osteocyte‐like cells affecting osteoclast precursor recruitment and differentiation.
  Marta Maycas, María Teresa Portolés, María Concepción Matesanz, Irene Buendía, Javier Linares, María José Feito, Daniel Arcos, María Vallet‐Regí, Lilian I. Plotkin, Pedro Esbrit, Arancha R. Gortázar.
  Journal of Cellular Physiology. April 25, 2017
  Diabetes mellitus (DM) induces bone deterioration, while mechanical stimulation promotes osteocyte‐driven bone formation. We aimed to evaluate the interaction of acute exposure (24 h) to high glucose (HG) with both the pro‐survival effect conferred to osteocytic MLO‐Y4 cells and osteoblastic MC3T3‐E1 cells by mechanical stimulation and the interaction of these cells with osteoclast precursor RAW264.7 cells. We found that 24 h of HG (25 mM) pre‐exposure prevented both cell survival and ERK and β‐catenin nuclear translocation upon mechanical stimulation by fluid flow (FF) (10 min) in both MLO‐Y4 and MC3T3‐E1 cells. However, migration of RAW 264.7 cells was inhibited by MLO‐Y4 cell‐conditioned medium (CM), but not by MC3T3‐E1 cell‐CM, with HG or FF. This inhibitory effect was associated with consistent changes in VEGF, RANTES, MIP‐1α, MIP‐1β MCP‐1, and GM‐CSF in MLO‐Y4 cell‐CM. RAW264.7 proliferation was inhibited by MLO‐Y4 CM under static or HG conditions, but it increased by FF‐CM with or without HG. In addition, both FF and HG abrogated the capacity of RAW 264.7 cells to differentiate into osteoclasts, but in a different manner. Thus, HG‐CM in static condition allowed formation of osteoclast‐like cells, which were unable to resorb hydroxyapatite. In contrast, FF‐CM prevented osteoclastogenesis even in HG condition. Moreover, HG did not affect basal RANKL or IL‐6 secretion or their inhibition induced by FF in MLO‐Y4 cells. In conclusion, this in vitro study demonstrates that HG exerts disparate effects on osteocyte mechanotransduction, and provides a novel mechanism by which DM disturbs skeletal metabolism through altered osteocyte‐osteoclast communication. This in vitro study demonstrates that HG exerts disparate effects on osteocyte mechanotransduction, and provides a novel mechanism by which DM disturbs skeletal metabolism through altered osteocyte‐osteoclast communication.

  April 25, 2017   doi: 10.1002/jcp.25829   open full text
• Diagnostic and Therapeutic Potential of Exosomes in Cancer: The Beginning of a New Tale?
  Hamed Mirzaei, Amirhossein Sahebkar, Mahmoud Reza Jaafari, Mohammad Goodarzi, Hamid Reza Mirzaei.
  Journal of Cellular Physiology. April 25, 2017
  Exosomes have emerged as one of the main players in intercellular communication. These small nano‐sized particles have many roles in various physiological pathways in normal and abnormal cells. Exosomes can carry various cargos such as proteins, mRNAs, and miRNAs to recipient cells. Uptake of exosomes and their cargo can induce and/or inhibit different cellular and molecular pathways that lead to the alteration of cell behavior. Multiple lines of evidence have indicated that exosomes released from cancer cells can effect development of cancer in different stages. These particles and their cargo could regulate different processes such as tumor growth, metastasis, drug resistance, angiogenesis, and immune system functioning. It has been observed that exosomes can be used as potential diagnostic biomarkers in various cancer types. Moreover, some studies have used these particles as biological vehicles for delivery of various drugs such as doxorubicin, siRNAs, and miRNAs. Here, we summarized the findings on the role of exosomes in different pathological processes involved in cancer. Moreover, application of these particles as diagnostic and therapeutic biomarkers in different types of cancers is discussed. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc.

  April 25, 2017   doi: 10.1002/jcp.25739   open full text
• sp2‐Iminosugar α‐glucosidase inhibitor 1‐C‐octyl‐2‐oxa‐3‐oxocastanospermine specifically affected breast cancer cell migration through Stim1, β1‐integrin, and FAK signaling pathways.
  Nahla Gueder, Ghada Allan, Marie‐Sophie Telliez, Frédéric Hague, José M. Fernandez, Elena M. Sanchez‐Fernandez, Carmen Ortiz‐Mellet, Ahmed Ahidouch, Halima Ouadid‐Ahidouch.
  Journal of Cellular Physiology. April 25, 2017
  Aberrant glycosylation changes on many glycoproteins are often related to cancer progression and metastasis. sp2‐Iminosugar‐type castanospermine analogues, inhibitors of α‐glucosidases, have been reported to exhibit antitumor activity. However, their effects on cell migration and the underlying molecular mechanism are not fully understood. Here, we investigated the effect of the pseudo‐C‐octyl glycoside 2‐oxa‐3‐oxocastanospermine derivatives (CO‐OCS) on breast cancer cells (MCF‐7 and MDA‐MB‐231 cells), and MCF‐10A mammary normal cell lines. We showed that CO‐OCS treatment results in the drastic decrease of breast cancer cell migration without affecting cell proliferation. Furthermore, CO‐OCS significantly reduced both the expression of β1‐integrin, which is a crucial interacting partner of Focal Adhesion Kinase (FAK), and the phosphorylation rates of FAK and ERK1/2. CO‐OCS also drastically reduced Ca2+ entry through Store Operated Channels (SOC). Orai1 and Stim1, two N‐glycosylated proteins, are involved in Store‐Operated Calcium Entry (SOCE), and are essential for breast tumor cell migration. Our results showed that CO‐OCS decreased the expression, at the protein level, of Stim1 without affecting that of Orai1. Moreover, cell migration and SOCE were attenuated by CO‐OCS as well as when Stim1 was silenced. In contrast, in MCF‐10A cells, CO‐OCS slightly reduced cell migration, but was without effect on gene expression of Stim1, Orai1, β1‐integrin, or FAK and ERK1/2 activation. Our results provide strong evidence for a significant effect of CO‐OCS on breast cancer cell migration and support that this effect was associated with β1‐integrin, Stim1, and FAK signaling pathways. CO‐OCS suppressed more drastically migration of breast cancer cells than normal cells CO‐OCS affected specifically breast cancer cell migration by targeting β1‐integrin and Stim1 CO‐OCS does not affect the expression of β1‐integrin, or Stim1 in MCF‐10A normal cells.

  April 25, 2017   doi: 10.1002/jcp.25832   open full text
• Up‐Regulation of Transient Receptor Potential Melastatin 6 Channel Expression by Tumor Necrosis Factor‐α in the Presence of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor.
  Chisa Furukawa, Naoko Fujii, Aya Manabe, Toshiyuki Matsunaga, Satoshi Endo, Hajime Hasegawa, Yoshinori Ito, Masahiko Yamaguchi, Yasuhiro Yamazaki, Akira Ikari.
  Journal of Cellular Physiology. April 25, 2017
  Anti‐epidermal growth factor receptor (EGFR) drugs such as erlotinib and gefitinib cause a side effect of hypomagnesemia, but chemotherapy to treat this has not yet been developed. The transient receptor potential melastatin 6 (TRPM6) channel is involved in the reabsorption of Mg2+ in the renal tubule. We reported previously that the expression of TRPM6 is up‐regulated by epidermal growth factor (EGF) in renal tubular epithelial NRK‐52E and HEK293 cells. EGF‐induced elevation of TRPM6 expression was inhibited by erlotinib, gefitinib, and lapatinib. We found that tumor necrosis factor‐α (TNF‐α) increases TRPM6 expression in the presence of erlotinib. Therefore, we investigated what molecules are involved in the up‐regulation of TRPM6 expression by TNF‐α. EGF increased the levels of phosphorylated extracellular signal‐regulated kinase 1 and 2 (p‐ERK1/2), which were inhibited by erlotinib. TNF‐α did not change p‐ERK1/2 levels, but increased the phosphorylation and nuclear localization of nuclear factor‐κB (NF‐κB), which were blocked by the NF‐κB inhibitors BAY 11–7082 and pyrrolidinedithiocarbamate ammonium. Similarly, luciferase reporter activity of human TRPM6 was increased by TNF‐α, which was blocked by NF‐κB inhibitors, and was inhibited by a mutation in the κB‐binding site in the proximal region of the TRPM6 promoter. A chromatin immunoprecipitation assay revealed that NF‐κB binds to the κB‐binding site, which was blocked by NF‐κB inhibitors. In the presence of erlotinib, TNF‐α increased Mg2+ influx, which was blocked by NF‐κB inhibitors. These results suggest that TNF‐α reverses the reduction in Mg2+ reabsorption caused by anti‐EGFR drugs. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc. Tumor necrosis factor‐α (TNF‐α) increases TRPM6 expression in the presence of anti‐epidermal growth factor receptor drug, erlotinib. TNF‐α did not change p‐ERκ1/2 levels, but increased the phosphorylation and nuclear localization of nuclear factor‐κB (NF‐κB), which were blocked by the NF‐κB inhibitors. In the presence of erlotinib, TNF‐α increased Mg2+ influx, which was blocked by NF‐κB inhibitors.

  April 25, 2017   doi: 10.1002/jcp.25709   open full text
• Brain‐derived neurotrophic factor improves proliferation of endometrial epithelial cells by inhibition of endoplasmic reticulum stress during early pregnancy.
  Whasun Lim, Hyocheol Bae, Fuller W. Bazer, Gwonhwa Song.
  Journal of Cellular Physiology. April 25, 2017
  Brain‐derived neurotrophic factor (BDNF) is a member of the neurotrophin family binds to two transmembrane receptors; neurotrophic receptor tyrosine kinase 2 (NTRK2) with high affinity and p75 with low affinity. Although BDNF‐NTRK2 signaling in the central nervous system is known, signaling in the female reproductive system is unknown. Therefore, we determined effects of BDNF on porcine endometrial luminal epithelial (pLE) cells isolated from Day 12 of pregnancy, as well as expression of BDNF and NTRK2 in endometria of cyclic and pregnant pigs. BDNF‐NTRK2 genes were expressed in uterine glandular (GE) and luminal (LE) epithelia during early pregnancy. In addition, their expression in uterine GE and LE decreased with increasing parity of sows. Recombinant BDNF increased proliferation in pLE cells in a dose‐dependent, as well as expression of PCNA and Cyclin D1 in nuclei of pLE cells. BDNF also activated phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, P38 proteins in pLE cells. In addition, cell death resulting from tunicamycin‐induced ER stress was prevented when pLE cells were treated with the combination of tunicamycin and BDNF which also decreased cells in the Sub‐G1 phase of the cell cycle. Furthermore, tunicamycin‐induced unfolded protein response genes were mostly down‐regulated to the basal levels as compared to non‐treated pLE cells. Our finding suggests that BDNF acts via NTRK2 to induce development of pLE cells for maintenance of implantation and pregnancy by activating cell signaling via the PI3K and MAPK pathways and by inhibiting ER stress. BDNF acts via NTRK2 to induce development of the uterine epithelial cells for maintenance of implantation and pregnancy by activating cell signaling via the PI3K and MAPK pathways and by inhibiting ER stress during early pregnancy.

  April 25, 2017   doi: 10.1002/jcp.25834   open full text
• Nap Counteracts Hyperglycemia/Hypoxia Induced Retinal Pigment Epithelial Barrier Breakdown Through Modulation Of HIFs and VEGF Expression.
  Agata Grazia D'Amico, Grazia Maugeri, Daniela Maria Rasà, Valentina La Cognata, Salvatore Saccone, Concetta Federico, Sebastiano Cavallaro, Velia D'Agata.
  Journal of Cellular Physiology. April 24, 2017
  Diabetic macular edema (DME) is a common complication leading to a central vision loss in patients with diabetes. In this eye pathology, the hyperglycaemic/hypoxic microenvironment of pigmented epithelium is responsible for outer blood retinal barrier integrity changes. More recently, we have shown that a small peptide derived from the activity‐dependent neuroprotective protein (ADNP), known as NAP, counteracts damages occurring during progression of diabetic retinopathy by modulating HIFs/VEGF pathway. Here, we have investigated for the first time the role of this peptide on outer blood retinal barrier (BRB) integrity exposed to hyperglycaemic/hypoxic insult mimicking a model in vitro of DME. To characterize NAP role on disease's pathogenesis, we have analysed its effect on HIFs /VEGF system in human retinal pigmented epithelial cells, ARPE‐19, grown in high glucose and low oxygen tension. The results have shown that NAP prevents outer BRB breakdown by reducing HIF1α /HIF2α, VEGF/VEGFRs and increasing HIF3 α expression, moreover it is able to reduce the percentage of apoptotic cells by modulating the expression of two death related genes, BAX and Bcl2. Further investigations are needed to determine the possible use of NAP in DME treatment. This article is protected by copyright. All rights reserved

  April 24, 2017   doi: 10.1002/jcp.25971   open full text
• WNT4 Acts Downstream of BMP2 to Mediate the Regulation of ATRA Signaling on RUNX1 Expression: Implications for Terminal Differentiation of Antler Chondrocytes.
  Hong‐Liang Zhang, Zhan‐Qing Yang, Cui‐Cui Duan, Shuang Geng, Kai Wang, Hai‐Fan Yu, Zhan‐Peng Yue, Bin Guo.
  Journal of Cellular Physiology. April 24, 2017
  Although ATRA is involved in regulating the proliferation and differentiation of chondrocytes, its underlying mechanism remains unknown. Here we showed that ATRA could stimulate the proliferation of antler chondrocytes and expression of COL X and MMP13 which were two well‐known markers for hypertrophic chondrocytes. Silencing of CRABP2 prevented the induction of ATRA on chondrocyte terminal differentiation, while overexpression of CRABP2 exhibited the opposite effects. CYP26A1 and CYP26B1 weakened the sensitivity of antler chondrocytes to ATRA. Further analysis evidenced that ATRA might induce chondrocyte terminal differentiation and modulate the expression of BMP2, WNT4 and RUNX1 through RARα/RXRα. Knockdown of BMP2 enhanced the induction of ATRA on the expression of COL X and MMP13, whereas overexpression of BMP2 abrogated this effectiveness. WNT4 might mediate the effects of ATRA and BMP2 on chondrocyte terminal differentiation. Dysregulation of BMP2 impaired the regulation of ATRA on WNT4 expression. Administration of ATRA to antler chondrocytes transfected with RUNX1 siRNA failed to induce the differentiation. Conversely, rRUNX1 strengthened the stimulation of ATRA on the expression of COL X and MMP13. Simultaneously, RUNX1 was a downstream effector of BMP2 and WNT4 in chondrocyte terminal differentiation. Moreover, WNT4 might play an important role in the crosstalk between BMP2 and RUNX1. Attenuation of BMP2 or WNT4 enhanced the interaction between ATRA and RUNX1, while constitutive expression of BMP2 or WNT4 reversed the regulation of ATRA on RUNX1. Collectively, WNT4 may act downstream of BMP2 to mediate the effects of ATRA on the terminal differentiation of antler chondrocytes through targeting RUNX1. This article is protected by copyright. All rights reserved

  April 24, 2017   doi: 10.1002/jcp.25972   open full text
• Generation of Insulin‐Producing Cells From Human‐Induced Pluripotent Stem Cells Using a Stepwise Differentiation Protocol Optimized With Platelet‐Rich Plasma.
  Seyed Ehsan Enderami, Yousef Mortazavi, Masoud Soleimani, Samad Nadri, Alireza Biglari, Reyhaneh Nassiri Mansour.
  Journal of Cellular Physiology. April 21, 2017
  Studies on patient‐specific human‐induced pluripotent stem cells (hiPSCs) as well as a series of autologous growth factors presumably will reveal their many benefits for cell base replacement therapy in type 1 diabetes mellitus (TIDM) patients in the future. For this purpose, we established a multistep protocol by adding platelet‐rich plasma (PRP) that induce the hiPSCs into insulin‐producing cells (IPCs). We present here a differentiation protocol consisting of five stages in two groups including protocol with PRP and without PRP. Charac­teristics of derived IPCs in both groups were evaluated at the mRNA and protein levels, cell cycle and viability in the end stage of cell differentiation. The in vitro studies indicated the treatment of hiPSCs in the protocol with PRP resulting in differentiated cells with strong characteristics of IPCs including islet‐like cells, the expression of mature and functional pancreatic beta cell specific marker genes. In addition to these pancreatic specific markers were detected by immunochemistry and Western blot. Our differentiated cells in two groups secreted insulin and C‐peptide in a glucose stimulation test by ELISA showing in vitro functional. The results of the cell cycle assay confirmed that differentiation has been done. We reported for the first time that PRP might be ideal additive in the culture medium to induce pancreatic differentiation in the hiPSCs. This study provides a new approach to investigate the role of PRP in pancreatic differentiation protocols and enhance the feasibility of using patient‐specific iPSCs and autologous PRP for future beta cells replacement therapies for T1DM. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc. We established a multistep protocol by adding platelet‐rich plasma (PRP) that induce the hiPSCs into insulin‐producing cells (IPCs). We reported for the first time that PRP might be ideal additive in the culture medium to induce pancreatic differentiation in the hiPSCs.

  April 21, 2017   doi: 10.1002/jcp.25721   open full text
• Purification of small molecule‐induced cardiomyocytes from human induced pluripotent stem cells using a reporter system.
  Geun Hye Hwang, So Mi Park, Ho Jae Han, Joong Sun Kim, Seung Pil Yun, Jung Min Ryu, Ho Jin Lee, Woochul Chang, Su‐Jin Lee, Jeong‐Hee Choi, Jin‐Sung Choi, Min Young Lee.
  Journal of Cellular Physiology. April 18, 2017
  In order to realize the practical use of human pluripotent stem cell (hPSC)‐derived cardiomyocytes for the purpose of clinical use or cardiovascular research, the generation of large numbers of highly purified cardiomyocytes should be achieved. Here, we show an efficient method for cardiac differentiation of human induced pluripotent stem cells (hiPSCs) in chemically defined conditions and purification of hiPSC‐derived cardiomyocytes using a reporter system. Regulation of the Wnt/β‐catenin signaling pathway is implicated in the induction of the cardiac differentiation of hPSCs. We increased cardiac differentiation efficiency of hiPSCs in chemically defined conditions through combined treatment with XAV939, a tankyrase inhibitor and IWP2, a porcupine inhibitor and optimized concentrations. Although cardiac differentiation efficiency was high (>80%), it was difficult to suppress differentiation into non‐cardiac cells, Therefore, we applied a lentiviral reporter system, wherein green fluorescence protein (GFP) and Zeocin‐resistant gene are driven by promoter activation of a gene (TNNT2) encoding cardiac troponin T (cTnT), a cardiac‐specific protein, to exclude non‐cardiomyocytes from differentiated cell populations. We transduced this reporter construct into differentiated cells using a lentiviral vector and then obtained highly purified hiPSC‐derived cardiomyocytes by treatment with the lowest effective dose of Zeocin. We significantly increased transgenic efficiency through manipulation of the cells in which the differentiated cells were simultaneously infected with virus and re‐plated after single‐cell dissociation. Purified cells specifically expressed GFP, cTnT, displayed typical properties of cardiomyocytes. This study provides an efficient strategy for obtaining large quantities of highly purified hPSC‐derived cardiomyocytes for application in regenerative medicine and biomedical research. Combined treatment with XAV939 and IWP2 enhances hiPSC cardiac differentiation. Large amount of highly purified hiPSC‐derived cardiomyocytes are obtained using a reporter system. Purified cells represent functional cardiomyocytes that possess electrophysiological properties and form cardiac junctions.

  April 18, 2017   doi: 10.1002/jcp.25783   open full text
• Establishment of a new conditionally immortalized human skeletal muscle microvascular endothelial cell line.
  Hironori Sano, Yasuteru Sano, Eri Ishiguchi, Fumitaka Shimizu, Masatoshi Omoto, Toshihiko Maeda, Hideaki Nishihara, Yukio Takeshita, Shiori Takahashi, Mariko Oishi, Takashi Kanda.
  Journal of Cellular Physiology. April 18, 2017
  In skeletal muscle, the capillaries have tight junctions (TJs) that are structurally similar to those in the blood‐brain barrier (BBB) and blood‐nerve barrier (BNB). Although many findings have been clarified in the territory of BBB and BNB, few have so far examined the TJs of capillaries in the skeletal muscle. In addition, no in vitro human skeletal muscle microvasculature models have been reported thus far. We newly established a new human skeletal muscle microvascular endothelial cell (HSMMEC) line. HSMMECs were isolated from human skeletal muscle and were infected with retroviruses harboring temperature‐sensitive SV40 T antigen and telomerase genes. This cell line, termed TSM15, showed a spindle fiber‐shaped morphology, an immunoreactivity to anti‐factor VIII and anti‐VE‐cadherin antibodies, and a temperature‐sensitive growth. TSM15 cells grew stably for more than 40 passages when they were cultured at 33°C, thereby retaining their spindle fiber‐shaped morphology and contact inhibition at confluence. The cells expressed tight junctional molecules such as claudin‐5, occludin, and zonula occludens‐1, as well as transporters such as a glucose transporter 1. The transendothelial electrical resistance of TSM15 was as high as those of the human brain microvascular endothelial cell line. This novel cell line might facilitate the analyses of the pathophysiology of inflammatory myopathy, such as dermatomyositis, and can improve our understanding of the physiological and biochemical properties of the microvasculature in human skeletal muscle. A new conditionally immortalized human skeletal muscle microvascular endothelial cell line was established. This cell line, termed TSM15, expressed tight junctional molecules as well as transporters and has a barrier integrity relevant to that of a human brain microvascular endothelial cell line.

  April 18, 2017   doi: 10.1002/jcp.25772   open full text
• GM13133 is a negative regulator in mouse white adipocytes differentiation and drives the characteristics of brown adipocytes.
  LiangHui You, YaHui Zhou, XianWei Cui, XingYun Wang, YaZhou Sun, Yao Gao, Xing Wang, Juan Wen, Kaipeng Xie, RanRan Tang, ChenBo Ji, XiRong Guo.
  Journal of Cellular Physiology. April 18, 2017
  Obesity is tightly associated with the disturbance of white adipose tissue storing excess energy. Thermogenic adipocytes (brown and beige) exert a critical role of oxidizing nutrients at the high rates through non‐shivering thermogenesis. The recruitment of brown characteristics in white adipocytes, termed browning, has been considered as a promising strategy for treating obesity and associated metabolic complications. Recently, long noncoding RNAs play a crucial role in regulating tissue development and participating in disease pathogenesis, yet their effects on the conversion of white into brown‐like adipocytes and thermogenic function were not totally understood. Here, we identified a mouse brown adipose specific expressed lncRNA, termed GM13133. Moreover, a considerable amount of GM13133 is expressed in adipocytes and actively modulated by cold, β3‐adrenergic agonist and cAMP stimuli, implying a potential role in the conversion from white to brown adipocytes. Overexpression of GM13133 did not affect the proliferation of mouse white pre‐adipocytes, but inhibited white adipocyte differentiation by decreasing lipid accumulation. The forced expression of GM13133 also significantly drove the conversion of white into brown‐like adipocytes with the enhanced mitochondrial biogenesis and the induced expression of brown adipocytes specific markers. A global mRNA analysis further indicated the possible regulatory role of cAMP signaling pathway in GM13133 mediated white‐to‐brown adipocytes conversion. Our results identified a lncRNA‐mediated modulation in primary mouse white adipocyte differentiation and indicate the functional significance of GM13133 in promoting browning of white adipocytes and maintenance of thermogenesis, further providing a potential strategy to treating obesity. Our results provide identified a lncRNA‐mediated modulation in primary mouse white adipocyte differentiation and further indicate the functional significance of GM13133 in promoting browning of white adipocytes and maintenance of thermogenesis, further providing a potential strategy to treating obesity.

  April 18, 2017   doi: 10.1002/jcp.25878   open full text
• Evaluation the ethno‐pharmacological studies in Iran during 2004–2016: A systematic review.
  Zahra Sadeghi, Maryam Akaberi, Alireza Sobhkhizi, Amirhossein Sahebkar, Seyed Ahmad Emami.
  Journal of Cellular Physiology. April 18, 2017
  Although Iran has a deep history in herbal medicine and great heritage of ancient medical scholars, few efforts have been made to evaluate ethnopharmacological aspects of medicinal plants in this country. In the present study, the authors have reviewed all important literature about the ethnopharmacological investigations on medicinal plants used in the last decade in Iran. All provinces of Iran were categorized according to a phytogeographical division. Information was collected through bibliographic investigations from scientific journals and books. Afterward, the data were analyzed through the construction of specific ecological regions of the country. Fifty‐five references reporting medicinal plants in five ecological zones were retrieved. The Irano‐Turanian subregion has produced the greatest number of publications in this field among others (47%). Results illustrate that the most reported botanical families were Lamiaceae and Asteraceae (28.57% and 27.73%, respectively). Among various illnesses reported for these plants, gastrointestinal (30.15%), respiratory problems (14.28%), diabetes (11.11%), and cold/flu (11.11%) were the most cited. The most frequently cited medicinal uses were attributed to decoction and infusion preparations. Iran has a rich history of knowledge about phytotherapy and has also a diverse geographical regions, and a plant flora that is a good candidate for drug discovery. Documentation of indigenous knowledge about herbal medicine used by Iranian tribes is vital for the future development of herbal drugs. Ethnopharmacological studies of Iranian folk medicine with quantitative analytical techniques are warranted to find drug candidates, and also to preserve the precious knowledge of the Iranian folk medicine. This is the first ethno‐pharmacological survery of medicinal plants used in the treatment of different diseases in Iran.

  April 18, 2017   doi: 10.1002/jcp.25803   open full text
• Osteogenic differentiation Potential of Mesenchymal Stem Cells cultured on Nanofibrous Scaffold Improved in the Presence of Pulsed Electromagnetic Field.
  Monireh Arjmand, Abdolreza Ardeshirylajimi, Hossein Maghsoudi, Esmaeel Azadian.
  Journal of Cellular Physiology. April 17, 2017
  Nowadays, tissue engineering by using stem cells in combination with scaffolds and bioactive molecules has made significant contributions to the regeneration of damaged bone tissues. Since the usage of bioactive molecules including, growth factors to induce differentiation is safety limited in clinical applications, and it has also been previously observed that extremely low frequency pulsed electromagnetic fields (PEMF) can be effective in the enhancement of proliferation rate and osteogenic differentiation of stem cells, the aim of this study was investigating the osteoinductive potential of PEMF in combination with Poly(caprolactone) (PCL) nanofibrous scaffold. To achieve this aim, Adipose‐derived mesenchymal stem cells (ADSCs) isolated and characterized and then osteogenic differentiation of them was investigated after culturing on the surface of PCL scaffold under treatments of PEMF, PEMF plus osteogenic medium (OM) and OM. Analysis of common osteogenic markers such as Alizarin red staining, ALP activity, calcium content and four important bone‐related genes in days of 7, 14 and 21 confirmed that the effects of PEMF on the osteogenic differentiation of ADSCs are very similar to the effects of osteogenic medium. Thus, regarding the immunological concerns about the application of bioactive molecules for tissue engineering, PEMF could be a good alternative for osteogenic medium. Although, results were showed a synergetic effect for simultaneous application of PEMF and PCL scaffold in the osteogenesis process of ADSCs. Taking together, ADSCs‐seeded PCL nanofibrous scaffold in combination with PEMF could be a great option for use in bone tissue engineering applications. This article is protected by copyright. All rights reserved

  April 17, 2017   doi: 10.1002/jcp.25962   open full text
• Activation of ClC‐3 chloride channel by 17β‐estradiol relies on the estrogen receptor α expression in breast cancer.
  Haifeng Yang, Lianshun Ma, Yawei Wang, Wanhong Zuo, Bingxue Li, Yaping Yang, Yehui Chen, Lixin Chen, Liwei Wang, Linyan Zhu.
  Journal of Cellular Physiology. April 17, 2017
  Although extensively studied, the mechanisms by which estrogen promotes breast cancer growth remain to be fully elucidated. Tamoxifen, an antiestrogen to treat ERα+ breast cancer, is also a high‐affinity blocker of the chloride channels. In this study, we explored the involvement of the chloride channels in the action of estrogen in breast cancer. We found that 17β‐estradiol (17β‐E2) concentration‐dependently activated the chloride currents in ERα+ breast cancer MCF‐7 cells. Extracellular hypertonic challenge and chloride channel blockers, NPPB and DIDS inhibited the 17β‐E2‐activated chloride currents. Decreased the ClC‐3 protein expression caused the depletion of the 17β‐E2‐activated chloride currents. 17β‐E2‐activated chloride currents which relied on the ERα expression were demonstrated by the following evidences. Firstly, 17β‐E2‐activated chloride currents could not be observed in ERα‐ breast cancer MDA‐MB‐231 cells. Secondly, ER antagonists, tamoxifen and ICI 182,780, and downregulation of ERα expression inhibited or abolished the 17β‐E2‐activated chloride currents. Thirdly, ERα expression was induced in MDA‐MB‐231 cells by ESR1 gene transfection, and then 17β‐E2‐activated chloride currents could be observed. In MCF‐7 cells, ERα and ClC‐3 mainly located in nucleus and translocated to cell plasma and membrane with respect to co‐localization following treatment of 17β‐E2. Downregulation of ERα expression could decrease the expression of ClC‐3 protein. Conversely, downregulation of ClC‐3 expression did not influence the ERα expression. Taken together, our findings demonstrated that ClC‐3 is a potential target of 17β‐E2 and modulates by the ERα in breast cancer cell. Pharmacological modulation of ClC‐3 may provide a deep understanding in antiestrogen treatment of breast cancer patients. This article is protected by copyright. All rights reserved

  April 17, 2017   doi: 10.1002/jcp.25963   open full text
• Platelet‐derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis.
  Yongzhi Wang, Su Zhang, Lingtao Luo, Eva Norström, Oscar Ö Braun, Matthias Mörgelin, Henrik Thorlacius.
  Journal of Cellular Physiology. April 14, 2017
  Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL‐6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL‐6 and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet‐derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild‐type recombinant annexin V abolished PMP‐induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild‐type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP‐induced formation of thrombin‐antithrombin complexes were reduced in platelet‐depleted mice and in animals pretreated with annexin V. PMP‐induced TG attenuated in FXII‐ and FVII‐deficient plasma. These findings suggest that sepsis‐induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis. This article is protected by copyright. All rights reserved

  April 14, 2017   doi: 10.1002/jcp.25959   open full text
• Spermicidal efficacy of VRP, a synthetic cationic antimicrobial peptide, inducing apoptosis and membrane disruption.
  Prasanta Ghosh, Arpita Bhoumik, Sudipta Saha, Sandipan Mukherjee, Sarfuddin Azmi, Jimut Kanti Ghosh, Sandhya Rekha Dungdung.
  Journal of Cellular Physiology. April 14, 2017
  Presently available contraceptives are mostly hormonal or detergent in nature with numerous side effects like irritation, lesion, inflammation in vagina, alteration of body homeostasis etc. Antimicrobial peptides with spermicidal activity but without adverse effects may be suitable alternatives. In the present study spermicidal activity of a cationic antimicrobial peptide VRP on human spermatozoa has been elucidated. Progressive forward motility of human spermatozoa was instantly stopped after 100 µM VRP treatment and at 350 µM all kinds of sperm motility ceased within 20 seconds as assessed by the Sander‐Cramer assay. The spermicidal effect was confirmed by eosin‐nigrosin assay and HOS test. VRP treatment (100µM) in human spermatozoa induced both the intrinsic and extrinsic pathways of apoptosis. TUNEL assay showed VRP treatment significantly disrupted the DNA integrity and changed the mitochondrial membrane permeability as evident from MPTP assay. AFM and SEM results depicted ultra structural changes including disruption of the acrosomal cap and plasma membrane of the head and midpiece region after treatment with 350 µM VRP. MTT assay showed after treatments with 100 µM and 350 µM of VRP for 24 hrs, a substantial amount of Lactobacillus acidophilus (about 90% and 75% respectively) remained viable. Hence, VRP being a small synthetic peptide with antimicrobial and spermicidal activity but tolerable to normal vaginal microflora, may be a suitable target for elucidating its contraceptive potentiality. This article is protected by copyright. All rights reserved

  April 14, 2017   doi: 10.1002/jcp.25958   open full text
• Metformin attenuates albumin‐induced alterations in renal tubular cells in vitro.
  Soumaya Allouch, Shankar Munusamy.
  Journal of Cellular Physiology. April 13, 2017
  Proteinuria (albuminuria) plays a crucial role in the etiology of chronic kidney disease (CKD) via alteration of multiple signaling pathways and cellular process in renal cells. The objectives of this study are to investigate the effects of activation of the energy‐sensing molecule AMP‐activated kinase (AMPK) in renal cells using metformin on endoplasmic reticulum (ER) stress, AKT, mTOR, epithelial‐to‐mesenchymal transition (EMT), autophagy, and apoptosis that are thought to mediate renal cell injury during proteinuria, and to dissect the AMPK‐ and non‐AMPK mediated effects of metformin using an in vitro model of albumin‐induced renal cell injury. Rat renal proximal tubular (NRK‐52E) cells were exposed to 10 and 15 mg/ml of albumin for 72 h in the presence of 1 mM Metformin and/or 0.5 µM compound C, and assessed for alterations in the aforementioned pathways. Metformin treatment restored AMPK phosphorylation and augmented autophagy in renal cells exposed to albumin. In addition, metformin treatment attenuated the albumin‐induced phosphorylation of AKT and the downstream targets of mTOR, and prevented albumin‐mediated inductions of EMT marker (α‐SMA), pro‐apoptotic ER stress marker CHOP, and apoptotic caspases ‐12 and ‐3 in renal cells. Blockade of metformin‐induced AMPK activation with compound C blunted the ER defense response and autophagy but had no effect on the markers of EMT and apoptosis in our model. Our studies suggest that metformin protects renal cells against proteinuric cytotoxicity via suppression of AKT and mTOR activation, inhibition of EMT and apoptosis, and augmentation of autophagy and ER defense response through AMPK‐independent and AMPK‐dependent mechanisms, respectively. Proteinuria (albuminuria) plays a crucial role in the etiology of chronic kidney disease via alteration of multiple signaling pathways in renal cells. The current study using an in vitro model of albumin‐induced cytotoxicity suggests that metformin protects renal cells against proteinuric cytotoxicity via suppression of AKT and mTOR activation, inhibition of epithelial‐to‐mesenchymal transition (EMT) and apoptosis, and augmentation of autophagy and ER defense response through AMPK‐independent and AMPK‐dependent mechanisms respectively.

  April 13, 2017   doi: 10.1002/jcp.25838   open full text
• Cytoskeletal remodeling and regulation of cell fate in the hypertensive neonatal pulmonary artery in response to stress.
  Dina Johar.
  Journal of Cellular Physiology. April 13, 2017
  Neonatal pulmonary hypertension (PHN) is a lethal progressive disease that occurs in prenatal circulatory transition. Mechanical wall strain caused by cardiac pulsation integrates with hypoxia to generate rapidly progressive myocyte cytoskeleton disassembly and failure to exert force generation. The physiological responses to such an interaction have not been investigated. The persistent phenotype does not respond to traditional vasodilator therapy; hence, there is a need for new treatment strategies to improve the morbidity and mortality outcomes. We reviewed the current research methods, models and markers of persistent PHN relevant to oxidative and nitrosative stress as well as cell fate commitment, with an emphasis on apoptosis and proliferation. We surveyed potential investigations into the role of senescence in neonatal PHN cell fate decision programming during vasodilator treatment and suggested putative drug targets to improve clinical outcomes. We identified important signaling intermediates of senescence and cell cycle entry regulation in hypertensive pulmonary arterial tissues. This article is protected by copyright. All rights reserved

  April 13, 2017   doi: 10.1002/jcp.25950   open full text
• Anti‐tumor effects of crocetin and related molecular targets.
  Maliheh Moradzadeh, Hamid Reza Sadeghnia, Alijan Tabarraei, Amirhossein Sahebkar.
  Journal of Cellular Physiology. April 13, 2017
  Natural products have gained a wide popularity as chemopreventive and anti‐cancer agents owing to their multi‐mechanistic mode of action, availability and synergism with several conventional chemotherapeutic agents. Crocetin is a carotenoid compound isolated from the stigma of Crocus sativus L. (saffron). Crocetin has shown promising effects as an anti‐tumor agent in animal models and cell culture systems. Crocetin retards the growth of cancer cells via inhibiting nucleic acid synthesis, enhancing anti‐oxidative system, and inducing apoptosis and differentiation pathways. The present review outlines natural sources of crocetin, and its pharmacokinetic and pharmacological properties relevant to the prevention and treatment of cancer. Also, we discuss molecular targets underlying the putative anti‐tumor effects of crocetin. This article is protected by copyright. All rights reserved

  April 13, 2017   doi: 10.1002/jcp.25953   open full text
• Leptin accelerates the pathogenesis of heterotopic ossification in rat tendon tissues via mTORC1 signaling.
  Huaji Jiang, Yuhui Chen, Guorong Chen, Xinggui Tian, Jiajun Tang, Lei Luo, Minjun Huang, Bin Yan, Xiang Ao, Wen Zhou, Liping Wang, Xiaochun Bai, Zhongmin Zhang, Liang Wang, Cory J. Xian.
  Journal of Cellular Physiology. April 13, 2017
  Leptin, an adipocyte‐derived cytokine associated with bone metabolism, is believed to play a critical role in the pathogenesis of heterotopic ossification (HO). The effect and underlying action mechanism of leptin were investigated on osteogenic differentiation of tendon‐derived stem cells (TDSCs) in vitro and the HO formation in rat tendons. Isolated rat TDSCs were treated with various concentrations of leptin in the presence or absence of mTORC1 signaling specific inhibitor rapamycin in vitro. A rat model with Achilles tenotomy was employed to evaluate the effect of leptin on HO formation together with or without rapamycin treatment. In vitro studies with TDSCs showed that leptin increased the expression of osteogenic biomarkers (alkaline phosphatase, runt‐related transcription factor 2, osterix, osteocalcin) and enhanced mineralization of TDSCs via activating the mTORC1 signal pathway (as indicated by phosphorylation of p70 ribosomal S6 kinase 1 and p70 ribosomal S6). However, mTORC1 signaling blockade with rapamycin treatment suppressed leptin‐induced osteogenic differentiation and mineralization. In vivo studies showed that leptin promoted HO formation in the Achilles tendon after tenotomy, and rapamycin treatment blocked leptin‐induced HO formation. In conclusion, leptin can promote TDSC osteogenic differentiation and heterotopic bone formation via mTORC1 signaling in both in vitro and in vivo models, which provides a new potential therapeutic target for HO prevention. This article is protected by copyright. All rights reserved

  April 13, 2017   doi: 10.1002/jcp.25955   open full text
• Complex Osteoclastogenic Inductive Effects of Nicotine Over Hydroxyapatite.
  J. Costa‐Rodrigues, I. Rocha, M.H. Fernandes.
  Journal of Cellular Physiology. April 13, 2017
  Cigarette smoke is associated to pathological weakening of bone tissue, being considered an important playmaker in conditions such as osteoporosis and periodontal bone loss. In addition, it is also associated with an increased risk of failure in bone regeneration strategies. The present work aimed to characterize the effects of nicotine on human osteoclastogenesis over a hydroxyapatite substrate. Osteoclast precursors were maintained in the absence or presence of the osteoclastogenesis enhancers M‐CSF and RANKL, and were further treated with nicotine levels representative of the concentrations observed in the plasma and saliva of smokers. It was observed that nicotine at low concentrations elicit an increase in osteoclast differentiation, but only in the presence of M‐CSF and RANKL it was also able to significantly increase the resorbing ability of osteoclasts. A slight downregulation of NFkB pathway and an increase in the production of TNF‐α and, particularly PGE2, were involved in the observed effects of nicotine. At high concentrations, nicotine revealed cytotoxic effects, causing a decrease in cell density. In conclusion, nicotine at levels found in the plasma of the smokers, has the ability to act directly on osteoclast precursors, inducing its osteoclastogenic differentiation. The stimulatory behavior appears to be dependent on the stage of osteoclastic differentiation of the precursor cells, which means, in the absence of M‐CSF and RANKL, it only favors the initial stages of osteoclast differentiation, while in the presence of the growth factors, a significant increase in their resorbing ability is also achieved. This article is protected by copyright. All rights reserved

  April 13, 2017   doi: 10.1002/jcp.25956   open full text
• Pin1, the Master Orchestrator of Bone Cell Differentiation.
  Rabia Islam, Won‐Joon Yoon, Hyun‐Mo Ryoo.
  Journal of Cellular Physiology. April 12, 2017
  Pin1 is an enzyme that specifically recognizes the peptide bond between phosphorylated serine or threonine (pS/pT‐P) and proline. This recognition causes a conformational change of its substrate, which further regulates downstream signaling. Pin1−/− mice show developmental bone defects and reduced mineralization. Pin1 targets RUNX2 (Runt‐Related Transcription Factor 2), SMAD1/5, and β‐catenin in the FGF, BMP, and WNT pathways, respectively. Pin1 has multiple roles in the crosstalk between different anabolic bone signaling pathways. For example, it controls different aspects of osteoblastogenesis and increases the transcriptional activity of Runx2, both directly and indirectly. Pin1 also influences osteoclastogenesis at different stages by targeting PU.1 (Purine‐rich nucleic acid binding protein 1), C‐FOS, and DC‐STAMP. The phenotype of Pin1−/− mice has led to the recent identification of multiple roles of Pin1 in different molecular pathways in bone cells. These roles suggest that Pin1 can be utilized as an efficient drug target in congenital and acquired bone diseases. J. Cell. Physiol. 232: 2339–2347, 2017. © 2016 Wiley Periodicals, Inc. Pin1 is a peptidyl prolyl cis–trans isomerase that isomerizes only phosphorylated Ser/Thr‐Pro motifs. Upon binding, Pin1 mediates structural modifications resulting in dramatic changes in substrate activity. In this review, we have summarized the known roles of Pin1 in molecular regulation of bone cell differentiation. Pin1 binds to Runx2, Smad1/5, and β‐catenin, which act downstream of the FGF, BMP, and Wnt signaling pathways, respectively. Binding of Pin1 increases the stability and transcriptional activity of Runx2 and Smad1/5. Pin1 also inhibits the nuclear export of β‐catenin and increases the level of transcriptionally active nuclear β‐catenin.

  April 12, 2017   doi: 10.1002/jcp.25442   open full text
• Human White Adipocytes Convert Into “Rainbow” Adipocytes In Vitro.
  Giulia Maurizi, Antonella Poloni, Domenico Mattiucci, Spartaco Santi, Angela Maurizi, Valerio Izzi, Angelica Giuliani, Stefania Mancini, Maria Cristina Zingaretti, Jessica Perugini, Ilenia Severi, Massimo Falconi, Marco Vivarelli, Maria Rita Rippo, Silvia Corvera, Antonio Giordano, Pietro Leoni, Saverio Cinti.
  Journal of Cellular Physiology. April 12, 2017
  White adipocytes are plastic cells able to reversibly transdifferentiate into brown adipocytes and into epithelial glandular cells under physiologic stimuli in vivo. These plastic properties could be used in future for regenerative medicine, but are incompletely explored in their details. Here, we focused on plastic properties of human mature adipocytes (MA) combining gene expression profile through microarray analysis with morphologic data obtained by electron and time lapse microscopy. Primary MA showed the classic morphology and gene expression profile of functional mature adipocytes. Notably, despite their committed status, MA expressed high levels of reprogramming genes. MA from ceiling cultures underwent transdifferentiation toward fibroblast‐like cells with a well‐differentiated morphology and maintaining stem cell gene signatures. The main morphologic aspect of the transdifferentiation process was the secretion of large lipid droplets and the development of organelles necessary for exocrine secretion further supported the liposecretion process. Of note, electron microscope findings suggesting liposecretion phenomena were found also in explants of human fat and rarely in vivo in fat biopsies from obese patients. In conclusion, both MA and post‐liposecretion adipocytes show a well‐differentiated phenotype with stem cell properties in line with the extraordinary plasticity of adipocytes in vivo. J. Cell. Physiol. 9999: 1–13, 2016. © 2016 Wiley Periodicals, Inc. Plastic properties of human mature adipocytes (MA) have been explored combining gene expression profile made by microarray analysis with morphologic data obtained by electron and time lapse microscopy. Mature adipocytes from ceiling cultures underwent transdifferentiation toward fibroblast‐like cells with a well‐differentiated morphology and maintaining stem cell gene signatures.

  April 12, 2017   doi: 10.1002/jcp.25743   open full text
• Serotonin transporter protects the placental cells against apoptosis in caspase 3‐independent pathway.
  Coedy Hadden, Tariq Fahmi, Anthonya Cooper, Alena V. Savenka, Vladimir V. Lupashin, Drucilla J. Roberts, Luc Maroteaux, Sylvie Hauguel‐de Mouzon, Fusun Kilic.
  Journal of Cellular Physiology. April 12, 2017
  Serotonin (5‐HT) and its specific transporter, SERT play important roles in pregnancy. Using placentas dissected from 18d gestational SERT‐knock out (KO), peripheral 5‐HT (TPH1)‐KO, and wild‐type (WT) mice, we explored the role of 5‐HT and SERT in placental functions in detail. An abnormal thick band of fibrosis and necrosis under the giant cell layer in SERT‐KO placentas appeared only moderately in TPH1‐KO and minimally present in WT placentas. The majority of the changes were located at the junctional zone of the placentas in SERT. The etiology of these findings was tested with TUNEL assays. The placentas from SERT‐KO and TPH1‐KO showed 49‐ and 8‐fold increase in TUNEL‐positive cells without a concurrent change in the DNA repair or cell proliferation compared to WT placentas. While the proliferation rate in the embryos of TPH1‐KO mice was 16‐fold lower than the rate in gestational age matched embryos of WT or SERT‐KO mice. These findings highlight an important role of continuous 5‐HT signaling on trophoblast cell viability. SERT may contribute to protecting trophoblast cells against cell death via terminating the 5‐HT signaling which changes cell death ratio in trophoblast as well as proliferation rate in embryos. However, the cell death in SERT‐KO placentas is in caspase 3‐independent pathway.

  April 12, 2017   doi: 10.1002/jcp.25812   open full text
• Dendritic Cell‐Specific Transmembrane Protein (DC‐STAMP) Regulates Osteoclast Differentiation via the Ca2+/NFATc1 Axis.
  Ya‐Hui Chiu, Edward Schwarz, Dongge Li, Yuexin Xu, Tzong‐Ren Sheu, Jinbo Li, Karen L. de Mesy Bentley, Changyong Feng, Baoli Wang, Jhih‐Cheng Wang, Liz Albertorio‐Saez, Ronald Wood, Minsoo Kim, Wensheng Wang, Christopher T. Ritchlin.
  Journal of Cellular Physiology. April 12, 2017
  DC‐STAMP is a multi‐pass transmembrane protein essential for cell–cell fusion between osteoclast precursors during osteoclast (OC) development. DC‐STAMP−/− mice have mild osteopetrosis and form mononuclear cells with limited resorption capacity. The identification of an Immunoreceptor Tyrosine‐based Inhibitory Motif (ITIM) on the cytoplasmic tail of DC‐STAMP suggested a potential signaling function. The absence of a known DC‐STAMP ligand, however, has hindered the elucidation of downstream signaling pathways. To address this problem, we engineered a light‐activatable DC‐STAMP chimeric molecule in which light exposure mimics ligand engagement that can be traced by downstream Ca2+ signaling. Deletion of the cytoplasmic ITIM resulted in a significant elevation in the amplitude and duration of intracellular Ca2+ flux. Decreased NFATc1 expression in DC‐STAMP−/− cells was restored by DC‐STAMP over‐expression. Multiple biological phenotypes including cell–cell fusion, bone erosion, cell mobility, DC‐STAMP cell surface distribution, and NFATc1 nuclear translocation were altered by deletion of the ITIM and adjacent amino acids. In contrast, mutations on each of the tyrosine residues surrounding the ITIM showed no effect on DC‐STAMP function. Collectively, our results suggest that the ITIM on DC‐STAMP is a functional motif that regulates osteoclast differentiation through the NFATc1/Ca2+ axis. J. Cell. Physiol. 232: 2538–2549, 2017. © 2016 Wiley Periodicals, Inc.

  April 12, 2017   doi: 10.1002/jcp.25638   open full text
• Contributions of 3D Cell Cultures for Cancer Research.
  Maddaly Ravi, Aarthi Ramesh, Aishwarya Pattabhi.
  Journal of Cellular Physiology. April 12, 2017
  Cancer cell lines have contributed immensely in understanding the complex physiology of cancers. They are excellent material for studies as they offer homogenous samples without individual variations and can be utilised with ease and flexibility. Also, the number of assays and end‐points one can study is almost limitless; with the advantage of improvising, modifying or altering several variables and methods. Literally, a new dimension to cancer research has been achieved by the advent of 3Dimensional (3D) cell culture techniques. This approach increased many folds the ways in which cancer cell lines can be utilised for understanding complex cancer biology. 3D cell culture techniques are now the preferred way of using cancer cell lines to bridge the gap between the ‘absolute in vitro’ and ‘true in vivo’. The aspects of cancer biology that 3D cell culture systems have contributed include morphology, microenvironment, gene and protein expression, invasion/migration/metastasis, angiogenesis, tumour metabolism and drug discovery, testing chemotherapeutic agents, adaptive responses and cancer stem cells. We present here, a comprehensive review on the applications of 3D cell culture systems for these aspects of cancers. J. Cell. Physiol. 9999: 1–19, 2016. © 2016 Wiley Periodicals, Inc. 3D cell cultures are gaining ground for several applications, of which cancer research is an important area. Several aspects of cancer physiology have been unraveled using these cultures. Also, 3D cell cultures provide realistic material for drug discovery and biomarker discovery.

  April 12, 2017   doi: 10.1002/jcp.25664   open full text
• The Role of Exercise and TFAM in Preventing Skeletal Muscle Atrophy.
  Nicholas T. Theilen, George H. Kunkel, Suresh C. Tyagi.
  Journal of Cellular Physiology. April 12, 2017
  Skeletal muscle atrophy is the consequence of protein degradation exceeding protein synthesis. This arises for a multitude of reasons including the unloading of muscle during microgravity, post‐surgery bedrest, immobilization of a limb after injury, and overall disuse of the musculature. The development of therapies prior to skeletal muscle atrophy settings to diminish protein degradation is scarce. Mitochondrial dysfunction is associated with skeletal muscle atrophy and contributes to the induction of protein degradation and cell apoptosis through increased levels of ROS observed with the loss of organelle function. ROS binds mtDNA, leading to its degradation and decreasing functionality. Mitochondrial transcription factor A (TFAM) will bind and coat mtDNA, protecting it from ROS and degradation while increasing mitochondrial function. Exercise stimulates cell signaling pathways that converge on and increase PGC‐1α, a well‐known activator of the transcription of TFAM and mitochondrial biogenesis. Therefore, in the present review we are proposing, separately, exercise and TFAM treatments prior to atrophic settings (muscle unloading or disuse) alleviate skeletal muscle atrophy through enhanced mitochondrial adaptations and function. Additionally, we hypothesize the combination of exercise and TFAM leads to a synergistic effect in targeting mitochondrial function to prevent skeletal muscle atrophy. J. Cell. Physiol. 232: 2348–2358, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by © 2016 Wiley Periodicals, Inc. Skeletal muscle atrophy via unloading and disuse of the musculature is the consequence of protein degradation exceeding protein synthesis. This state is correlated with increased markers of mitochondrial dysfunction. We hypothesize targeting increased mitochondrial function through exercise and mitochondrial transcription factor A treatments will diminish skeletal muscle atrophy after a period of muscle unloading and disuse.

  April 12, 2017   doi: 10.1002/jcp.25737   open full text
• Homocysteine as a Pathological Biomarker for Bone Disease.
  Jyotirmaya Behera, Jyoti Bala, Mohammed Nuru, Suresh C. Tyagi, Neetu Tyagi.
  Journal of Cellular Physiology. April 12, 2017
  In the last few decades, perturbation in methyl‐group and homocysteine (Hcy) balance have emerged as independent risk factors in a number of pathological conditions including neurodegenerative disease, cardiovascular dysfunction, cancer development, autoimmune disease, and kidney disease. Recent studies report Hcy to be a newly recognized risk factor for osteoporosis. Elevated Hcy levels are known to modulate osteoclastgenesis by causing detrimental effects on bone via oxidative stress induced metalloproteinase‐mediated extracellular matrix degradation and decrease in bone blood flow. Evidence from previous studies also suggests that the decreased chondrocytes mediated bone mineralization in chick limb‐bud mesenchymal cells and during the gestational period of ossification in rat model. However, Hcy imbalance and its role in bone loss, regression in vascular invasion, and osteoporosis, are not clearly understood. More investigations are required to explore the complex interplay between Hcy imbalance and onset of bone disease progression. This article reviews the current body of knowledge on regulation of Hcy mediated oxidative stress and its role in bone remodeling, vascular blood flow and progression of bone disease. J. Cell. Physiol. 9999: 1–6, 2016. © 2016 Wiley Periodicals, Inc. This article reviews the current body of knowledge on regulation of Hcy‐mediated oxidative stress and its role in bone remodeling, vascular blood flow, and progression of bone disease.

  April 12, 2017   doi: 10.1002/jcp.25693   open full text
• Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is Involved in Cell Cycle Progression.
  Alessandro Poli, Roberta Fiume, Gianluca Baldanzi, Daniela Capello, Stefano Ratti, Marco Gesi, Lucia Manzoli, Andrea Graziani, Pann‐Ghill Suh, Lucio Cocco, Matilde Y. Follo.
  Journal of Cellular Physiology. April 10, 2017
  Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550–2557, 2017. © 2016 Wiley Periodicals, Inc.

  April 10, 2017   doi: 10.1002/jcp.25642   open full text
• Arabinogalactan Proteins From Baobab and Acacia Seeds Influence Innate Immunity of Human Keratinocytes In Vitro.
  Abderrakib Zahid, Julie Despres, Magalie Benard, Eric Nguema‐Ona, Jerome Leprince, David Vaudry, Christophe Rihouey, Maité Vicré‐Gibouin, Azeddine Driouich, Marie‐Laure Follet‐Gueye.
  Journal of Cellular Physiology. April 10, 2017
  Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However, little is currently known regarding their potential activity toward skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability. Moreover, real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD‐2, TLR‐5, and IL1‐α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. J. Cell. Physiol. 232: 2558–2568, 2017. © 2016 Wiley Periodicals, Inc. Real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis showed that AGP extracted from acacia or baobab seeds induced a substantial overexpression of hBD‐2, TLR‐5, and IL1‐α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses.

  April 10, 2017   doi: 10.1002/jcp.25646   open full text
• Anti‐Tumor Effects of Cardiac Glycosides on Human Lung Cancer Cells and Lung Tumorspheres.
  Vivek Kaushik, Juan Sebastian Yakisich, Neelam Azad, Yogesh Kulkarni, Rajkumar Venkatadri, Clayton Wright, Yon Rojanasakul, Anand Krishnan V. Iyer.
  Journal of Cellular Physiology. April 10, 2017
  Lung cancer is a leading cause of cancer‐related death in the United States. Although several drugs have been developed that target individual biomarkers, their success has been limited due to intrinsic or acquired resistance for the specific targets of such drugs. A more effective approach is to target multiple pathways that dictate cancer progression. Cardiac glycosides demonstrate such multimodal effects on cancer cell survival, and our aim was to evaluate the effect of two naturally occurring monosaccaridic cardiac glycosides—Convallatoxin and Peruvoside on lung cancer cells. Although both drugs had significant anti‐proliferative effects on H460 and Calu‐3 lung cancer cells, Convallatoxin demonstrated twofold higher activity as compared to Peruvoside using both viability and colony forming assays, suggesting a role for the aglycone region in dictating drug potency. The tumor suppressor p53 was found to be important for action of both drugs—p53‐underexpressing cells were less sensitive as compared to p53‐positive H460 cells. Further, assessment of p53‐underexpressing H460 cells showed that drugs were able to arrest cells in the G0/G1 phase of the cell cycle in a dose‐dependent manner. Both drugs significantly inhibited migration and invasion of cancer cells and decreased the viability of floating tumorspheres. An assessment of intracellular pathways indicated that both drugs were able to modulate proteins that are involved in apoptosis, autophagy, cell cycle, proliferation, and EMT. Our data suggest, a promising role for cardiac glycosides in lung cancer treatment, and provides impetus for further investigation of the anti‐cancer potential of this class of drugs. J. Cell. Physiol. 232: 2497–2507, 2017. © 2016 Wiley Periodicals, Inc. The cardiac glycosides convallatoxin and peruvoside exert potent anti‐cancer effects in lung cancer cells. A significant inhibition of cell migration, invasion, and lung tumorsphere formation was observed in response to both drugs.

  April 10, 2017   doi: 10.1002/jcp.25611   open full text
• The Role of Calcium‐Sensing Receptors in Endothelin‐1‐Dependent Effects on Adult Rat Ventricular Cardiomyocytes: Possible Contribution to Adaptive Myocardial Hypertrophy.
  Elena Dyukova, Rolf Schreckenberg, Christoph Arens, Guzel Sitdikova, Klaus‐Dieter Schlüter.
  Journal of Cellular Physiology. April 10, 2017
  Nitric oxide (NO)‐deficiency as it occurs during endothelial dysfunction activates the endothelin‐1 (ET‐1) system and increases the expression of receptor activity modifying protein (RAMP)‐1 that acts as a chaperon for calcium‐sensing receptors (CaR) that have recently been identified to improve cardiac function. Here, we hypothesized that ET‐1 increases the cardiac expression of CaR and thereby induces an adaptive type of hypertrophy. Expressions of RAMP‐1, endothelin receptors, and CaR were analyzed by RT‐PCR in left ventricular tissues of L‐NAME‐treated rats. Effects of ET‐1 on CaR expression and cell function (load free cell shortening) were analyzed in adult rat ventricular cardiomyocytes. siRNA directed against CaR and RAMP‐1 was used to investigate a causal relationship. PD142893 and BQ788 were used to dissect the contribution of ETB1, ETB2, and ETA receptors. Non‐specific NO synthase inhibition with L‐Nitro arginine methyl ester (L‐NAME) caused a cardiac upregulation of ETB receptors and CaR suggesting a paracrine effect of ET‐1 on cardiomyocytes. Indeed, ET‐1 induced the expression of CaR in cultured cardiomyocytes. Under these conditions, cardiomyocytes increased cell size (hypertrophy) but maintained normal function. Inhibition of ETA and ETB1 receptors led to ET‐1‐dependent reduction in cell shortening and attenuated up‐regulation of CaR. Down‐regulation of RAMP‐1 reduced CaR responsiveness. In conclusion, ET‐1 causes an adaptive type of hypertrophy by up‐regulation of CaR in cardiomyocytes via ETA and/or ETB1 receptors. J. Cell. Physiol. 232: 2508–2518, 2017. © 2016 Wiley Periodicals, Inc. Current manuscript is the first report indicating the ET‐1‐dependent upregulation of CaR receptor in cardiomyocytes and it shows that this upregulation allows cardiac cells to maintain proper function. Second new finding indicates involvement of ETA and /or ETB1 receptors in adaptive hypertrophy in cardiomyocytes, whereas ETB2 receptors activation leads to mal‐adaptive effect. Finally, chaperone activity of RAMP1 is required for the functional coupling of CaR in cardiomyocytes.

  April 10, 2017   doi: 10.1002/jcp.25612   open full text
• Lkb1 regulation of skeletal muscle development, metabolism and muscle progenitor cell homeostasis.
  Tizhong Shan, Ziye Xu, Jiaqi Liu, Weiche Wu, Yizhen Wang.
  Journal of Cellular Physiology. April 10, 2017
  Liver kinase B1 (Lkb1), also named as Serine/Threonine protein kinase 11 (STK11), is a serine/threonine kinase that plays crucial roles in various cellular processes including cell survival, cell division, cellular polarity, cell growth, cell differentiation, and cell metabolism. In metabolic tissues, Lkb1 regulates glucose homeostasis and energy metabolism through phosphorylating and activating the AMPK subfamily proteins. In skeletal muscle, Lkb1 affects muscle development and postnatal growth, lipid and fatty acid oxidation, glucose metabolism, and insulin sensitivity. Recently, the regulatory roles of Lkb1 in regulating division, self‐renew, proliferation, and differentiation of skeletal muscle progenitor cells have been reported. In this review, we discuss the roles of Lkb1 in regulating skeletal muscle progenitor cell homeostasis and skeletal muscle development and metabolism. In skeletal muscle, Lkb1 affects muscle development and postnatal growth, lipid, and fatty acid oxidation, glucose metabolism and insulin sensitivity. In skeletal muscle progenitor cells, Lkb1 regulates cell division, self‐renew, proliferation, and differentiation. Here, we review the role of Lkb1 in skeletal muscle development, metabolism and muscle progenitor cell homeostasis.

  April 10, 2017   doi: 10.1002/jcp.25786   open full text
• Stimulatory actions of a novel thiourea derivative on large‐conductance, calcium‐activated potassium channels.
  Sheng‐Nan Wu, Jyh‐Haur Chern, Santai Shen, Hwei‐Hisen Chen, Ying‐Ting Hsu, Chih‐Chin Lee, Ming‐Huan Chan, Ming‐Chi Lai, Feng‐Shiun Shie.
  Journal of Cellular Physiology. April 10, 2017
  In this study, we examine whether an anti‐inflammatory thiourea derivative, compound #326, actions on ion channels. The effects of compound #326 on Ca2+‐activated K+ channels were evaluated by patch‐clamp recordings obtained in cell‐attached, inside‐out or whole‐cell configuration. In pituitary GH3 cells, compound #326 increased the amplitude of Ca2+‐activated K+ currents (IK(Ca)) with an EC50 value of 11.6 μM, which was reversed by verruculogen, but not tolbutamide or TRAM‐34. Under inside‐out configuration, a bath application of compound #326 raised the probability of large‐conductance Ca2+‐activated K+ (BKCa) channels. The activation curve of BKCa channels was shifted to less depolarised potential with no modification of the gating charge of the curve; consequently, the difference of free energy was reduced in the presence of this compound. Compound #326‐stimulated activity of BKCa channels is explained by a shortening of mean closed time, despite its inability to alter single‐channel conductance. Neither delayed‐rectifier nor erg‐mediated K+ currents was modified. Compound #326 decreased the peak amplitude of voltage‐gated Na+ current with no clear change in the overall current–voltage relationship of this current. In HEK293T cells expressing α‐hSlo, compound #326 enhanced BKCa channels effectively. Intriguingly, the inhibitory actions of compound #326 on interleukin 1β in lipopolysaccharide‐activated microglia were significantly reversed by verruculogen, whereas BKCa channel inhibitors suppressed the expressions of inducible nitric oxide synthase. The BKCa channels could be an important target for compound #326 if similar in vivo results occur, and the multi‐functionality of BKCa channels in modulating microglial immunity merit further investigation. Compound #326‐stimulated activity of BKCa channels is explained by a shortening of mean closed time, despite its inability to alter single‐channel conductance. Intriguingly, the inhibitory actions of compound #326 on interleukin 1β in lipopolysaccharide‐activated microglia were significantly reversed by verruculogen, whereas BKCa channel inhibitors suppressed the expressions of inducible nitric oxide synthase.

  April 10, 2017   doi: 10.1002/jcp.25788   open full text
• Inhibitory effect of blue light emitting diode on migration and invasion of cancer cells.
  Phil‐Sun Oh, Hyun‐Soo Kim, Eun‐Mi Kim, Hyosook Hwang, Hyang Hwa Ryu, SeokTae Lim, Myung‐Hee Sohn, Hwan‐Jeong Jeong.
  Journal of Cellular Physiology. April 10, 2017
  The aim of this study was to determine the effects and molecular mechanism of blue light emitting diode (LED) in tumor cells. A migration and invasion assay for the metastatic behavior of mouse colon cancer CT‐26 and human fibrosarcoma HT‐1080 cells was performed. Cancer cell migration‐related proteins were identified by obtaining a 2‐dimensional gel electrophoresis (2‐DE) in total cellular protein profile of blue LED‐irradiated cancer cells, followed by matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) analysis of proteins. Protein levels were examined by immunoblotting. Irradiation with blue LED inhibited CT‐26 and HT‐1080 cell migration and invasion. The anti‐metastatic effects of blue LED irradiation were associated with inhibition of matrix metalloproteinase (MMP)‐2 and MMP‐9 expression. P38 MAPK phosphorylation was increased in blue LED‐irradiated CT‐26 and HT‐1080 cells, but was inhibited after pretreatment with SB203580, a specific inhibitor of p38 MAPK. Inhibition of p38 MAPK phosphorylation by SB203580 treatment increased number of migratory cancer cells in CT‐26 and HT‐1080 cells, indicating that blue LED irradiation inhibited cancer cell migration via phosphorylation of p38 MAPK. Additionally blue LED irradiation of mice injected with CT‐26 cells expressing luciferase decreased early stage lung metastasis compared to untreated control mice. These results indicate that blue LED irradiation inhibits cancer cell migration and invasion in vitro and in vivo.

  April 10, 2017   doi: 10.1002/jcp.25805   open full text
• Let‐7i‐Induced Atg4B Suppression Is Essential for Autophagy of Placental Trophoblast in Preeclampsia.
  Yinyan Xu, Xinyan Huang, Juan Xie, Yanni Chen, Jing Fu, Li Wang.
  Journal of Cellular Physiology. April 10, 2017
  Autophagy, identified as type II programmed cell death, has already been known to be involved in the pathophysiology of preeclampsia (PE), which is a gestational disease with high morbidity. The present study aims to investigate the functional role of let‐7i, a miRNA, in trophoblastic autophagy. Placental tissue used in this study was collected from patients with severe preeclampsia (SPE) or normal pregnant women. A decreased level of let‐7i was found in placenta of SPE. In addition, autophagic vacuoles were observed in SPE and the expression of microtubule associated protein 1 light chain 3 (LC3) II/I was elevated. In vitro, let‐7i mimics suppressed the autophagic activities in human HTR‐8/SVneo trophoblast cell line (HTR‐8) and human placental choriocarcinoma cell line JEG‐3, whereas let‐7i inhibitor enhanced the activities. As a potential target of let‐7i, autophagy‐related 4B cysteine peptidase (Atg4B) had an increased expression level in SPE. As expected, the increased expression of Atg4B was negatively regulated by let‐7i using dual luciferase reporter assay. Furthermore, these trophoblast‐like cells transfected with the let‐7i mimic or inhibitors resulted in a significant change of Atg4B in both mRNA and protein level. More importantly, Atg4B overexpression could partly reverse let‐7i mimic‐reduced LC3II/I levels; whereas Atg4B silencing partly attenuated let‐7i inhibitor‐induced the level of LC3II/I expression. Taken together, these findings suggest that let‐7i is able to regulate autophagic activity via regulating Atg4B expression, which might contribute to the pathogenesis of PE. J. Cell. Physiol. 232: 2581–2589, 2017. © 2016 Wiley Periodicals, Inc. This study provided insight into the role of let‐7i in the pathophysiology of PE. Our study demonstrates for the first time that a novel let‐7i‐Atg4B‐autophagy signaling pathway plays a key role in PE. This finding suggests that miRNAs are key molecules involved in autophagic activity, and might be regard as a new therapeutic target against PE.

  April 10, 2017   doi: 10.1002/jcp.25661   open full text
• KCNQ1 variants associate with hypertension in type 2 diabetes and affect smooth muscle contractility in vitro.
  Kuo‐Chin Huang, Te‐Mao Li, Xiang Liu, Jin‐Hua Chen, Wen‐Kuei Chien, Yi‐Tzone Shiao, Hsinyi Tsang, Ting‐Hsu Lin, Chiu‐Chu Liao, Shao‐Mei Huang, Ju‐Pi Li, Cheng‐Wen Lin, Jung‐Chun Lin, Chih‐Chien Lin, Chih‐Ho Lai, Chi‐Fung Cheng, Wen‐Miin Liang, Chien‐Hui Hung, Ching‐Chu Chen, Ying‐Ju Lin, Fuu‐Jen Tsai.
  Journal of Cellular Physiology. April 10, 2017
  KCNQ1 encodes a potassium voltage‐gated channel and represents a susceptibility locus for type 2 diabetes mellitus (T2DM). Here, we explored the association between KCNQ1 polymorphisms and hypertension risk in individuals with T2DM, as well as the role of KCNQ1 in vascular smooth muscle cell contraction in vitro. To investigate the relationship between KCNQ1 and the risk of developing hypertension in patients with T2DM, we divided the T2DM cohort into hypertension (n = 452) and non‐hypertension (n = 541) groups. The Mann–Whitney U test, chi‐square test, and multivariate regression analyses were used to assess the clinical characteristics and genotypic frequencies. In vitro studies utilized the rat aortic smooth muscle A10 cell line. Patients in the hypertension group were significantly older at the time of enrollment and had higher levels of body mass index, waist‐to‐hip ratio, and triglyceride than those in the non‐hypertension group. The KCNQ1 rs3864884 and rs12576239 genetic variants were associated with hypertension in T2DM. KCNQ1 expression was lower in the individuals with the CC versus the CT and TT genotypes. Smooth muscle cell contractility was inhibited by treatment with a KCNQ1 inhibitor. These results suggest that KCNQ1 might be associated with hypertension in individuals with T2DM. The KCNQ1 rs3864884 and rs12576239 genetic variants were associated with hypertension in T2DM. Smooth muscle cell contractility was inhibited by treatment with a KCNQ1 inhibitor. KCNQ1 might be associated with hypertension in individuals with T2DM.

  April 10, 2017   doi: 10.1002/jcp.25775   open full text
• Irx3 and Bmp2 regulate mouse mesenchymal cell chondrogenic differentiation in both a Sox9‐dependent and ‐independent manner.
  Yoshihiro Tamamura, Kenichi Katsube, Hisashi Mera, Maki Itokazu, Shigeyuki Wakitani.
  Journal of Cellular Physiology. April 10, 2017
  Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage‐specific proteoglycans. The existence of Sox9‐independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois‐related homeobox 3 (Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9‐independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2‐mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9‐mediated regulation. Irx3 cooperates with Bmp2 to regulate the expression of type II collagen and aggrecan in a Sox9‐dependent manner, whereas that of epiphycan and lumican in a Sox9‐independent manner in C3H10T1/2 cells.

  April 10, 2017   doi: 10.1002/jcp.25776   open full text
• Testosterone Rescues the De‐Differentiation of Smooth Muscle Cells Through Serum Response Factor/Myocardin.
  Carolina Leimgruber, Amado A. Quintar, Nahuel Peinetti, María V. Scalerandi, Juan P. Nicola, Joseph M. Miano, Cristina A. Maldonado.
  Journal of Cellular Physiology. April 10, 2017
  Prostatic smooth muscle cells (pSMCs) differentiation is a key factor for prostatic homeostasis, with androgens exerting multiple effects on these cells. Here, we demonstrated that the myodifferentiator complex Srf/Myocd is up‐regulated by testosterone in a dose‐dependent manner in primary cultures of rat pSMCs, which was associated to the increase in Acta2, Cnn1, and Lmod1 expressions. Blocking Srf or Myocd by siRNAs inhibited the myodifferentiator effect of testosterone. While LPS led to a dedifferentiated phenotype in pSMCs, characterized by down‐regulation of Srf/Myocd and smooth muscle cell (SMC)‐restricted genes, endotoxin treatment on Myocd‐overexpressing cells did not result in phenotypic alterations. Testosterone at a physiological dose was able to restore the muscular phenotype by normalizing Srf/Myocd expression in inflammation‐induced dedifferentiated pSMCs. Moreover, the androgen reestablished the proliferation rate and IL‐6 secretion increased by LPS. These results provide novel evidence regarding the myodifferentiating role of testosterone on SMCs by modulating Srf/Myocd. Thus, androgens preserve prostatic SMC phenotype, which is essential to maintain the normal structure and function of the prostate. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc. Androgens favor a contractile phenotype on smooth muscle cells that relies on the Myocd/SRF myodifferentiator complex, with this role being critical for cellular homeostasis and pathophysiology.

  April 10, 2017   doi: 10.1002/jcp.25679   open full text
• TNF‐α promotes osteoclastogenesis through JNK signaling‐dependent induction of Semaphorin3D expression in estrogen‐deficiency induced osteoporosis.
  Chenglin Sang, Jiefeng Zhang, Yongxian Zhang, Fangjing Chen, Xuecheng Cao, Lei Guo.
  Journal of Cellular Physiology. April 10, 2017
  Tumor necrosis factor α (TNF‐α)‐induced osteoclast formation have been demonstrated to play an important role in the pathogenesis of estrogen deficiency‐mediated bone loss, but the exact mechanisms by which TNF‐α enhanced osteoclast differentiation were not fully elucidated. The class III semaphorins members were critical to regulate bone homeostasis. Here, we identified a novel mechanism whereby TNF‐α increasing Semaphorin3D expression contributes to estrogen deficiency‐induced osteoporosis. In this study, we found that Semaphorin3D expression was upregulated by TNF‐α during the process of RANKL‐induced osteoclast differentiation. Inhibition of Semaphorin3D in pre‐osteoclasts could attenuate the stimulatory effects of TNF‐α on osteoclast proliferation and differentiation. Mechanistically, blocking of the Jun N‐terminal kinase (JNK) signaling markedly rescued TNF‐α‐induced Semaphorin3D expression, suggesting that JNK signaling was involved in the regulation of Semaphorin3D expression by TNF‐α. In addition, silencing of Semaphorin3D in vivo could alleviate estrogen deficiency‐induced osteoporosis. Our results revealed a novel function for Semaphorin3D and suggested that increased Semaphorin3D may contribute to enhanced bone loss by increased TNF‐α in estrogen deficiency‐induced osteoporosis. Thus, Semaphorin3D may provide a potential therapeutic target for the treatment of estrogen‐deficiency induced osteoporosis. We found that TNF‐a could increase Sema3D expression in RANKL‐induced osteoclast formation. Upregulation of Sema3D expression was required for TNF‐a‐mediated osteoclast proliferation and differentiation. JNK signaling was involved in the regulation of Sema3D expression by TNF‐a.

  April 10, 2017   doi: 10.1002/jcp.25784   open full text
• Sensitivity of TP53‐Mutated Cancer Cells to the Phytoestrogen Genistein Is Associated With Direct Inhibition of Plk1 Activity.
  Sol‐Bi Shin, Sang‐Uk Woo, Young‐Won Chin, Young‐Joo Jang, Hyungshin Yim.
  Journal of Cellular Physiology. April 10, 2017
  Polo‐like kinase 1 (Plk1), a conserved Ser/Thr mitotic kinase, has been identified as a promising target for anticancer drug development because its overexpression is correlated with malignancy. Here, we found that genistein, an isoflavone, inhibits Plk1 kinase activity directly. Previously the mitotic disturbance phenomenon induced by treatment with genistein was not fully explained by its inhibitory effect on EGFR. In kinase profiling assays, it showed selectivity relative to a panel of kinases, including EGFR. Treatment with genistein induced cell death in a concentration‐dependent manner in cancer cells from diverse tissue origins, but not in non‐transformed cells such as hTERT‐RPE or MCF10A cells. We also observed that genistein tended to be more selective against cancer cells with mutations in the TP53 gene. TP53‐depeleted LNCaP and NCI‐H460 cells using shRNA targeting human TP53 were more sensitive to cell death by treatment of genistein. Furthermore, genistein induced mitotic arrest by inhibiting Plk1 activity and, consequently, led to mitotic catastrophe and apoptosis. These data suggest that genistein may be a promising anticancer drug candidate due to its inhibitory activity against Plk1 as well as EGFR and effectiveness toward cancer cells, especially those with p53‐mutation. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc. The inhibitory activity of genistein on the Plk1 kinase was relatively selective to its activity against a panel of other kinases, including EGFR. Genistein had a cytotoxic effect in human cancer cells from diverse tissue origins, but not in non‐transformed cells. The sensitivity of cancer cells containing mutations in the TP53 gene to genistein tended to be higher when compared with that of cells with wild‐type TP53. Since around 50% of solid tumors have mutant TP53, the development of genistein as an anticancer drug would be valuable for treatment of such tumors.

  April 10, 2017   doi: 10.1002/jcp.25680   open full text
• A critical role for adiponectin‐mediated development of endometrial luminal epithelial cells during the peri‐implantation period of pregnancy.
  Whasun Lim, Myung Jin Choi, Hyocheol Bae, Fuller W. Bazer, Gwonhwa Song.
  Journal of Cellular Physiology. April 10, 2017
  Adiponectin is one of the adipokines in the collagen superfamily. It is secreted primarily by white adipocytes and influences reproductive processes including ovarian and uterine functions. Adiponectin regulates energy homeostasis, insulin sensitivity, and is anti‐inflammatory in various tissues. Its receptors (ADIPOR1 and ADIPOR2) are widely expressed in mammalian tissues, including porcine conceptuses and endometrial during the estrous cycle and peri‐implantation period of pregnancy. However, regulatory effects of adiponectin on endometrial epithelial cells are unknown. Therefore, we investigated the effects of parity on expression of ADIPOR1 and ADIPOR2 and the effects of adiponectin in the porcine endometrium during early pregnancy. Results of this study revealed robust expression of ADIPOR1 and ADIPOR2 in uterine luminal (LE) and glandular (GE) epithelia during early pregnancy and expression decreased as with increasing parity. For porcine luminal epithelial (pLE) cells, adiponectin enhanced proliferation, and increased phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, P38, and P90RSK in a time‐dependent manner. Moreover, the abundance of adiponectin‐activated signaling molecules were suppressed by pharmacological inhibitors including wortmannin, U0126, SP600125, and SB203580, respectively, in pLE cells. Furthermore, inhibition of each targeted signal transduction molecule influenced proliferation of adiponectin‐stimulated pLE cells. In addition, adiponectin inhibited tunicamycin‐induced endoplasmic reticulum (ER)‐stress through effects on ER stress regulated proteins in pLE cells. Collectively, these results suggest that adiponectin affects development of porcine uterine epithelia and reproductive performance through modulation of PI3K/AKT and MAPK cell signaling pathways. Adiponectin enhance uterine receptivity to implantation and placentation through PI3K/AKT and MAPK pathways for development of the porcine uterus and reproductive performance.

  April 10, 2017   doi: 10.1002/jcp.25768   open full text
• Anti‐Atherosclerotic Effects of Vitamins D and E in Suppression of Atherogenesis.
  Bahman Rashidi, Zahra Hoseini, Amirhossein Sahebkar, Hamed Mirzaei.
  Journal of Cellular Physiology. April 10, 2017
  Atherosclerosis is a progressive and multifactorial disease which occurs under the influence of various risk factors including endothelial dysfunction (ED), oxidative stress, and low‐density lipoprotein (LDL) oxidation. In contract to the initial hypotheses on the usefulness of vitamin E supplementation for cardiovascular disease prevention, large outcome trials showed consumption of vitamin E has no obvious effect on cardiovascular disease and, in some cases, it may even increase the rate of mortality. This seemingly unexpected finding may be due to the opposite effects of vitamin E compounds. Vitamin E is a group of compounds which have different and even opposing effects, yet in most of the studies, the exact consumed component of vitamin E is not determined. It appears that the combined consumption of gamma‐tocopherol, vitamin C, D, and tetrahydrobiopterin (BH4) may be extremely effective in both preventing atherogenesis and suppressing plaque development. In this regard, one of main issues is effect of vitamins E and D deficiency on microRNAs network in atherosclerosis. Various studies have indicated that miRNAs have key roles in atherosclerosis pathogenesis. The deficiency of vitamins E and D could provide a deregulation for miRNAs network and these events could lead to progression of atherosclerosis. Here, we highlighted a variety of mechanisms involve in the progression of atherosclerosis and effects of vitamins D and E on these mechanisms. Moreover, we summarized miRNAs involve in atherosclerosis and their regulation by vitamins E and D deficiency. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.

  April 10, 2017   doi: 10.1002/jcp.25738   open full text
• Regulation of chondrocyte functions by transient receptor potential cation channel V6 in osteoarthritis.
  Tengfei Song, Jun Ma, Lei Guo, Peng Yang, Xuhui Zhou, Tianwen Ye.
  Journal of Cellular Physiology. April 10, 2017
  Transient receptor potential vanilloid (TRPV) channels function to maintain the dynamic balance of calcium signaling and calcium metabolism in bones. The goal of this study was to determine the potential role of TRPV6 in regulation of chondrocytes. The level of TRPV6 expression was analyzed by western blot in articular cartilage derived from the knee joints of osteoarthritis (OA) rat models and OA patients. Bone structure and osteoarthritic changes in the knee joints of TRPV6 knockout mice were examined using micro‐computed and histological analysis at the age of 6 and 12 months old. Furthermore, to investigate the effects of TRPV6 on chondrocyte extracellular matrix secretion, the release of matrix degrading enzymes, cell proliferation, and apoptosis, we decreased and increased TRPV6 expression in chondrocytes with lentiviral constructs encoding shRNA targeting TRPV6 and encoding TRPV6, respectively. The results showed that the level of TRPV6 expression in an OA rat model was markedly down‐regulated. TRPV6 knockout mice showed severe osteoarthritis changes, including cartilage fibrillation, eburnation, and loss of proteoglycans. In addition, deficiency of TRPV6 clearly affected chondrocyte function, such as extracellular matrix secretion, the release of matrix degrading enzymes, cell proliferation, and apoptosis. Taken together, our results implicated that TRPV6 channel, as a chondro‐protective factor, was involved in the pathogenesis of OA. The level of TRPV6 expression in an OA rat model was markedly down‐regulated. TRPV6 knockout mice showed severe osteoarthritic changes. Deficiency of TRPV6 clearly affected chondrocyte function.

  April 10, 2017   doi: 10.1002/jcp.25770   open full text
• Live Cell Imaging: Assessing the Phototoxicity of 488 and 546 nm Light and Methods to Alleviate it.
  Stephen Douthwright, Greenfield Sluder.
  Journal of Cellular Physiology. April 10, 2017
  In live cell imaging of fluorescent proteins, phototoxicity of the excitation light can be problematical. Cell death is obvious, but reduced cell viability can make the interpretation of observations error prone. We characterized the phototoxic consequences of 488 and 546 nm light on untransformed human cells and tested methods that have or could be used to alleviate photodamage. Unlabeled RPE1 cells were given single 0.5–2.5 min irradiations in early G1 from a mercury arc lamp on a fluorescence microscope. Four hundred eighty‐eight nanometer light produced a dose‐dependent decrease in the percentage of cells that progressed to mitosis, slowing of the cell cycle for some of those entering mitosis, and a ∼12% incidence of cell death for the highest dose. For 546 nm light we found a 10–15% reduction in the percentage of cells entering mitosis, no strong dose dependency, and a ∼2% incidence of cell death for the longest irradiations. For cells expressing GFP‐centrin1 or mCherry‐centrin1, fewer entered mitosis for each dose than unlabeled cells. For constant total dose 488 nm light irradiations of unlabeled cells, reducing the intensity 10‐fold or spreading the exposures out as a series of 10 sec pulses at 1 min intervals produced a minor and not consistent improvement in the percentage of cells entering mitosis. Reducing oxidative processes, by culturing at ∼3% oxygen or adding the reducing agent Trolox noticeably increased the fraction of cells entering mitosis. Thus, for long‐term imaging there can be value to using RFP constructs and for GFP‐tagged proteins reducing oxidative processes. J. Cell. Physiol. 232: 2461–2468, 2017. © 2016 Wiley Periodicals, Inc. We characterized the phototoxic consequences of 488 and 546 nm light on untransformed human cells. Four hundred eighty‐eight nanometer light produced a dose‐dependent decrease in the percentage of cells that progressed to mitosis, and slowing of the cell cycle for some of those entering mitosis and for 546 nm light, we found a 10–15% reduction in the percentage of cells entering mitosis, but no strong dose dependency. Reducing oxidative processes, by culturing at ∼3% oxygen or adding the reducing agent Trolox noticeably increased the fraction of cells entering mitosis.

  April 10, 2017   doi: 10.1002/jcp.25588   open full text
• Cardiotrophin‐1 Regulates Adipokine Production in 3T3‐L1 Adipocytes and Adipose Tissue From Obese Mice.
  Miguel López‐Yoldi, Beatriz Marcos‐Gomez, María Asunción Romero‐Lozano, Neira Sáinz, Jesús Prieto, Jose Alfredo Martínez, Matilde Bustos, Maria J. Moreno‐Aliaga.
  Journal of Cellular Physiology. April 10, 2017
  Cardiotrophin‐1 (CT‐1) belongs to the IL‐6 family of cytokines. Previous studies of our group revealed that CT‐1 is a key regulator of glucose and lipid metabolism. The aim of the present study was to analyze the in vitro and in vivo effects of CT‐1 on the production of several adipokines involved in body weight regulation, nutrient metabolism, and inflammation. For this purpose, 3T3‐L1 adipocytes were incubated with recombinant protein CT‐1 (rCT‐1) (1–40 ng/ml) for 1 and 18 h. Moreover, the acute effects of rCT‐1 administration (0.2 mg/kg, i.v.) for 30 min and 3 h on adipokines levels were also evaluated in high‐fat fed obese mice. In 3T3‐L1 adipocytes, rCT‐1 treatment downregulated the expression and secretion of leptin, resistin, and visfatin. However, rCT‐1 significantly stimulated apelin mRNA and secretion. rCT‐1 (18 h) also promoted the activation by phosphorylation of AKT, ERK 1/2, and STAT3. Interestingly, pre‐treatment with the PI3K inhibitor LY294002 reversed the stimulatory effects of rCT‐1 on apelin expression, suggesting that this pathway could be mediating the effects of rCT‐1 on apelin production. In contrast, acute administration of rCT‐1 (30 min and 3 h) to diet‐induced obese mice downregulated leptin and resistin, without significantly modifying apelin or visfatin mRNA in adipose tissue. Furthermore, CT‐1 null mice exhibited altered expression of adipokines in adipose tissue. The present study demonstrates that rCT‐1 modulates the production of adipokines in vitro and in vivo, suggesting that the regulation of the secretory function of adipocytes could be involved in the metabolic actions of this cytokine. J. Cell. Physiol. 232: 2469–2477, 2017. © 2016 Wiley Periodicals, Inc. The current study demonstrates that CT‐1 is capable of regulating the secretory pattern of adipokines by adipocytes, decreasing the production of pro‐inflammatory adipokines, such as leptin, resistin, and visfatin, while stimulating the secretion of apelin. These suggest that the beneficial actions of CT‐1 on glucose and lipid metabolism could be also related to its ability to control adipokine secretion and therefore the cross‐talk between adipose tissue and other key metabolic organs.

  April 10, 2017   doi: 10.1002/jcp.25590   open full text
• Overexpression of Large‐Conductance Calcium‐Activated Potassium Channels in Human Glioblastoma Stem‐Like Cells and Their Role in Cell Migration.
  Paolo Rosa, Luigi Sforna, Silvia Carlomagno, Giorgio Mangino, Massimo Miscusi, Mauro Pessia, Fabio Franciolini, Antonella Calogero, Luigi Catacuzzeno.
  Journal of Cellular Physiology. April 10, 2017
  Glioblastomas (GBMs) are brain tumors characterized by diffuse invasion of cancer cells into the healthy brain parenchyma, and establishment of secondary foci. GBM cells abundantly express large‐conductance, calcium‐activated potassium (BK) channels that are thought to promote cell invasion. Recent evidence suggests that the GBM high invasive potential mainly originates from a pool of stem‐like cells, but the expression and function of BK channels in this cell subpopulation have not been studied. We investigated the expression of BK channels in GBM stem‐like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87‐MG cells, BK channel expression and function were markedly upregulated by growth conditions that enriched the culture in GBM stem‐like cells (U87‐NS). Cytofluorimetric analysis further confirmed the appearance of a cell subpopulation that co‐expressed high levels of BK channels and CD133, as well as other stem cell markers. A similar association was also found in cells derived from freshly resected GBM biopsies. Finally, transwell migration tests showed that U87‐NS cells migration was much more sensitive to BK channel block than U87‐MG cells. Our data show that BK channels are highly expressed in GBM stem‐like cells, and participate to their high migratory activity. J. Cell. Physiol. 232: 2478–2488, 2017. © 2016 Wiley Periodicals, Inc. We investigated the expression of BK channels in GBM stem‐like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87‐MG cells, BK channel expression and function were markedly upregulated in GBM stem‐like cells expressing CD133, as well as other stem cell markers. In addition, transwell migration tests showed that GBM stem‐like cell migration was very sensitive to BK channel block.

  April 10, 2017   doi: 10.1002/jcp.25592   open full text
• TRIM33 is essential for osteoblast proliferation and differentiation via BMP pathway.
  Jia Guo, Wei Qin, Quan Xing, Manman Gao, Fuxin Wei, Zhi Song, Lingling Chen, Ying Lin, Xianling Gao, Zhengmei Lin.
  Journal of Cellular Physiology. April 10, 2017
  Tripartite motif containing 33 (TRIM33) functions both as a positive and negative regulator of the TGF‐β/BMP pathway in tumors; however, its effect and mechanism during osteoblast proliferation and differentiation, which involves the TGF‐β/BMP pathway is not defined. In this study, we used mouse C3H10T1/2 mesenchymal stem cell line and MC3T3‐E1 preosteoblasts to investigate the role of TRIM33 during this process. The results demonstrated that the expression of TRIM33 increased during the differentiation. Moreover, the overexpression or knockdown of TRIM33 resulted in both an augmentation or decrease in osteoblast differentiation, which were measured by the expression of alkaline phosphatase (ALP) at the mRNA level, both Runt‐related transcription factor 2 (Runx2) and osteocalcin (OCN) at the protein level, and the formation of mineral modules. To further demonstrate the mechanism of TRIM33 in this process, we found that TRIM33 could positively mediate the BMP pathway by forming TRIM33‐Smad1/5 complex. This interaction between TRIM33 and Smad1/5 triggered the phosphorylation of Smad1/5. In addition, the essential role of TRIM33 in osteoblast proliferation was determined in this study by CellCounting Kit (CCK) −8 and cell cycle assays. In summary, we establish the function of TRIM33 as a positive regulator of osteoblast differentiation in BMP pathway, which mediates its effect through its interaction with and activation of Smad1/5. In addition, the results clearly demonstrate that TRIM33 is necessary for osteoblast proliferation by regulating cell cycle. These results suggest that TRIM33 can be a positive target of osteoblast proliferation and differentiation through BMP pathway. We establish the function of TRIM33 as a positive regulator of osteoblast differentiation in BMP pathway, which mediates its effect through its interaction with and activation of Smad1/5. In addition, the results clearly demonstrate that TRIM33 is necessary for osteoblast proliferation by regulating cell cycle. These results suggest that TRIM33 can be a positive target of osteoblast proliferation and differentiation through BMP pathway.

  April 10, 2017   doi: 10.1002/jcp.25769   open full text
• Epithelial–mesenchymal transition (EMT): A biological process in the development, stem cell differentiation, and tumorigenesis.
  Tong Chen, Yanan You, Hua Jiang, Zack Z. Wang.
  Journal of Cellular Physiology. April 10, 2017
  The lineage transition between epithelium and mesenchyme is a process known as epithelial–mesenchymal transition (EMT), by which polarized epithelial cells lose their adhesion property and obtain mesenchymal cell phenotypes. EMT is a biological process that is often involved in embryogenesis and diseases, such as cancer invasion and metastasis. The EMT and the reverse process, mesenchymal–epithelial transition (MET), also play important roles in stem cell differentiation and de‐differentiation (or reprogramming). In this review, we will discuss current research progress of EMT in embryonic development, cellular differentiation and reprogramming, and cancer progression, all of which are representative models for researches of stem cell biology in normal and in diseases. Understanding of EMT and MET may help to identify specific markers to distinguish normal stem cells from cancer stem cells in future. In this review, we discuss current research progress of EMT in embryonic development, cellular differentiation and reprogramming, and cancer progression, all of which are representative models for researches of stem cell biology in normals and in diseases.

  April 10, 2017   doi: 10.1002/jcp.25797   open full text
• Neurotransmitters: The Critical Modulators Regulating Gut–Brain Axis.
  Rahul Mittal, Luca H. Debs, Amit P. Patel, Desiree Nguyen, Kunal Patel, Gregory O'Connor, M'hamed Grati, Jeenu Mittal, Denise Yan, Adrien A. Eshraghi, Sapna K. Deo, Sylvia Daunert, Xue Zhong Liu.
  Journal of Cellular Physiology. April 10, 2017
  Neurotransmitters, including catecholamines and serotonin, play a crucial role in maintaining homeostasis in the human body. Studies on these neurotransmitters mainly revolved around their role in the “fight or flight” response, transmitting signals across a chemical synapse and modulating blood flow throughout the body. However, recent research has demonstrated that neurotransmitters can play a significant role in the gastrointestinal (GI) physiology. Norepinephrine (NE), epinephrine (E), dopamine (DA), and serotonin have recently been a topic of interest because of their roles in the gut physiology and their potential roles in GI and central nervous system pathophysiology. These neurotransmitters are able to regulate and control not only blood flow, but also affect gut motility, nutrient absorption, GI innate immune system, and the microbiome. Furthermore, in pathological states, such as inflammatory bowel disease (IBD) and Parkinson's disease, the levels of these neurotransmitters are dysregulated, therefore causing a variety of GI symptoms. Research in this field has shown that exogenous manipulation of catecholamine serum concentrations can help in decreasing symptomology and/or disease progression. In this review article, we discuss the current state‐of‐the‐art research and literature regarding the role of neurotransmitters in regulation of normal GI physiology, their impact on several disease processes, and novel work focused on the use of exogenous hormones and/or psychotropic medications to improve disease symptomology. J. Cell. Physiol. 232: 2359–2372, 2017. © 2016 Wiley Periodicals, Inc. Neurotransmitters affect microbiota in the gut: Neurotransmitters, including serotonin, alter the microbiota in the gut that can modulate the production of cytokines and bacterial by‐products, leading to either healthy or diseased state.

  April 10, 2017   doi: 10.1002/jcp.25518   open full text
• MAP Kinase‐Dependent RUNX2 Phosphorylation Is Necessary for Epigenetic Modification of Chromatin During Osteoblast Differentiation.
  Yan Li, Chunxi Ge, Renny T. Franceschi.
  Journal of Cellular Physiology. April 10, 2017
  RUNX2, an essential transcription factor for osteoblast differentiation and bone formation is activated by ERK/MAP kinase‐dependent phosphorylation. However, relationship between these early events and specific epigenetic modifications of chromatin during osteoblast differentiation have not been previously examined. Here, we explore these relationships using chromatin immunoprecipitation (ChIP) to detect chromatin modifications in RUNX2‐binding regions of Bglap2 and Ibsp. Growth of MC3T3‐E1c4 preosteoblast cells in differentiation conditions rapidly induced Bglap2 and lbsp mRNAs. For both genes, osteogenic stimulation increased chromatin‐bound P‐ERK, P‐RUNX2, p300, and RNA polymerase II as well as histone H3K9 and H4K5 acetylation. The level of H3K4 di‐methylation, another gene activation‐associated histone mark, also increased. In contrast, levels of the gene repressive marks, H3K9 mono‐, di‐, and tri‐methylation in the same regions were reduced. Inhibition of MAP kinase signaling blocked differentiation‐dependent chromatin modifications and Bglap2 and Ibsp expression. To evaluate the role of RUNX2 phosphorylation in these responses, RUNX2‐deficient C3H10T1/2 cells were transduced with adenovirus encoding wild type or phosphorylation site mutant RUNX2 (RUNX2 S301A/S319A). Wild type RUNX2, but not the non‐phosphorylated mutant, increased H3K9 and H4K5 acetylation as well as chromatin‐associated P‐ERK, p300, and polymerase II. Thus, RUNX2 phosphorylation is necessary for subsequent epigenetic changes required for osteoblast gene expression. Taken together, this study reveals a molecular mechanism through which osteogenic genes are controlled by a MAPK and P‐RUNX2‐dependent process involving epigenetic modifications of specific promoter regions. J. Cell. Physiol. 232: 2427–2435, 2017. © 2016 Wiley Periodicals, Inc. The RUNX2 transcription factor requires MAP kinase‐dependent phosphorylation to become transcriptionally active. Here, we show that phosphorylation is required for transcription‐related changes in histone acetylation and methylation and recruitment of cofactors such as P300/CBP and RNA polymerase II to promoter regions of bone‐related genes.

  April 10, 2017   doi: 10.1002/jcp.25517   open full text
• Bone Shaft Revascularization After Marrow Ablation Is Dramatically Accelerated in BSP‐/‐ Mice, Along With Faster Hematopoietic Recolonization.
  Wafa Bouleftour, Renata Neves Granito, Arnaud Vanden‐Bossche, Odile Sabido, Bernard Roche, Mireille Thomas, Marie Thérèse Linossier, Jane E. Aubin, Marie‐Hélène Lafage‐Proust, Laurence Vico, Luc Malaval.
  Journal of Cellular Physiology. April 10, 2017
  The bone organ integrates the activity of bone tissue, bone marrow, and blood vessels and the factors ensuring this coordination remain ill defined. Bone sialoprotein (BSP) is with osteopontin (OPN) a member of the small integrin binding ligand N‐linked glycoprotein (SIBLING) family, involved in bone formation, hematopoiesis and angiogenesis. In rodents, bone marrow ablation induces a rapid formation of medullary bone which peaks by ∼8 days (d8) and is blunted in BSP‐/‐ mice. We investigated the coordinate hematopoietic and vascular recolonization of the bone shaft after marrow ablation of 2 month old BSP+/+ and BSP‐/‐ mice. At d3, the ablated area in BSP‐/‐ femurs showed higher vessel density (×4) and vascular volume (×7) than BSP+/+. Vessel numbers in the shaft of ablated BSP+/+ mice reached BSP‐/‐ values only by d8, but with a vascular volume which was twice the value in BSP‐/‐, reflecting smaller vessel size in ablated mutants. At d6, a much higher number of Lin− (×3) as well as LSK (Lin− IL‐7Rα− Sca‐1hi c‐Kithi, ×2) and hematopoietic stem cells (HSC: Flt3− LSK, ×2) were counted in BSP‐/‐ marrow, indicating a faster recolonization. However, the proportion of LSK and HSC within the Lin− was lower in BSP‐/‐ and more differentiated stages were more abundant, as also observed in unablated bone, suggesting that hematopoietic differentiation is favored in the absence of BSP. Interestingly, unablated BSP‐/‐ femur marrow also contains more blood vessels than BSP+/+, and in both intact and ablated shafts expression of VEGF and OPN are higher, and DMP1 lower in the mutants. In conclusion, bone marrow ablation in BSP‐/‐ mice is followed by a faster vascular and hematopoietic recolonization, along with lower medullary bone formation. Thus, lack of BSP affects the interplay between hematopoiesis, angiogenesis, and osteogenesis, maybe in part through higher expression of VEGF and the angiogenic SIBLING, OPN. J. Cell. Physiol. 232: 2528–2537, 2017. © 2016 Wiley Periodicals, Inc. In mice with a knockout of the SIBLING gene, Bone Sialoprotein (BSP‐/‐) vascular and hematopoietic recolonization are faster than in wild type, despite lower medullary bone formation. This goes with enhanced expression of osteopontin and VEGF, and downregulation of Dentin Matrix Protein 1, another SIBLING shown to inhibit angiogenesis. Lack of BSP thus affects the interplay between hematopoiesis, angiogenesis, and osteogenesis, in part through the mediation of pro‐ and antiangiogenic factors.

  April 10, 2017   doi: 10.1002/jcp.25630   open full text
• Use of a Collagen Membrane to Enhance the Survival of Primary Intestinal Epithelial Cells.
  Fiorella Di Claudio, Cecilia I. Muglia, Paola L. Smaldini, María Lucía Orsini Delgado, Fernando M. Trejo, J. Raúl Grigera, Guillermo H. Docena.
  Journal of Cellular Physiology. April 10, 2017
  Intestinal epithelial cell culture is important for biological, functional, and immunological studies. Since enterocytes have a short in vivo life span due to anoikis, we aimed to establish a novel and reproducible method to prolong the survival of mouse and human cells. Cells were isolated following a standard procedure, and cultured on ordered‐cow's collagen membranes. A prolonged cell life span was achieved; cells covered the complete surface of bio‐membranes and showed a classical enterocyte morphology with high expression of enzymes supporting the possibility of cryopreservation. Apoptosis was dramatically reduced and cultured enterocytes expressed cytokeratin and LGR5 (low frequency). Cells exposed to LPS or flagellin showed the induction of TLR4 and TLR5 expression and a functional phenotype upon exposure to the probiotic Bifidobacterium bifidum or the pathogenic Clostridium difficile. The secretion of the homeostatic (IL‐25 and TSLP), inhibitory (IL‐10 and TGF‐β), or pro‐inflammatory mediators (IL‐1β and TNF) were induced. In conclusion, this novel protocol using cow's collagen‐ordered membrane provides a simple and reproducible method to maintain intestinal epithelial cells functional for cell‐microorganism interaction studies and stem cell expansion. J. Cell. Physiol. 232: 2489–2496, 2017. © 2016 Wiley Periodicals, Inc. We have developed a method for culturing primary intestinal epithelial cells useful for the study of their interaction with microbes.

  April 10, 2017   doi: 10.1002/jcp.25594   open full text
• Cre Recombinase Strains Used for the Study of Adipose Tissues and Adipocyte Progenitors.
  Jiaqi Liu, Ziye Xu, Weiche Wu, Yizhen Wang, Tizhong Shan.
  Journal of Cellular Physiology. April 10, 2017
  Adipose tissues play important roles in whole body energy homeostasis and lifer span. Understanding the mechanisms of controlling adipose tissues development is significant for providing useful information to treat the worldwide epidemic of obesity and its associated metabolic diseases. Several different Cre transgenes have been generated and used for determining the origin of adipose tissues and the function of individual gene in regulating adipose growth and development. Here, we mainly review and discuss the efficiency and specific of those Cre recombinase mouse strains used for the study of adipose tissues and adipocyte progenitors. J. Cell. Physiol. 9999: 1–6, 2016. © 2016 Wiley Periodicals, Inc. We mainly review and discuss the efficiency and specific of those Cre recombinase mouse strains used for determining the origin of adipose tissues and the function of individual gene in regulating adipose growth and development.

  April 10, 2017   doi: 10.1002/jcp.25675   open full text
• HSF1 phosphorylation by ERK/GSK3 suppresses RNF126 to sustain IGF‐IIR expression for hypertension‐induced cardiomyocyte hypertrophy.
  Chih‐Yang Huang, Fa‐Lun Lee, Shu‐Fen Peng, Kuan‐Ho Lin, Ray‐Jade Chen, Tsung‐Jung Ho, Fu‐Jen Tsai, V. Vijaya Padma, Wei‐Wen Kuo, Chih‐Yang Huang.
  Journal of Cellular Physiology. April 06, 2017
  Hypertension‐induced cardiac hypertrophy and apoptosis are major characteristics of early‐stage heart failure (HF). Inhibition of extracellular signal‐regulated kinases (ERK) efficaciously suppressed angiotensin II (ANG II)‐induced cardiomyocyte hypertrophy and apoptosis by blocking insulin‐like growth factor II receptor (IGF‐IIR) signaling. However, the detailed mechanism by which ANG II induces ERK‐mediated IGF‐IIR signaling remains elusive. Here, we found that ANG II activated ERK to upregulate IGF‐IIR expression via the angiotensin II type I receptor (AT1R). ERK activation subsequently phosphorylates HSF1 at serine 307, leading to a secondary phosphorylation by glycogen synthase kinase III (GSK3) at serine 303. Moreover, we found that ANG II mediated ERK/GSK3‐induced IGF‐IIR protein stability by downregulating the E3 ubiquitin ligase of IGF‐IIR RING finger protein CXXVI (RNF126). The expression of RNF126 decreased following ANG II‐induced HSF1S303 phosphorylation, resulting in IGF‐IIR protein stability and increased cardiomyocyte injury. Inhibition of GSK3 significantly alleviated ANG II‐induced cardiac hypertrophy in vivo and in vitro. Taken together, these results suggest that HSF1 phosphorylation stabilizes IGF‐IIR protein stability by downregulating RNF126 during cardiac hypertrophy. ANG II activates ERK/GSK3 to phosphorylate HSF1, resulting in RNF126 degradation, which stabilizes IGF‐IIR protein expression and eventually results in cardiac hypertrophy. HSF1 could be a valuable therapeutic target for cardiac diseases among hypertensive patients. This article is protected by copyright. All rights reserved

  April 06, 2017   doi: 10.1002/jcp.25945   open full text
• Maintenance of Bone Homeostasis by DLL1‐Mediated Notch Signaling.
  Yukari Muguruma, Katsuto Hozumi, Hiroyuki Warita, Takashi Yahata, Tomoko Uno, Mamoru Ito, Kiyoshi Ando.
  Journal of Cellular Physiology. March 31, 2017
  Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta‐like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast‐specific manner, we investigated the ligand‐specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1‐induced Notch signaling was responsible for the expansion of the bone‐forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1‐Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast‐specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast‐osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1‐mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand‐specific activation of Notch signaling in the maintenance of bone homeostasis. J. Cell. Physiol. 232: 2569–2580, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc. The proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1‐Notch signaling directly controls the physiological expansion and differentiation of osteoblasts and indirectly affects the maturation and function of osteoclasts, thereby ensuring the homeostasis of bone mass.

  March 31, 2017   doi: 10.1002/jcp.25647   open full text
• Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria.
  Stephen D. Thorpe, Silvia Gambassi, Clare L. Thompson, Charmilie Chandrakumar, Annalisa Santucci, Martin M. Knight.
  Journal of Cellular Physiology. March 31, 2017
  Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2‐dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog‐related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU. This study investigates whether the rare inherited disease alkaptonuria (AKU) is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling, which is known to mediate cartilage degradation in osteoarthritis. AKU results in alterations to primary cilia structure which are associated with ciliary trafficking defects with the potential to impact a range of fundamental signaling pathways. In particular, AKU chondrocytes demonstrated a complete inability to activate Hedgehog signaling in response to endogenous ligand; which we propose may be the result of the observed dysfunction of cilia structure and trafficking. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU.

  March 31, 2017   doi: 10.1002/jcp.25839   open full text
• Crosstalk of autophagy and apoptosis: Involvement of the dual role of autophagy under ER stress.
  Shuling Song, Jin Tan, Yuyang Miao, Mengmeng Li, Qiang Zhang.
  Journal of Cellular Physiology. March 31, 2017
  Endoplasmic reticulum (ER) stress is a common cellular stress response that is triggered by a variety of conditions that disturb cellular homeostasis, and induces cell apoptosis. Autophagy, an important and evolutionarily conserved mechanism for maintaining cellular homeostasis, is closely related to the apoptosis induced by ER stress. There are common upstream signaling pathways between autophagy and apoptosis induced by ER stress, including PERK/ATF4, IRE1α, ATF6, and Ca2+. Autophagy can not only block the induction of apoptosis by inhibiting the activation of apoptosis‐associated caspase which could reduce cellular injury, but also help to induce apoptosis. In addition, the activation of apoptosis‐related proteins can also inhibit autophagy by degrading autophagy‐related proteins, such as Beclin‐1, Atg4D, Atg3, and Atg5. Although the interactions of different autophagy‐ and apoptosis‐related proteins, and also common upstream signaling pathways have been found, the potential regulatory mechanisms have not been clearly understood. In this review, we summarize the dual role of autophagy, and the interplay and potential regulatory mechanisms between autophagy and apoptosis under ER stress condition. The dura role of autophagy, pro‐survival, and ‐death, may be dependent on the extent of ER stress, and it implements by the regulation of apoptosis. Autophagy can not only block the induction of apoptosis by inhibiting the activation of apoptosis‐associated caspase which could reduce cellular injury, but also help to induce apoptosis. In addition, the activation of apoptosis‐related proteins can also inhibit autophagy by degrading autophagy‐related proteins, such as Beclin‐1, Atg4D, Atg3, and Atg5.

  March 31, 2017   doi: 10.1002/jcp.25785   open full text
• Staphylococcal protein A promotes osteoclastogenesis through MAPK signaling during bone infection.
  Yuan Wang, Xin Liu, Ce Dou, Zhen Cao, Chuan Liu, Shiwu Dong, Jun Fei.
  Journal of Cellular Physiology. March 31, 2017
  Bone infection is a common and serious complication in the orthopedics field, which often leads to excessive bone destruction and non‐union. Osteoclast is the only type of cells which have the function of bone resorption. Its over activation is closely related to excessive bone loss. Staphylococcus aureus (S. aureus) is a major pathogen causing bone infection, which can produce a large number of strong pathogenic substances staphylococcal protein A (SPA). However, few studies were reported about the effects of SPA on osteoclastogenesis. In our study, we observed that S. aureus activated osteoclasts and promoted bone loss in bone infection specimens. Then, we investigated the effects of SPA on RANKL‐induced osteoclastogenesis in vitro, the results revealed that SPA promoted osteoclastic differentiation and fusion, and enhanced osteoclastic bone resorption. In addition, we also showed that SPA upregulated the expression of NFATc1 and c‐FOS through the activation of MAPK signaling to promote osteoclastogenesis. Our findings might help us better understand the pathogenic role of S. aureus in bone infection and develop new therapeutic strategies for infectious bone diseases. Staphylococcal protein A (SPA) is one of the most important pathogenic components of Staphylococcus aureus (S. aureus). Here we reported that S. aureus caused bone loss in bone infection specimens. In vitro study showed that SPA promoted osteoclastic differentiation, fusion and bone resorption activity by upregulating NFATc1 and c‐FOS through MAPK signaling.

  March 31, 2017   doi: 10.1002/jcp.25774   open full text
• Challenging of AS160/TBC1D4 Alters Intracellular Lipid milieu in L6 Myotubes Incubated With Palmitate.
  Agnieszka Mikłosz, Bartłomiej Łukaszuk, Małgorzata Żendzian‐Piotrowska, Justyna Brańska‐Januszewska, Halina Ostrowska, Adrian Chabowski.
  Journal of Cellular Physiology. March 31, 2017
  The Akt substrate of 160 kDa (AS160) is a key regulator of GLUT4 translocation from intracellular depots to the plasma membrane in myocytes. Likely, AS160 also controls LCFAs transport, which requires relocation of fatty acid transporters. The aim of the present study was to determine the impact of AS160 knockdown on lipid milieu in L6 myotubes incubated with palmitate (PA). Therefore, we compared two different settings, namely: 1) AS160 knockdown prior to palmitate incubation (pre‐PA‐silencing, AS160−/PA); 2) palmitate incubation with subsequent AS160 knockdown (post‐PA‐silencing, PA/AS160−). The efficiency of AS160 silencing was checked at mRNA and protein levels. The expression and localization of FA transporters were determined using Western Blot and immunofluorescence analyses. Intracellular lipid content (FFA, DAG, TAG, and PL) and FA composition were estimated by GLC, whereas basal palmitate uptake was analyzed by means of scintigraphy. Both groups with silenced AS160 were characterized by a greater expression of FA transporters (FAT/CD36, FATP‐1, 4) which had contributed to an increased FA cellular influx. Accordingly, we observed that post‐PA‐silencing of AS160 resulted in a marked decrement in DAG, TAG, and PL contents, but increased FFA content (PA/AS160− vs. PA). The opposite effect was observed in the group with pre‐PA‐silencing of AS160 in which AS160 knockdown did not affect the lipid pools (AS160−/PA vs. PA). Our results indicate that post‐PA‐silencing of AS160 has a capacity to decrease the lipotoxic effect(s) of PA by decreasing the content of lipids (DAG and PL) that promote insulin resistance in myotubes. J. Cell. Physiol. 232: 2373–2386, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc. AS160/TBC1D4 is a key regulator of GLUT4 translocation to the plasma membrane in myocytes, moreover, it role in the control of protein mediated fatty acids transport is also likely. The current study indicates that AS160/TBC1D4 may influence not only FA transporters content, and therefore their influx, but also their storage and oxidation. However, the effect differs depending on the events sequence, i.e., AS160 knock‐down prior to or after palmitate incubation.

  March 31, 2017   doi: 10.1002/jcp.25632   open full text
• Pyk2 inhibition promotes contractile differentiation in arterial smooth muscle.
  Mario Grossi, Anirban Bhattachariya, Ina Nordström, Karolina M. Turczyńska, Daniel Svensson, Sebastian Albinsson, Bengt‐Olof Nilsson, Per Hellstrand.
  Journal of Cellular Physiology. March 31, 2017
  Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage‐dependent toward voltage‐independent regulation of the intracellular calcium level, and inhibition of non‐voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium‐dependent signaling is the FAK‐family non‐receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium‐dependent manner. We used the Pyk2 inhibitor PF‐4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF‐BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long‐term (24–96 hr) treatment with PF‐4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L‐type calcium channel and large‐conductance calcium‐activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store‐operated calcium influx were reduced after Pyk2 inhibition of growth‐stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions. The Pyk2 inhibitor PF‐4594755 potently inhibits Pyk2 phosphorylation of vascular smooth muscle cells stimulated by FBS or PDGF‐BB. This is accompanied by increased expression of smooth muscle marker proteins without concomitant effects on cell proliferation. Pyk2 inhibition upregulates SERCA2a expression and may preserve the contractile phenotype by lowering basal intracellular calcium and promoting voltage‐dependent control of calcium influx over the plasma membrane.

  March 31, 2017   doi: 10.1002/jcp.25760   open full text
• Mactosylceramide prevents glial cell overgrowth by inhibiting insulin and fibroblast growth factor receptor signaling.
  Stine Gerdøe‐Kristensen, Viktor K. Lund, Hans H. Wandall, Ole Kjaerulff.
  Journal of Cellular Physiology. March 31, 2017
  Receptor tyrosine kinase (RTK) signaling controls key aspects of cellular differentiation, proliferation, survival, metabolism, and migration. Deregulated RTK signaling also underlies many cancers. Glycosphingolipids (GSL) are essential elements of the plasma membrane. By affecting clustering and activity of membrane receptors, GSL modulate signal transduction, including that mediated by the RTK. GSL are abundant in the nervous system, and glial development in Drosophila is emerging as a useful model for studying how GSL modulate RTK signaling. Drosophila has a simple GSL biosynthetic pathway, in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3‐kinase (PI3K). However, to what extent this effect involves changes in upstream signaling events is unresolved. We show here that glial overgrowth in egh is strongly linked to increased activation of Insulin and fibroblast growth factor receptors (FGFR). Glial hypertrophy is phenocopied when overexpressing gain‐of‐function mutants of the Drosophila insulin receptor (InR) and the FGFR homolog Heartless (Htl) in wild type SPG, and is suppressed by inhibiting Htl and InR activity in egh. Knockdown of GlcCer synthase in the SPG fails to suppress glial overgrowth in egh nerves, and slightly promotes overgrowth in wild type, suggesting that RTK hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. We conclude that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of insulin and fibroblast growth factor receptors in Drosophila glia. Receptor tyrosine kinase signaling controls key aspects of cellular differentiation, proliferation, survival, metabolism, and migration. Glycosphingolipids modulate signal transduction, including that mediated by the receptor tyrosine kinases. We report that an early product in glycosphingolipid biosynthesis, mactosylceramide, prevents inappropriate activation of insulin and fibroblast growth factor receptors in Drosophila glial cells.

  March 31, 2017   doi: 10.1002/jcp.25762   open full text
• Acetoacetate induces hepatocytes apoptosis by the ROS‐mediated MAPKs pathway in ketotic cows.
  Xiliang Du, Zhen Shi, Zhicheng Peng, Chenxu Zhao, Yuming Zhang, Zhe Wang, Xiaobing Li, Guowen Liu, Xinwei Li.
  Journal of Cellular Physiology. March 31, 2017
  Dairy cows with ketosis are characterized by oxidative stress, hepatic damage, and hyperketonemia. Acetoacetate (AA) is the main component of ketone bodies in ketotic cows, and is associated with the above pathological process. However, the potential mechanism was not illuminated. Therefore, the aim of this study was to investigate the mechanism of AA‐induced hepatic oxidative damage in ketotic cows. Compared with healthy cows, ketotic cows exhibited severe oxidative stress and hepatic damage. Moreover, the extent of hepatic damage and oxidative stress had a positive relationship with the AA levels. In vitro, AA treatment increased reactive oxygen species (ROS) content and further induced oxidative stress and apoptosis of bovine hepatocytes. In this process, AA treatment increased the phosphorylation levels of JNK and p38MAPK and decreased the phosphorylation level of ERK, which could increase p53 and inhibit nuclear factor E2‐related factor 2 (Nrf2) expression, nuclear localization, and DNA‐binding affinity, thereby inducing the overexpression of pro‐apoptotic molecules Bax, Caspase 3, Caspase 9, PARP and inhibition of anti‐apoptotic molecule Bcl‐2. Antioxidant N‐acetylcysteine (NAC) treatment or interference of MAPKs pathway could attenuate the hepatocytes apoptosis induced by AA. Collectively, these results indicate that AA triggers hepatocytes apoptosis via the ROS‐mediated MAPKs pathway in ketotic cows. Excess acetoacetate induced imbalance of oxidation and antioxidation, resulting in overproduction of reactive oxygen species (ROS) in the mitochondria. High concentration of ROS increased the phosphorylation levels of p38MAPK and JNK and decreased the phosphorylation levels of ERK1/2, which could activate p53 and inhibit Nrf2 expression, nuclear localization, and DNA‐binding affinity. Consequently, the expression of pro‐apoptotic molecules Bax, Casepase9, Casepase3, and PARP was significantly upregulated and anti‐apoptotic molecules Bcl‐2 were markedly downregulated, resulting in hepatocytes apoptosis in ketotic cows.

  March 31, 2017   doi: 10.1002/jcp.25773   open full text
• Biological and pharmacological evaluation of dimethoxycurcumin: A metabolically stable curcumin analogue with a promising therapeutic potential.
  Manouchehr Teymouri, Nastaran Barati, Matteo Pirro, Amirhosein Sahebkar.
  Journal of Cellular Physiology. March 31, 2017
  Dimethoxycurcumin (DiMC) is a synthetic analog of curcumin with superior inter‐related pro‐oxidant and anti‐cancer activity, and metabolic stability. Numerous studies have shown that DiMC reserves the biologically beneficial features, including anti‐inflammatory, anti‐carcinogenic, and cytoprotective properties, almost to the same extent as curcumin exhibits. DiMC lacks the phenolic‐OH groups as opposed to curcumin, dimethoxycurcumin, and bis‐demethoxycurcumin that all vary in the number of methoxy groups per molecule, and has drawn the attentions of researchers who attempted to discover the structure‐activity relationship (SAR) of curcumin. In this regard, tetrahydrocurcumin (THC), the reduced and biologically inert metabolite of curcumin, denotes the significance of the conjugated α,β diketone moiety for the curcumin activity. DiMC exerts unique molecular activities compared to curcumin, including induction of androgen receptor (AR) degradation and suppression of the transcription factor activator protein‐1 (AP‐1). The enhanced AR degradation on DiMC treatment suggests it as a novel anticancer agent against resistant tumors with androgenic etiology. Further, DiMC might be a potential treatment for acne vulgaris. DiMC induces epigenetic alteration more effectively than curcumin, although both showed no direct DNA hypomethylating activity. Given the metabolic stability, nanoparticulation of DiMC is more promising for in vivo effectiveness. However, studies in this regard are still in its infancy. In the current review, we portray the various molecular and biological functions of DiMC reported so far. Whenever possible, the efficiency is compared with curcumin and the reasons for DiMC being more metabolically stable are elaborated. We also provide future perspective investigations with respect to varying DiMC‐nanoparticles. This review, being the first of its kind, is a comprehensive evaluation of the molecular, cellular, and clinical properties of dimethoxycurcumin as a promising analog of curcumin. We have discussed various molecular and biological functions of DiMC reported so far. Whenever possible, the efficiency compared with curcumin and the reasons for DiMC being more metabolically stable than curcumin has been explained. We have also provided future perspective investigations with respect to varying DiMC‐nanoparticles.

  March 31, 2017   doi: 10.1002/jcp.25749   open full text
• Therapeutic effects of curcumin in inflammatory and immune‐mediated diseases: A nature‐made jack‐of‐all‐trades?
  Elham Abdollahi, Amir Abbas Momtazi, Thomas P. Johnston, Amirhossein Sahebkar.
  Journal of Cellular Physiology. March 31, 2017
  Curcumin is a dietary polyphenol from turmeric with numerous pharmacological activities. Novel animal and human studies indicate that curcumin can affect different immune cells, such as various T lymphocyte subsets, macrophages, dendritic cells, B lymphocytes and natural killer cells, which results in decreasing severity of various diseases with immunological etiology. The present review provides a comprehensive overview of the effects of curcumin on different immune cells and immune system‐related diseases. The present review provides a comprehensive overview of the effects of curcumin on different immune cells and immune system‐related diseases.

  March 31, 2017   doi: 10.1002/jcp.25778   open full text
• Cyclic‐glycine‐proline accelerates mammary involution by promoting apoptosis and inhibiting IGF‐1 function.
  Gagandeep Singh‐Mallah, Christopher D. McMahon, Jian Guan, Kuljeet Singh.
  Journal of Cellular Physiology. March 31, 2017
  In rodents, post‐lactational involution of mammary glands is characterized by the loss of mammary epithelial cells via apoptosis, which is associated with a decline in the expression of insulin‐like growth factor‐1 (IGF‐1). Overexpression of IGF‐1 delays involution by inhibiting apoptosis of epithelial cells and preserving the remaining secretory alveoli. Cyclic‐glycine‐proline (cGP), a metabolite of IGF‐1, normalizes IGF‐1 function under pathological conditions by regulating the bioavailability of IGF‐1. The present study investigated the effect of cGP on the physiological decline in IGF‐1 function during post‐lactational mammary involution. Rat dams were gavaged with either cGP (3 mg/kg) or saline once per day from post‐natal d8‐22. Before collecting tissue on post‐natal d23, a pair of mammary glands were sealed on d20 (72 hr‐engorgement, thus representative of late‐involution) and d22 (24 hr‐engorgement, thus representative of mid‐involution), while the remaining glands were allowed to involute naturally (early‐involution). During early‐involution, cGP accelerated the loss of mammary cells through apoptosis, resulting in an earlier clearance of intact secretory alveoli compared with the control group. This coincided with an earlier up‐regulation of the cell survival factors, Bcl‐xl and IGF‐1R, in the early‐involution cGP glands compared with the control glands. During late‐involution, cGP reduced the bioactivity of IGF‐1, which was evident through decreased phosphorylation of IGF‐1R in the regressed alveoli. Maternal administration of cGP did not alter milk production and composition during early‐, peak‐, or late‐stage of lactation. These data show that cGP accelerates post‐lactational involution by promoting apoptosis and the physiological decline in IGF‐1 function. Role of cGP in regulating IGF‐1 function during brain development and in neurological conditions is well known. This study shows that cGP accelerates the onset of post‐lactational involution in mammary glands by promoting the physiological decline in IGF‐1 function.

  March 31, 2017   doi: 10.1002/jcp.25782   open full text
• Effects of Carbocysteine and Beclomethasone on Histone Acetylation/Deacetylation Processes in Cigarette Smoke Exposed Bronchial Epithelial Cells.
  Elisabetta Pace, Serena Di Vincenzo, Maria Ferraro, Liboria Siena, Giuseppina Chiappara, Paola Dino, Patrizio Vitulo, Alessandro Bertani, Federico Saibene, Luigi Lanata, Mark Gjomarkaj.
  Journal of Cellular Physiology. March 31, 2017
  Histone deacetylase expression/activity may control inflammation, cell senescence, and responses to corticosteroids. Cigarette smoke exposure, increasing oxidative stress, may negatively affect deacetylase expression/activity. The effects of cigarette smoke extracts (CSE), carbocysteine, and beclomethasone dipropionate on chromatin remodeling processes in human bronchial epithelial cells are largely unknown. The present study was aimed to assess the effects of cigarette smoke, carbocysteine, and beclomethasone dipropionate on histone deacetylase 3 (HDAC3) expression/activity, N‐CoR (nuclear receptor corepressor) expression, histone acetyltransferases (HAT) (p300/CBP) expression, p‐CREB and IL‐1 m‐RNA expression, neutrophil chemotaxis. Increased p‐CREB expression was observed in the bronchial epithelium of smokers. CSE increased p‐CREB expression and decreased HDAC3 expression and activity and N‐CoR m‐RNA and protein expression. At the same time, CSE increased the expression of the HAT, p300/CBP. All these events increased acetylation processes within the cells and were associated to increased IL‐1 m‐RNA expression and neutrophil chemotaxis. The incubation of CSE exposed cells with carbocysteine and beclomethasone counteracted the effects of cigarette smoke on HDAC3 and N‐CoR but not on p300/CBP. The increased deacetylation processes due to carbocysteine and beclomethasone dipropionate incubation is associated to reduced p‐CREB, IL‐1 m‐RNA expression, neutrophil chemotaxis. These findings suggest a new role of combination therapy with carbocysteine and beclomethasone dipropionate in restoring deacetylation processes compromised by cigarette smoke exposure. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc. Cigarette smoke exposure, increasing oxidative stress, may negatively affect deacetylase expression/activity, thus, supporting increased pro‐inflammatory responses.The findings here provided suggest a new role of combination therapy with carbocysteine and beclomethasone dipropionate in restoring deacetylation processes compromised by cigarette smoke exposure.

  March 31, 2017   doi: 10.1002/jcp.25710   open full text
• Cholinergic and Dopaminergic Neuronal Differentiation of Human Adipose Tissue Derived Mesenchymal Stem Cells.
  HE Marei, A El‐Gamal, A Althani, N Afifi, A Abd‐Elmaksoud, A Farag, C Cenciarelli, Thomas Caceci, Anwarul Hasan.
  Journal of Cellular Physiology. March 30, 2017
  Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into various cell types such as cartilage, bone, and fat cells. Recent studies have shown that induction of MSCs in vitro by growth factors including epidermal growth factor (EGF) and fibroblast growth factor (FGF2) causes them to differentiate into neural like cells. These cultures also express ChAT, a cholinergic marker; and TH, a dopaminergic marker for neural cells. To establish a protocol with maximum differentiation potential, we examined MSCs under three experimental culture conditions using neural induction media containing FGF2, EGF, BMP‐9, retinoic acid and heparin. Adipose‐derived MSCs were extracted and expanded in vitro for 3 passages after reaching >80% confluency, for a total duration of 9 days. Cells were then characterized by flow cytometry for CD markers as CD44 positive and CD45 negative. MSCs were then treated with neural induction media and were characterized by morphological changes and Q‐PCR. Differentiated MSCs expressed markers for immature and mature neurons; β Tubulin III (TUBB3) and MAP2, respectively showing the neural potential of these cells to differentiate into functional neurons. Improved protocols for MSCs induction will facilitate and ensure the reproducibility and standard production of MSCs for therapeutic applications in neurodegenerative diseases. This article is protected by copyright. All rights reserved

  March 30, 2017   doi: 10.1002/jcp.25937   open full text
• Smoothened‐antagonists reverse homogentisic acid‐induced alterations of Hedgehog signaling and primary cilium length in alkaptonuria.
  Silvia Gambassi, Michela Geminiani, Stephen D. Thorpe, Giulia Bernardini, Lia Millucci, Daniela Braconi, Maurizio Orlandini, Clare L. Thompson, Elena Petricci, Fabrizio Manetti, Maurizio Taddei, Martin M. Knight, Annalisa Santucci.
  Journal of Cellular Physiology. March 29, 2017
  Alkaptonuria (AKU) is an ultra‐rare genetic disease, in which the accumulation of a toxic metabolite, homogentisic acid (HGA) leads to the systemic development of ochronotic aggregates. These aggregates cause severe complications mainly at the level of joints with extensive degradation of the articular cartilage. Primary cilia have been demonstrated to play an essential role in development and the maintenance of articular cartilage homeostasis, through their involvement in mechanosignaling and Hedgehog signaling pathways. Hedgehog signaling has been demonstrated to be activated in osteoarthritis (OA) and to drive cartilage degeneration in vivo. The numerous similarities between OA and AKU suggest that primary cilia Hedgehog signaling may also be altered in AKU. Thus, we characterized an AKU cellular model in which healthy chondrocytes were treated with HGA (66 µM) to replicate AKU cartilage pathology. We investigated the degree of activation of the Hedgehog signaling pathway and how treatment with inhibitors of the receptor Smoothened (Smo) influenced Hedgehog activation and primary cilia structure. The results obtained in this work provide a further step in the comprehension of the pathophysiological features of AKU, suggesting a potential therapeutic approach to modulate AKU cartilage degradation processes through manipulation of the Hedgehog pathway. Primary cilia of alkaptonuric (AKU) chondrocytes are shorter as compared to healthy cells and HGA treatment leads to the same phenotype. Primary cilia alterations are correlated to an aberrant Hedgehog (Hh) signaling activation. Treatment of HGA‐treated chondrocytes with antagonists of Smoothened reported cilia lengths and Hh activation to the normal levels.

  March 29, 2017   doi: 10.1002/jcp.25761   open full text
• Signaling in the Auditory System: Implications in Hair Cell Regeneration and Hearing Function.
  Rahul Mittal, Luca H. Debs, Desiree Nguyen, Amit P. Patel, M'hamed Grati, Jeenu Mittal, Denise Yan, Adrien A. Eshraghi, Xue Zhong Liu.
  Journal of Cellular Physiology. March 29, 2017
  Ear is a sensitive organ involved in hearing and balance function. The complex signaling network in the auditory system plays a crucial role in maintaining normal physiological function of the ear. The inner ear comprises a variety of host signaling pathways working in synergy to deliver clear sensory messages. Any disruption, as minor as it can be, has the potential to affect this finely tuned system with temporary or permanent sequelae including vestibular deficits and hearing loss. Mutations linked to auditory symptoms, whether inherited or acquired, are being actively researched for ways to reverse, silence, or suppress them. In this article, we discuss recent advancements in understanding the pathways involved in auditory system signaling, from hair cell development through transmission to cortical centers. Our review discusses Notch and Wnt signaling, cell to cell communication through connexin and pannexin channels, and the detrimental effects of reactive oxygen species on the auditory system. There has been an increased interest in the auditory community to explore the signaling system in the ear for hair cell regeneration. Understanding signaling pathways in the auditory system will pave the way for the novel avenues to regenerate sensory hair cells and restore hearing function. J. Cell. Physiol. 9999: 1–12, 2017. © 2016 Wiley Periodicals, Inc. Wnt signaling influences hair cell differentiation. The presence of Wnt causes progenitor cells to differentiate into hair cell. Without Wnt, the progenitor cells become supporting cells.

  March 29, 2017   doi: 10.1002/jcp.25695   open full text
• LIPUS suppressed LPS‐induced IL‐1α through the inhibition of NF‐κB nuclear translocation via AT1‐PLCβ pathway in MC3T3‐E1 cells.
  Mayu Nagao, Natsuko Tanabe, Soichiro Manaka, Masako Naito, Jumpei Sekino, Tadahiro Takayama, Takayuki Kawato, Go Torigoe, Shunichiro Kato, Naoya Tsukune, Masao Maeno, Naoto Suzuki, Shuichi Sato.
  Journal of Cellular Physiology. March 29, 2017
  Inflammatory cytokines, interleukin (IL)‐1, IL‐6, and TNF‐α, are involved in inflammatory bone diseases such as rheumatoid osteoarthritis and periodontal disease. Particularly, periodontal disease, which destroys alveolar bone, is stimulated by lipopolysaccharide (LPS). Low‐intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics and dental treatments. However, the mechanism underlying effects of LIPUS on LPS‐induced inflammatory cytokine are not well understood. We therefore aimed to investigate the role of LIPUS on LPS‐induced IL‐1α production. Mouse calvaria osteoblast‐like cells MC3T3‐E1 were incubated in the presence or absence of LPS (Porphyromonas gingivalis), and then stimulated with LIPUS for 30 min/day. To investigate the role of LIPUS, we determined the expression of IL‐1α stimulated with LIPUS and treated with an angiotensin II receptor type 1 (AT1) antagonist, Losartan. We also investigate to clarify the pathway of LIPUS, we transfected siRNA silencing AT1 (siAT1) in MC3T3‐E1. LIPUS inhibited mRNA and protein expression of LPS‐induced IL‐1α. LIPUS also reduced the nuclear translocation of NF‐κB by LPS‐induced IL‐1α. Losartan and siAT1 blocked all the stimulatory effects of LIPUS on IL‐1α production and IL‐1α‐mediated NF‐κB translocation induced by LPS. Furthermore, PLCβ inhibitor U73122 recovered NF‐κB translocation. These results suggest that LIPUS inhibits LPS‐induced IL‐1α via AT1‐PLCβ in osteoblasts. We exhibit that these findings are in part of the signaling pathway of LIPUS on the anti‐inflammatory effects of IL‐1α expression. LIPUS reduced IL‐1α production induced by LPS. LPS increased NF‐κB nuclear translocation. LIPUS stimulates AT1 receptor that acts inhibition of NF‐κB nuclear translocation.

  March 29, 2017   doi: 10.1002/jcp.25777   open full text
• Production of Macrophage Inhibitory Factor (MIF) by Primary Sertoli Cells; Its Possible Involvement in Migration of Spermatogonial Cells.
  Mahmoud Huleihel, Maram Abofoul‐Azab, Yael Abarbanel, Iris Einav, Elyahu Levitas, Eitan Lunenfeld.
  Journal of Cellular Physiology. March 29, 2017
  Macrophage migration inhibitory factor (MIF) is a multifunctional molecule. MIF was originally identified as a T‐cell‐derived factor responsible for the inhibition of macrophage migration. In testicular tissue of adult rats, MIF is constitutively expressed by Leydig cells under physiological conditions. The aim of this study was to examine MIF levels in testicular homogenates from different aged mice, and the capacity of Sertoli cells to produce it. We also examined MIF involvement in spermatogonial cell migration. Similar levels of MIF protein were detected in testicular homogenates of mice of different ages (1–8‐week‐old). However, the RNA expression levels of MIF were high in 1‐week‐old mice and significantly decreased with age compared to 1‐week‐old mice. MIF was stained in Sertoli, Leydig cells, and developed germ cells in the seminiferous tubules. Isolated Sertoli cells from 1‐week‐old mice stained to MIF. Cultures of Sertoli cells from 1‐week‐old mice produced and expressed high levels of MIF which significantly decreased with age. MIF was localized in the cytoplasm and nucleus of Sertoli cell cultures isolated from 1‐week‐old mice; however, it was localized only in the cytoplasm and branches of cultures isolated from 8‐week‐old mice. MIFR was detected in GFRα1 and Sertoli cells. MIF could induce migration of spermatogonial cells, and this effect was synergistic with glial cell‐line neurotrophic factor. Our results show, for the first time, the capacity of Sertoli cells to produce MIF under normal conditions and that MIFR expressed in GFRα1 and Sertoli cells. We also showed that MIF induced spermatogonial cell migration. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc. Our study showed that Sertoli cells and spermatogonial cells generate MIF and express MIF receptor. MIF could induce spermatogonial cell migration.

  March 29, 2017   doi: 10.1002/jcp.25718   open full text
• Epigenetic Signatures at the RUNX2‐P1 and Sp7 Gene Promoters Control Osteogenic Lineage Commitment of Umbilical Cord‐Derived Mesenchymal Stem Cells.
  Hugo Sepulveda, Rodrigo Aguilar, Catalina P. Prieto, Francisco Bustos, Sócrates Aedo, José Lattus, Brigitte van Zundert, Veronica Palma, Martin Montecino.
  Journal of Cellular Physiology. March 28, 2017
  Wharton's Jelly mesenchymal stem cells (WJ‐MSCs) are an attractive potential source of multipotent stem cells for bone tissue replacement therapies. However, the molecular mechanisms involved in their osteogenic conversion are poorly understood. Particularly, epigenetic control operating at the promoter regions of the two master regulators of the osteogenic program, RUNX2/P57 and SP7 has not yet been described in WJ‐MSCs. Via quantitative PCR profiling and chromatin immunoprecipitation (ChIP) studies, here we analyze the ability of WJ‐MSCs to engage osteoblast lineage. In undifferentiated WJ‐MSCs, RUNX2/P57 P1, and SP7 promoters are found deprived of significant levels of the histone post‐translational marks that are normally associated with transcriptionally active genes (H3ac, H3K27ac, and H3K4me3). Moreover, the RUNX2 P1 promoter lacks two relevant histone repressive marks (H3K9me3 and H3K27me3). Importantly, RUNX2 P1 promoter is found highly enriched in the H3K4me1 mark, which has been shown recently to mediate gene repression of key regulatory genes. Upon induction of WJ‐MSCs osteogenic differentiation, we found that RUNX2/P57, but not SP7 gene expression is strongly activated, in a process that is accompanied by enrichment of activating histone marks (H3K4me3, H3ac, and H3K27ac) at the P1 promoter region. Histone mark analysis showed that SP7 gene promoter is robustly enriched in epigenetic repressive marks that may explain its poor transcriptional response to osteoblast differentiating media. Together, these results point to critical regulatory steps during epigenetic control of WJ‐MSCs osteogenic lineage commitment that are relevant for future applications in regenerative medicine. J. Cell. Physiol. 232: 2519–2527, 2017. © 2016 Wiley Periodicals, Inc. Wharton's Jelly Mesenchymal Stem Cells (WJ‐MSCs) are an attractive potential source of multipotent cells for tissue replacement therapies. Here, we study the ability of WJ‐MSCs to engage the osteoblast differentiation by analyzing epigenetic signatures at the promoter regions of the two master regulators of this process, RUNX2/P57 and SP7. We find that the RUNX2/p57, but not SP7, gene is expressed in WJ‐MSCs stimulated to differentiate to osteoblasts. This process is accompanied by the enrichment of activating histone marks at the RUNX2‐P57 P1 promoter and maintenance of epigenetic silencing histone marks at the SP7 promoter.

  March 28, 2017   doi: 10.1002/jcp.25627   open full text
• Super‐resolution structure of DNA significantly differs in buccal cells of controls and Alzheimer's patients.
  Angeles Garcia, David Huang, Amanda Righolt, Christiaan Righolt, Maria Carmela Kalaw, Shubha Mathur, Elizabeth McAvoy, James Anderson, Angela Luedke, Justine Itorralba, Sabine Mai.
  Journal of Cellular Physiology. March 28, 2017
  The advent of super‐resolution microscopy allowed for new insights into cellular and physiological processes of normal and diseased cells. In this study, we report for the first time on the super‐resolved DNA structure of buccal cells from patients with Alzheimer's disease (AD) versus age‐ and gender‐matched healthy, non‐caregiver controls. In this super‐resolution study cohort of 74 participants, buccal cells were collected and their spatial DNA organization in the nucleus examined by 3D Structured Illumination Microscopy (3D‐SIM). Quantitation of the super‐resolution DNA structure revealed that the nuclear super‐resolution DNA structure of individuals with AD significantly differs from that of their controls (p < 0.05) with an overall increase in the measured DNA‐free/poor spaces. This represents a significant increase in the interchromatin compartment. We also find that the DNA structure of AD significantly differs in mild, moderate, and severe disease with respect to the DNA‐containing and DNA‐free/poor spaces. We conclude that whole genome remodeling is a feature of buccal cells in AD. Figure 1 summarizes the super‐resolved DNA structure of buccal cells in Alzheimer's patients and their age‐ and gender‐matched controls.

  March 28, 2017   doi: 10.1002/jcp.25751   open full text
• MicroRNA‐1 overexpression blunts cardiomyocyte hypertrophy elicited by thyroid hormone.
  Gabriela Placoná Diniz, Caroline Antunes Lino, Camila Rodrigues Moreno, Nathalia Senger, Maria Luiza Morais Barreto‐Chaves.
  Journal of Cellular Physiology. March 28, 2017
  It is well‐known that increased thyroid hormone (TH) levels induce cardiomyocyte growth. MicroRNAs (miRNAs) have been identified as key players in cardiomyocyte hypertrophy, which is associated with increased risk of heart failure. In this study, we evaluated the miR‐1 expression in TH‐induced cardiac hypertrophy, as well as the potential involvement of miR‐1 in cardiomyocyte hypertrophy elicited by TH in vitro. The possible role of type 1 angiotensin II receptor (AT1R) in the effect promoted by TH in miR‐1 expression was also evaluated. Neonatal rat cardiac myocytes (NRCMs) were treated with T3 for 24 hr and Wistar rats were subjected to hyperthyroidism for 14 days combined or not with AT1R blocker. Real Time RT‐PCR analysis indicated that miR‐1 expression was decreased in cardiac hypertrophy in response to TH in vitro and in vivo, and this effect was unchanged by AT1R blocker. In addition, HDAC4, which is target of miR‐1, was increased in NRCMs after T3 treatment. A gain‐of‐function study revealed that overexpression of miR‐1 prevented T3‐induced cardiomyocyte hypertrophy and reduced HADC4 mRNA levels in NRCMs. In vivo experiments confirmed the downregulation of miR‐1 in cardiac tissue from hyperthyroid animals, which was accompanied by increased HDAC4 mRNA levels. In addition, HDAC inhibitor prevented T3‐induced cardiomyocyte hypertrophy. Our data reveal a new mechanistic insight into cardiomyocyte growth in response to TH, suggesting that miR‐1 plays a role in cardiomyocyte hypertrophy induced by TH potentially via targeting HADC4. A gain‐of‐function study revealed that overexpression of miR‐1 prevented T3‐induced cardiomyocyte hypertrophy and reduced HADC4 mRNA levels in neonatal cardiomyocytes. In vivo experiments confirmed the downregulation of miR‐1 in cardiac tissue from hyperthyroid animals, which was accompanied by increased HDAC4 mRNA levels. HDAC inhibition prevented T3‐induced cardiomyocyte hypertrophy revealing a new mechanistic insight into cardiomyocyte growth in response to TH.

  March 28, 2017   doi: 10.1002/jcp.25781   open full text
• Novel circulating biomarkers for non‐alcoholic fatty liver disease: A systematic review.
  Amirhossein Sahebkar, Elena Sancho, David Abelló, Jordi Camps, Jorge Joven.
  Journal of Cellular Physiology. March 28, 2017
  Currently, a liver biopsy remains the only reliable way to precisely diagnose non‐alcoholic fatty liver disease (NAFLD) and establish the severity of liver injury, presence of fibrosis, and architecture remodeling. However, the cost and the intrinsic invasive procedure of a liver biopsy rules it out as a gold standard diagnostic test, and the imaging test are not the best choice due to the price, and currently is being refined. The lack of a biomarker of NAFLD pushes to develop this new line of research. The aim of the present systematic review is to clarify and update all the NAFLD biomarkers described in the literature until recently. We highlight α‐ketoglutarate and CK18‐F as currently the best potential biomarker of NAFLD. However, due to methodological differences, we propose the implementation of international, multicenter, multiethnic studies with larger population size, and biopsy proven NAFLD diagnosis to analyze and compare α‐ketoglutarate and CK18‐F as potential biomarkers of the silent evolution of NAFLD. The lack of a biomarker of NAFLD pushes to develop this new line of research. The aim of the present systematic review is to clarify and update all the NAFLD biomarkers described in the literature until recently. We highlight α‐ketoglutarate and CK18‐F as currently the best potential biomarker of NAFLD.

  March 28, 2017   doi: 10.1002/jcp.25779   open full text
• Steady state peripheral blood provides cells with functional and metabolic characteristics of real hematopoietic stem cells.
  Antonin Bourdieu, Maryse Avalon, Véronique Lapostolle, Sadek Ismail, Margaux Mombled, Christelle Debeissat, Marianne Guérinet, Pascale Duchez, Jean Chevaleyre, Marija Vlaski‐Lafarge, Arnaud Villacreces, Vincent Praloran, Zoran Ivanovic, Philippe Brunet de la Grange.
  Journal of Cellular Physiology. March 28, 2017
  Hematopoietic stem cells (HSCs), which are located in the bone marrow, also circulate in cord and peripheral blood. Despite high availability, HSCs from steady state peripheral blood (SSPB) are little known and not used for research or cell therapy. We thus aimed to characterize and select HSCs from SSPB by a direct approach with a view to delineating their main functional and metabolic properties and the mechanisms responsible for their maintenance. We chose to work on Side Population (SP) cells which are highly enriched in HSCs in mouse, human bone marrow, and cord blood. However, no SP cells from SSBP have as yet been characterized. Here we showed that SP cells from SSPB exhibited a higher proliferative capacity and generated more clonogenic progenitors than non‐SP cells in vitro. Furthermore, xenotransplantation studies on immunodeficient mice demonstrated that SP cells are up to 45 times more enriched in cells with engraftment capacity than non‐SP cells. From a cell regulation point of view, we showed that SP activity depended on O2 concentrations close to those found in HSC niches, an effect which is dependent on both hypoxia‐induced factors HIF‐1α and HIF‐2α. Moreover SP cells displayed a reduced mitochondrial mass and, in particular, a lower mitochondrial activity compared to non‐SP cells, while they exhibited a similar level of glucose incorporation. These results provided evidence that SP cells from SSPB displayed properties of very primitive cells and HSC, thus rendering them an interesting model for research and cell therapy. We provided evidence that Side Population (SP) cells from Steady State Peripheral Blood (SSPB) displayed properties of very primitive cells and HSC based on in vitro and in vivo functional assays. Moreover these SP cells are better maintained during culture at low O2 concentrations, and they displayed a reduced mitochondrial mass and mitochondrial activity compared to non‐SP cells, while exhibiting a similar level of glucose incorporation. These cells are thus an interesting model for research and cell therapy.

  March 28, 2017   doi: 10.1002/jcp.25881   open full text
• Advanced glycation end products (AGE) potentiates cell death in p53 negative cells via upregulaion of NF‐kappa B and impairment of autophagy.
  Neeharika Verma, Sunil K. Manna.
  Journal of Cellular Physiology. March 27, 2017
  Accumulation of advanced glycation end products (AGE) in diabetic patients and ageing people due to excess availability of simple 3‐ or 4‐carbon sugars, is well‐known. AGE has multiple deleterious effects including age‐related disorders, apoptosis, inflammation, and obesity. We have found that AGE increases autophagy but the sustained amount of autophagosomes is observed till 3 days without maturation. It is important to understand the underlying mechanism of AGE‐mediated signaling responsible for impairment of autophagy and its correlation to the induction of several adverse effects. We have identified cross talk between autophagy and apoptosis upon AGE stimulation, specifically in p53 negative cells. AGE impairs autophagosomes' clearance in p53 negative cells as observed with an autophagosome maturation blocker‐bafilomycinA1 treated cells. This autophagy impairment is well supported by upregulation and overexpression of NF‐κB in these p53 negative cells. Autophagy impairment acts as a switch to initiate apoptosis via regulation of NF‐κB and its dependent genes. Increase in the expression of NF‐κB‐dependent NEDD4, an E3 ubiquitin ligase, which targets Beclin1 for cleavage is also evident. Beclin1 interacts with Bcl‐2, an anti‐apoptotic protein thereby engaging it to facilitate apoptosis upon AGE stimulation. For the first time, we are providing data that NF‐κB targeted cell signaling is involved in AGE‐mediated autophagy impairment in p53 negative/null cells. The p53 acts antagonistically to prevent this impairment. This study will help to control the AGE‐mediated detrimental effects associated with ageing and lysosomal storage disorders. NF‐κB targeted cell signaling is involved in AGE‐mediated autophagy impairment in p53 negative/null cells. The p53 acts antagonistically to prevent this impairment. This study will help to control the AGE‐mediated detrimental effects associated with ageing and lysosomal storage disorders.

  March 27, 2017   doi: 10.1002/jcp.25828   open full text
• Distinctive expression pattern of cystathionine‐β‐synthase and cystathionine‐γ‐lyase identifies mesenchymal stromal cells transition to mineralizing osteoblasts.
  Laura Gambari, Gina Lisignoli, Elena Gabusi, Cristina Manferdini, Francesca Paolella, Anna Piacentini, Francesco Grassi.
  Journal of Cellular Physiology. March 27, 2017
  Mesenchymal stromal cells (MSCs) are key players in the repair or regeneration of the damaged bone tissue. However, heterogeneity exists between MSCs derived from different donors in their bone formation ability both in vitro and in vivo. The identification of markers defining MSCs with different functional phenotypes is fundamental to maximize their clinical potential. In our previous in vivo study, impaired expression in MSCs of cystathionine‐β‐synthase (CBS) and cystathionine‐γ‐lyase (CSE), the two key enzymes in the catabolic pathway of homocysteine, was associated to decreased bone formation and to the onset of osteoporosis in mice. Here, we investigated whether osteogenic differentiation of human MSCs (hMSCs) modulates the expression of CBS and CSE. The expression of CBS and CSE was also assessed during chondrogenesis to confirm the specificity of their expression during osteogenesis. hMSCs displayed a heterogeneous mineralizing capacity between donors (70% of the samples mineralized, while 30% did not mineralize). Inducible expression of CBS and CSE was found to be associated with a mineralizing phenotype in hMSCs. In particular, up‐regulation of CSE was restricted to hMSCs undergoing mineralization. During chondrogenesis, CBS was significantly up‐regulated while CSE expression was not affected. Ex‐vivo findings confirmed that mature h‐osteoblasts (hOBs) show consistently higher expression of CBS and CSE than hMSCs. Our data provide the first evidence that the expression of CBS and CSE in hMSCs closely correlates with the transition of hMSCs toward the osteoblastic phenotype and that CSE may constitute a novel marker of osteogenic differentiation. Human mesenchymal stromal cells (hMSCs) play a pivotal role in the regeneration of bone tissue, but are affected by a marked heterogeneity in functional response to osteogenic stimuli. Inducible expression of CBS and CSE was found to be associated with a mineralizing phenotype in hMSCs and their expression correlates with the transition of hMSCs towards the osteoblastic phenotype. CSE may constitute a novel marker of osteogenic differentiation.

  March 27, 2017   doi: 10.1002/jcp.25825   open full text
• Roles of Notch1 Signaling in Regulating Satellite Cell Fates Choices and Postnatal Skeletal Myogenesis.
  Tizhong Shan, Ziye Xu, Weiche Wu, Jiaqi Liu, Yizhen Wang.
  Journal of Cellular Physiology. March 27, 2017
  Adult skeletal muscle stem cells, also called satellite cells, are indispensable for the growth, maintenance, and regeneration of the postnatal skeletal muscle. Satellite cells, predominantly quiescent in mature resting muscles, are activated after skeletal muscle injury or degeneration. Notch1 signaling is an evolutionarily conserved pathway that plays crucial roles in satellite cells homeostasis and postnatal skeletal myogenesis and regeneration. Activation of Notch1 signaling promotes the muscle satellite cells quiescence and proliferation, but inhibits differentiation of muscle satellite cells. Notably, the new roles of Notch1 signaling during late‐stage of skeletal myogenesis including in post‐differentiation myocytes and post‐fusion myotubes have been recently reported. Here, we mainly review and discuss the regulatory roles of Notch1 in regulating satellite cell fates choices and skeletal myogenesis. J. Cell. Physiol. 9999: 1–4, 2016. © 2016 Wiley Periodicals, Inc. Notch1 signaling is an evolutionarily conserved pathway that plays crucial roles in satellite cells homeostasis and postnatal skeletal myogenesis. Recently, the new roles of Notch1 signaling during late‐stage of skeletal myogenesis including in post‐differentiation myocytes and post‐fusion myotubes have been reported. Here, we mainly review and discuss the regulatory roles of Notch1 in regulating satellite cell fates choices and skeletal myogenesis.

  March 27, 2017   doi: 10.1002/jcp.25730   open full text
• The role of various peroxisome proliferator‐activated receptors and their ligands in clinical practice.
  Giuseppe Derosa, Amirhossein Sahebkar, Pamela Maffioli.
  Journal of Cellular Physiology. March 27, 2017
  Peroxisome proliferator‐activated receptors (PPARs) are ligand‐activated transcription factors involved in several physiological processes including modulation of cellular differentiation, development, metabolism of carbohydrates, lipids, proteins, and tumorigenesis. The aim of this review is to examine how different PPAR ligands act, and discuss their use in clinical practice. PPAR ligands have a lot of effects and applications in clinical practice. Some PPAR ligands such as fibrates (PPAR‐α ligands) are currently used for the treatment of dyslipidemia, while pioglitazone and rosiglitazone (PPAR‐γ ligands) are anti‐diabetic and insulin‐sensitizing agents. Regarding new generation drugs, acting on both α/γ, β/δ, or α/δ receptors simultaneously, preliminary data on PPAR‐α/γ dual agonists revealed a positive effect on lipid profile, blood pressure, atherosclerosis, inflammation, and anti‐coagulant effects, while the overexpression of PPAR‐β/δ seems to prevent obesity and to decrease lipid storage in cardiac cells. Finally, PPAR‐α/δ dual agonist induces resolution of nonalcoholic steatohepatitis without fibrosis worsening. Peroxisome proliferator‐activated receptors (PPARs) are ligand‐activated transcription factors involved in several physiological processes. PPAR ligands have a lot of effects and applications in clinical practice. The aim of this review is to examine how different PPAR ligands act, and discuss their use in clinical practice.

  March 27, 2017   doi: 10.1002/jcp.25804   open full text
• NOR1 Suppresses Cancer Stem‐Like Cells Properties of Tumor Cells via the Inhibition of the AKT‐GSK‐3β‐Wnt/β‐catenin‐ALDH1A1 Signal Circuit.
  Wei Wang, Mei Yi, Shengnan Chen, Junjun Li, Haijing Zhang, Wei Xiong, Guiyuan Li, Xiaoling Li, Bo Xiang.
  Journal of Cellular Physiology. March 27, 2017
  Cancer stem cells (CSCs) play a key role in tumor radiotherapy and chemotherapy resistance, relapse, and metastasis, and are primarily maintained in a resting state in vivo. The failure of conventional therapies to target CSCs is the main cause of treatment failure. The discovery of CSCs in nasopharyngeal carcinoma (NPC) tumors is becoming more prevalent; however, the understanding of the mechanisms underlying the maintenance of tumor stemness is still limited. We previously cloned NOR1, a tumor suppressor gene downregulated in NPC cell lines and tissues. In this study, we demonstrate that Wnt/β‐catenin and ALDH1A1 form a signal circuit and that NOR1 antagonizes the tumor stem cell‐like phenotype in NPC cell lines: the ectopic overexpression of NOR1 reduced β‐catenin and ALDH1A1 expression; β‐catenin/TCF4 targeted the regulation of ALDH1A1 transcription in NPC cells; silencing ALDH1A1 reduced AKT (total and phosphorylated) and GSK‐3β (phosphorylated) expression; and eventually feedback decreased β‐catenin expression levels. We also found that NOR1 expression decreased cancer stem‐like cell properties of NPC cells, reduced their ability to form tumor spheroids in vitro, reduced tumorigenicity in nude mice in vivo, and increased sensitivity to chemotherapy agents. Taken together, our findings illustrated a new function of NOR1 that suppresses cancer stem‐like cell properties in tumor cells by inhibiting the AKT‐GSK‐3β‐Wnt/β‐catenin‐ALDH1A1 signal circuit. The study suggests that NOR1 deletion expression in NPC cells may be a potential molecular target for cancer stem cell therapy. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc. Cancer stem cells (CSCs) play a key role in tumor radiotherapy and chemotherapy resistance, relapse, and metastasis. Our findings illustrated a new function of NOR1 that suppresses cancer stem‐like cell properties in tumor cells by inhibiting the AKT‐GSK‐3β‐Wnt/β‐catenin‐ALDH1A1 signal circuit.

  March 27, 2017   doi: 10.1002/jcp.25706   open full text
• Methylated of genes behaving as potential biomarkers in evaluating malignant degree of glioblastoma.
  Wan‐Shun Wen, Sheng‐Li Hu, Zhibing Ai, Lin Mou, Jing‐Min Lu, Sen Li.
  Journal of Cellular Physiology. March 27, 2017
  Abnormal methylation genes usually act as oncogenes or anti‐oncogenes in the occurrence and development of tumor, indicating their potential role as biomarkers in the evaluation of malignant tumor. However, the research on methylation's association with glioblastoma was rare. We attempted to figure out whether the methylation of genes could serve as the biomarker in evaluating the malignant degree of GBM. Methylation microarray data of 275 GBM patients have been downloaded from The Cancer Genome Atlas (TCGA) dataset. Logistic regression was used to find the methylated genes associated with the malignant degree of patients with the tumor. Functional enrichment analysis and network analysis were further performed on these selected genes. A total of 668, 412, 470, and 620 genes relevant with the methylation or demethylation were found to be associated with the malignant degree, Grades 1–4 of tumor. The higher the degree of malignant tumor, the higher of its methylation degree of its corresponding genes. GO and KEGG analysis results showed that these methylated genes were enriched in many functions as cell adhesion, abnormal transcription, and cell cycle disorder, etc. Of note, CCL11 and LCN11 were found to be significantly related to the progression of GBM. Critical genes associated with cell cycle as CCL11 and LCN1 may play essential roles in the occurrence, development, and transition of glioblastoma. More research was needed to explore its potential molecular mechanism. Abnormal methylation genes usually act as oncogenes or anti‐oncogenes in the occurrence and development of tumor, indicating their potential role as biomarkers in the evaluation of malignant tumor.A total of 668, 412, 470 and 620 genes relevant with the methylation or demethylation were found to be associated with the malignant degree, Grades 1–4 of glioblastoma. Of note, CCL11 and LCN11 were found to be significantly related to the progression of GBM.

  March 27, 2017   doi: 10.1002/jcp.25831   open full text
• Mouse Oocytes Acquire Mechanisms That Permit Independent Cell Volume Regulation at the End of Oogenesis.
  Samantha Richard, Alina P. Tartia, Detlev Boison, Jay M. Baltz.
  Journal of Cellular Physiology. March 27, 2017
  Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1. In fully‐grown oocytes, release of adhesion and GLYT1 activation also occur spontaneously in oocytes removed from the follicle. It is unknown, however, whether the capacity to release oocyte‐ZP adhesion or activate GLYT1 first arises in the oocyte after ovulation is triggered or instead growing oocytes already possess these capabilities but they are suppressed in the follicle. Here, we assessed when during oogenesis oocyte‐ZP adhesion can be released and when GLYT1 can be activated, with adhesion assessed by an osmotic assay and GLYT1 activity determined by [3H]‐glycine uptake. Oocyte‐ZP adhesion could not be released by growing oocytes until they were nearly fully grown. Similarly, the amount of GLYT1 activity that can be elicited in oocytes increased sharply at the end of oogenesis. The SLC6A9 protein that is responsible for GLYT1 activity and Slc6a9 transcripts are present in growing oocytes and increased over the course of oogenesis. Furthermore, SLC6A9 becomes localized to the oocyte plasma membrane as the oocyte grows. Thus, oocytes acquire the ability to regulate their cell volume by releasing adhesion to the ZP and activating GLYT1 as they approach the end of oogenesis. J. Cell. Physiol. 232: 2436–2446, 2017. © 2016 Wiley Periodicals, Inc. Independent cell volume regulation in the mouse oocyte normally becomes active after ovulation is triggered in a two‐step process: the strong adhesion of the oocyte to the surrounding zona pellucida is released and glycine transport via the GLYT1 transporter is activated. Both mechanisms develop at the end of oocyte growth but remain suppressed within the ovarian follicle.

  March 27, 2017   doi: 10.1002/jcp.25581   open full text
• ­NOD Mice Having a Lyn Tyrosine Kinase Mutation Exhibit Abnormal Neutrophil Chemotaxis.
  Yue Wu, Michael Hannigan, Lijun Zhan, Joseph A. Madri, Chi‐Kuang Huang.
  Journal of Cellular Physiology. March 27, 2017
  Neutrophils from NOD (Non‐Obese Diabetic) mice exhibited reduced migration speed, decreased frequency of directional changes, and loss of directionality during chemotaxis (compared to wild‐type [WT] C57BL/6 mice). Additionally, F‐actin of chemotaxing NOD neutrophils failed to orient toward the chemoattractant gradient and NOD neutrophil adhesion was impaired. A point mutation near the autophosphorylation site of Lyn in NOD mice was identified. Point mutations of G to A (G1412 in LynA and G1199 in LynB) cause a change of amino acid E393 (glutamic acid) to K (lysine) in LynA (E393→K) (E372 of LynB), affecting fMLP‐induced tyrosine phosphorylation. These data indicate that the Lyn mutation in NOD neutrophils is likely responsible for dysregulation of neutrophil adhesion and directed migration, implying the role of Lyn in modulating diabetic patient's susceptibility to bacterial and fungal infections. J. Cell. Physiol. 232: 1689–1695, 2017. © 2016 Wiley Periodicals, Inc. A specific Lyn kinase mutation has been documented in NOD mice which affects neutrophil directed migration. This may thus affect response to infections in this murine model of diabetes.

  March 27, 2017   doi: 10.1002/jcp.25583   open full text
• Vertebrate Lonesome Kinase Regulated Extracellular Matrix Protein Phosphorylation, Cell Shape, and Adhesion in Trabecular Meshwork Cells.
  Rupalatha Maddala, Nikolai P. Skiba, Ponugoti Vasantha Rao.
  Journal of Cellular Physiology. March 27, 2017
  Glaucoma, a leading cause of irreversible blindness, is commonly associated with elevated intraocular pressure (IOP) due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although dysregulated production and organization of extracellular matrix (ECM) is presumed to increase resistance to AH outflow and elevate IOP by altering TM cell contractile and adhesive properties, it is not known whether regulation of ECM protein phosphorylation via the secretory vertebrate lonesome kinase (VLK) influences TM cellular characteristics. Here, we tested this possibility. Experiments carried out in this study reveal that the 32 kDa protein is a prominent VLK isoform detectable in lysates and conditioned media (CM) of human TM cells. Increased levels of VLK were observed in CM of TM cells subjected to cyclic mechanical stretch, or treated with dexamethasone, TGF‐β2, and TM cells expressing constitutively active RhoA GTPase. Downregulation of VLK expression in TM cells using siRNA decreased tyrosine phosphorylation (TyrP) of ECM proteins and focal adhesions, and induced changes in cell shape in association with reduced levels of actin stress fibers and phospho‐paxillin. VLK was also demonstrated to regulate TGF‐β2‐induced TyrP of ECM proteins. Taken together, these results suggest that VLK secretion can be regulated by external cues, intracellular signal proteins, and mechanical stretch, and VLK can in turn regulate TyrP of ECM proteins secreted by TM cells and control cell shape, actin stress fibers, and focal adhesions. These observations indicate a potential role for VLK in homeostasis of AH outflow and IOP, and in the pathobiology of glaucoma. J. Cell. Physiol. 232: 2447–2460, 2017. © 2016 Wiley Periodicals, Inc. Our results suggest that VLK secretion can be regulated by external cues, intracellular signal proteins, and mechanical stretch, and VLK can in turn regulate TyrP of ECM proteins secreted by TM cells and control cell shape, actin stress fibers, and focal adhesions. These observations indicate a potential role for VLK in homeostasis of AH outflow and IOP, and in the pathobiology of glaucoma.

  March 27, 2017   doi: 10.1002/jcp.25582   open full text
• Mechanisms involved in enhancement of matrix metalloproteinase‐9 expression in macrophages by interleukin‐33.
  Wataru Ariyoshi, Toshinori Okinaga, Wichida Chaweewannakorn, Sumio Akifusa, Tatsuji Nisihara.
  Journal of Cellular Physiology. March 27, 2017
  Endothelial transmigration of macrophages is accomplished by matrix metalloproteinase (MMP)‐induced degradation of the basement membrane and extracellular matrix components. Macrophages upregulate MMP‐9 expression and secretion upon immunological challenges and require its activity for migration during inflammatory responses. Interleukin (IL)‐33 is a recently discovered pro‐inflammatory cytokine that belongs to the IL‐1 family. The aim of this study was to elucidate the mechanisms underlying IL‐33‐induced MMP‐9 expression in the mouse monocyte/macrophage line RAW264.7. IL‐33 increased MMP‐9 mRNA and protein expression in RAW264.7 cells. Blockage of IL‐33‐IL‐33 receptor (ST2L) binding suppressed IL‐33‐mediated induction of MMP‐9. IL‐33 induced phosphorylation and nuclear translocation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and nuclear factor‐kappa B (NF‐κB). Chromatin immunoprecipitation indicated that IL‐33 increased c‐fos recruitment to the MMP‐9 promoter. Reporter assay findings also revealed that IL‐33 stimulated the transcriptional activity of activator protein 1 (AP‐1). Pre‐treatment of the cells with a specific inhibitor of ERK1/2 and NF‐κB attenuated the IL‐33‐induced activation of AP‐1 subunits, transcriptional activity of AP‐1, and expression of MMP‐9. We also demonstrated that ERK‐dependent activation of cAMP response element binding protein (CREB) is a key step for AP‐1 activation by IL‐33. These results indicate an essential role of ERK/CREB and NF‐κB cascades in the induction of MMP‐9 in monocytes/macrophages through AP‐1 activation. IL‐33‐ST2L interaction increased MMP‐9 expression in RAW264.7 cells. ERK/CREB and NF‐κB cascades have an essential role in the induction of MMP‐9 in monocytes/macrophages through AP‐1 activation.

  March 27, 2017   doi: 10.1002/jcp.25809   open full text
• Meta‐Analysis of Transcriptome Regulation During Induction to Cardiac Myocyte Fate From Mouse and Human Fibroblasts.
  Shima Rastegar‐Pouyani, Niusha Khazaei, Ping Wee, Moein Yaqubi, Abdulshakour Mohammadnia.
  Journal of Cellular Physiology. March 24, 2017
  Ectopic expression of a defined set of transcription factors (TFs) can directly convert fibroblasts into a cardiac myocyte cell fate. Beside inefficiency in generating induced cardiomyocytes (iCMs), the molecular mechanisms that regulate this process remained to be well defined. The main purpose of this study was to provide better insight on the transcriptome regulation and to introduce a new strategy for candidating TFs for the transdifferentiation process. Eight mouse and three human high quality microarray data sets were analyzed to find differentially expressed genes (DEGs), which we integrated with TF‐binding sites and protein–protein interactions to construct gene regulatory and protein–protein interaction networks. Topological and biological analyses of constructed gene networks revealed the main regulators and most affected biological processes. The DEGs could be categorized into two distinct groups, first, up‐regulated genes that are mainly involved in cardiac‐specific processes and second, down‐regulated genes that are mainly involved in fibroblast‐specific functions. Gata4, Mef2a, Tbx5, Tead4 TFs were identified as main regulators of cardiac‐specific gene expression program; and Trp53, E2f1, Myc, Sfpi1, Lmo2, and Meis1 were identified as TFs which mainly regulate the expression of fibroblast‐specific genes. Furthermore, we compared gene expression profiles and identified TFs between mouse and human to find the similarities and differences. In summary, our strategy of meta‐analyzing the data of high‐throughput techniques by computational approaches, besides revealing the mechanisms involved in the regulation of the gene expression program, also suggests a new approach for increasing the efficiency of the direct reprogramming of fibroblasts into iCMs. J. Cell. Physiol. 232: 2053–2062, 2017. © 2016 Wiley Periodicals, Inc. The DEGs could be categorized into two distinct groups, first, up‐regulated genes that are mainly involved in cardiac‐specific processes and second, down‐regulated genes that are mainly involved in fibroblast‐specific functions. Gata4, Mef2a, Tbx5, Tead4 TFs were identified as main regulators of cardiac‐specific gene expression program; and Trp53, E2f1, Myc, Sfpi1, Lmo2, and Meis1 were identified as TFs which mainly regulate the expression of fibroblast‐specific genes. Furthermore, we compared gene expression profiles and identified TFs between mouse and human to find the similarities and differences.

  March 24, 2017   doi: 10.1002/jcp.25580   open full text
• Molecular Imaging of Huntington's Disease.
  Andrea Ciarmiello, Giampiero Giovacchini, Elisabetta Giovannini, Patrizia Lazzeri, Elisa Borsò, Antonio Mannironi, Luigi Mansi.
  Journal of Cellular Physiology. March 24, 2017
  The onset and the clinical progression of Huntington Disease (HD) is influenced by several events prompted by a genetic mutation that affects several organs tissues including different regions of the brain. In the last decades years, Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI) helped to deepen the knowledge of neurodegenerative mechanisms that guide to clinical symptoms. Brain imaging with PET represents a tool to investigate the physiopathology occurring in the brain and it has been used to predict the age of onset of the disease and to evaluate the therapeutic efficacy of new drugs. This article reviews the contribution of PET and MRI in the research field on Huntington's disease, focusing in particular on some most relevant achievements that have helped recognize the molecular changes, the clinical symptoms and evolution of the disease. J. Cell. Physiol. 232: 1988–1993, 2017. © 2016 Wiley Periodicals, Inc. The onset and the clinical progression of Huntington Disease (HD) is influenced by several events prompted by a genetic mutation that affects several organs tissues including different regions of the brain. In the last decades years, Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI) helped to deepen the knowledge of neurodegenerative mechanisms that guide to clinical symptoms. Brain imaging with PET represents a tool to investigate the physiopathology occurring in the brain and it has been used to predict the age of onset of the disease and to evaluate the therapeutic efficacy of new drugs. This article reviews the contribution of PET and MRI in the research field on Huntington's disease, focusing in particular on some most relevant achievements that have helped recognize the molecular changes, the clinical symptoms, and evolution of the disease.

  March 24, 2017   doi: 10.1002/jcp.25666   open full text
• Interactive Effect of Corticosterone and Lactate on Regulation of Testosterone Production in Rat Leydig Cells.
  Chih‐Chieh Chen, Chien‐Wei Chen, Po‐Han Lin, Jou‐Chun Chou, Ting‐Chun Weng, Cai‐Yun Jian, Sindy Hu, Wei‐Ho Lai, Fu‐Kong Lieu, Shyi‐Wu Wang, Paulus S. Wang.
  Journal of Cellular Physiology. March 24, 2017
  The increasing intensity of exercise enhanced corticosterone and lactate production in both humans and rodents. Our previous studies also demonstrated that lactate could stimulate testosterone production in vivo and in vitro. However, the production of testosterone in response to combined corticosterone and lactate on Leydig cells, and underlying molecular mechanisms are remained unclear. This study investigated the changes in testosterone levels of Leydig cells upon exposure to lactate, corticosterone or combination of both, and revealed the detailed mechanisms. Leydig cells were isolated from rat testes, and treated with different concentrations of lactate (2.5–20 mM), cortiosterone (10−9–10−4 M) and lactate plus corticosterone. The production of testosterone were assayed by radioimmunoassay, and the key molecular proteins, including luteinizing hormone receptor (LHR), protein kinase A (PKA), steroidogenic acute regulatory protein (StAR), and cholesterol P450 side‐chain cleavage enzyme (P450scc) involved in testosterone production were performed by Western blot. Results showed that testosterone levels were significantly increased with lactate, while decresed with corticosterone and lactate plus corticosterone treatment. Protein expressions of LHR and P450scc were upregulated with lactate treatment. However, PKA and P450scc were downregulated by lactate plus corticosterone treatment. This downregulation was followed by decreased testoterone levels in Leydig cells. Furthermore, acetylated cAMP, which activates testosterone production was increased with lactate, but not altered by conrtiosterone. Our findings conclude that corticosterone may interfere with lactate, and restrict lactate‐stimulated testosterone production in Leydig cells. J. Cell. Physiol. 232: 2135–2144, 2017. © 2016 Wiley Periodicals, Inc. Lactate activates the LHR‐cAMP‐P450scc pathway to increase endogenous testosterone production. Treatment of CORT down‐regulates LHR‐cAMP‐PKA‐P450scc pathway to diminish testosterone production. Activation of intracellular adenylyl cyclase, PKA and P450scc pathways are critical to increase endogenous testosterone production in Leydig cells.

  March 24, 2017   doi: 10.1002/jcp.25700   open full text
• Palmitoleic Acid Improves Metabolic Functions in Fatty Liver by PPARα‐Dependent AMPK Activation.
  Camila O. de Souza, Alexandre A.S. Teixeira, Luana A. Biondo, Edson A. Lima Junior, Helena A.P. Batatinha, Jose C. Rosa Neto.
  Journal of Cellular Physiology. March 24, 2017
  Background: Palmitoleic acid, since described as lipokine, increases glucose uptake by modulation of 5′AMP‐activated protein kinase (AMPK), as well as increasing lipolysis by activation of peroxisome proliferator‐activated receptor‐α (PPARα), in adipose tissue. However, in liver, the effects of palmitoleic acid on glucose metabolism and the role of PPARα remain unknown. Objective: To investigate whether palmitoleic acid improved the hepatic insulin sensitivity of obese mice. Methods: C57BL6 and PPARα knockout (KO) mice were fed for 12 weeks with a standard diet (SD) or high‐fat diet (HF), and in the last 2 weeks were treated with oleic or palmitoleic acid. Results: Palmitoleic acid promoted a faster uptake of glucose in the body, associated with higher insulin concentration; however, even when stimulated with insulin, palmitoleic acid did not modulate the insulin pathway (AKT, IRS). Palmitoleic acid increased the phosphorylation of AMPK, upregulated glucokinase and downregulated SREBP‐1. Regarding AMPK downstream, palmitoleic acid increased the production of FGF‐21 and stimulated the expression of PPARα. Palmitoleic acid treatment did not increase AMPK phosphorylation, modulate glucokinase or increase FGF‐21 in liver of PPARα KO mice. Conclusions: In mice fed with a high‐fat diet, palmitoleic acid supplementation stimulated the uptake of glucose in liver through activation of AMPK and FGF‐21, dependent on PPARα. J. Cell. Physiol. 232: 2168–2177, 2017. © 2016 Wiley Periodicals, Inc. Palmitoleic acid (PM) increased glucose uptake and modulated glucokinase in liver; Those effects are linked to higher PPARα expression, activation of AMPK and FGF‐21. Similar beneficial effects were not observed in liver of PPARα KO mice. Thus, activation of AMPK and FGF‐21 by PM in liver, are dependent of PPARα.

  March 24, 2017   doi: 10.1002/jcp.25715   open full text
• Opposite Effects of Mechanical Action of Fluid Flow on Proangiogenic Factor Secretion From Human Adipose‐Derived Stem Cells With and Without Oxidative Stress.
  Beatriz Bravo, Cira García de Durango, Álvaro González, Arancha R. Gortázar, Xavier Santos, Jerónimo Forteza‐Vila, Fernando Vidal‐Vanaclocha.
  Journal of Cellular Physiology. March 24, 2017
  Mechanical forces, hypoxia, and oxidative stress contribute to skin renewal, perfusion, and wound healing, but how are they regulating subcutaneous adipose‐derived stem cells (ASCs) in the inflammatory microenvironment associated to skin repair and disorders is unknown. In this study, ASCs were isolated from lipoaspirate samples from plastic surgery patients, primary cultured and their differentiation and secretion of a panel of cytokines with pronounced effects on skin repair and angiogenesis were studied under mechanical stimulation by intermittent fluid flow, 1% hypoxia and oxidative stress by glutathione (GSH) depletion with buthionine sulfoximine (BSO) treatment. Mechanical action of fluid flow did not alter mesenchymal phenotype of CD90+/CD29+/CD44+/CD34−/CD106−/CD45−ASCs; however, it remarkably induced ASC secretion of human umbilical vein endothelial cell (HUVEC) migration‐stimulating factors. Multiplex Luminex assay further confirmed an increased secretion of VEGF, G‐CSF, HGF, Leptin, IL‐8, PDGF‐BB, Angiopoietin‐2, and Follistatin from mechanically‐stimulated ASCs via cyclooxygenase‐2. Consistent with this mechanism, GSH depletion and hypoxia also increased ASC secretion of VEGF, IL‐8, leptin, Angiopoitein‐2, and PDGF‐BB. However, mechanical action of fluid flow abrogated VEGF and HUVEC migration‐stimulating activity from GSH‐depleted and hypoxic ASCs. Conversely, GSH depletion and hypoxia abrogated VEGF and HUVEC migration‐stimulating activity from mechano‐stimulated ASCs. Although mechanical action of fluid flow, hypoxia, and GSH‐depletion had independent proangiogenic‐stimulating activity on ASCs, mechanical stimulation had opposite effects on proangiogenic factor secretion from ASCs with and without oxidative stress. These data uncover the role of hypoxia and endogenous redox balance during the proangiogenic response of ASCs and other mesenchymal‐derived cell types to mechanical action of interstitial fluid flow. J. Cell. Physiol. 232: 2158–2167, 2017. © 2016 Wiley Periodicals, Inc. In this original work, we described the critical role of endogenous redox balance during the proangiogenic response of ASCs to mechanical forces. These data uncover the role of hypoxia and endogenous redox balance during the proangiogenic response of ASCs and other mesenchymal‐derived cell types to mechanical action of interstitial fluid flow.

  March 24, 2017   doi: 10.1002/jcp.25712   open full text
• Aging Selectively Modulates Vitamin C Transporter Expression Patterns in the Kidney.
  Katherine Forman, Fernando Martínez, Manuel Cifuentes, Romina Bertinat, Katterine Salazar, Francisco Nualart.
  Journal of Cellular Physiology. March 24, 2017
  In the kidney, vitamin C is reabsorbed from the glomerular ultrafiltrate by sodium‐vitamin C cotransporter isoform 1 (SVCT1) located in the brush border membrane of the proximal tubules. Although we know that vitamin C levels decrease with age, the adaptive physiological mechanisms used by the kidney for vitamin C reabsorption during aging remain unknown. In this study, we used an animal model of accelerated senescence (SAMP8 mice) to define the morphological alterations and aging‐induced changes in the expression of vitamin C transporters in renal tissue. Aging induced significant morphological changes, such as periglomerular lymphocytic infiltrate and glomerular congestion, in the kidneys of SAMP8 mice, although no increase in collagen deposits was observed using 2‐photon microscopy analysis and second harmonic generation. The most characteristic histological alteration was the dilation of intracellular spaces in the basolateral region of proximal tubule epithelial cells. Furthermore, a combination of laser microdissection, qRT‐PCR, and immunohistochemical analyses allowed us to determine that SVCT1 expression specifically increased in the proximal tubules from the outer strip of the outer medulla (segment S3) and cortex (segment S2) during aging and that these tubules also express GLUT1. We conclude that aging modulates vitamin C transporter expression and that renal over‐expression of SVCT1 enhances vitamin C reabsorption in aged animals that may synthesize less vitamin C. J. Cell. Physiol. 232: 2418–2426, 2017. © 2016 Wiley Periodicals, Inc. Although we know that vitamin C levels decrease with age, the adaptive physiological mechanisms used by the kidney for vitamin C reabsorption during aging remain unknown. In this study, we used an animal model of accelerated senescence (SAMP8 mice) to define the morphological alterations and aging‐induced changes in the expression of vitamin C transporters in renal tissue.

  March 24, 2017   doi: 10.1002/jcp.25504   open full text
• Lipocalin‐2 (NGAL) Attenuates Autophagy to Exacerbate Cardiac Apoptosis Induced by Myocardial Ischemia.
  Hye Kyoung Sung, Yee Kwan Chan, Meng Han, James Won Suk Jahng, Erfei Song, Eric Danielson, Thorsten Berger, Tak W. Mak, Gary Sweeney.
  Journal of Cellular Physiology. March 24, 2017
  Lipocalin‐2 (Lcn2; also termed neutrophil gelatinase‐associated lipocalin (NGAL)) levels correlate positively with heart failure (HF) yet mechanisms via which Lcn2 contributes to the pathogenesis of HF remain unclear. In this study, we used coronary artery ligation surgery to induce ischemia in wild‐type (wt) mice and this induced a significant increase in myocardial Lcn2. We then compared wt and Lcn2 knockout (KO) mice and observed that wt mice showed greater ischemia‐induced caspase‐3 activation and DNA damage measured by TUNEL than Lcn2KO mice. Analysis of autophagy by LC3 and p62 Western blotting, LC3 immunohistochemistry and transmission electron microscopy (TEM) indicated that Lcn2 KO mice had a greater ischemia‐induced increase in autophagy. Lcn2KO were protected against ischemia‐induced cardiac functional abnormalities measured by echocardiography. Upon treating a cardiomyocyte cell line (h9c2) with Lcn2 and examining AMPK and ULK1 phosphorylation, LC3 and p62 by Western blot as well as tandem fluorescent RFP/GFP‐LC3 puncta by immunofluorescence, MagicRed assay for lysosomal cathepsin activity and TEM we demonstrated that Lcn2 suppressed autophagic flux. Lcn2 also exacerbated hypoxia‐induced cytochromc c release from mitochondria and caspase‐3 activation. We generated an autophagy‐deficient H9c2 cell model by overexpressing dominant‐negative Atg5 and found significantly increased apoptosis after Lcn2 treatment. In summary, our data indicate that Lcn2 can suppress the beneficial cardiac autophagic response to ischemia and that this contributes to enhanced ischemia‐induced cell death and cardiac dysfunction. J. Cell. Physiol. 232: 2125–2134, 2017. © 2016 Wiley Periodicals, Inc. This study examined the regulation of autophagy by Lcn2 and the functional significance in the heart. Our data led us to conclude that, at least in part, via inhibition of cardiomyocyte autophagy Lcn2 exacerbated stress‐induced cell death and cardiac dysfunction.

  March 24, 2017   doi: 10.1002/jcp.25672   open full text
• MAPK3/1 participates in the activation of primordial follicles through mTORC1‐KITL signaling.
  Yu Zhao, Yu Zhang, Jia Li, Nana Zheng, Xiaoting Xu, Jing Yang, Guoliang Xia, Meijia Zhang.
  Journal of Cellular Physiology. March 24, 2017
  The majority of ovarian primordial follicles are preserved in a dormant state to maintain the female reproductive lifespan, and only a few primordial follicles are activated to enter the growing follicle pool in each wave. Recent studies have shown that primordial follicular activation depends on mammalian target of rapamycin complex 1 (mTORC1)‐KIT ligand (KITL) signaling in pre‐granulosa cells and its receptor (KIT)‐phosphoinositol 3 kinase (PI3K) signaling in oocytes. However, the upstream regulator of mTORC1 signaling is unclear. The results of the present study showed that the phosphorylated mitogen‐activated protein kinase3/1 (MAPK3/1) protein is expressed in some primordial follicles and all growing follicles. Culture of 3 days post‐parturition (dpp) ovaries with the MAPK3/1 signaling inhibitor U0126 significantly reduced the number of activated follicles and was accompanied by dramatically reduced granulosa cell proliferation and increased oocyte apoptosis. Western blot and immunofluorescence analyses showed that U0126 significantly decreased the phosphorylation levels of Tsc2, S6K1, and rpS6 and the expression of KITL, indicating that U0126 inhibits mTORC1‐KITL signaling. Furthermore, U0126 decreased the phosphorylation levels of Akt, resulting in a decreased number of oocytes with Foxo3 nuclear export. To further investigate MAPK3/1 signaling in primordial follicle activation, we used phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor bpV(HOpic) to promote primordial follicle activation. In this model, U0126 also inhibited the activation of primordial follicles and mTORC1 signaling. Thus, these results suggest that MAPK3/1 participates in primordial follicle activation through mTORC1‐KITL signaling. Primordial follicular activation depends on mammalian target of rapamycin complex 1 (mTORC1)‐KIT ligand (KITL) signaling in pre‐granulosa cells and its receptor (KIT)‐phosphoinositol 3 kinase (PI3K) signaling in oocytes. Here, we have shown that MAPK3/1 signaling participates in the activation of primordial follicles through mTORC1 signaling and the expression of KITL in pre‐granulosa cells to activate the PI3K‐Akt pathway.

  March 24, 2017   doi: 10.1002/jcp.25868   open full text
• p62 Promotes Amino Acid Sensitivity of mTOR Pathway and Hepatic Differentiation in Adult Liver Stem/Progenitor Cells.
  Masakazu Sugiyama, Tomoharu Yoshizumi, Yoshihiro Yoshida, Yuki Bekki, Yoshihiro Matsumoto, Shohei Yoshiya, Takeo Toshima, Toru Ikegami, Shinji Itoh, Norifumi Harimoto, Shinji Okano, Yuji Soejima, Ken Shirabe, Yoshihiko Maehara.
  Journal of Cellular Physiology. March 24, 2017
  Autophagy is a homeostatic process regulating turnover of impaired proteins and organelles, and p62 (sequestosome‐1, SQSTM1) functions as the autophagic receptor in this process. p62 also functions as a hub for intracellular signaling such as that in the mammalian target of rapamycin (mTOR) pathway. Liver stem/progenitor cells have the potential to differentiate to form hepatocytes or cholangiocytes. In this study, we examined effects of autophagy, p62, and associated signaling on hepatic differentiation. Adult stem/progenitor cells were isolated from the liver of mice with chemically induced liver injury. Effects of autophagy, p62, and related signaling pathways on hepatic differentiation were investigated by silencing the genes for autophagy protein 5 (ATG5) and/or SQSTM1/p62 using small interfering RNAs. Hepatic differentiation was assessed based on increased albumin and hepatocyte nuclear factor 4α, as hepatocyte markers, and decreased cytokeratin 19 and SOX9, as stem/progenitor cell markers. These markers were measured using quantitative RT‐PCR, immunofluorescence, and Western blotting. ATG5 silencing decreased active LC3 and increased p62, indicating inhibition of autophagy. Inhibition of autophagy promoted hepatic differentiation in the stem/progenitor cells. Conversely, SQSTM1/p62 silencing impaired hepatic differentiation. A suggested mechanism for p62‐dependent hepatic differentiation in our study was activation of the mTOR pathway by amino acids. Amino acid activation of mTOR signaling was enhanced by ATG5 silencing and suppressed by SQSTM1/p62 silencing. Our findings indicated that promoting amino acid sensitivity of the mTOR pathway is dependent on p62 accumulated by inhibition of autophagy and that this process plays an important role in the hepatic differentiation of stem/progenitor cells. J. Cell. Physiol. 232: 2112–2124, 2017. © 2016 Wiley Periodicals, Inc. Inhibition of autophagy by siRNA for ATG5 promoted hepatic differentiation in liver stem/progenitor cells. Autophagic adaptor protein, p62, was an important molecule to promote hepatic differentiation by inhibition of autophagy. p62 promoted amino acid sensitivity of the mTOR pathway and hepatic differentiation in liver stem/progenitor cells.

  March 24, 2017   doi: 10.1002/jcp.25653   open full text
• T11TS repress gliomagenic apoptosis of bone marrow hematopoietic stem cells.
  Somnath Mondal, Iman Hazra, Ankur Datta, Omar Faruk Sk Md, Saibal Moitra, Santanu Kumar Tripathi, Swapna Chaudhuri.
  Journal of Cellular Physiology. March 24, 2017
  Combating gliomagenic global immunosuppression is one of the emerging key for improving prognosis in malignant glioma. Apoptosis plays a pivotal role within the adult hematopoietic system particularly in regulating the cells of immune system. Gliomagenic regulation of apoptotic mediators within bone marrow milieu has not been elucidated. We previously demonstrated that administration of membrane glycopeptides T11 target structure (T11TS) not only rejuvenate bone marrow hematopoietic stem cells (BMHSCs) from glioma mediated hibernation by inhibiting gliomagenic overexpression of Ang‐1/Tie‐2 but also stimulate glioma mediated diminution of expression CD34, c‐kit, and Sca‐1 markers. In the present study, we investigated the impact of glioma on apoptotic signaling cascades of BMHSCs and consequences following T11TS therapy. Bone marrow smear and Annexin V staining confirm gliomagenic acceleration of apoptotic fate of BMHSCs whereas T11TS treatment in glioma‐bearing rats disrupted apoptosis of BMHSCs. Flowcytometry, immunoblotting, and immunofluorescence imagining results revealed multi potent T11TS not only significantly downregulates gliomagenic overexpression of Fas, Fas L, Bid, and caspase‐8, the pro‐apoptotic extrinsic mediators but also strongly inhibits cytosolic release of cytochrome‐c, Apf‐1, and Bax to deactivate gliomagenic caspase‐9, 3 the key intrinsic apoptotic mediators followed by up modulation of anti‐apoptotic Bcl‐2 in glioma associated HSCs. T11TS is also able to diminish the perforin‐granzyme B mediated apoptotic verdict of BMHSCs during gliomagenesis. The anti‐apoptotic action of T11TS on glioma associated BMHSCs provide a crucial insight into how T11TS exerts its immunomodulatory action against glioma mediated immune devastation. T11TS inhibits gliomagenic acceleration of premature apoptosis of bone marrow hematopoietic stem cells by repressing extrinsic, intrinsic, and granzyme B mediated apoptogenic cascades. Flowcytometry, immunoblotting experiments and immunofluorescence imagining results revealed multi potent T11TS significantly downregulates gliomaogenic overexpression of the key apoptotic mediators of all the above three pathways.

  March 24, 2017   doi: 10.1002/jcp.25874   open full text
• Mild Aerobic Exercise Training Hardly Affects the Diaphragm of mdx Mice.
  Giuseppe Morici, Monica Frinchi, Alessandro Pitruzzella, Valentina Di Liberto, Rosario Barone, Andrea Pace, Valentina Di Felice, Natale Belluardo, Francesco Cappello, Giuseppa Mudò, Maria R. Bonsignore.
  Journal of Cellular Physiology. March 24, 2017
  In the mdx mice model of Duchenne Muscular Dystrophy (DMD), mild endurance exercise training positively affected limb skeletal muscles, whereas few and controversial data exist on the effects of training on the diaphragm. The diaphragm was examined in mdx (C57BL/10ScSn‐Dmdmdx) and wild‐type (WT, C57BL/10ScSc) mice under sedentary conditions (mdx‐SD, WT‐SD) and during mild exercise training (mdx‐EX, WT‐EX). At baseline, and after 30 and 45 days (training: 5 d/wk for 6 weeks), diaphragm muscle morphology and Cx39 protein were assessed. In addition, tissue levels of the chaperonins Hsp60 and Hsp70 and the p65 subunit of nuclear factor‐kB (NF‐kB) were measured in diaphragm, gastrocnemius, and quadriceps in each experimental group at all time points. Although morphological analysis showed unchanged total area of necrosis/regeneration in the diaphragm after training, there was a trend for larger areas of regeneration than necrosis in the diaphragm of mdx‐EX compared to mdx‐SD mice. However, the levels of Cx39, a protein associated with active regeneration in damaged muscle, were similar in the diaphragm of mdx‐EX and mdx‐SD mice. Hsp60 significantly decreased at 45 days in the diaphragm, but not in limb muscles, in both trained and sedentary mdx compared to WT mice. In limb muscles, but not in the diaphragm, Hsp70 and NF‐kB p65 levels were increased in mdx mice irrespective of training at 30 and 45 days. Therefore, the diaphragm of mdx mice showed little inflammatory and stress responses over time, and appeared hardly affected by mild endurance training. J. Cell. Physiol. 232: 2044–2052, 2017. © 2016 Wiley Periodicals, Inc. Differently from limb muscles, the diaphragm of mdx mice showed no evidence of decreased muscle damage over time, as measured by Cx39 protein, in exercised compared to sedentary mice. Such findings were associated with progressive decrease of Hsp60 expression, and unchanged Hsp70 and NF‐kB expression in the diaphragm, irrespective of training. The data suggest that mild exercise training hardly affects the diaphragm muscle in the mdx mice model of Duchenne Muscular Dystrophy (DMD).

  March 24, 2017   doi: 10.1002/jcp.25573   open full text
• Mimicking exercise in three‐dimensional bioengineered skeletal muscle to investigate cellular and molecular mechanisms of physiological adaptation.
  Andreas M. Kasper, Daniel C. Turner, Neil R. W. Martin, Adam P. Sharples.
  Journal of Cellular Physiology. March 21, 2017
  Bioengineering of skeletal muscle in vitro in order to produce highly aligned myofibres in relevant three dimensional (3D) matrices have allowed scientists to model the in vivo skeletal muscle niche. This review discusses essential experimental considerations for developing bioengineered muscle in order to investigate exercise mimicking stimuli. We identify current knowledge for the use of electrical stimulation and co‐culture with motor neurons to enhance skeletal muscle maturation and contractile function in bioengineered systems in vitro. Importantly, we provide a current opinion on the use of acute and chronic exercise mimicking stimuli (electrical stimulation and mechanical overload) and the subsequent mechanisms underlying physiological adaptation in 3D bioengineered muscle. We also identify that future studies using the latest bioreactor technology, providing simultaneous electrical and mechanical loading and flow perfusion in vitro, may provide the basis for advancing knowledge in the future. We also envisage, that more studies using genetic, pharmacological, and hormonal modifications applied in human 3D bioengineered skeletal muscle may allow for an enhanced discovery of the in‐depth mechanisms underlying the response to exercise in relevant human testing systems. Finally, 3D bioengineered skeletal muscle may provide an opportunity to be used as a pre‐clinical in vitro test‐bed to investigate the mechanisms underlying catabolic disease, while modelling disease itself via the use of cells derived from human patients without exposing animals or humans (in phase I trials) to the side effects of potential therapies. This review discusses the current understanding, advances, and future directions for the stimulation of three‐dimensional bioengineered skeletal muscle to investigate the mechanisms of physiological adaptation to exercise.

  March 21, 2017   doi: 10.1002/jcp.25840   open full text
• TGF‐β Inhibits Osteogenesis by Upregulating the Expression of Ubiquitin Ligase SMURF1 via MAPK‐ERK Signaling.
  Xuewu Sun, Ziang Xie, Yan Ma, Xin Pan, Jiying Wang, Zhijun Chen, Peihua Shi.
  Journal of Cellular Physiology. March 21, 2017
  High incidence of osteoporotic fractures emphasizes the necessity of developing effective measures to promote osteogenesis. In our study, we investigated a possible role of MAPK‐ERK signaling in the TGF‐β‐mediated osteoblastic differentiation. Our results indicated that TGF‐β activated the MAPK‐ERK pathway and inhibited osteogenesis in mesenchymal pluripotent cell line, C3H10T1/2, and preosteoblastic cell line, MC3T3 cells. And the downregulation of MAPK‐ERK signaling using pharmacological inhibitor U0126 and RNA interference rescued osteoblast differentiation suppressed by TGF‐β, which was confirmed by Alkaline phosphatase (ALP) staining and alizarrn red staining and the enhanced expression of osteogenesic markers. Western blotting analysis indicated that TGF‐β induced protein expression of E3 ubiquitin‐protein ligase SMURF1, which contributed to the degradation of RUNX2 and SMAD1 as evidenced by SMURF1 inhibition using RNA interference and proteasome inhibitor MG132. Moreover, we observed that the expression of SMURF1 was decreased, while that of SMAD1 and RUNX2 increased by MAPK‐ERK inhibitor U0126 in TGF‐β‐treated differentiating preosteoblasts, suggesting that MAPK‐ERK regulated the transcription of osteogenesis‐related genes. Furthermore, a synergistic effect between U0126 and bone morphogenic protein (BMP)‐2 on osteoblast differentiation and bone formation was observed both in cell cultures and experimental animals. In conclusion, our results revealed that TGF‐β inhibited osteoblastic differentiation by inducing the MAPK‐ERK pathway which upregulated the expression of ubiquitin ligase SMURF1 and resulted in reduced presence of osteogenic proteins. In addition, the potentiation of BMP‐2 on osteogenic activity by ERK1/2 inhibitor U0126 suggests that it may have potential clinical utility for promoting osteogenesis in bone fracture repair. This article is protected by copyright. All rights reserved

  March 21, 2017   doi: 10.1002/jcp.25920   open full text
• Artesunate Inhibits RANKL‐induced Osteoclastogenesis and Bone Resorption In Vitro and Prevents LPS‐induced Bone Loss In Vivo.
  Cheng‐Ming Wei, Qian Liu, Fang‐Ming Song, Xi‐Xi Lin, Yi‐Ji Su, Jiake Xu, Lin Huang, Shao‐Hui Zong, Jin‐Min Zhao.
  Journal of Cellular Physiology. March 15, 2017
  Osteoclasts are multinuclear giant cells responsible for bone resorption in lytic bone diseases such as osteoporosis, arthritis, periodontitis, and bone tumors. Due to the severe side‐effects caused by the currently available drugs, a continuous search for novel bone‐protective therapies is essential. Artesunate (Art), the water‐soluble derivative of artemisinin has been investigated owing to its anti‐malarial properties. However, its effects in osteoclastogenesis have not yet been reported. In this study, Art was shown to inhibit the nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis, the mRNA expression of osteoclastic‐specific genes, and resorption pit formation in a dose‐dependent manner in primary bone marrow‐derived macrophages cells (BMMs). Furthermore, Art markedly blocked the RANKL‐induced osteoclastogenesis by attenuating the degradation of IκB and phosphorylation of NF‐κB p65. Consistent with the in vitro results, Art inhibited lipopolysaccharide (LPS)‐induced bone resorption by suppressing the osteoclastogenesis. Together our data demonstrated that Art inhibits RANKL‐induced osteoclastogenesis by suppressing the NF‐κB signaling pathway and that it is a promising agent for the treatment of osteolytic diseases. This article is protected by copyright. All rights reserved

  March 15, 2017   doi: 10.1002/jcp.25907   open full text
• Visualization of Stimulus‐specific Heterogeneous Activation of Individual Vascular Smooth Muscle Cells in Aortic Tissues.
  Satoshi Komatsu, Toshio Kitazawa, Mitsuo Ikebe.
  Journal of Cellular Physiology. March 11, 2017
  Intercellular communication among autonomic nerves, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) plays a central role in an uninterrupted regulation of blood flow through vascular contractile machinery. Impairment of this communication is linked to development of vascular diseases such as hypertension, cerebral/coronary vasospasms, aortic aneurism, and erectile dysfunction. Although the basic concept of the communication as a whole has been studied, the spatiotemporal correlation of ECs/VSMCs in tissues at the cellular level is unknown. Here we show a unique VSMC response to ECs during contraction and relaxation of isolated aorta tissues through visualization of spatiotemporal activation patterns of smooth muscle myosin II. ECs in the intimal layer dictate the stimulus‐specific heterogeneous activation pattern of myosin II in VSMCs within distinct medial layers. Myosin light chain (MLC) phosphorylation (active form of myosin II) gradually increases towards outer layers (approximately 3 fold higher MLC phosphorylation at the outermost layer than that of the innermost layer), presumably by release of an intercellular messenger, nitric oxide (NO). Our study also demonstrates that the MLC phosphorylation at the outermost layer in spontaneously hypertensive rats (SHR) during NO‐induced relaxation is quite high and approximately 10 fold higher than that of its counterpart, the Wister‐Kyoto rats (WKY), suggesting that the distinct pattern of myosin II activation within tissues is important for vascular protection against elevated blood pressure. This article is protected by copyright. All rights reserved

  March 11, 2017   doi: 10.1002/jcp.25903   open full text
• HMGB1 down‐regulation mediates terameprocol vascular anti‐proliferative effect in experimental pulmonary hypertension.
  Rita Nogueira‐Ferreira, Manuel J. Ferreira‐Pinto, Ana Filipa Silva, Rui Vitorino, Joana Justino, Raquel Costa, Daniel Moreira‐Gonçalves, Jean‐François Quignard, Thomas Ducret, Jean‐Pierre Savineau, Adelino F. Leite‐Moreira, Rita Ferreira, Tiago Henriques‐Coelho.
  Journal of Cellular Physiology. March 09, 2017
  Pulmonary arterial hypertension (PAH) is a progressive disease with a poor prognosis. Pulmonary artery smooth muscle cells (PASMCs) play a crucial role in PAH pathophysiology, displaying a hyperproliferative, and apoptotic‐resistant phenotype. In the present study, we evaluated the potential therapeutic role of terameprocol (TMP), an inhibitor of cellular proliferation and promoter of apoptosis, in a well‐established pre‐clinical model of PAH induced by monocrotaline (MCT) and studied the biological pathways modulated by TMP in PASMCs. Wistar rats injected with MCT or saline (SHAM group) were treated with TMP or vehicle. On day 21 after injection, we assessed bi‐ventricular hemodynamics and cardiac and pulmonary morphometry. The effects of TMP on PASMCs were studied in a primary culture isolated from SHAM and MCT‐treated rats, using an iTRAQ‐based proteomic approach to investigate the molecular pathways modulated by this drug. In vivo, TMP significantly reduced pulmonary and cardiac remodeling and improved cardiac function in PAH. In vitro, TMP inhibited proliferation and induced apoptosis of PASMCs. A total of 65 proteins were differentially expressed in PASMCs from MCT rats treated with TMP, some of which involved in the modulation of transforming growth factor beta pathway and DNA transcription. Anti‐proliferative effect of TMP seems to be explained, at least in part, by the down‐regulation of the transcription factor HMGB1. Our findings support the beneficial role of TMP in PAH and suggest that it may be an effective therapeutic option to be considered in the clinical management of PAH. TMP‐reduced pulmonary and cardiac remodeling and hemodynamic features of PAH. Its vascular anti‐proliferative effect seems to be mediated by the protein HMGB1. TMP may be an effective therapeutic option in the clinical management of PAH.

  March 09, 2017   doi: 10.1002/jcp.25763   open full text
• Phosphoregulation of K+‐Cl− Cotransporters During Cell Swelling: Novel Insights.
  Rachelle Frenette‐Cotton, Andrée‐Anne Marcoux, Alexandre P. Garneau, Micheline Noel, Paul Isenring.
  Journal of Cellular Physiology. March 09, 2017
  The K+‐Cl− cotransporters (KCCs) belong to the cation‐Cl− cotransporter family and consist of 4 isoforms and many splice variants. Their main role is to promote electroneutral efflux of K+ and Cl− ions across the surface of many cell types and, thereby, to regulate intracellular ion concentration, cell volume and epithelial salt movement. These transport systems are induced by an increase in cell volume and are less active at lower intracellular [Cl−] (Cli), but the mechanisms at play are still ill‐defined. In this work, we have exploited the Xenopus laevis expression system to study the role of lysine‐deficient protein kinases (WNKs), protein phosphatases 1 (PP1s) and SPS1‐related proline/alanine‐rich kinase (SPAK) in KCC4 regulation during cell swelling. We have found that WNK4 and PP1 regulate KCC4 activity as part of a common signalling module, but that they do not exert their effects through SPAK or carrier dephosphorylation. We have also found that the phosphatases at play include PP1α and PP1γ1, but that WNK4 acts directly on the PP1s instead of the opposite. Unexpectedly, however, both cell swelling and a T926A substitution in the C‐terminus of full‐length KCC4 led to higher levels of heterologous K+‐Cl− cotransport and overall carrier phosphorylation. These results imply that the response to cell swelling must also involve allosteric‐sensitive kinase‐dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation. This article is protected by copyright. All rights reserved

  March 09, 2017   doi: 10.1002/jcp.25899   open full text
• Cytokine networks and their association with Helicobacter pylori infection in gastric carcinoma.
  Vahid Bagheri, Bahram Memar, Amir Abbas Momtazi, Amirhossein Sahebkar, Mehran Gholamin, Mohammad Reza Abbaszadegan.
  Journal of Cellular Physiology. March 07, 2017
  Cytokine networks as dynamic networks are pivotal aspects of tumor immunology, especially in gastric cancer (GC), in which infection, inflammation, and antitumor immunity are key elements of disease progression. In this review, we describe functional roles of well‐known GC‐modulatory cytokines, highlight the functions of cytokines with more recently described roles in GC, and emphasize the therapeutic potential of targeting the complex cytokine milieu. We also focus on the role of Helicobacter pylori (HP)‐induced inflammation in GC and discuss how HP‐induced chronic inflammation can lead to the induction of stem cell hyperplasia, morphological changes in gastric mucosa and GC development. In this review, we describe functional roles of well‐known gastric cancer (GC)‐modulatory cytokines, highlight the functions of cytokines with more recently described roles in GC, and emphasize the therapeutic potential of targeting the complex cytokine milieu. We also focus on the role of Helicobacter pylori (HP)‐induced inflammation in GC and discuss how HP‐induced chronic inflammation can lead to GC.

  March 07, 2017   doi: 10.1002/jcp.25822   open full text
• Bone Loss in C57BL/6J‐OlaHsd Mice, a Substrain of C57BL/6J Carrying Mutated Alpha‐Synuclein and Multimerin‐1 Genes.
  Tamar Liron, Bitya Raphael, Sahar Hiram‐Bab, Itai A Bab, Yankel Gabet.
  Journal of Cellular Physiology. March 07, 2017
  The inbred mouse strain C57BL/6 is commonly used for the generation of transgenic mouse and is a well‐established strain in bone research. Different vendors supply different substrains of C57BL/6J as wild‐type animals when genetic drift did not incur any noticeable phenotype. However, we sporadically observed drastic differences in the bone phenotype of ‘WT’ C57BL/6J mice originating from different labs and speculated that these variations are attributable, at least in part, to the variation between C57BL/6J substrains, which is often overlooked. C57BL/6J‐OlaHsd is a commonly used substrain that despite a well‐defined deletion in the alpha‐synuclein (Snca) and multimerin‐1 (Mmrn1) genes, was reported to display no obvious phenotype and is used as WT control. Here we compared the bone phenotype of C57BL/6J‐OlaHsd (6J‐OLA) to C57BL/6J‐RccHsd (6J‐RCC) and to the original C57BL/6J (6J‐JAX). Using µCT analysis, we found that 6J‐OLA mice display a significantly lower trabecular bone mass compared to 6J‐RCC and 6J‐JAX. PCR analysis revealed that both the Snca and Mmrn1 genes are expressed in bone tissue of 6J‐RCC animals but not of 6J‐OLA mutants, suggesting either or both genes play a role in bone metabolism. In vitro analysis demonstrated increase in osteoclasts number and decreased osteoblast mineralization in cells derived from 6J‐OLA compared with 6J‐RCC. Our data may shed light on unexplained differences in basal bone measurements between different research centers and reiterate the importance of specifying the exact substrain type. In addition, our findings describe the physiological role for Mmrn1 and/or Snca in bone remodeling. This article is protected by copyright. All rights reserved

  March 07, 2017   doi: 10.1002/jcp.25895   open full text
• 2‐Methoxyestradiol Impacts on Amino Acids‐mediated Metabolic Reprogramming in Osteosarcoma Cells by Interaction with NMDA Receptor.
  Magdalena Gorska‐Ponikowska, Ugo Perricone, Alicja Kuban‐Jankowska, Giosue Lo Bosco, Giampaolo Barone.
  Journal of Cellular Physiology. March 06, 2017
  Deregulation of serine and glycine metabolism, have been identified to function as metabolic regulators in supporting tumor cell growth. The role of serine and glycine in regulation of cancer cell proliferation is complicated, dependent on concentrations of amino acids and tissue‐specific. D‐serine and glycine are coagonists of N‐methyl‐D‐aspartate receptor subunit GRIN1. Importantly, NMDA receptors are widely expressed in cancer cells and play an important role in regulation of cell death, proliferation and metabolism of numerous malignancies. The aim of the present work was to associate the metabolism of glycine and D‐serine with the anticancer activity of 2‐methoxyestradiol. 2‐methoxyestradiol is a potent anticancer agent but also a physiological 17β‐ estradiol metabolite. In the study we have chosen two malignant cell lines expressing functional GRIN1 receptors, i.e. osteosarcoma 143B and breast cancer MCF7. We used MTS assay, migration assay, flow cytometric analyses, western blotting and immunoprecipitation techniques as well as molecular modeling studies. We have demonstrated the extensive crosstalk between the deregulated metabolic network and cancer cell signaling. Herein, we observed an anticancer effect of high concentrations of glycine and D‐serine in osteosarcoma cells. In contrast, the amino acids when used at low, physiological concentrations induced the proliferation and migration of osteosarcoma and breast cancer cells. Importantly, the pro‐cancergogenic effects of both glycine and D‐serine where abrogated by the usage of 2‐methoxyestradiol at both physiological and pharmacological relevant concentrations. The obtained data confirmed that 2‐methoxyestradiol may be a physiological anticancer molecule. This article is protected by copyright. All rights reserved

  March 06, 2017   doi: 10.1002/jcp.25888   open full text
• Swelling‐induced Chloride Current in Glioblastoma Proliferation, Migration and Invasion.
  Raymond Wong, Wenliang Chen, Xiao Zhong, James T. Rutka, Zhong‐Ping Feng, Hong‐Shuo Sun.
  Journal of Cellular Physiology. March 06, 2017
  Glioblastoma (GBM) remains as the most common and aggressive brain tumor. The survival of GBM has been linked to the aberrant activation of swelling‐induced chloride current ICl,swell. In this study, we investigated the effects of ICl,swell on cell viability, proliferation and migration in the human GBM cell lines, U251 and U87, using a combination of patch clamp electrophysiology, MTT, colony formation, wound healing assays and Western immunoblotting. First, we showed that the specific inhibitor of ICl,swell, DCPIB, potently reduced the ICl,swell in U87 cells. Next, in both U87 and U251 cells, we found that DCPIB reduced GBM viability, proliferation, colony formation, migration and invasion. In addition, our Western immunoblot assay showed that DCPIB‐treated U251 cells had a reduction in JAK2, STAT3, and Akt phosphorylation, thus suggesting that DCPIB potentially suppresses GBM functions through inhibition of the JAK2/STAT3 and PI3K/Akt signaling pathways. Therefore, the ICl,swell may be a potential drug target for GBM. This article is protected by copyright. All rights reserved

  March 06, 2017   doi: 10.1002/jcp.25891   open full text
• Reciprocal regulation between βTrCP and Smurf1 suppresses proliferative capacity of liver cancer cells.
  Yue Zhang, Wenhua Wang, Si Cai, Yan Chen, Qinwan Wang, Qiuhui Pan, Fenyong Sun, Jiayi Wang.
  Journal of Cellular Physiology. March 06, 2017
  We previously reported that both the ubiquitin E3 ligases βTrCP (beta‐transducin repeat‐containing E3 ubiquitin protein ligase) and Smurf1 (SMAD‐specific E3 ubiquitin protein ligase 1) play similar antitumorigenic roles in liver cancer cells. However, whether and how they are reciprocally regulated remains elusive. Here, we show that βTrCP interacts with Smurf1 through the 7 × tryptophan (W) aspartic acid (D)(WD) 40 and the region homologous to the E6‐AP carboxyl terminus (HECT) domains, which are the E3 ligase domains of βTrCP and Smurf1, respectively. The E3 ligase domains of βTrCP and Smurf1 are also critical for maintaining the protein expressions of Smurf1 and βTrCP. Moreover, a positive correlation between βTrCP and Smurf1 was also revealed by tissue microarray analysis, indicating that this relationship might be important in liver cancer. Further, we found that Smurf1 increases the protein stability of βTrCP, possibly by reducing autoubiquitination of βTrCP, and vice versa. Interestingly, such effects depended on the presence of E3 ligase domains. Importantly, depletion of Smurf1‐ or βTrCP‐enhanced proliferative capacity of liver cancer cells could be partially reversed by overexpression of wild‐type βTrCP or Smurf1 but not their E3 ligase‐dead mutants. Collectively, a reciprocal post‐translational regulation between βTrCP and Smurf1 has been uncovered in this study. Simultaneous enhancement of βTrCP and Smurf1 functions might be helpful in the treatment of liver cancer. In the present study, we identified a close interaction between βTrCP and Smurf1, uncovering a reciprocal post‐translational regulation between RING and HECT E3s in liver cancer cells. Simultaneous stimulation of βTrCP and Smurf1 functions may help to conquer liver cancer.

  March 06, 2017   doi: 10.1002/jcp.25780   open full text
• TUDCA: An Agonist of the Bile Acid Receptor GPBAR1/TGR5 With Anti‐Inflammatory Effects in Microglial Cells.
  Natalia Yanguas‐Casás, M. Asunción Barreda‐Manso, Manuel Nieto‐Sampedro, Lorenzo Romero‐Ramírez.
  Journal of Cellular Physiology. March 03, 2017
  Bile acids are steroid acids found in the bile of mammals. The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is neuroprotective in different animal models of stroke and neurological diseases. We have previously shown that TUDCA has anti‐inflammatory effects on glial cell cultures and in a mouse model of acute neuroinflammation. We show now that microglial cells (central nervous system resident macrophages) express the G protein‐coupled bile acid receptor 1/Takeda G protein‐coupled receptor 5 (GPBAR1/TGR5) in vivo and in vitro. TUDCA binding to GPBAR1/TGR5 caused an increase in intracellular cAMP levels in microglia that induced anti‐inflammatory markers, while reducing pro‐inflammatory ones. This anti‐inflammatory effect of TUDCA was inhibited by small interference RNA for GPBAR1/TGR5 receptor, as well as by treatment with a protein kinase A (PKA) inhibitor. In the mouse model of acute neuroinflammation, treating the animals with TUDCA was clearly anti‐inflammatory. TUDCA biased the microglial phenotype in vivo and in vitro toward the anti‐inflammatory. The bile acid receptor GPBAR1/TGR5 could be a new therapeutic target for pathologies coursing with neuroinflammation and microglia activation, such as traumatic brain injuries, stroke, or neurodegenerative diseases. TUDCA and other GPBAR1/TGR5 agonists need to be further investigated, to determine their potential in attenuating the neuropathologies associated with microglia activation. J. Cell. Physiol. 232: 2231–2245, 2017. © 2016 Wiley Periodicals, Inc. We provide evidence that tauroursodeoxycholic acid (TUDCA) exerts anti‐inflammatory effects through its binding to GPBAR1/TGR5, and the consequent increase in intracellular cAMP levels in microglia. Besides, TUDCA treatment biased microglia towards the anti‐inflammatory phenotype under pro‐inflammatory conditions in vitro and in vivo. TUDCA and other GPBAR1/TGR5 agonists are potential therapeutic targets for the treatment of neuropathologies associated with microglia activation.

  March 03, 2017   doi: 10.1002/jcp.25742   open full text
• Association between AXL, Hippo Transducers, and Survival Outcomes in Male Breast Cancer.
  Anna Di Benedetto, Marcella Mottolese, Francesca Sperati, Cristiana Ercolani, Luigi Di Lauro, Laura Pizzuti, Patrizia Vici, Irene Terrenato, Abeer M. Shaaban, Matthew P. Humphries, Sreekumar Sundara‐Rajan, Maddalena Barba, Valerie Speirs, Ruggero De Maria, Marcello Maugeri‐Saccà.
  Journal of Cellular Physiology. March 03, 2017
  Male breast cancer (MBC) is an uncommon malignancy. We have previously reported that the expression of the Hippo transducers TAZ/YAP and their target CTGF was associated with inferior survival in MBC patients. Preclinical evidence demonstrated that Axl is a transcriptional target of TAZ/YAP. Thus, we herein assessed AXL expression to further investigate the significance of active TAZ/YAP‐driven transcription in MBC. For this study, 255 MBC samples represented in tissue microarrays were screened for AXL expression, and 116 patients were included. The association between categorical variables was verified by the Pearson's Chi‐squared test of independence (2‐tailed) or the Fisher Exact test. The relationship between continuous variables was tested with the Pearson's correlation coefficient. The Kaplan–Meier method was used for estimating survival curves, which were compared by log‐rank test. Factors potentially impacting 10‐year and overall survival were verified in Cox proportional regression models. AXL was positively associated with the TAZ/CTGF and YAP/CTGF phenotypes (P = 0.001 and P = 0.002, respectively). Patients with TAZ/CTGF/AXL‐ or YAP/CTGF/AXL‐expressing tumors had inferior survival compared with non‐triple‐positive patients (log rank P = 0.042 and P = 0.048, respectively). The variables TAZ/CTGF/AXL and YAP/CTGF/AXL were adverse factors for 10‐year survival in the multivariate Cox models (HR 2.31, 95%CI:1.02–5.22, P = 0.045, and HR 2.27, 95%CI:1.00–5.13, P = 0.050). Nearly comparable results were obtained from multivariate analyses of overall survival. The expression pattern of AXL corroborates the idea of the detrimental role of TAZ/YAP activation in MBC. Overall, Hippo‐linked biomarkers deserve increased attention in this rare disease. J. Cell. Physiol. 232: 2246–2252, 2017. © 2016 Wiley Periodicals, Inc. (1) The expression pattern of AXL corroborates the idea of TAZ/YAP activation in male breast cancer; (2) patients whose tumors had the TAZ/CTGF/AXL and YAP/CTGF/AXL molecular backgrounds experienced adverse survival outcomes compared with their negative counterparts.

  March 03, 2017   doi: 10.1002/jcp.25745   open full text
• Exhaustive Training Leads to Hepatic Fat Accumulation.
  Alisson L. da Rocha, Ana P. Pinto, Giovana R. Teixeira, Bruno C. Pereira, Luciana C. Oliveira, Adriana C. Silva, Gustavo P. Morais, Dennys E. Cintra, José R. Pauli, Adelino S. R. da Silva.
  Journal of Cellular Physiology. March 03, 2017
  Recently, we demonstrated that an overtraining (OT) protocol for mice based on downhill running sessions increased the hepatic phosphorylation of 70‐kDa ribosomal protein S6 kinase 1 (S6K1; Thr389), a downstream target of the mammalian target of rapamycin complex 1 (mTORC1). In liver, the overactivation of the Akt/mTORC1 pathway induces lipogenesis via regulation of the action of sterol regulatory element binding protein‐1 (SREBP‐1) at multiple steps. Herein, we verified the effects of three running OT models with same external load (i.e., the product between intensity and volume of training), but performed in downhill, uphill and without inclination, on the proteins related to the mTORC1 signaling pathway, the protein content of the SREBP‐1, ACC, and FAS, and the morphological characteristics of C57BL/6 mouse livers. In summary, the downhill running‐induced OT model up‐regulated the levels of major proteins of the mTORC1 signaling pathway, the protein levels of SREBP‐1 (p125 precursor) and induced signs of cell swelling accompanied by acute inflammation. The other two OT protocols performed uphill and without inclination did not modulate the most analyzed molecular proteins, but induced hepatic morphological alterations, suggesting an acute pathological adaptation. The three OT models induced hepatic fat accumulation. J. Cell. Physiol. 232: 2094–2103, 2017. © 2016 Wiley Periodicals, Inc. The downhill running‐induced overtraining model increased the levels of major proteins of the mTOR signaling pathway in mouse livers despite high levels of pAMPK (Thr172). The three overtraining models increased the percentage of fat in liver.

  March 03, 2017   doi: 10.1002/jcp.25625   open full text
• Tumor Necrosis Factor‐α Increases Claudin‐1, 4, and 7 Expression in Tubular Cells: Role in Permeability Changes.
  Yasaman Amoozadeh, Qinghong Dan, Shaista Anwer, Hsiao Han Huang, Vanessa Barbieri, Faiza Waheed, Mazharul Maishan, Katalin Szászi.
  Journal of Cellular Physiology. March 03, 2017
  Tumor necrosis factor‐α (TNFα), is a pathogenic cytokine in kidney disease that alters expression of claudins in tubular cells. Previously we showed that in LLC‐PK1 cells TNFα caused a biphasic change in transepithelial resistance (TER) consisting of an early drop and recovery, followed by a late increase. However, the underlying mechanisms and the role of specific claudins in the TER effect remained incompletely understood. Here we sought to define how TNFα affects claudins 1, 4, and 7 in tubular cells and to correlate their changes with the TER effect. We show that TNFα elevates total and surface levels of Cldn‐1, 4, and 7, and increases their mRNA expression through the ERK and JNK pathways. Further, JNK is also important for TNFα‐induced changes in claudin‐2 expression. Continuous monitoring of TER using Electric cell‐substrate impedance sensing (ECIS) reveals that the two phases of the TNFα effect are differently regulated. Specifically, inhibition of the ERK or JNK pathways prevent the late TER increase, but not the early TER effect. Silencing experiments also show that Cldn‐1 is necessary for the early TNFα‐induced TER change, while all three claudins appear to contribute to the late TER increase. In summary, we define a central role for ERK and JNK in TNFα‐induced altered claudin expression and barrier tightening. Together, our current and previous works show that the TNFα‐induced early TER effect requires claudin‐1, while claudin‐2 decrease is a significant mediator of the late TER increase, and elevation in claudin‐1, 4, and 7 contribute to a smaller extent. J. Cell. Physiol. 232: 2210–2220, 2017. © 2016 Wiley Periodicals, Inc. We demonstrate that TNFα elevates levels of claudins 1,4, and 7 and define a central role for ERK and JNK in this effect. Using ECIS we correlate these signalling pathways and claudins with the biphasic TER changes induced by TNFα and show their differential roles in the early and late phases.

  March 03, 2017   doi: 10.1002/jcp.25736   open full text
• Dlx3 and GCM‐1 functionally coordinate the regulation of placental growth factor in human trophoblast‐derived cells.
  Sha Li, Mark S. Roberson.
  Journal of Cellular Physiology. March 01, 2017
  Placental growth factor (PGF) is abundantly expressed by trophoblast cells within human placentae and is important for trophoblast development and placental vascularization. Circulating maternal serum levels of PGF are dynamically upregulated across gestation in normal pregnancies, whereas low circulating levels and placental production of PGF have been implicated in the pathogenesis of preeclampsia and other gestational diseases. However, the underlying molecular mechanism of regulating PGF expression in the human placenta remains poorly understood. In this study, we demonstrated that transcription factors Distal‐less 3 (DLX3) and Glial cell missing‐1 (GCM1) were both sufficient and required for PGF expression in human trophoblast‐derived cells by overexpression and knockdown approaches. Surprisingly, while DLX3 and GCM1 were both positive regulators of PGF, co‐overexpression of DLX3 and GCM1 led to an antagonist effect on PGF expression on the endogenous gene and a luciferase reporter. Further, deletion and site‐directed mutagenesis studies identified a novel regulatory element on the PGF promoter mediating both DLX3‐ and GCM1‐dependent PGF expression. This regulatory region was also found to be essential for the basal activity of the PGF promoter. Finally, Chromatin‐immunoprecipitation (ChIP) assays revealed colocalization of DLX3 and GCM1 at the identified regulatory region on the PGF promoter. Taken together, our studies provide important insights into intrinsic regulation of human placental PGF expression through the functional coordination of DLX3 and GCM1, and are likely to further the understanding of pathogenesis of PGF dysregulation in preeclampsia and other disease conditions. Our studies examine the role and requirement for the transcription factors Dlx3 and GCM‐1 in the regulation of placental growth factor (PGF) in human placental trophoblast cell lines. Extensive over‐expression and knockdown studies reveal a requirement for both factors; however, the combined action of Dlx3 and GCM‐1 is antagonistic to PGF expression. Our studies identify a novel cis‐acting regulatory element in the proximal region of the PGF gene promoter that binds both factors and is required for the antagonist effects of Dlx3 on GCM‐1.

  March 01, 2017   doi: 10.1002/jcp.25752   open full text
• Tolerogenic probiotics: potential immunoregulators in Systemic Lupus Erythematosus.
  Seyed‐Alireza Esmaeili, Mahmoud Mahmoudi, Amir Abbas Momtazi, Amirhossein Sahebkar, Hassan Doulabi, Maryam Rastin.
  Journal of Cellular Physiology. March 01, 2017
  Probiotics are commensal or nonpathogenic microbes that colonize the gastrointestinal tract and confer beneficial effects on the host through several mechanisms such as competitive exclusion, anti‐bacterial effects, and modulation of immune responses. There is growing evidence supporting the immunomodulatory ability of some probiotics. Several experimental and clinical studies have been shown beneficial effect of some probiotic bacteria, particularly Lactobacillus and Bifidobacteria strains, on inflammatory and autoimmune diseases. Systemic lupus erythematosus (SLE) is an autoimmune disease that is mainly characterized by immune intolerance towards self‐antigens. Some immunomodulatory probiotics have been found to regulate immune responses via tolerogenic mechanisms. Dendritic and T regulatory (Treg) cells, IL‐6, IFN‐γ, IL‐17, and IL‐23 can be considered as the most determinant dysregulated mediators in tolerogenic status. As demonstrated by documented experimental and clinical trials on inflammatory and autoimmune diseases, a number of probiotic bacterial strains can restore tolerance in host through modification of such dysregulated mediators. Since there are limited reports regarding to impact of probiotic supplementation in SLE patients, the preset review was aimed to suggest a number of probiotics bacteria, mainly from Bifidobacteria and Lactobacillus strains that are able to ameliorate immune responses. The aim was followed through literature survey on immunoregulatory probiotics that can restore tolerance and also modulate the important dysregulated pro/anti‐inflammatory cytokines contributing to the pathogenesis of SLE. Since there are limited reports regarding to impact of probiotic supplementation in SLE patients, the preset review was aimed to suggest a number of probiotics bacteria, mainly from Bifidobacteria and Lactobacillus strains that are able to ameliorate immune responses. The aim was followed through literature survey on immunoregulatory probiotics that can restore tolerance and also modulate the important dysregulated pro/anti‐inflammatory cytokines contributing to the pathogenesis of SLE.

  March 01, 2017   doi: 10.1002/jcp.25748   open full text
• Uremic Serum Impairs Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stromal Cells.
  Elena Della Bella, Stefania Pagani, Gianluca Giavaresi, Irene Capelli, Giorgia Comai, Chiara Donadei, Maria Cappuccilli, Gaetano La Manna, Milena Fini.
  Journal of Cellular Physiology. March 01, 2017
  Chronic Kidney Disease‐Mineral and Bone Disorder (CKD‐MBD) is characterized by an increased fracture risk. Bone marrow mesenchymal stromal cells (BMSCs) may be involved in the pathogenesis of bone disease and, in view of their promising potential applications in bone tissue engineering, the effect of uremia on BMSCs regenerative potential represents a central issue. The present study evaluated in vitro the effect of a serum pool from hemodialysis patients on BMSCs to observe its influence on osteogenic differentiation. Besides alterations in spatial organization and cytotoxicity along with hyperproliferation, gene expression analysis suggested an impairment in the osteogenic differentiation. More importantly, Receptor activator of nuclear factor kappa‐B ligand (RANKL) was upregulated with a mild reduction in osteoprotegerin levels. In summary, uremic environment seems to impair BMSCs osteogenic differentiation. Moreover BMSCs themselves may enhance osteoclastogenesis, feasibly contributing to the altered bone remodeling in CKD‐MBD patients. J. Cell. Physiol. 232: 2201–2209, 2017. © 2016 Wiley Periodicals, Inc. BMSCs were cultured in presence of a serum pool from hemodialysis patients (HUS) to study the effect of uremia on BMSC behavior. HUS induced both hyperproliferation and cytotoxicity, with an alteration in the spatial organization of cells. Moreover, osteogenic differentiation was impaired: a reduced mineralization and an altered pattern of gene expression were observed, in particular upregulation of RANKL and reduction in OPG levels.

  March 01, 2017   doi: 10.1002/jcp.25732   open full text
• Impact of Cryopreservation on Caprine Fetal Adnexa Derived Stem Cells and Its Evaluation for Growth Kinetics, Phenotypic Characterization, and Wound Healing Potential in Xenogenic Rat Model.
  Anjali Somal, Irfan A. Bhat, Indu B. , Anuj P. Singh, Bibhudatta S.K. Panda, Perumal A. Desingu, Sriti Pandey, Mukesh K. Bharti, Amar Pal, Guttula Saikumar, Vikash Chandra, Guttula Taru Sharma.
  Journal of Cellular Physiology. March 01, 2017
  This study was conducted to know the impact of cryopreservation on caprine fetal adnexa derived mesenchymal stem cells (MSCs) on the basic stem cell characteristics. Gravid caprine uteri (2–3 months) were collected from local abattoir to derive (amniotic fluid [cAF], amniotic sac [cAS], Wharton's jelly [cWJ], and cord blood [cCB]) MSCs and expanded in vitro. Cells were cryopreserved at 3rd passage (P3) using 10% DMSO. Post‐thaw viability and cellular properties were assessed. Cells were expanded to determine growth kinetics, tri‐lineage differentiation, localization, and molecular expression of MSCs and pluripotency markers; thereafter, these cells were transplanted in the full‐thickness (2 × 2cm2) rat skin wound to determine their wound healing potential. The post‐thaw (pt) growth kinetics study suggested that cWJ MSCs expanded more rapidly with faster population doubling time (PDT) than that of other fetal adnexa MSCs. The relative mRNA expression of surface antigens (CD73, CD90, and CD 105) and pluripotency markers (Oct4, KLF, and cMyc) was higher in cWJ MSCs in comparison to cAS, cAF, and cCB MSCs post‐thaw. The percent wound contraction on 7th day was more than 50% for all the MSC‐treated groups (pre and post‐thaw), against 39.55% in the control group. On day 28th, 99% and more wound contraction was observed in cAF, cAF‐pt, cAS‐pt, cWJ, cWJ‐pt, and cCB, MSCs with better scores for epithelization, neovascularization, and collagen characteristics at a non‐significant level. It is concluded that these MSCs could be successfully cryopreserved without altering their stemness and wound healing properties. J. Cell. Physiol. 232: 2186–2200, 2017. © 2016 Wiley Periodicals, Inc. The present study showed that cWJ MSCs have better pluripotency and post thaw survivability than other fetal adnexa derived MSCs and all the adnexa derived MSCs were found to heal excision wound faster and quality of healed wound was also better than control, indicating that these cells can be cryopreserved and may be used for regenerative cell therapy.

  March 01, 2017   doi: 10.1002/jcp.25731   open full text
• Notch Signaling Participates in TGF‐β‐Induced SOST Expression Under Intermittent Compressive Stress.
  Jeeranan Manokawinchoke, Piyamas Sumrejkanchanakij, Prasit Pavasant, Thanaphum Osathanon.
  Journal of Cellular Physiology. March 01, 2017
  Notch signaling is regulated by mechanical stimuli in various cell types. It has previously been reported that intermittent compressive stimuli enhanced sclerostin (SOST) expression in human periodontal ligament cells (hPDLs) by regulating transforming growth factor‐β (TGF‐β) expression. The aim of the present study was to determine the involvement of Notch signaling in the TGF‐β‐induced SOST expression in hPDLs. Cells were treated with intermittent compressive stress in a computer‐controlled apparatus for 24 h. The mRNA and protein expression of the cells were determined by real‐time polymerase chain reaction and Western blot analysis, respectively. In some experiments, the target signaling pathway was impeded by the addition of a TGF‐β receptor kinase inhibitor (SB431542) or a γ‐secretase inhibitor (DAPT). The results demonstrated that hPDLs under intermittent compressive stress exhibited significantly higher NOTCH2, NOTCH3, HES1, and HEY1 mRNA expression compared with control, indicating that mechanical stress induced Notch signaling. DAPT pretreatment markedly reduced the intermittent stress‐induced SOST expression. The expression of NOTCH2, NOTCH3, HES1, and HEY1 mRNA under compressive stress was significantly reduced after pretreatment with SB431542, coinciding with a reduction in SOST expression. Recombinant human TGF‐β1 enhanced SOST, Notch receptor, and target gene expression in hPDLs. Further, DAPT treatment attenuated rhTGF‐β1‐induced SOST expression. In summary, intermittent compressive stress regulates Notch receptor and target gene expression via the TGF‐β signaling pathway. In addition, Notch signaling participates in TGF‐β‐induced SOST expression in hPDLs. J. Cell. Physiol. 232: 2221–2230, 2017. © 2016 Wiley Periodicals, Inc. Intermittent stress stimulates TGF‐β signaling via an unknown mechanism. TGF‐β, in turn, induces the expression of SOST, NOTCH2, NOTCH3, HES1, and HEY1, whose expression was reduced by SB431542. Inhibition SOST expression, but not that of TGF‐β by DAPT and NOTCH2 shRNA indicate that NOTCH signaling is a downstream participant in the TGF‐β‐induced SOST expression.

  March 01, 2017   doi: 10.1002/jcp.25740   open full text
• Hyaluronic Acid Nanohydrogel Loaded With Quercetin Alone or in Combination to a Macrolide Derivative of Rapamycin RAD001 (Everolimus) as a New Treatment for Hormone‐Responsive Human Breast Cancer.
  Vincenzo Quagliariello, Rosario Vincenzo Iaffaioli, Emilia Armenia, Ottavia Clemente, Manlio Barbarisi, Guglielmo Nasti, Massimiliano Berretta, Alessandro Ottaiano, Alfonso Barbarisi.
  Journal of Cellular Physiology. March 01, 2017
  The aim of this study is based on the evaluation of anticancer, anti‐inflammatory activities, and cellular uptake of hyaluronic acid nanohydrogel of quercetin tested alone and in combination to a macrolide derivative of rapamycin RAD001 (everolimus) on hormone‐responsive breast cancer cell line MCF‐7. Biological investigations were focused on the receptor mediated cellular internalization of the nanohydrogel and its abilities to reduce secretion of several cytokines (IL‐8, IL‐6, IL‐19), VEGF, and metalloproteases (MMP‐2, MMP‐9) under pro‐inflammatory conditions. Nanohydrogel show a CD44 dependent endocytosis with evident time dependent cytoplasmatic accumulation with abilities to reduce secretion of all cytokines of ∼60% compared to untreated cells. Combination of formulated quercetin and everolimus leads to a synergistic cytotoxic effects with a Combination Index of 0.38. These results highlights the importance of synergistic effect of the hyaluronic acid nanohydrogel of quercetin with everolimus in the regulation of human breast cancer cell proliferation and emphasize the antitumor and anti‐inflammatory properties of the nanocarrier. J. Cell. Physiol. 232: 2063–2074, 2017. © 2016 Wiley Periodicals, Inc. Hyaluronic acid nanohydrogel of Quercetin has anti‐anticancer and antinflammatory activity in human breast cancer cells. Combination of Everolimus and hyaluronic acid nanohydrogel of Quercetin leads to synergistic cytotoxicity in MCF‐7 call line.

  March 01, 2017   doi: 10.1002/jcp.25587   open full text
• Deregulated ALG‐2/HEBP2 axis alters microtubule dynamics and mitotic spindle behavior to stimulate cancer development.
  Juan Qin, Yang Yang, Siqi Gao, Yang Liu, Fan Yu, Yunqiang Zhou, Rui Lyu, Min Liu, Xinqi Liu, Dengwen Li, Jun Zhou.
  Journal of Cellular Physiology. March 01, 2017
  Cancer cells are characterized by genomic instability, resulting in the accumulation of mutations that promote cancer progression. One way that genomic instability can arise is through improper regulation of the microtubule cytoskeleton that impacts the function of the mitotic spindle. In this study, we have identified a critical role for the interaction between apoptosis‐linked gene 2 (ALG‐2) and heme‐binding protein 2 (HEBP2) in the above processes. Our data show that the gene copy numbers and mRNA levels for both ALG‐2 and HEBP2 are significantly upregulated in breast and lung cancer. Coexpression of ALG‐2 and HEBP2 markedly increases the cytoplasmic pool of ALG‐2 and alters the subcellular distribution of HEBP2. Our data further reveal that abnormality in the ALG‐2/HEBP2 interaction impairs spindle orientation and positioning during mitosis. In addition, this complex appears to modulate the dynamic properties of microtubules in cancer cells. These finding thus uncover an important function for deregulated ALG‐2/HEBP2 axis in cancer development by influencing microtubule dynamics and spindle behavior, providing novel insight into the etiology and pathogenesis of cancer. In this study, we uncover a previously unrecognized role for deregulated ALG‐2/HEBP2 axis in cancer development. Mechanistically, this is mediated by altered microtubule dynamics and impaired spindle orientation and positioning.

  March 01, 2017   doi: 10.1002/jcp.25754   open full text
• Integrated Analysis and MicroRNA Expression Profiling Identified Seven miRNAs Associated With Progression of Oral Squamous Cell Carcinoma.
  Zhong‐Yi Yan, Zhi‐Qing Luo, Lai‐Jian Zhang, Jia Li, Jia‐Qiang Liu.
  Journal of Cellular Physiology. March 01, 2017
  MicroRNAs have been used as diagnostic and prognostic biomarkers for many cancers including oral squamous cell carcinoma (OSCC). Several studies have been shown that microRNA (miRNA) play important roles during the progression of OSCC. However, the results vary largely in different studies due to different platforms and sample sizes. In this study, we systematically evaluated a large scale of miRNA profiles from current qualified OSCC samples, and further investigated the functions of genes regulated by these key miRNAs as well as the signaling pathways through which these miRNA effect carcinogenesis. Seven key miRNAs were identified, and of which three were significantly upregulated, including hsa‐miR‐21, hsa‐miR‐31, hsa‐miR‐338, and four were downregulated, namely hsa‐miR‐125b, hsa‐miR‐133a, hsa‐miR‐133b, and hsa‐miR‐139. The function enrichment analysis revealed that target genes of upregulated miRNAs were associated with cellular protein metabolic process, macromolecule metabolic process, and TGF‐beta pathway, while the targets of downregulated were enriched in negative regulation of macromolecule biosynthetic process and gene expression, and p53, long‐term potentiation and adherens junction pathways. Transcription factor analysis revealed that there were 67 (51.1%) transcription factors influenced by both up and downregulated miRNAs. In summary, seven key miRNAs were found to play essential role in progression of OSCC, as well as the target genes and transcription factors of these miRNAs. The potential functions of these target genes identified in our study may be profitable to diagnosis and prognostic prediction of OSCC as biomarkers. J. Cell. Physiol. 232: 2178–2185, 2017. © 2016 Wiley Periodicals, Inc. Seven key miRNAs were found to play essential role in progression of OSCC, as well as the target genes and transcription factors of these miRNAs. The potential functions of these target genes identified in our study may be profitable to diagnosis and prognostic prediction of OSCC as biomarkers.

  March 01, 2017   doi: 10.1002/jcp.25728   open full text
• Human Telomerase Reverse Transcriptase (hTERT) Positively Regulates 26S Proteasome Activity.
  Eunju Im, Jong Bok Yoon, Han‐Woong Lee, Kwang Chul Chung.
  Journal of Cellular Physiology. February 28, 2017
  Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, an RNA‐dependent DNA polymerase that elongates telomeric DNA. hTERT displays several extra‐telomeric functions that are independent of its telomere‐regulatory function, including tumor progression, and neuronal cell death regulation. In this study, we evaluated these additional hTERT non‐telomeric functions. We determined that hTERT interacts with several 19S and 20S proteasome subunits. The 19S regulatory particle and 20S core particle are part of 26S proteasome complex, which plays a central role in ubiquitin‐dependent proteolysis. In addition, hTERT positively regulated 26S proteasome activity independent of its enzymatic activity. Moreover, hTERT enhanced subunit interactions, which may underlie hTERT's ability of hTERT to stimulate the 26S proteasome. Furthermore, hTERT displayed cytoprotective effect against ER stress via the activation of 26S proteasome in acute myeloid leukemia cells. Our data suggest that hTERT acts as a novel chaperone to promote 26S proteasome assembly and maintenance. J. Cell. Physiol. 232: 2083–2093, 2017. © 2016 Wiley Periodicals, Inc. The present study demonstrated that hTERT positively regulates 26S proteasome activity independent of its enzymatic activity. Moreover, hTERT displayed cytoprotective effect against ER stress via the activation of 26S proteasome in acute myeloid leukemia cells. Our data suggest that hTERT acts as a novel chaperone to promote 26S proteasome assembly and maintenance.

  February 28, 2017   doi: 10.1002/jcp.25607   open full text
• Chemoresistance of Lung and Breast Cancer Cells Growing Under Prolonged Periods of Serum Starvation.
  Juan Sebastian Yakisich, Rajkumar Venkatadri, Neelam Azad, Anand Krishnan V. Iyer.
  Journal of Cellular Physiology. February 28, 2017
  The efficacy of chemotherapy is hindered by both tumor heterogeneity and acquired or intrinsic multi‐drug resistance caused by the contribution of multidrug resistance proteins and stemness‐associated prosurvival markers. Therefore, targeting multi‐drug resistant cells would be much more effective against cancer. In this study, we characterized the chemoresistance properties of adherent (anchorage‐dependent) lung H460 and breast MCF‐7 cancer cells growing under prolonged periods of serum starvation (PPSS). We found that under PPSS, both cell lines were highly resistant to Paclitaxel, Colchicine, Hydroxyurea, Obatoclax, Wortmannin, and LY294002. Levels of several proteins associated with increased stemness such as Sox2, MDR1, ABCG2, and Bcl‐2 were found to be elevated in H460 cells but not in MCF‐7 cells. While pharmacological inhibition of either MDR1, ABCG2, Bcl‐2 with Verapamil, Sorafenib, or Obatoclax, respectively decreased the levels of their target proteins under routine culture conditions as expected, such inhibition did not reverse PX resistance in PPSS conditions. Paradoxically, treatment with inhibitors in serum‐starved conditions produced an elevation of their respective target proteins. In addition, we found that Digitoxin, an FDA approved drug that decrease the viability of cancer cells growing under PPSS, downregulates the expression of Sox2, MDR1, phospho‐ AKT, Wnt5a/b, and β‐catenin. Our data suggest that PPSS‐induced chemoresistance is the result of extensive rewiring of intracellular signaling networks and that multi‐resistance can be effectively overcome by simultaneously targeting multiple targets of the rewired network. Furthermore, our PPSS model provides a simple and useful tool to screen drugs for their ability to target multiple pathways of cancer resistance. J. Cell. Physiol. 232: 2033–2043, 2017. © 2016 Wiley Periodicals, Inc. Cancer cells growing under prolonged periods of serum starvation (PPSS) become multidrug resistant. Under PPSS cancer cells respond aberrantly and paradoxically to some anticancer drugs likely by rewire signaling pathway networks.

  February 28, 2017   doi: 10.1002/jcp.25514   open full text
• Isolation, identification, and characterization of cancer stem cells: A review.
  Mohammad Reza Abbaszadegan, Vahid Bagheri, Mahya Shariat Razavi, Amir Abbas Momtazi, Amirhossein Sahebkar, Mehran Gholamin.
  Journal of Cellular Physiology. February 28, 2017
  Cancer stem cells (CSCs) or tumor‐initiating cells (TICs) as a small subset of neoplastic cells are able to produce a tumor (tumorigenesis), maintain the population of tumorigenic cells (self‐renewal), and generate the heterogeneous cells constructing the entire tumor (pluripotency). The research on stationary and circulating CSCs due to resistance to conventional therapies and inability in complete eradication of cancer is critical for developing novel therapeutic strategies for a more effective reduction in the risk of tumor metastasis and cancer recurrence. This review compiles information about different methods of detection and dissociation, side population, cellular markers, and establishment culture of CSCs, as well as characteristics of CSCs such as tumorigenicity, and signaling pathways associated with self‐renewal and the capability of the same histological tumor regeneration in various cancers. This review compiles information about detection, dissociation, side population, cellular markers, and establishment culture of cancer stem cells, as well as characteristics of CSCs such as tumorigenicity, signaling pathways associated with self‐renewal and the capability of the same histological tumor regeneration in various cancers.

  February 28, 2017   doi: 10.1002/jcp.25759   open full text
• Celastrol Attenuates Cadmium‐Induced Neuronal Apoptosis via Inhibiting Ca2+‐CaMKII‐Dependent Akt/mTOR Pathway.
  Ruijie Zhang, Yu Zhu, Xiaoqing Dong, Beibei Liu, Nana Zhang, Xiaoxue Wang, Lei Liu, Chong Xu, Shile Huang, Long Chen.
  Journal of Cellular Physiology. February 28, 2017
  Cadmium (Cd), an environmental and industrial pollutant, affects the nervous system and consequential neurodegenerative disorders. Recently, we have shown that celastrol prevents Cd‐induced neuronal cell death partially by suppressing Akt/mTOR pathway. However, the underlying mechanism remains to be elucidated. Here, we show that celastrol attenuated Cd‐elevated intracellular‐free calcium ([Ca2+]i) level and apoptosis in neuronal cells. Celastrol prevented Cd‐induced neuronal apoptosis by inhibiting Akt‐mediated mTOR pathway, as inhibition of Akt with Akt inhibitor X or ectopic expression of dominant negative Akt reinforced celastrol's prevention of Cd‐induced phosphorylation of S6K1/4E‐BP1 and cell apoptosis. Furthermore, chelating intracellular Ca2+ with BAPTA/AM or preventing [Ca2+]i elevation using EGTA potentiated celastrol's repression of Cd‐induced [Ca2+]i elevation and consequential activation of Akt/mTOR pathway and cell apoptosis. Moreover, celastrol blocked Cd‐elicited phosphorylation of CaMKII, and pretreatment with BAPTA/AM or EGTA enhanced celastrol's suppression of Cd‐increased phosphorylation of CaMKII in neuronal cells, implying that celastrol hinders [Ca2+]i‐mediated CaMKII phosphorylation. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of Akt/mTOR pathway and cell apoptosis, and this was strengthened by celastrol. Taken together, these data demonstrate that celastrol attenuates Cd‐induced neuronal apoptosis via inhibiting Ca2+‐CaMKII‐dependent Akt/mTOR pathway. Our findings underscore that celastrol may act as a neuroprotective agent for the prevention of Cd‐induced neurodegenerative disorders. J. Cell. Physiol. 232: 2145–2157, 2017. © 2016 Wiley Periodicals, Inc. Celastrol prevents Cd‐induced elevation of [Ca2+]i, which results in inhibition of CaMKII phosphorylation. This leads to suppression of CaMKII‐dependent Akt/mTOR pathway, thereby preventing Cd‐induced apoptosis in neuronal cells.

  February 28, 2017   doi: 10.1002/jcp.25703   open full text
• Differential Expression of Estrogen Receptor Variants in Response to Inflammation Signals in Human Airway Smooth Muscle.
  Bharathi Aravamudan, Katelyn J. Goorhouse, Ghanashyam Unnikrishnan, Michael A. Thompson, Christina M. Pabelick, John R. Hawse, Y. S. Prakash, Venkatachalem Sathish.
  Journal of Cellular Physiology. February 24, 2017
  The prevalence of asthma is higher in pre‐pubescent and aging males, and in post‐pubertal females, strongly indicating that sex steroids (especially estrogen) may be an important modulator in lung disease. We recently demonstrated that airway smooth muscle (ASM) expresses both alpha and beta forms of the estrogen receptor (ERα and ERβ) in males and females, and that these receptors regulate intracellular [Ca2+] and ASM contractility. Although both ERα and ERβ have multiple splice variants, it is unclear if and how the expression of these variants is modulated under conditions such as chronic inflammation/asthma. In order to test the hypothesis that the differential expression of ERα and ERβ variants contributes to the pathogenesis of asthma, we profiled the expression of various ERα and ERβ genes in asthmatic and inflamed (TNFα‐ or IL‐13‐treated) ASM. Gene expression was assessed at both the mRNA and protein levels in asthmatic ASM cells or non‐asthmatic cells treated with TNFα (20 ng/ml) or IL‐13 (50 ng/ml). We observed marked variation in the expression of ER isoforms in response to inflammatory stimuli, and in non‐asthmatic versus asthmatic ASM. Changes in protein levels of ERα and ERβ corresponded with the observed differential mRNA patterns. Pharmacological studies implicate cytosolic (p42/44 MAPK and PI3 K) and nuclear (NFκB, STAT6, and AP‐1) signaling pathways as putative mechanisms that mediate and/or regulate effects of inflammation on ER expression. We conclude that variations in ASM ER expression profiles occur with inflammation and that ER variants could contribute to estrogen signaling in airway diseases such as asthma. J. Cell. Physiol. 232: 1754–1760, 2017. © 2016 Wiley Periodicals, Inc.

  February 24, 2017   doi: 10.1002/jcp.25674   open full text
• Cytoplasmic Localization of RUNX3 via Histone Deacetylase‐Mediated SRC Expression in Oxidative‐Stressed Colon Cancer Cells.
  Kyoung Ah Kang, Mei Jing Piao, Yea Seong Ryu, Young Hee Maeng, Jin Won Hyun.
  Journal of Cellular Physiology. February 21, 2017
  Runt domain transcription factor 3 (RUNX3) is a transcription factor that functions as a tumor suppressor. RUNX3 is frequently inactivated by epigenetic silencing or its protein mislocalization (cytoplasmic localization) in many cancer types. This study investigated whether oxidative stress induces redistribution of RUNX3 from the nucleus to the cytoplasm. The cytoplasmic localization of RUNX3 was associated with oxidative stress‐induced RUNX3 phosphorylation at tyrosine residues via SRC activation. Moreover, oxidative stress increased expression of histone deacetylases (HDACs). RUNX3 phosphorylation and SRC expression induced by oxidative stress were inhibited by knockdown of HDAC1, restoring the nuclear localization of RUNX3 under oxidative stress. In conclusion, these results demonstrate that HDAC1‐ and SRC‐mediated phosphorylation of RUNX3 induced by oxidative stress is associated with the cytoplasmic localization of RUNX3 and can lead to RUNX3 inactivation and carcinogenesis. J. Cell. Physiol. 232: 1914–1921, 2017. © 2016 Wiley Periodicals, Inc. RUNX3 cytoplasmic localization induced by oxidative stress in colon cancer cells. Oxidative stress induces HDAC1 expression and then activates SRC. Activated SRC induces expression of phospho‐RUNX3. Phospho‐RUNX3 is retained in the cytoplasm, leading to degradation of cytoplasmic RUNX3 by the proteasome system and finally resulting carcinogenesis.

  February 21, 2017   doi: 10.1002/jcp.25746   open full text
• Na+/H+ exchanger NHE1 and NHE2 have opposite effects on migration velocity in rat gastric surface cells.
  Anja Paehler vor der Nolte, Giriprakash Chodisetti, Zhenglin Yuan, Florian Busch, Brigitte Riederer, Min Luo, Yan Yu, Manoj B. Menon, Andreas Schneider, Renata Stripecke, Katerina Nikolovska, Sunil Yeruva, Ursula Seidler.
  Journal of Cellular Physiology. February 21, 2017
  Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na+/H+ exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1‐dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1‐dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ∼10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady‐state pHi and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process. Genetic deletion of the Na+/H+ exchanger NHE2 leads to compromised wound healing in the gastrointestinal tract of mice. We studied the roles of NHE1 and NHE2 in gastrointestinal wound healing in vitro, and while NHE1 activity supported wound healing velocity, NHE2/GFP‐overexpression dramatically reduced wound healing velocity compared to GFP expression alone. This effect was abolished by a NHE2‐ but not a NHE1 inhibitory concentration of HOE642. Thus, NHE1 and NHE2 may be important for the initiation and the resolution of gastrointestinal wound healing.

  February 21, 2017   doi: 10.1002/jcp.25758   open full text
• Activin‐SMAD signaling is required for maintenance of porcine iPS cell self‐renewal through upregulation of NANOG and OCT4 expression.
  Fan Yang, Ning Wang, Yaxian Wang, Tong Yu, Huayan Wang.
  Journal of Cellular Physiology. February 21, 2017
  Porcine induced pluripotent stem cells (piPSCs) retain the enormous potential for farm animal reproduction and translational medicine, and have been reported by many laboratories worldwide. Some piPSC lines were bFGF‐dependence and showed mouse EpiSC‐like morphology; other lines were LIF‐dependence and showed mouse ESC‐like morphology. Metastable state of piPSC line that required both LIF and bFGF was also reported. Because bona fide pig embryonic stem cells were not available, uncovering piPSC state‐specific regulatory circuitries was the most important task. In this study, we explored the function of Activin‐SMAD signaling pathway and its downstream activated target genes in piPSCs. Transcriptome analysis showed that genes involved in Activin‐SMAD signaling pathway were evidently activated during porcine somatic cell reprogramming, regardless piPSCs were LIF‐ or bFGF‐dependent. Addition of Activin A and overexpression of SMAD2/3 significantly promoted expressions of porcine NANOG and OCT4, whereas inhibition of Activin‐SMAD signaling by SB431542 and SMAD7 reduced NANOG and OCT4 expressions, and induced piPSCs differentiation exiting from pluripotent state. Our data demonstrate that activation of Activin‐SMAD signaling pathway by addition of Activin A in culture medium is necessary for maintenance of self‐renewal in porcine pluripotent stem cells. Our data demonstrate that activation of Activin‐SMAD signaling pathway by addition of Activin A in culture medium is necessary for maintenance of self‐renewal in porcine pluripotent stem cells.

  February 21, 2017   doi: 10.1002/jcp.25747   open full text
• LCN2 overexpression in bone enhances the hematopoietic compartment via modulation of the bone marrow microenvironment.
  Delfina Costa, Elisa Principi, Edoardo Lazzarini, Fiorella Descalzi, Ranieri Cancedda, Patrizio Castagnola, Sara Tavella.
  Journal of Cellular Physiology. February 21, 2017
  Lipocalin‐2 (LCN2) is a member of the lipocalin family whose expression is modulated in several conditions, including cell differentiation, innate immunity, stress, and cancer. Although it is known that it is expressed in bone, its function in this tissue remains poorly studied. To this end, we took advantage of transgenic mice lines that expressed LCN2 driven by a bone specific type I collagen (LCN2‐Tg). In the bone marrow (BM) of LCN2‐Tg mice we observed an increased number of phenotypically long‐term hematopoietic stem cells (LT‐HSC) that also displayed a higher proliferation rate compared to wild‐type controls (Wt). Furthermore, hematopoietic progenitor cells, obtained from LCN2‐Tg BM showed an increased clonogenic capacity compared to those obtained from LCN2‐Tg spleen, a higher concentration of serum erythropoietin and a higher number of mature erythrocytes in the peripheral blood of old LCN2‐Tg animals compared to aged‐matched wt. The findings of a combined increase in the BM of the LCN2‐Tg mice of SDF‐1, SCF, and TIMP‐1 levels along with the reduction of both MMP‐9 activity and cathepsin K concentration may explain the observed effects on the HSC compartment. This study shows that LCN2 overexpression in bones modifies the BM microenvironment via modulation of the expression of key secreted factors and cytokines, which in turn regulate the HSC niche behavior enhancing both HSC homing in young mice and erythrocytes production in older mice. The overexpression of LCN2 in bone stimulates the expansion of the HSC by inducing pro‐retention factors and inhibiting pro‐egress signals for hematopoietic stem cells.

  February 21, 2017   doi: 10.1002/jcp.25755   open full text
• STIM‐1 and ORAI‐1 channel mediate angiotensin‐II‐induced expression of Egr‐1 in vascular smooth muscle cells.
  Estelle R. Simo‐Cheyou, Ju Jing Tan, Ryszard Grygorczyk, Ashok K. Srivastava.
  Journal of Cellular Physiology. February 21, 2017
  An upregulation of Egr‐1 expression has been reported in models of atherosclerosis and intimal hyperplasia and, various vasoactive peptides and growth promoting stimuli have been shown to induce the expression of Egr‐1 in vascular smooth muscle cells (VSMC). Angiotensin‐II (Ang‐II) is a key vasoactive peptide that has been implicated in the pathogenesis of vascular diseases. Ang‐II elevates intracellular Ca2+ through activation of the store‐operated calcium entry (SOCE) involving an inositol‐3‐phosphate receptor (IP3R)‐coupled depletion of endoplasmic reticular Ca2+ and a subsequent activation of the stromal interaction molecule 1 (STIM‐1)/Orai‐1 complex. However, the involvement of IP3R/STIM‐1/Orai‐1‐Ca2+‐dependent signaling in Egr‐1 expression in VSMC remains unexplored. Therefore, in the present studies, we have examined the role of Ca2+ signaling in Ang‐II‐induced Egr‐1 expression in VSMC and investigated the contribution of STIM‐1 or Orai‐1 in mediating this response. 2‐aminoethoxydiphenyl borate (2‐APB), a dual non‐competitive antagonist of IP3R and inhibitor of SOCE, decreased Ang‐II‐induced Ca2+ release and attenuated Ang‐II‐induced enhanced expression of Egr‐1 protein and mRNA levels. Egr‐1 upregulation was also suppressed following blockade of calmodulin and CaMKII. Furthermore, RNA interference‐mediated depletion of STIM‐1 or Orai‐1 attenuated Ang‐II‐induced Egr‐1 expression as well as Ang‐II‐induced phosphorylation of ERK1/2 and CREB. In addition, siRNA‐induced silencing of CREB resulted in a reduction in the expression of Egr‐1 stimulated by Ang‐II. In summary, our data demonstrate that Ang‐II‐induced Egr‐1 expression is mediated by STIM‐1/Orai‐1/Ca2+‐dependent signaling pathways in A‐10 VSMC. Angiotensin‐II‐induced intracellular calcium release depletes the endoplasmic reticulum store, resulting in the activation of the stromal interaction molecule 1 (STIM1) known to cooperate with Orai‐1 transmembrane channel in mediating store‐operated calcium entry (SOCE) in the cells. In this study, we demonstrate that intact SOCE and unaltered expression of STIM‐1 and Orai‐1, in association with an involvement of calcium signaling molecules such as calmodulin and CaMKII, are required for Ang‐II‐dependent induction of the early growth response protein‐1 in VSMC. We also provide evidence of the critical role played by ERK1/2 and CREB activation in mediating this response.

  February 21, 2017   doi: 10.1002/jcp.25810   open full text
• The Role of Cardiac Myosin Binding Protein C3 in Hypertrophic Cardiomyopathy‐Progress and Novel Therapeutic Opportunities.
  Iman A. Mohamed, Navaneethakrishnan T. Krishnamoorthy, Gheyath K. Nasrallah, Sahar I. Da'as.
  Journal of Cellular Physiology. February 16, 2017
  Hypertrophic cardiomyopathy (HCM) is a common autosomal dominant genetic cardiovascular disorder marked by genetic and phenotypic heterogeneity. Mutations in the gene encodes the cardiac myosin‐binding protein C, cMYBPC3 is amongst the various sarcomeric genes that are associated with HCM. These mutations produce mutated mRNAs and truncated cMyBP‐C proteins. In this review, we will discuss the implications and molecular mechanisms involved in MYBPC3 different mutations. Further, we will highlight the novel targets that can be developed into potential therapeutics for the treatment of HMC. J. Cell. Physiol. 232: 1650–1659, 2017. © 2016 Wiley Periodicals, Inc. This review discusses the implications and the and molecular mechanisms of MYBPC3 different mutations. Further, we will highlight the novel targets that can be developed into potential therapeutics for the treatment of Hypertrophic cardiomyopathy.

  February 16, 2017   doi: 10.1002/jcp.25639   open full text
• Subclinical alteration of the cervical–vaginal microbiome in women with idiopathic infertility.
  Giuseppina Campisciano, Fiorella Florian, Angela D'Eustacchio, David Stanković, Giuseppe Ricci, Francesco De Seta, Manola Comar.
  Journal of Cellular Physiology. February 16, 2017
  Biomarkers have a wide application in research and clinic, they help to choose the correct treatment for diseases. Recent studies, addressing the vaginal microbiome using next generation sequencing (NGS), reported the involvement of bacterial species in infertility. We compared the vaginal microbiome of idiopathic infertile women with that of healthy, including bacterial vaginosis affected women and non‐idiopathic infertile women, to identify bacterial species suitable as biomarkers. Information on microorganisms was obtained from the V3‐16S rDNA sequencing of cervical–vaginal fluids of 96 women using the Ion Torrent platform. Data were processed with QIIME and classified against the Vaginal 16S rDNA Reference Database. The analysis revealed a significant beta‐diversity variation (p < 0.001) between the four groups included in the study. L. iners, L. crispatus, and L. gasseri distinguished idiopathic infertile women from the other groups. In these women, a microbial profile similar to that observed in bacterial vaginosis women has been detected. Our results suggest that the quantitative assessment and identification of specific microorganisms of the cervical–vaginal microflora could increase the accuracy of available tools for the diagnosis of infertility and improve the adoption of therapeutic protocols. Women with repeated failed in vitro fertilization cycles may benefit from microbial screening by NGS. NGS screening could increase the accuracy of available tools for the diagnosis of infertility and improve therapeutic protocols.

  February 16, 2017   doi: 10.1002/jcp.25806   open full text
• Dact1, a Wnt‐Pathway Inhibitor, Mediates Human Mesangial Cell TGF‐β1‐Induced Apoptosis.
  Daniele Pereira Jardim, Paula Cristina Eiras Poço, Alexandre Holthausen Campos.
  Journal of Cellular Physiology. February 16, 2017
  Chronic kidney disease (CKD) is a worldwide public health problem that affects millions of men and women of all ages and racial groups. Loss of mesangial cells (MC) represents an early common feature in the pathogenesis of CKD. Transforming growth factor‐β1 (TGF‐β1) is a key inducer of kidney damage and triggers several pathological changes in renal cells, notably MC apoptosis. However, the mechanism of MC apoptosis induced by TGF‐β1 remains elusive. Here, we demonstrate for the first time a novel regulatory pathway in which the disheveled‐binding antagonist of β‐catenin 1 (Dact1) gene is upregulated by TGF‐β1, inducing MC apoptosis. We also show that the inhibitory effect of Dact1 and TGF‐β1 on the transcriptional activation of the pro‐survival Wnt pathway is the mechanism of death induction. In addition, Dact1 mRNA/protein levels are increased in kidney remnants from 5/6 nephrectomized rats and strongly correlate with TGF‐β1 expression. Together, our results point to Dact1 as a novel element controlling MC survival that is causally related to CKD progression. J. Cell. Physiol. 232: 2104–2111, 2017. © 2016 Wiley Periodicals, Inc. Here, we demonstrate for the first time a novel regulatory pathway in which the disheveled‐binding antagonist of β‐catenin 1 (Dact1) gene is upregulated by TGF‐β1, inducing MC apoptosis.

  February 16, 2017   doi: 10.1002/jcp.25636   open full text
• Identification of MSX1 and DCLK1 as mRNA Biomarkers for Colorectal Cancer Detection Through DNA Methylation Information.
  Ai‐Jun Sun, Hai‐Bo Gao, Gao Liu, Heng‐Fa Ge, Zun‐Ping Ke, Sen Li.
  Journal of Cellular Physiology. February 16, 2017
  Colorectal cancer is the second most deadly malignancy in the United States. However, the currently screening options had their limitation. Novel biomarkers for colorectal cancer detections are necessary to reduce the mortality. The clinical information, mRNA expression levels and DNA methylation information of colorectal cancer were downloaded from TCGA. The patients were separated into training group and testing group based on their platforms for DNA methylation. Beta values of DNA methylation from tumor tissues and normal tissues were utilized to figure out the position that were differentially methylated. The expression levels of mRNA of thirteen genes, whose CpG islands were differentially methylated, were extracted from the RNA‐Seq results from TCGA. The probabilities whether the mRNA was differentially expressed between tumor and normal samples were calculated using Student's t‐test. Logistic regression and decision tree were built for cancer detection and their performances were evaluated by the area under the curve (AUC). Twenty‐four genomic locations were differentially methylated, which could be mapped to eleven genes. Nine out of eleven genes had differentially expressed mRNA levels, which were used to build the model for cancer detection. The final detection models consisting of mRNA expression levels of these nine genes had great performances on both training group and testing group. The model that constructed in this study suggested MSX1 and DCLK1 might be used in colorectal cancer detection or as target of cancer therapies. J. Cell. Physiol. 232: 1879–1884, 2017. © 2016 Wiley Periodicals, Inc. Nine out of eleven genes had differentially expressed mRNA levels, which were used to build the model for cancer detection. The final detection models consisting of mRNA expression levels of these nine genes had great performances on both training group and testing group. The model that constructed in this study suggested MSX1 and DCLK1 might be used in colorectal cancer detection or as target of cancer therapies.

  February 16, 2017   doi: 10.1002/jcp.25733   open full text
• Mesenchyme Homeobox 2 Enhances Migration of Endothelial Colony Forming Cells Exposed to Intrauterine Diabetes Mellitus.
  Cassandra R. Gohn, Emily K. Blue, BreAnn M. Sheehan, Kaela M. Varberg, Laura S. Haneline.
  Journal of Cellular Physiology. February 16, 2017
  Diabetes mellitus (DM) during pregnancy has long‐lasting implications for the fetus, including cardiovascular morbidity. Previously, we showed that endothelial colony forming cells (ECFCs) from DM human pregnancies have decreased vasculogenic potential. Here, we evaluate whether the molecular mechanism responsible for this phenotype involves the transcription factor, Mesenchyme Homeobox 2 (MEOX2). In human umbilical vein endothelial cells, MEOX2 upregulates cyclin‐dependent kinase inhibitor expression, resulting in increased senescence and decreased proliferation. We hypothesized that dysregulated MEOX2 expression in neonatal ECFCs from DM pregnancies decreases network formation through increased senescence and altered cell cycle progression. Our studies show that nuclear MEOX2 is increased in ECFCs from DM pregnancies. To determine if MEOX2 is sufficient and/or required to induce impaired network formation, MEOX2 was overexpressed and depleted in ECFCs from control and DM pregnancies, respectively. Surprisingly, MEOX2 overexpression in control ECFCs resulted in increased network formation, altered cell cycle progression, and increased senescence. In contrast, MEOX2 knockdown in ECFCs from DM pregnancies led to decreased network formation, while cell cycle progression and senescence were unaffected. Importantly, migration studies demonstrated that MEOX2 overexpression increased migration, while MEOX2 knockdown decreased migration. Taken together, these data suggest that altered migration may be mediating the impaired vasculogenesis of ECFCs from DM pregnancies. While initially believed to be maladaptive, these data suggest that MEOX2 may serve a protective role, enabling increased vessel formation despite exposure to a DM intrauterine environment. J. Cell. Physiol. 232: 1885–1892, 2017. © 2016 Wiley Periodicals, Inc. Endothelial colony forming cells (ECFCs) from diabetic human pregnancies have decreased vasculogenic potential both in vitro and in vivo. The goal of this study was to evaluate whether the molecular mechanism responsible for this phenotype involves the transcription factor, Mesenchyme Homeobox 2 (MEOX2). While initially believed to be maladaptive, these data suggest that MEOX2 may serve a protective role, enabling increased vessel formation despite exposure to a DM intrauterine environment.

  February 16, 2017   doi: 10.1002/jcp.25734   open full text
• Protective Role of GPER Agonist G‐1 on Cardiotoxicity Induced by Doxorubicin.
  Ernestina M. De Francesco, Carmine Rocca, Francesco Scavello, Daniela Amelio, Teresa Pasqua, Damiano C. Rigiracciolo, Andrea Scarpelli, Silvia Avino, Francesca Cirillo, Nicola Amodio, Maria C. Cerra, Marcello Maggiolini, Tommaso Angelone.
  Journal of Cellular Physiology. February 16, 2017
  The use of Doxorubicin (Dox), a frontline drug for many cancers, is often complicated by dose‐limiting cardiotoxicity in approximately 20% of patients. The G‐protein estrogen receptor GPER/GPR30 mediates estrogen action as the cardioprotection under certain stressful conditions. For instance, GPER activation by the selective agonist G‐1 reduced myocardial inflammation, improved immunosuppression, triggered pro‐survival signaling cascades, improved myocardial mechanical performance, and reduced infarct size after ischemia/reperfusion (I/R) injury. Hence, we evaluated whether ligand‐activated GPER may exert cardioprotection in male rats chronically treated with Dox. 1 week of G‐1 (50 μg/kg/day) intraperitoneal administration mitigated Dox (3 mg/kg/day) adverse effects, as revealed by reduced TNF‐α, IL‐1β, LDH, and ROS levels. Western blotting analysis of cardiac homogenates indicated that G‐1 prevents the increase in p‐c‐jun, BAX, CTGF, iNOS, and COX2 expression induced by Dox. Moreover, the activation of GPER rescued the inhibitory action elicited by Dox on the expression of BCL2, pERK, and pAKT. TUNEL assay indicated that GPER activation may also attenuate the cardiomyocyte apoptosis upon Dox exposure. Using ex vivo Langendorff perfused heart technique, we also found an increased systolic recovery and a reduction of both infarct size and LDH levels in rats treated with G‐1 in combination with Dox respect to animals treated with Dox alone. Accordingly, the beneficial effects induced by G‐1 were abrogated in the presence of the GPER selective antagonist G15. These data suggest that GPER activation mitigates Dox‐induced cardiotoxicity, thus proposing GPER as a novel pharmacological target to limit the detrimental cardiac effects of Dox treatment. J. Cell. Physiol. 232: 1640–1649, 2017. © 2016 Wiley Periodicals, Inc. 1. The potential beneficial effects of GPER activation in Doxorubicin (Dox)‐induced cardiotoxicity was evaluated. 2. In male rat heart, the treatment with the selective GPER ligand G‐1 prevented the increase in TNF‐α, IL‐1β, and LDH plasma levels induced by Dox. In Langendorff perfused rat heart, G‐1 also increased the systolic recovery at the end of ischemia and reduced infarct size and LDH levels triggered by Dox. Moreover, G‐1 attenuated the Dox‐induced myocyte apoptosis, as well as the inhibition of pro‐survival signaling cascades like ERK1/2 and AKT. Next, G‐1 counteracted the down‐regulation of BCL2 as well as the up‐regulation of apoptotic, inflammatory and fibrotic targets, such as BAX, iNOS, COX2, and CTGF induced by Dox. 3. Our study paves the way for further analyzing the role of GPER as pharmacological target in novel therapeutic strategies aimed to prevent the cardiotoxicity exerted by Dox.

  February 16, 2017   doi: 10.1002/jcp.25585   open full text
• Tight Junction Protein Abundance and Apoptosis During Involution of Rat Mammary Glands.
  Claire V. C. Phyn, Kerst Stelwagen, Stephen R. Davis, Christopher D. McMahon, Joanne M. Dobson, Kuljeet Singh.
  Journal of Cellular Physiology. February 16, 2017
  To examine tight junction protein abundance and apoptosis of epithelial cells at the onset of involution in rodent mammary glands, milk accumulation and mammary engorgement were induced by teat‐sealing with an adhesive for 0, 6, 12, 18, 24, and 36 h (n = 6 per group) at peak lactation. In non‐sealed control glands, histological analysis confirmed a lactating phenotype, indicating suckling by pups throughout the experiment. In contrast, alveoli of teat‐sealed glands were distended within 6 h, with maximal luminal size observed by 12 h of non‐suckling. By 18 h following teat‐sealing, an involuting phenotype was observed, indicated by alveolar lumina engorged with milk vesicles and increased leukocytes. Relative to non‐sealed glands, mammary apoptosis was increased in engorged glands 18 h following teat‐sealing. The abundance of ZO‐1 and occludin proteins was decreased in engorged glands by 12 and 18 h, respectively, following teat‐sealing. In contrast, the claudin‐1 22 kDa band was increased by 6 h and peaked at 12–18 h, whereas the 28 kDa band declined by 36 h, relative to controls. There were no temporal changes in ZO‐1, occludin, and claudin‐1 22 kDa proteins within control glands, although there were minor differences in claudin‐1 28 kDa. These data indicate that intramammary milk accumulation due to cessation of milk removal is associated with mammary apoptosis. The apoptotic event is preceded by a rapid loss of abundance of ZO‐1, occludin and an initial increase in claudin‐1. The loss of cell–cell communication may initiate involution and apoptosis of mammary epithelial cells and is a localized intramammary event, occurring only in non‐suckled glands. J. Cell. Physiol. 232: 2075–2082, 2017. © 2016 Wiley Periodicals, Inc. To examine tight junction protein abundance and apoptosis of epithelial cells at the onset of involution in rodent mammary glands, milk accumulation and mammary engorgement was induced by teat‐sealing for 0, 6, 12, 18, 24, and 36 h (n = 6 per group) at peak lactation. The loss of cell–cell communication may initiate involution and apoptosis of mammary epithelial cells and is a localized intramammary event, occurring only in non‐suckled glands.

  February 16, 2017   doi: 10.1002/jcp.25591   open full text
• Postnatal Hyperplasic Effects of ActRIIB Blockade in a Severely Dystrophic Muscle.
  Cory Nielsen, Ross M. Potter, Christopher Borowy, Kimberly Jacinto, Ravi Kumar, C. George Carlson.
  Journal of Cellular Physiology. February 16, 2017
  The efficacy of two ActRIIB ligand‐trapping agents (RAP‐031 and RAP‐435) in treating muscular dystrophy was examined by determining their morphological effects on the severely dystrophic triangularis sterni (TS) muscle of the mdx mouse, a model for Duchenne muscular dystrophy. These agents trap all endogenous ligands to the ActRIIB receptor and thereby block myostatin signaling in a highly selective manner. Short‐term (1 month) and long‐term (3 months) in vivo treatment of 1‐month‐old mdx mice increased myonuclei and fiber cross section (FCS) density but did not alter individual fiber size. Vehicle‐treated mdx mice exhibited age‐dependent increases in myonuclei and FCS density, and age‐dependent reductions in centronucleation that were each enhanced by treatment with RAP‐435. Distributions of FCS area (FCSA) in the mdx TS were 90% identical to those from untreated age‐matched nondystrophic mice and were unaltered by the substantial fiber hyperplasia observed with age and RAP‐435 treatment. These results were inconsistent with injury‐induced fiber regeneration which produces altered FCSA distributions characterized by a distinct class of smaller regenerated fibers. Nondystrophic mice exhibited a constant postnatal density of fiber cross sections and myonuclei, and RAP‐435 treatment of nondystrophic mice increased TS mean FCSA but had no effects on myonuclei or FCS density. These results demonstrating a continual postnatal proliferation and fusion of satellite cells and a response to myostatin blockade characteristic of developing prenatal muscle suggest that the lack of dystrophin directly results in unrestrained postnatal satellite cell activation that is not necessarily dependent upon prior fiber degeneration. J. Cell. Physiol. 232: 1774–1793, 2017. © 2016 Wiley Periodicals, Inc. We examined the efficacy of two ActRIIB ligand‐trapping agents in altering the morphology of a severely dystrophic muscle in the mdx mouse. The results indicate that severely dystrophic muscle exhibits monotonic postnatal increases in myonuclei and fiber density that are not associated with alterations in the distribution of fiber cross‐sectional areas. Long‐term treatment with an ActRIIB ligand‐trapping agent substantially enhanced this postnatal hyperplasia and reduced centronucleation. These results demonstrating a continual postnatal proliferation and fusion of satellite cells and a response to myostatin blockade characteristic of developing prenatal muscle suggest that the lack of dystrophin directly results in unrestrained postnatal satellite cell activation that is not necessarily dependent upon prior fiber degeneration.

  February 16, 2017   doi: 10.1002/jcp.25694   open full text
• MicroRNA‐125a‐5p Is a Downstream Effector of Sorafenib in Its Antiproliferative Activity Toward Human Hepatocellular Carcinoma Cells.
  Nicoletta Potenza, Nicola Mosca, Silvia Zappavigna, Filomena Castiello, Marta Panella, Carmela Ferri, Daniela Vanacore, Antonio Giordano, Paola Stiuso, Michele Caraglia, Aniello Russo.
  Journal of Cellular Physiology. February 16, 2017
  Sorafenib is an antitumor drug for treatment of advanced hepatocellular carcinoma (HCC). It acts as a multikinase inhibitor suppressing cell proliferation and angiogenesis. Human microRNA‐125a‐5p (miR‐125a) is endowed with similar activities and is frequently downregulated in HCC. Looking for a potential microRNA‐based mechanism of action of the drug, we found that sorafenib increases cellular expression of miR‐125a in cultured HuH‐7 and HepG2 HCC cells. Upregulation of the microRNA inhibited cell proliferation by suppression of sirtuin‐7, a NAD(+)‐dependent deacetylase, and p21/p27‐dependent cell cycle arrest in G1. Later, recruitment of miR‐125a in the antiproliferative activity of sorafenib was inquired by modulating its expression in combination with the drug treatment. This analysis showed that intracellular delivery of miR‐125a had no additive effect on the antiproliferative activity of sorafenib, whereas a miR‐125a inhibitor could counteract it. Finally, evaluation of other oncogenic targets of miR‐125a revealed its ability to interfere with the expression of matrix metalloproteinase‐11, Zbtb7a proto‐oncogene, and c‐Raf, possibly contributing to the antiproliferative activity of the drug. J. Cell. Physiol. 232: 1907–1913, 2017. © 2016 Wiley Periodicals, Inc. Upregulation of the microRNA inhibited cell proliferation by suppression of sirtuin‐7, a NAD(+)‐dependent deacetylase, and p21/p27‐dependent cell cycle arrest in G1. Intracellular delivery of miR‐125a had no additive effect on the antiproliferative activity of sorafenib, whereas a miR‐125a inhibitor could counteract it. miR‐125a interfered with the expression of matrix metalloproteinase‐11, Zbtb7a proto‐oncogene, and c‐Raf, possibly contributing to the antiproliferative activity of the drug.

  February 16, 2017   doi: 10.1002/jcp.25744   open full text
• Knockdown of REV3 synergizes with ATR inhibition to promote apoptosis induced by cisplatin in lung cancer cells.
  He‐Guo Jiang, Ping Chen, Jin‐Yu Su, Ming Wu, Hai Qian, Yi Wang, Jian Li.
  Journal of Cellular Physiology. February 13, 2017
  It has been demonstrated that REV3, the catalytic subunit of the translesion synthesis (TLS) polymerase ζ, play an important role in DNA damage response (DDR) induced by cisplatin, and Ataxia‐telangietasia mutated and Rad‐3‐related (ATR) knase is a central player in activating cell cycle checkpoint, stabilizing replication forks, regulating DDR, and promoting repair of DNA damage caused by cisplatin. Cancer cells deficient in either one of REV3 and ATR are more sensitive to cisplatin. However, whether co‐inhibition of REV3 and ATR can further increase sensitivity of non‐small cell lung cancer (NSCLC) cells to cisplatin is not clear. In this study, we show that REV3 knockdown combined with ATR inhibition further enhance cytotoxicity of cisplatin in NSCLC cells, including cisplatin‐sensitive and ‐resistant cell lines, compared to individual knockdown of REV3 or ATR, which are accompanied by markedly caspase‐dependent apoptosis response, pronounced DNA damage accumulation and severe impediment of interstrand crosslink (ICL), and double strand break (DSB) repair. Our results suggest that REV3 knockdown synergize strongly with ATR inhibition to significantly increase sensitivity of cisplatin in NSCLC cells by inhibiting ICL and DSB repair. Thus simultaneously targeting REV3 and ATR may represent one approach to overcome cisplatin resistance and improve chemotherapeutic efficacy in NSCLC treatment. Herein, we showed that depletion of REV3 combined with ATR inhibition significantly potentiates the cytotoxicity of cisplatin to non‐small cell lung cancer (NSCLC) cells, which is concomitant with pronounced DNA damage accumulation and severe impediment of interstrand crosslink (ICL) and double strand break (DSB) repair, compared to individual depletion of REV3 or ATR. Our data demonstrate that REV3 depletion can synergize with ATR inhibition to increase suppression effect of cisplatin on NSCLC cells through inhibition of ICL and DSB repair.

  February 13, 2017   doi: 10.1002/jcp.25792   open full text
• M2‐like macrophages induce colon cancer cell invasion via matrix metalloproteinases.
  Katyayni Vinnakota, Yuan Zhang, Benson Chellakkan Selvanesan, Geriolda Topi, Tavga Salim, Janna Sand‐Dejmek, Gunilla Jönsson, Anita Sjölander.
  Journal of Cellular Physiology. February 13, 2017
  The inflammatory milieu plays an important role in colon cancer development and progression. Previously, we have shown that tumor‐associated macrophages (TAMs), an important component of the tumor microenvironment, are enriched in tumors compared with normal tissue and confer a poorer prognosis. In the present study, we found that matrix metallopeptidase‐9 (MMP‐9), which degrades extracellular matrix proteins, was increased in biopsies from colon cancer patients and in mouse xenografts with SW480 cell‐derived tumors. SW480 colon cancer cells exposed to M2‐like macrophage‐conditioned medium (M2‐medium) exhibited increased MMP‐9 mRNA, protein expression and gelatinase activity. A similar effect was obtained by the addition of tumor necrosis factor‐α (TNFα) and leukotriene D4 (LTD4). MMP‐9 expression and activity were reduced by a TNFα blocking antibody adalimumab and a cysteinyl leukotriene receptor 1 (CysLTR1, the receptor for LTD4) antagonist montelukast. M2‐medium also induced changes in the epithelial–mesenchymal transition (EMT) markers E‐cadherin, β‐catenin, vimentin, and snail in SW480 cells. We also found that both M2‐medium and TNFα and LTD4 induced stabilization/nuclear translocation of β‐catenin. Furthermore, we also observed an elongated phenotype that may indicate increased invasiveness, as confirmed in a collagen I invasion assay. M2‐medium increased the invasive ability, and a similar effect was also obtained by the addition of TNFα and LTD4. The specific MMP inhibitor I or adalimumab and montelukast reduced the number of invasive cells. In conclusion, our findings show that M2‐medium enriched in TNFα and LTD4 promote colon cancer cell invasion via MMP‐9 expression and activation and the induction of EMT. Our findings show that M2‐medium enriched in TNFalfa and LTD4 promote colon cancer cell invasion via MMP‐9 expression and activation and the induction of EMT in SW480 colon cancer cells.

  February 13, 2017   doi: 10.1002/jcp.25808   open full text
• Lipid profiling of parkin‐mutant human skin fibroblasts.
  Simona Lobasso, Paola Tanzarella, Daniele Vergara, Michele Maffia, Tiziana Cocco, Angela Corcelli.
  Journal of Cellular Physiology. February 10, 2017
  Parkin mutations are a major cause of early‐onset Parkinson's disease (PD). The impairment of protein quality control system together with defects in mitochondria and autophagy process are consequences of the lack of parkin, which leads to neurodegeneration. Little is known about the role of lipids in these alterations of cell functions. In the present study, parkin‐mutant human skin primary fibroblasts have been considered as cellular model of PD to investigate on possible lipid alterations associated with the lack of parkin protein. Dermal fibroblasts were obtained from two unrelated PD patients with different parkin mutations and their lipid compositions were compared with that of two control fibroblasts. The lipid extracts of fibroblasts have been analyzed by combined matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF/MS) and thin‐layer chromatography (TLC). In parallel, we have performed direct MALDI‐TOF/MS lipid analyses of intact fibroblasts by skipping lipid extraction steps. Results show that the proportions of some phospholipids and glycosphingolipids were altered in the lipid profiles of parkin‐mutant fibroblasts. The detected higher level of gangliosides, phosphatidylinositol, and phosphatidylserine could be linked to dysfunction of autophagy and mitochondrial turnover; in addition, the lysophosphatidylcholine increase could represent the marker of neuroinflammatory state, a well‐known component of PD. In the present study, parkin‐mutant human skin primary fibroblasts have been considered as cellular model of Parkinson's disease (PD) in order to investigate on possible lipid alterations associated with the lack of parkin protein. Dermal fibroblasts were obtained from two unrelated PD patients with different parkin mutations and their lipid compositions were compared with that of two control fibroblasts. The lipid extracts of fibroblasts have been analyzed by combined MALDI‐TOF/MS and TLC. In parallel, we have performed direct MALDI‐TOF/MS lipid analyses of intact fibroblasts. Results show that the proportions of some phospholipids and glycosphingolipids were altered in the lipid profiles of parkin‐mutant fibroblasts.

  February 10, 2017   doi: 10.1002/jcp.25815   open full text
• Mesenchymal stem cells improves survival in LPS‐induced acute lung injury acting through inhibition of NETs formation.
  Leonardo Pedrazza, Aline Andrea Cunha, Carolina Luft, Nailê Karine Nunes, Felipe Schimitz, Rodrigo Benedetti Gassen, Ricardo Vaz Breda, Marcio Vinícius Fagundes Donadio, Angela Terezinha de Souza Wyse, Paulo Marcio Condessa Pitrez, Jose Luis Rosa, Jarbas Rodrigues de Oliveira.
  Journal of Cellular Physiology. February 09, 2017
  Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are syndromes of acute hypoxemic respiratory failure resulting from a variety of direct and indirect injuries to the gas exchange parenchyma of the lungs. During the ALI, we have an increase release of proinflammatory cytokines and high reactive oxygen species (ROS) formation. These factors are responsible for the release and activation of neutrophil‐derived proteases and the formation of neutrophil extracellular traps (NETs). The excessive increase in the release of NETs cause damage to lung tissue. Recent studies have studies involving the administration of mesenchymal stem cells (MSCs) for the treatment of experimental ALI has shown promising results. In this way, the objective of our study is to evaluate the ability of MSCs, in a lipopolysaccharide (LPS)‐induced ALI model, to reduce inflammation, oxidative damage, and consequently decrease the release of NETs. Mice were submitted lung injury induced by intratracheal instillation of LPS and subsequently treated or not with MSCs. Treatment with MSCs was able to modulate pulmonary inflammation, decrease oxidative damage, and reduce the release of NETs. These benefits from treatment are evident when we observe a significant increase in the survival curve in the treated animals. Our results demonstrate that MSCs treatment is effective for the treatment of ALI. For the first time, it is described that MSCs can reduce the formation of NETs and an experimental model of ALI. This finding is directly related to these cells modulate the inflammatory response and oxidative damage in the course of the pathology. Treatment with MSCs was able to modulate pulmonary inflammation, decrease oxidative damage, and reduce the release of NETs. For the first time, it is described that MSCs can reduce the formation of NETs and an experimental model of ALI.

  February 09, 2017   doi: 10.1002/jcp.25816   open full text
• Impact of lysosomal storage disorders on biology of mesenchymal stem cells: Evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha‐galactosidase A (GLA) enzymes.
  Tiziana Squillaro, Ivana Antonucci, Nicola Alessio, Anna Esposito, Marilena Cipollaro, Mariarosa Anna Beatrice Melone, Gianfranco Peluso, Liborio Stuppia, Umberto Galderisi.
  Journal of Cellular Physiology. February 07, 2017
  Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha‐galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia–telangiectasia‐mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma‐H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity. Gaucher disease (GD) and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha‐galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected; thus, we evaluated whether GBA and GLA silencing could affect the biology of mesenchymal stem cells (MSCs) isolated from bone marrow and amniotic fluid. Our data suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity.

  February 07, 2017   doi: 10.1002/jcp.25807   open full text
• LSP1, a responsive protein from Meyerozyma guilliermondii, elicits defence response and improves glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis Fisch adventitious roots.
  Juan Wang, Jianli Li, Jing Li, Jinxin Li, Shujie Liu, Wenyuan Gao.
  Journal of Cellular Physiology. February 07, 2017
  This research explored the effects of protein and polysaccharide in Meyerozyma guilliermondii on active compounds in Glycyrrhiza uralensis Fisch adventitious roots. In this study, a responsive protein LSP1 was purified from the Meyerozyma guilliermondii since the excellent induction. The contents of total flavonoids (3.46 mg · g−1), glycyrrhizic acid (0.41 mg · g−1), glycyrrhetinic acid (0.41 mg · g−1), and polysaccharide (94.49 mg · g−1) in adventitious root peaked at LSP1 group, which were 1.6, 3.4, 2.4, 2.0‐fold that of control, respectively. Besides, the responsive protein LSP1 significantly activated the defense signaling, mitogen‐activated protein kinases and extremely up‐regulated the expression of defense‐related genes and functional genes involved in glycyrrhizic acid biosynthesis. This research explored the effects of protein and polysaccharide in Meyerozyma guilliermondii on active compounds in Glycyrrhiza uralensis Fisch adventitious roots. In this study, a responsive protein LSP1 was purified from the M. guilliermondii since the excellent induction. Besides, the responsive protein LSP1 significantly activated the defense signaling, mitogen‐activated protein kinases and extremely up‐regulated the expression of defense‐related genes and functional genes involved in glycyrrhizic acid biosynthesis.

  February 07, 2017   doi: 10.1002/jcp.25811   open full text
• Involvement of CX3CL1 in the Migration of Osteoclast Precursors Across Osteoblast Layer Stimulated by Interleukin‐1ß.
  Tsuyoshi Matsuura, Shizuko Ichinose, Masako Akiyama, Yuki Kasahara, Noriko Tachikawa, Ken‐ichi Nakahama.
  Journal of Cellular Physiology. February 03, 2017
  The trigger for bone remodeling is bone resorption by osteoclasts. Osteoclast differentiation only occurs on the old bone, which needs to be repaired under physiological conditions. However, uncontrolled bone resorption is often observed in pro‐inflammatory bone diseases, such as rheumatoid arthritis. Mature osteoclasts are multinuclear cells that differentiate from monocyte/macrophage lineage cells by cell fusion. Although Osteoclast precursors should migrate across osteoblast layer to reach bone matrix before maturation, the underlying mechanisms have not yet been elucidated in detail. We herein found that osteoclast precursors utilize two routes to migrate across osteoblast layer by confocal‐ and electro‐microscopic observations. The osteoclast supporting activity of osteoblasts inversely correlated with osteoblast density and was positively related to the number of osteoclast precursors under the osteoblast layer. Osteoclast differentiation was induced by IL‐1ß, but not by PGE2 in high‐density osteoblasts. Osteoblasts and osteoclast precursors expressed CX3CL1 and CX3CR1, respectively, and the expression of CX3CL1 increased in response to interleukin‐1ß. An anti‐CX3CL1‐neutralizing antibody inhibited the migration of osteoclast precursors and osteoclast differentiation. These results strongly suggest the involvement of CX3CL1 in the migration of osteoclast precursors and osteoclastogenesis, and will contribute to the development of new therapies for bone diseases. J. Cell. Physiol. 232: 1739–1745, 2017. © 2016 Wiley Periodicals, Inc. Transcellular and paracellular migration of osteoclast precursors across osteoblast layer were observed. CX3CL1 was involved in the migration of osteoclast precursors and osteoclastogenesis.

  February 03, 2017   doi: 10.1002/jcp.25577   open full text
• DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro.
  Li Duan, Yujie Liang, Bin Ma, Daming Wang, Wei Liu, Jianghong Huang, Jianyi Xiong, Liangquan Peng, Jielin Chen, Weimin Zhu, Daping Wang.
  Journal of Cellular Physiology. January 31, 2017
  DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full‐scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome‐wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5‐azacytidine (5‐AzaC). This research revealed that the general methylation level of CpG was increased while the COL‐1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5‐AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL‐1A1 promoter was increased after 5‐AzaC treatment. The COL‐1A1 expression level was increased while that of SOX‐9 was decreased during the chondrocyte dedifferentiation. 5‐AzaC treatment up‐regulated the SOX‐9 expression while down‐regulated the COL‐1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage‐specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. J. Cell. Physiol. 232: 1708–1716, 2017. © 2016 Wiley Periodicals, Inc. Differential regulation of the DNA methylation level of cartilage‐specific genes might contribute the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation.

  January 31, 2017   doi: 10.1002/jcp.25486   open full text
• Cytoskeleton Aberrations in Alkaptonuric Chondrocytes.
  Michela Geminiani, Silvia Gambassi, Lia Millucci, Pietro Lupetti, Giulia Collodel, Lucia Mazzi, Bruno Frediani, Daniela Braconi, Barbara Marzocchi, Marcella Laschi, Giulia Bernardini, Annalisa Santucci.
  Journal of Cellular Physiology. January 31, 2017
  Alkaptonuria (AKU) is an ultra‐rare autosomal genetic disorder caused by a defect in the activity of the enzyme homogentisate 1,2‐dioxygenase (HGD) that leads to the accumulation of homogentisic acid (HGA) and its oxidized product, benzoquinone acetic acid (BQA), in the connective tissues causing a pigmentation called “ochronosis.” The consequent progressive formation of ochronotic aggregates generate a severe condition of oxidative stress and inflammation in all the affected areas. Experimental evidences have also proved the presence of serum amyloid A (SAA) in several AKU tissues and it allowed classifying AKU as a secondary amyloidosis. Although AKU is a multisystemic disease, the most affected system is the osteoarticular one and articular cartilage is the most damaged tissue. In this work, we have analyzed for the first time the cytoskeleton of AKU chondrocytes by means of immunofluorescence staining. We have shown the presence of SAA within AKU chondrocytes and finally we have demonstrated the co‐localization of SAA with three cytoskeletal proteins: actin, vimentin, and β‐tubulin. Furthermore, in order to observe the ultrastructural features of AKU chondrocytes we have performed TEM analysis, focusing on the Golgi apparatus structure and, to demonstrate that pigmented areas in AKU cartilage are correspondent to areas of oxidation, 4‐HNE presence has been evaluated by means of immunofluorescence. J. Cell. Physiol. 232: 1728–1738, 2017. © 2016 Wiley Periodicals, Inc. The cytoskeleton of alkaptonuric chondrocytes is severely alterated. Serum amyloid A is demonstrated to be present within alkaptonuric chondrocytes and to co‐localize with the three main cytoskeletal proteins: actin, vimentin, and β‐tubulin. These findings lead to hypothesize a role of SAA in cytoskeleton alterations of alkaptonuric chondrocytes.

  January 31, 2017   doi: 10.1002/jcp.25500   open full text
• Exosomes: Nanoparticulate tools for RNA interference and drug delivery.
  Fahimeh Shahabipour, Nastaran Barati, Thomas P. Johnston, Giuseppe Derosa, Pamela Maffioli, Amirhossein Sahebkar.
  Journal of Cellular Physiology. January 31, 2017
  Exosomes are naturally occurring extracellular vesicles released by most mammalian cells in all body fluids. Exosomes are known as key mediators in cell‐cell communication and facilitate the transfer of genetic and biochemical information between distant cells. Structurally, exosomes are composed of lipids, proteins, and also several types of RNAs which enable these vesicles to serve as important disease biomarkers. Moreover, exosomes have emerged as novel drug and gene delivery tools owing to their multiple advantages over conventional delivery systems. Recently, increasing attention has been focused on exosomes for the delivery of drugs, including therapeutic recombinant proteins, to various target tissues. Exosomes are also promising vehicles for the delivery of microRNAs and small interfering RNAs, which is usually hampered by rapid degradation of these RNAs, as well as inefficient tissue specificity of currently available delivery strategies. This review highlights the most recent accomplishments and trends in the use of exosomes for the delivery of drugs and therapeutic RNA molecules. Exosomes are naturally occurring extracellular vesicles released by most mammalian cells in all body fluids. Recently, increasing attention has been focused on exosomes for the delivery of drugs, including therapeutic recombinant proteins, to various target tissues. This review highlights the most recent accomplishments and trends in the use of exosomes for the delivery of drugs and therapeutic RNA molecules.

  January 31, 2017   doi: 10.1002/jcp.25766   open full text
• Extracellular Hsp70 Enhances Mesoangioblast Migration via an Autocrine Signaling Pathway.
  Maria M. Barreca, Walter Spinello, Vincenzo Cavalieri, Giuseppina Turturici, Gabriella Sconzo, Punit Kaur, Rosaria Tinnirello, Alexzander A. A. Asea, Fabiana Geraci.
  Journal of Cellular Physiology. January 31, 2017
  Mouse mesoangioblasts are vessel‐associated progenitor stem cells endowed with the ability of multipotent mesoderm differentiation. Therefore, they represent a promising tool in the regeneration of injured tissues. Several studies have demonstrated that homing of mesoangioblasts into blood and injured tissues are mainly controlled by cytokines/chemokines and other inflammatory factors. However, little is known about the molecular mechanisms regulating their ability to traverse the extracellular matrix (ECM). Here, we demonstrate that membrane vesicles released by mesoangioblasts contain Hsp70, and that the released Hsp70 is able to interact by an autocrine mechanism with Toll‐like receptor 4 (TLR4) and CD91 to stimulate migration. We further demonstrate that Hsp70 has a positive role in regulating matrix metalloproteinase 2 (MMP2) and MMP9 expression and that MMP2 has a more pronounced effect on cell migration, as compared to MMP9. In addition, the analysis of the intracellular pathways implicated in Hsp70 regulated signal transduction showed the involvement of both PI3K/AKT and NF‐κB. Taken together, our findings present a paradigm shift in our understanding of the molecular mechanisms that regulate mesoangioblast stem cells ability to traverse the extracellular matrix (ECM). J. Cell. Physiol. 232: 1845–1861, 2017. © 2016 Wiley Periodicals, Inc. Extracellular Hsp70 released through extracellular vesicles regulates mesoangioblast migration via the activation of NF‐κB and PI3K/AKT pathways.

  January 31, 2017   doi: 10.1002/jcp.25722   open full text
• HDAC1, HDAC4, and HDAC9 Bind to PC3/Tis21/Btg2 and Are Required for Its Inhibition of Cell Cycle Progression and Cyclin D1 Expression.
  Laura Micheli, Giorgio D'Andrea, Luca Leonardi, Felice Tirone.
  Journal of Cellular Physiology. January 27, 2017
  PC3/Tis21 is a transcriptional cofactor that inhibits proliferation in several cell types, including neural progenitors. Here, we report that PC3/Tis21 associates with HDAC1, HDAC4, and HDAC9 in vivo, in fibroblast cells. Furthermore, when HDAC1, HDAC4, or HDAC9 are silenced in fibroblasts or in a line of cerebellar progenitor cells, the ability of PC3/Tis21 to inhibit proliferation is significantly reduced. Overexpression of HDAC1, HDAC4, or HDAC9 in fibroblasts and in cerebellar precursor cells synergizes with PC3/Tis21 in inhibiting the expression of cyclin D1, a cyclin selectively inhibited by PC3/Tis21. Conversely, the depletion of HDAC1 or HDAC4 (but not HDAC9) in fibroblasts and in cerebellar precursor cells significantly impairs the ability of PC3/Tis21 to inhibit cyclin D1 expression. An analysis of HDAC4 deletion mutants shows that both the amino‐terminal moiety and the catalytic domain of HDAC4 associate to PC3/Tis21, but neither alone is sufficient to potentiate the inhibition of cyclin D1 by PC3/Tis21. As a whole, our findings indicate that PC3/Tis21 inhibits cell proliferation in a way dependent on the presence of HDACs, in fibroblasts as well as in neural cells. Considering that several reports have demonstrated that HDACs can act as transcriptional corepressors on the cyclin D1 promoter, our data suggest that the association of PC3/Tis21 to HDACs is functional to recruit them to target genes, such as cyclin D1, for repression of their expression. J. Cell. Physiol. 232: 1696–1707, 2017. © 2016 Wiley Periodicals, Inc. By silencing HDAC1, HDAC4, and HDAC9 in fibroblast as well as in neural cells, we reveal that HDAC1, HDAC4, and HDAC9 are each required for the negative control of the cell cycle exerted by PC3/Tis21. Such effect is likely dependent on the ability of HDAC1, HDAC4, and HDAC9 to associate with PC3/Tis21, that we demonstrate here. Moreover, we find that HDAC1 and HDAC4—but not HDAC9—are required for the inhibition exerted by PC3/Tis21 of cyclin D1 expression.

  January 27, 2017   doi: 10.1002/jcp.25467   open full text
• State of the Art in MicroRNA as Diagnostic and Therapeutic Biomarkers in Chronic Lymphocytic Leukemia.
  Hamed Mirzaei, Sima Fathullahzadeh, Razieh Khanmohammadi, Mansoreh Darijani, Fatemeh Momeni, Aria Masoudifar, Mohammad Goodarzi, Omid Mardanshah, Jan Stanveng, Mahmoud Reza Jaafari, Hamid Reza Mirzaei.
  Journal of Cellular Physiology. January 13, 2017
  Early diagnostic is one of the most important steps in cancer therapy which helps to design and choose a better therapeutic approach. The finding of biomarkers in various levels including genomics, transcriptomics and proteomics levels could provide better treatment for various cancers such as chronic lymphocytic leukemia (CLL). The CLL is the one of main lymphoid malignancies which is specified by aggregation of mature B lymphocytes. Among different biomarkers (e.g. CD38, chromosomes abnormalities, ZAP‐70, TP53 and miRNA), microRNAs (miRNAs) have appeared as new diagnostic and therapeutic biomarkers in patients with the CLL disease. Multiple lines of evidence indicated that deregulation of miRNAs could be associated with pathological events which are present in the CLL. These molecules have an effect on a variety of targets such as Bcl2, c‐fos, c‐Myc, TP53, TCL1 and STAT3 which play critical roles in the CLL pathogenesis. It has been shown that expression of miRNAs could lead to the activation of B cells and BCR. Moreover, exosomes containing miRNAs are one of the other molecules which could contribute to BCR stimulation and progression of CLL cells. Hence, miRNAs and exosomes released from CLL cells could be used as potential diagnostic and therapeutic biomarkers for CLL. This critical review focuses on a very important aspect of CLL based on biomarker discovery covers the pros and cons of using miRNAs as important diagnostics and therapeutics biomarkers for this deadly disease. This article is protected by copyright. All rights reserved

  January 13, 2017   doi: 10.1002/jcp.25799   open full text
• Gating Modulation of the Tumor‐Related Kv10.1 Channel by Mibefradil.
  Froylán Gómez‐Lagunas, Elisa Carrillo, Luis A. Pardo, Walter Stühmer.
  Journal of Cellular Physiology. January 11, 2017
  Several reports credit mibefradil with tumor suppressing properties arising from its known inhibition of Ca2+ currents. Given that mibefradil (Mb) is also known to inhibit K+ channels, we decided to study the interaction between this organic compound and the tumor‐related Kv10.1 channel. Here we report that Mb modulates the gating of Kv10.1. Mb induces an apparent inactivation from both open and early closed states where the channels dwell at hyperpolarized potentials. Additionally, Mb accelerates the kinetics of current activation, in a manner that depends on initial conditions. Our observations suggest that Mb binds to the voltage sensor domain of Kv10.1 channels, thereby modifying the gating of the channels in a way that in some, but not all, aspects opposes to the gating effects exerted by divalent cations. J. Cell. Physiol. 232: 2019–2032, 2017. © 2016 Wiley Periodicals, Inc. Several reports credit mibefradil, a T‐type calcium channel inhibitor, with tumor suppressing properties. Given that mibefradil also inhibits some potassium channels, we studied the interaction of this compound with the tumor related Kv10.1 channel. We found that mibefradil induces an apparent inactivation both from the open and early closed states, and accelerates current activation in a manner that depends on the holding potential. We hypothesize that mibefradil binds to the voltage‐sensor module altering the gating of the channels.

  January 11, 2017   doi: 10.1002/jcp.25448   open full text
• MicroRNA: Relevance to Stroke Diagnosis, Prognosis and Therapy.
  Hamed Mirzaei, Fatemeh Momeni, Leila Saadatpour, Amirhossein Sahebkar, Mohammad Goodarzi, Aria Masoudifar, Shirin Kouhpayeh, Hossein Salehi, Hamid Reza Mirzaei, Mahmoud Reza Jaafari.
  Journal of Cellular Physiology. January 09, 2017
  Stroke is a life‐threatening disease that accounts for a considerable burden of mortality in both developing and developed world. Identification of specific biomarkers for stroke and its outcomes can greatly contribute to improved care of patients. MicroRNAs (miRNAs) are known as novel biomarkers that could be used as diagnostic, prognostic and therapeutic biomarkers. Various studies have shown that miRNAs have key roles in the pathogenesis of stroke, and its complications and outcomes. In addition, there is evidence showing that mesenchaymal stromal cell‐derived exosomes containing miRNAs can be used for monitoring and treatment of various diseases such as stroke. Here, we summarized various aspects of miRNA applications in different stages of stroke. This article is protected by copyright. All rights reserved

  January 09, 2017   doi: 10.1002/jcp.25787   open full text
• HOTAIR Role in Melanoma Progression and its Identification in the Blood of Patients with Advanced Disease.
  Monica Cantile, Giosuè Scognamiglio, Laura Marra, Gabriella Aquino, Chiara Botti, Maria Rosaria Falcone, Maria Gabriella Malzone, Giuseppina Liguori, Maurizio Di Bonito, Renato Franco, Paolo Antonio Ascierto, Gerardo Botti.
  Journal of Cellular Physiology. January 09, 2017
  Objectives The molecular mechanisms responsible for the metastatic progression of melanoma have not been fully defined yet. We have recently shown that an important role in this process is certainly played by HOX genes, whose regulation is under control of particular non‐coding RNAs, some of which are present within the HOX locus. HOTAIR is the most studied among them, whose aberrant expression is associated with the metastatic progression of many malignancies. The aim of this study was to verify the role played by HOTAIR in metastatic progression of melanoma and to evaluate the circulating levels of HOTAIR in the blood of patients with metastatic melanoma. Methods A series of melanocytic lesions were selected to evaluate the potential changes in the expression of HOTAIR during the evolution of the disease through in situ and molecular approaches. Results None of the benign melanocytic lesions showed the presence of HOTAIR. The staining of HOTAIR resulted very weak in the primary pT1 lesions, while it was very strong in all pairs of primary tissues and corresponding metastases. Surprisingly, we found the presence of HOTAIR in some intratumoral lymphocytes, while this positivity decreased in lymphocyte component further away from the tumor. HOTAIR was also detected in the serum of selected metastatic patients. Conclusion These data allowed us to speculate on the fundamental role played by HOTAIR in tumor evolution of melanoma. Its presence in intratumoral lymphocytes might suggest that its involvement in the modulation of tumor microenvironment and the detection in the serum could be used in the management of melanoma patients. This article is protected by copyright. All rights reserved

  January 09, 2017   doi: 10.1002/jcp.25789   open full text
• Biological Roles of Glial Fibrillary Acidic Protein as a Biomarker in Cartilage Regenerative Medicine.
  Sanshiro Kanazawa, Satoru Nishizawa, Tsuyoshi Takato, Kazuto Hoshi.
  Journal of Cellular Physiology. January 07, 2017
  Glial fibrillary acidic protein (GFAP) is an intermediate filament that is expressed in specifically expressed auricular chondrocytes, which are good cell sources of cartilage regenerative medicine. Although our group uses GFAP as a biomarker of matrix production in the cultured auricular chondrocytes, the biological roles of GFAP in auricular chondrocytes has remained unknown. In this study, we demonstrated the biological functions of GFAP in the human and mouse derived auricles to clarify the significance and role with the chondrocytes of GFAP in order to provide useful information for reliable and safe regenerative medicine. We examined the cell responses to stretch stress for these chondrocytes and completed a nuclear morphological analysis. Based on these results, GFAP seems to support the resistance to severe mechanical stress in the tissue which physiologically suffers from a stretch overload, and plays pivotal roles in the conservation of cell structures and functions through the maintenance of nuclear morphology. This article is protected by copyright. All rights reserved

  January 07, 2017   doi: 10.1002/jcp.25771   open full text
• Optimized Method for Isolating Highly Purified and Functional Porcine Aortic Endothelial and Smooth Muscle Cells.
  Farideh Beigi, Mitalben Patel, Marco A. Morales‐Garza, Caitlin Winebrenner, Andrea S. Gobin, Eric Chau, Luiz C. Sampaio, Doris A. Taylor.
  Journal of Cellular Physiology. January 06, 2017
  Background Numerous protocols exist for isolating aortic endothelial and smooth muscle cells from small animals. However, establishing a protocol for isolating pure cell populations from large animal vessels that are more elastic has been challenging. We developed a simple sequential enzymatic approach to isolate highly purified populations of porcine aortic endothelial and smooth muscle cells. Methods The lumen of a porcine aorta was filled with 25 U/mL dispase solution and incubated at 37°C to dissociate the endothelial cells. The smooth muscle cells were isolated by mincing the tunica media of the treated aorta and incubating the pieces in 0.2% and then 0.1% collagenase type I solution. Results The isolated endothelial cells stained positive for von Willebrand factor, and 97.2% of them expressed CD31. Early‐ and late‐passage endothelial cells had a population doubling time of 38 hours and maintained a capacity to take up DiI‐Ac‐LDL and form tubes in Matrigel®. The isolated smooth muscle cells stained highly positive for alpha‐smooth muscle actin, and an impurities assessment showed that only 1.8% were endothelial cells. Population doubling time for the smooth muscle cells was ∼70 hours at passages 3 and 7; and the cells positively responded to endothelin‐1, as shown by a 66% increase in the intracellular calcium level. Conclusions This simple protocol allows for the isolation of highly pure populations of endothelial and smooth muscle cells from porcine aorta that can survive continued passage in culture without losing functionality or becoming overgrown by fibroblasts. This article is protected by copyright. All rights reserved

  January 06, 2017   doi: 10.1002/jcp.25764   open full text
• SIRT1‐SIRT3 Axis Regulates Cellular Response to Oxidative Stress and Etoposide.
  Ilaria Carnevale, Laura Pellegrini, Patrizia D'Aquila, Serena Saladini, Emanuela Lococo, Lucia Polletta, Enza Vernucci, Eleonora Foglio, Stefano Coppola, Luigi Sansone, Giuseppe Passarino, Dina Bellizzi, Matteo A. Russo, Massimo Fini, Marco Tafani.
  Journal of Cellular Physiology. January 06, 2017
  Sirtuins are conserved NAD+‐dependent deacylases. SIRT1 is a nuclear and cytoplasmic sirtuin involved in the control of histones a transcription factors function. SIRT3 is a mitochondrial protein, which regulates mitochondrial function. Although, both SIRT1 and SIRT3 have been implicated in resistance to cellular stress, the link between these two sirtuins has not been studied so far. Here we aimed to unravel: i) the role of SIRT1‐SIRT3 axis for cellular response to oxidative stress and DNA damage; ii) how mammalian cells modulate such SIRT1‐SIRT3 axis and which mechanisms are involved. Therefore, we analyzed the response to different stress stimuli in WT or SIRT1‐silenced cell lines. Our results demonstrate that SIRT1‐silenced cells are more resistant to H2O2 and etoposide treatment showing decreased ROS accumulation, γ‐H2AX phosphorylation, caspase‐3 activation and PARP cleavage. Interestingly, we observed that SIRT1‐silenced cells show an increased SIRT3 expression. To explore such a connection, we carried out luciferase assays on SIRT3 promoter demonstrating that SIRT1‐silencing increases SIRT3 promoter activity and that such an effect depends on the presence of SP1 and ZF5 recognition sequences on SIRT3 promoter. Afterwards, we performed co‐immunoprecipitation assays demonstrating that SIRT1 binds and deacetylates the transcription inhibitor ZF5 and that there is a decreased interaction between SP1 and ZF5 in SIRT1‐silenced cells. Therefore, we speculate that acetylated ZF5 cannot bind and sequester SP1 that is free, then, to increase SIRT3 transcription. In conclusion, we demonstrate that cells with low SIRT1 levels can maintain their resistance and survival by increasing SIRT3 expression. J. Cell. Physiol. 232: 1835–1844, 2017. © 2016 Wiley Periodicals, Inc. Our article demonstrate that cells with low SIRT1 levels can maintain their resistance and survival to exogenous stresses by increasing SIRT3 expression.

  January 06, 2017   doi: 10.1002/jcp.25711   open full text
• Calprotectin Induces IL‐6 and MCP‐1 Production via Toll‐Like Receptor 4 Signaling in Human Gingival Fibroblasts.
  Yasufumi Nishikawa, Yukari Kajiura, Jung Hwan Lew, Jun‐ichi Kido, Toshihiko Nagata, Koji Naruishi.
  Journal of Cellular Physiology. January 06, 2017
  Calprotectin, a heterodimer of S100A8 and S100A9 molecules, is associated with inflammatory diseases such as inflammatory bowel disease. We have reported that calprotectin levels in gingival crevicular fluids of periodontitis patients are significantly higher than in healthy subjects. However, the functions of calprotectin in pathophysiology of periodontitis are still unknown. The aim of this study is to investigate the effects of calprotectin on the productivity of inflammatory cytokines in human gingival fibroblasts (HGFs). The HGFs cell line CRL‐2014® (ATCC) were cultured, and total RNAs were collected to examine the expression of TLR2/4 and RAGE mRNA using RT‐PCR. After the cells were treated with S100A8, S100A9, and calprotectin, supernatants were collected and the levels of IL‐6 and MCP‐1 were measured using ELISA methods. To examine the intracellular signals involved in calprotectin‐induced cytokine production, several chemical inhibitors were used. Furthermore, after the siRNA‐mediated TLR4 down‐regulated cells were treated with S100A8, S100A9, and calprotectin, the levels of IL‐6 and MCP‐1 were also measured. HGFs showed greater expression of TLR4 mRNA, but not TLR2 and RAGE mRNA compared with human oral epithelial cells. Calprotectin increased significantly the production of MCP‐1 and IL‐6 in HGFs, and the cytokine productions were significantly suppressed in the cells treated with MAPKs, NF‐κB, and TLR4 inhibitors. Furthermore, calprotectin‐mediated MCP‐1 and IL‐6 production were significantly suppressed in TLR4 down‐regulated cells. Taken together, calprotectin induces IL‐6 and MCP‐1 production in HGFs via TLR4 signaling that involves MAPK and NF‐κB, resulting in the progression of periodontitis. J. Cell. Physiol. 232: 1862–1871, 2017. © 2016 Wiley Periodicals, Inc.

  January 06, 2017   doi: 10.1002/jcp.25724   open full text
• Cyclic Tensile Strain Reduces TNF‐α Induced Expression of MMP‐13 by Condylar Temporomandibular Joint Cells.
  Hessam Tabeian, Astrid D. Bakker, Beatriz F. Betti, Frank Lobbezoo, Vincent Everts, Teun J. de Vries.
  Journal of Cellular Physiology. January 06, 2017
  To investigate whether the disproportionate degradation of mandibular condyle cartilage in arthritic juvenile temporomandibular joint (TMJ) is related to distinctive responses of TMJ‐derived cells to tumor necrosis factor‐α (TNF‐α), and whether mechanical loading affects this response. The effect of TNF‐α (0.1–10 ng/ml) was tested on juvenile porcine TMJ cells isolated from the condyle, fossa, and disc, grown in 3D agarose gels. Expression of anabolic and catabolic factors was quantified by RT‐qPCR and/or immunohistochemistry. Condylar cells were stimulated for 12 h with TNF‐α (10 ng/ml), followed by 8 h of 6% cyclic tensile strain, and gene expression of MMPs was quantified. TNF‐α (10 ng/ml) reduced the expression of the matrix proteins collagen types I and II after 6 h of incubation. Aggrecan gene expression was increased in the presence of 0.1 ng/ml TNF‐α. The fossa and disc cells responded to TNF‐α with an increased expression of the aggrecanase ADAMTS4. TNF‐α enhanced MMP‐13 gene and protein expression only by condylar cells. Mechanical loading reduced this effect. Cells isolated from the different cartilaginous structures reacted differently to TNF‐α. Since the disc and fossa contain a very low level of proteoglycans in comparison to the condyle, the role played by ADAMTS4 in degradation of the fossa and disc might be limited. TNF‐α induced MMP‐13 expression by condylar cells might be involved in the degradation of the juvenile condyle. Since this expression was reduced by mechanical loading, functional loading with oral physiotherapy or orthodontic activators may help to reduce the catabolic effect of TNF‐α. J. Cell. Physiol. 232: 1287–1294, 2017. © 2016 Wiley Periodicals, Inc. Cells isolated from the different cartilaginous structures reacted differently to TNF‐α. TNF‐α enhanced MMP‐13 gene and protein expression only by condylar cells. Mechanical loading reduced this effect.

  January 06, 2017   doi: 10.1002/jcp.25593   open full text
• A MicroRNA Cluster miR‐23–24–27 Is Upregulated by Aldosterone in the Distal Kidney Nephron Where it Alters Sodium Transport.
  Xiaoning Liu, Robert S. Edinger, Christine A. Klemens, Yu L. Phua, Andrew J. Bodnar, William A. LaFramboise, Jacqueline Ho, Michael B. Butterworth.
  Journal of Cellular Physiology. January 05, 2017
  The epithelial sodium channel (ENaC) is expressed in the epithelial cells of the distal convoluted tubules, connecting tubules, and cortical collecting duct (CCD) in the kidney nephron. Under the regulation of the steroid hormone aldosterone, ENaC is a major determinant of sodium (Na+) and water balance. The ability of aldosterone to regulate microRNAs (miRs) in the kidney has recently been realized, but the role of miRs in Na+ regulation has not been well established. Here we demonstrate that expression of a miR cluster mmu‐miR‐23–24–27, is upregulated in the CCD by aldosterone stimulation both in vitro and in vivo. Increasing the expression of these miRs increased Na+ transport in the absence of aldosterone stimulation. Potential miR targets were evaluated and miR‐27a/b was verified to bind to the 3′‐untranslated region of intersectin‐2, a multi‐domain protein expressed in the distal kidney nephron and involved in the regulation of membrane trafficking. Expression of Itsn2 mRNA and protein was decreased after aldosterone stimulation. Depletion of Itsn2 expression, mimicking aldosterone regulation, increased ENaC‐mediated Na+ transport, while Itsn2 overexpression reduced ENaC's function. These findings reinforce a role for miRs in aldosterone regulation of Na+ transport, and implicate miR‐27 in aldosterone's action via a novel target. J. Cell. Physiol. 232: 1306–1317, 2017. © 2016 Wiley Periodicals, Inc. The steroid hormone aldosterone alters the expression of microRNAs in distal kidney nephron epithelial cells, and coordinately upregulates the miR‐23–24–27 cluster. MiR‐27 targets intersectin 2 to reduce its expression. Decreased intersectin 2 levels results in an increase in the activity of the epithelial sodium channel to augment sodium transport.

  January 05, 2017   doi: 10.1002/jcp.25599   open full text
• Early Subchondral Bone Loss at Arthritis Onset Predicted Late Arthritis Severity in a Rat Arthritis Model.
  Guillaume Courbon, Damien Cleret, Marie‐Thérèse Linossier, Laurence Vico, Hubert Marotte.
  Journal of Cellular Physiology. January 05, 2017
  Synovitis is usually observed before loss of articular function in rheumatoid arthritis (RA). In addition to the synovium and according to the “Inside–Outside” theory, bone compartment is also involved in RA pathogenesis. Then, we investigated time dependent articular bone loss and prediction of early bone loss to late arthritis severity on the rat adjuvant‐induced arthritis (AIA) model. Lewis female rats were longitudinally monitored from arthritis induction (day 0), with early (day 10) and late (day 17) steps. Trabecular and cortical microarchitecture parameters of four ankle bones were assessed by microcomputed tomography. Gene expression was determined at sacrifice. Arthritis occurred at day 10 in AIA rats. At this time, bone erosions were detected on four ankle bones, with cortical porosity increase (+67%) and trabecular alterations including bone volume fraction (BV/TV: −13%), and trabecular thickness decrease. Navicular bone assessment was the most reproducible and sensitive. Furthermore, strong correlations were observed between bone alterations at day 10 and arthritis severity or bone loss at day 17, including predictability of day 10 BV/TV to day 17 articular index (R2 = 0.76). Finally, gene expression at day 17 confirmed massive osteoclast activation and interestingly provided insights on strong activation of bone formation inhibitor markers at the joint level. In rat AIA, bone loss was already observed at synovitis onset and was predicted late arthritis severity. Our results reinforced the key role of subchondral bone in arthritis pathogenesis, in favour to the “Inside–Outside” theory. Mechanisms of bone loss in rat AIA involved resorption activation and formation inhibition changes. J. Cell. Physiol. 232: 1318–1325, 2017. © 2016 Wiley Periodicals, Inc. We explored time dependent articular bone loss and prediction of early bone loss to late arthritis severity on the rat adjuvant‐induced arthritis (AIA) model. At clinical synovitis onset, bone porosity and microarchitecture alterations were recorded in the ankle bones. These early modifications were predictive of arthritis outcome, underlining the importance of early bone loss investigation.

  January 05, 2017   doi: 10.1002/jcp.25601   open full text
• Calretinin Immunoreactivity in the Human Testis Throughout Fetal Life.
  Giovanna G. Altobelli, Francesca Pentimalli, Mariarosaria D'Armiento, Susan Van Noorden, Vincenzo Cimini.
  Journal of Cellular Physiology. January 05, 2017
  The main functions of the testis are sex hormone and sperm cell production. Steroidogenesis occurs in the Leydig interstitial cells and spermatogenesis in the seminiferous tubules. Male gonad morphogenesis is a finely orchestrated process, mainly coordinated by hormones, whose actions can significantly affect post‐pubertal testicular function. Calcium is a key intracellular messenger, which regulates many signal transduction pathways, and is also implicated in steroidogenesis. Calcium homeostasis and signaling rely on many calcium‐binding proteins including calretinin, of the “EF‐hand” protein family. Calretinin is a highly conserved protein mainly expressed in the nervous system but also detected in rat and human adult and fetal testis as well as in pathological conditions. Calretinin expression in the fetal testis, however, has not been thoroughly analyzed probably owing to limited availability and paucity of tissues. Here, we examined by immunocytochemistry the expression of calretinin in human fetal testis specimens, obtained from natural and therapeutic abortions, at various developmental ages. We found that calretinin‐immunoreactive Leydig cells were visible throughout the timeframe studied (14th–27th week). Immunoreactivity was also observed in Sertoli cells and in the germ cells of the immature seminiferous tubules. Overall our data indicate that calretinin expression parallels the decline in Leydig cell number, suggesting that its presence is indeed correlated to their steroidogenic activity. They also suggest that the intratubular positivity of calretinin could be linked to the ability of Sertoli cells to produce locally acting hormones contributing to the histodifferentiation of the male genital tract. J. Cell. Physiol. 232: 1872–1878, 2017. © 2016 Wiley Periodicals, Inc. Human fetal testis specimens, obtained upon natural and therapeutic abortions at various developmental ages (weeks 14–27), show calretinin expression in the Leydig interstitial cells, in the epididymis and in the immature seminiferous tubules. Calretinin expression parallels the decline in Leydig cell number. The presence of calretinin seems correlated to the Leydig cells steroidogenic activity, which is consistent with the role that calcium intracellular signaling plays in the steroidogenic process. Our findings, by adding calretinin to the list of players underlying fetal testis development, are particularly timely and prompt further assessment of calretinin role in this context both in normal and pathological conditions.

  January 05, 2017   doi: 10.1002/jcp.25727   open full text
• Fibroblast Growth Factor 21 (FGF21) Promotes Formation of Aerobic Myofibers via the FGF21‐SIRT1‐AMPK‐PGC1α Pathway.
  Xinyi Liu, Yongliang Wang, Liming Hou, Yuanzhu Xiong, Shuhong Zhao.
  Journal of Cellular Physiology. January 05, 2017
  Fibroblast growth factor 21(FGF21) is a pivotal regulator of energy metabolism, which is currently being assessed as a potential drug target for the treatment of insulin‐resistant conditions. However, the cellular mechanisms by which FGF21 affects myogenesis remain unclear. In this study, we explored the function of FGF21 in myogenesis both in vitro and in vivo. Our experiments showed for the first time that FGF21 promotes myoblast differentiation and serves as a switch of molecular transformation from anaerobic myofibers to aerobic myofibers via the FGF21‐SIRT1‐AMPK‐PGC1α axis. Furthermore, we employed the Dual‐Luciferase Reporter Assay System and Electrophoretic Mobility Shift Assay (EMSA) and demonstrated that MYOD, a major myogenic transcription factor, binds directly to the promoter region of Fgf21, leading to the activation of Fgf21 expression in mouse C2C12 myoblasts. Our study revealed a novel mechanism of myogenesis and muscle fiber transformation and indicated that FGF21 serves as a vital regulator of muscle development and important contributor to the pathogenesis of myopathy. J. Cell. Physiol. 232: 1893–1906, 2017. © 2016 Wiley Periodicals, Inc. (1) The mouse FGF21 gene expression is positively regulated by the transcription factor MyoD. (2) FGF21 promotes myogenic differentiation and facilitates the switch of the muscle fiber type from anaerobic myofibers to aerobic myofibers via stimulation the FGF21‐SIRT1‐AMPK‐PGC1α axis. (3) The increased FGF21 protein induced by fasting is likely to change the expression of the PGC‐1α protein from a physiological to an abnormally excessive level via the activation of AMPK signaling, which results in muscle atrophy.

  January 05, 2017   doi: 10.1002/jcp.25735   open full text
• Roles of Notch Signaling in Adipocyte Progenitor Cells and Mature Adipocytes.
  Tizhong Shan, Jiaqi Liu, Weiche Wu, Ziye Xu, Yizhen Wang.
  Journal of Cellular Physiology. January 05, 2017
  Adipose tissues, composed with mature adipocytes and preadipocytic stromal/stem cells, play crucial roles in whole body energy metabolism and regenerative medicine. Mature adipocytes are derived and differentiated from mesenchymal stem cells (MSCs) or preadipocytes. This differentiation process, also called adipogenesis, is regulated by several signaling pathways and transcription factors. Notch1 signaling is a highly conserved pathway that is indispensable for stem cell hemostasis and tissue development. In adipocyte progenitor cells, Notch1 signaling regulates the adipogenesis process including proliferation and differentiation of the adipocyte progenitor cells in vitro. Notably, the roles of Notch1 signaling in beige adipocytes formation, adipose development, and function, and the whole body energy metabolism have been recently reported. Here, we mainly review and discuss the roles of Notch1 signaling in adipogenesis in vitro as well as in beige adipocytes formation, adipocytes dedifferentiation, and function in vivo. J. Cell. Physiol. 232: 1258–1261, 2017. © 2016 Wiley Periodicals, Inc. Notch1 signaling not only regulates the adipogenesis process including proliferation and differentiation of the adipocyte progenitor cells in vitro, but also affects beige adipocytes formation, adipose development, and function, and the whole body energy metabolism in vivo.

  January 05, 2017   doi: 10.1002/jcp.25697   open full text
• Modulation of Chloride Currents in Human Lung Epithelial Cells Exposed to Exogenous Oxidative Stress.
  Rita Canella, Marta Martini, Roberta Borriello, Carlotta Cavicchio, Ximena M. Muresan, Mascia Benedusi, Franco Cervellati, Giuseppe Valacchi.
  Journal of Cellular Physiology. January 05, 2017
  Air pollution continues to be a major public health concern affecting 9 out of 10 individuals living in urban areas worldwide. Respiratory tract is the organ most exposed to gas pollution, and ozone has been shown to be one of the most noxious pollutants to which living organisms are exposed. In the present work, we have investigated the effects of 0.1 ppm of ozone on chloride currents in human lung epithelial cells (A549 line) and whether this effect could be modulated by vitamin E pre‐treatment. Whole‐cell patch clamp technique was applied to not excitable cells in order to obtain information about chloride currents behavior, important for epithelial lung cells homeostasis. Significant alteration of the I–V curve after ozone treatment was observed, with the appearance of a large outward rectifier component decreasing over time and returning to the basal state levels after 24 h. Statistical analysis indicated a modification of the amount of ions passing the membrane in the unit of time as a possible cause of this difference. RT‐qPCR analysis showed an increase in ClC‐2 and ORCC mRNA after ozone exposure. In addition, pre‐treatment with vitamin E was able to suppress the outward rectifier component induced by ozone, bringing back the current values to the control level and preventing ozone induced chloride channels up regulation. Our data suggest that ozone exposure is able to modify chloride current density and the use of vitamin E can prevent the above‐mentioned damage. J. Cell. Physiol. 232: 1817–1825, 2017. © 2016 Wiley Periodicals, Inc. Pollutant ozone is able to induce oxidative stress to lung epithelial cells and this affect chloride currents and the expression of ClC2 and ORCC channel genes levels. Vitamin E pre‐treatment is able to rescue this noxious effect.

  January 05, 2017   doi: 10.1002/jcp.25705   open full text
• A Human 3D In Vitro Model to Assess the Relationship Between Osteoporosis and Dissemination to Bone of Breast Cancer Tumor Cells.
  Francesca Salamanna, Veronica Borsari, Silvia Brogini, Paola Torricelli, Simona Cepollaro, Matteo Cadossi, Milena Fini.
  Journal of Cellular Physiology. January 05, 2017
  Despite consistent improvements in diagnostic and therapeutic strategies for breast cancer, up to 40% of patients will develop bone metastases. To reduce the morbidity and complications related with bone metastases, it is imperative to reduce their etiological factors. Osteoporosis, being characterized by a sudden estrogen deficiency, may provide a favorable condition for bone metastasis. This work, using a humanized 3D in vitro model, aims at evaluating the relationship between osteoporosis and breast cancer‐derived bone metastases. Bone tissue discarded from total hip replacement surgery of healthy and osteoporotic patients was cultured in a rolling apparatus system in hypoxic environment. Protein levels (i.e., vascular endothelial growth factor (VEGF), VEGF receptor 1, VEGF receptor 2, interleukin (IL)‐6, IL‐1β, IL‐8 IL‐10, tumor necrosis factor α (TNF‐α), osteoprotegerin (OPG), receptor activator for nuclear factor KB ligand (RANKL)) and histological and immunohistochemical (i.e., cytokeratin 8 and 18) analyses showed a noticeable specificity of breast cancer cells for the colonization of osteoporotic bone. These data are the first to demonstrate that using humanized 3D in vitro systems, which individually model the pre‐ and postmenopausal bone microenvironment, it is possible to recognize major differences in tumor growth and colonization between healthy and osteoporotic status. Thus, this system might help to develop a shared system between basic and clinical sciences where a personalized diagnosis is associated to a therapeutic strategy designed for a single patient: a model able to achieve a translational research approach in the clinical setting, which may lead to the application and dissemination of personalized medicine. J. Cell. Physiol. 232: 1826–1834, 2017. © 2016 Wiley Periodicals, Inc. Humanized 3D in vitro model; relationship between osteoporosis and breast cancer‐derived bone metastases; differences in tumor growth and colonization between healthy and osteoporotic status.

  January 05, 2017   doi: 10.1002/jcp.25708   open full text
• Identifying Nuclear Matrix‐Attached DNA Across the Genome.
  Jason R. Dobson, Deli Hong, A. Rasim Barutcu, Hai Wu, Anthony N. Imbalzano, Jane B. Lian, Janet L. Stein, Andre J. van Wijnen, Jeffrey A. Nickerson, Gary S. Stein.
  Journal of Cellular Physiology. January 05, 2017
  Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method‐specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR‐Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF‐10A mammary epithelial‐like cells and MDA‐MB‐231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene‐regulatory histone modifications (ChIP‐Seq). In the normal‐like cells, nuclear matrix‐attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non‐expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR‐Seq approach, we provide the first genome‐wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix‐associated genome is highly cell‐context dependent. J. Cell. Physiol. 232: 1295–1305, 2017. © 2016 Wiley Periodicals, Inc. MAR‐Seq is a next‐generation sequencing strategy to identify matrix attachment regions (MARs) that is based on crosslinking chromatin to its nuclear attachment sites. Comparison of MCF‐10A mammary epithelial‐like cells and MDA‐MB‐231 breast cancer cells showed that nuclear matrix‐attached DNA in normal‐like MCF‐10A cells is enriched in expressed genes, while in the breast cancer cells, it is enriched in non‐expressed genes. This new MAR‐Seq approach reveals that the nuclear matrix‐associated genome is highly cell‐context dependent.

  January 05, 2017   doi: 10.1002/jcp.25596   open full text
• Primary Cilium‐Regulated EG‐VEGF Signaling Facilitates Trophoblast Invasion.
  Chia‐Yih Wang, Hui‐Ling Tsai, Jhih‐Siang Syu, Ting‐Yu Chen, Mei‐Tsz Su.
  Journal of Cellular Physiology. December 29, 2016
  Trophoblast invasion is an important event in embryo implantation and placental development. During these processes, endocrine gland‐derived vascular endothelial growth factor (EG‐VEGF) is the key regulator mediating the crosstalk at the feto‐maternal interface. The primary cilium is a cellular antenna receiving environmental signals and is crucial for proper development. However, little is known regarding the role of the primary cilium in early human pregnancy. Here, we demonstrate that EG‐VEGF regulates trophoblast cell invasion via primary cilia. We found that EG‐VEGF activated ERK1/2 signaling and subsequent upregulation of MMP2 and MMP9, thereby facilitating cell invasion in human trophoblast HTR‐8/SVneo cells. Inhibition of ERK1/2 alleviated the expression of MMPs and trophoblast cell invasion after EG‐VEGF treatment. In addition, primary cilia were observed in all the trophoblast cell lines tested and, more importantly, in human first‐trimester placental tissue. The receptor of EG‐VEGF, PROKR1, was detected in primary cilia. Depletion of IFT88, the intraflagellar transporter required for ciliogenesis, inhibited primary cilium growth, thereby ameliorating ERK1/2 activation, MMP upregulation, and trophoblast cell invasion promoted by EG‐VEGF. These findings demonstrate a novel function of primary cilia in controlling EG‐VEGF‐regulated trophoblast invasion and reveal the underlying molecular mechanism. J. Cell. Physiol. 232: 1467–1477, 2017. © 2016 Wiley Periodicals, Inc. Primary cilium is a cellular antenna that conveys extracellular signals into a cellular response. EG‐VEGF binds to PROKR1 locating on the cilium, and activates ERK1/2 signaling followed by upregulating MMP2 and MMP9 for facilitating cell invasion. We demonstrated that EG‐VEGF regulated trophoblast cell invasion via primary cilium in the present study.

  December 29, 2016   doi: 10.1002/jcp.25649   open full text
• Identification of a Novel Human E‐Cadherin Splice Variant and Assessment of Its Effects Upon EMT‐Related Events.
  María Laura Matos, Lara Lapyckyj, Marina Rosso, María José Besso, María Victoria Mencucci, Clara Isabel Marín Briggiler, Silvina Giustina, Laura Inés Furlong, Mónica Hebe Vazquez‐Levin.
  Journal of Cellular Physiology. December 29, 2016
  Epithelial Cadherin (E‐cadherin) is involved in calcium‐dependent cell–cell adhesion and signal transduction. The E‐cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E‐cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E‐cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense‐Mediated mRNA Decay (NMD). The novel E‐cadherin variant was detected in 29/42 (69%) human tumor cell lines, expressed at variable levels (E‐cadherin variant expression relative to the wild type mRNA = 0.05–11.6%). Stable transfection of the novel E‐cadherin variant in MCF‐7 cells (MCF7Ecadvar) resulted in downregulation of wild type E‐cadherin expression (transcript/protein) and EMT‐related changes, among them acquisition of a fibroblastic‐like cell phenotype, increased expression of Twist, Snail, Zeb1, and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N‐cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell–cell adhesion (Hanging‐drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF‐7 cells incubated with culture medium supplemented with conditioned medium from HEK‐293 cells transfected with the E‐cadherin variant mRNA. Further characterization of the novel E‐cadherin variant will help understanding the molecular basis of tumor progression and improve cancer diagnosis. J. Cell. Physiol. 232: 1368–1386, 2017. © 2016 Wiley Periodicals, Inc. This report describes the identification and partial characterization of a novel human E‐cadherin variant mRNA produced by alternative splicing. Stable transfection of the E‐cadherin variant mRNA in MCF‐7 cells (MCF7Ecadvar cells) resulted in downregulation of wild type E‐cadherin and EMT‐related changes, that is, acquisition of a fibroblast‐like cellular phenotype, increased expression of E‐cadherin transcriptional repressors, vimentin and N‐cadherin, and loss of cytokeratins. MCF7Ecadvar cells depicted dramatic changes in cellular behavior, showing reduced cell–cell adhesion and increased cell migration and invasiveness.

  December 29, 2016   doi: 10.1002/jcp.25622   open full text
• Exploring the Role of PGC‐1α in Defining Nuclear Organisation in Skeletal Muscle Fibres.
  Jacob Alexander Ross, Adam Pearson, Yotam Levy, Bettina Cardel, Christoph Handschin, Julien Ochala.
  Journal of Cellular Physiology. December 29, 2016
  Muscle fibres are multinucleated cells, with each nucleus controlling the protein synthesis in a finite volume of cytoplasm termed the myonuclear domain (MND). What determines MND size remains unclear. In the present study, we aimed to test the hypothesis that the level of expression of the transcriptional coactivator PGC‐1α and subsequent activation of the mitochondrial biogenesis are major contributors. Hence, we used two transgenic mouse models with varying expression of PGC‐1α in skeletal muscles. We isolated myofibres from the fast twitch extensor digitorum longus (EDL) and slow twitch diaphragm muscles. We then membrane‐permeabilised them and analysed the 3D spatial arrangements of myonuclei. In EDL muscles, when PGC‐1α is over‐expressed, MND volume decreases; whereas, when PGC‐1α is lacking, no change occurs. In the diaphragm, no clear difference was noted. This indicates that PGC‐1α and the related mitochondrial biogenesis programme are determinants of MND size. PGC‐1α may facilitate the addition of new myonuclei in order to reach MND volumes that can support an increased mitochondrial density. J. Cell. Physiol. 232: 1270–1274, 2017. © 2016 Wiley Periodicals, Inc. Muscle fibres are multinucleated cells, with individual nuclei controlling the protein synthesis in a finite volume of cytoplasm termed myonuclear domain. The present study indicates that one important regulator of myonuclear domain size is PGC‐1α and related mitochondrial biogenesis programme.

  December 29, 2016   doi: 10.1002/jcp.25678   open full text
• SV40 Infection of Mesenchymal Stromal Cells From Wharton's Jelly Drives the Production of Inflammatory and Tumoral Mediators.
  Carolina Cason, Giuseppina Campisciano, Nunzia Zanotta, Erica Valencic, Serena Delbue, Ramona Bella, Manola Comar.
  Journal of Cellular Physiology. December 29, 2016
  The Mesenchymal Stromal Cells from umbilical cord Wharton's jelly (WJSCs) are a source of cells with high potentiality for the treatment of human immunological disorders. Footprints of the oncogenic viruses Simian Virus 40 (SV40) and JC Virus (JCPyV) have been recently detected in human WJSCs specimens. The aim of this study is to evaluate if WJSCs can be efficiently infected by these Polyomaviruses and if they can potentially exert tumoral activity. Cell culture experiments indicated that WJSCs could sustain both SV40 and JCPyV infections. A transient and lytic replication was observed for JCPyV, while SV40 persistently infected WJSCs over a long period of time, releasing a viral progeny at low titer without evident cytopathic effect (CPE). Considering the association between SV40 and human tumors and the reported ability of the oncogenic viruses to drive the host innate immune response to cell transformation, the expression profile of a large panel of immune mediators was evaluated in supernatants by the Bioplex platform. RANTES, IL‐3, MIG, and IL‐12p40, involved in chronic inflammation, cells differentiation, and transformation, were constantly measured at high concentration comparing to control. These findings represent a new aspect of SV40 biological activity in the humans, highlighting its interaction with specific host cellular pathways. In view of these results, it seems to be increasingly urgent to consider Polyomaviruses in the management of WJSCs for their safely use as promising therapeutic source. J. Cell. Physiol. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc. This study aims to evaluate if WJSCs can be efficiently infected by the Polyomaviruses SV40 and JCPyV and if they can exert their potential tumoral activity. Results reported a transient and lytic replication for JCPyV, while SV40 persistently infected WJSCs over a long period of time, releasing viral progeny. Specific factors, including RANTES, IL‐3, MIG, and IL‐12p40, involved in chronic inflammation, cells differentiation, and transformation, were constantly produced at high concentration. These findings represent a new aspect of SV40 biological activity in human host. In view of these results, it seems to be increasingly urgent to consider Polyomaviruses in the management of WJSCs for their safely use as promising therapeutic source.

  December 29, 2016   doi: 10.1002/jcp.25723   open full text
• Lgr4 Expression in Osteoblastic Cells Is Suppressed by Hydrogen Peroxide Treatment.
  Chantida Pawaputanon Na Mahasarakham, Yayoi Izu, Katsuhiko Nishimori, Yuichi Izumi, Masaki Noda, Yoichi Ezura.
  Journal of Cellular Physiology. December 29, 2016
  LGR4 is expressed in bone and has been shown to be involved in bone metabolism. Oxidative stress is one of the key issues in pathophysiology of osteoporosis. However, the link between Lgr4 and oxidative stress has not been known. Therefore, effects of hydrogen peroxide on Lgr4 expression in osteoblasts were examined. Hydrogen peroxide treatment suppressed the levels of Lgr4 mRNA expression in an osteoblastic cell line, MC3T3‐E1. The suppressive effects were not obvious at 0.1 mM, while 1 mM hydrogen peroxide suppressed Lgr4 expression by more than 50%. Hydrogen peroxide treatment suppressed Lgr4 expression within 12 h and this suppression lasted at least up to 48 h. Hydrogen peroxide suppression of Lgr4 expression was still observed in the presence of a transcription inhibitor but was no longer observed in the presence of a protein synthesis inhibitor. Although Lgr4 expression in osteoblasts is enhanced by BMP2 treatment as reported before, hydrogen peroxide treatment suppressed Lgr4 even in the presence of BMP2. Finally, hydrogen peroxide suppressed Lgr4 expression in primary cultures of osteoblasts similarly to MC3T3‐E1 cells. These date indicate that hydrogen peroxide suppresses Lgr4 expression in osteoblastic cells. J. Cell. Physiol. 232: 1761–1766, 2017. © 2016 Wiley Periodicals, Inc.

  December 29, 2016   doi: 10.1002/jcp.25684   open full text
• Curcumin as a Potential Candidate for Treating Hyperlipidemia: A Review of Cellular and Metabolic Mechanisms.
  Yunes Panahi, Yasin Ahmadi, Manouchehr Teymouri, Thomas P. Johnston, Amirhossein Sahebkar.
  Journal of Cellular Physiology. December 24, 2016
  Curcumin is an herbal polyphenol extensively investigated for antioxidant, anti‐inflammatory and hypolipidaemic properties. In the present review, the efficacy of curcumin for improving a plasma lipid profile has been evaluated and compared with statins, a well‐known class of medicines for treating hypercholesterolemia and hyperlipidaemia. Curcumin is presumably most effective in reducing triglyceride (TG), while statins are most efficient in lowering low‐density lipoproteins‐cholesterol (LDL‐C). Additionally, various molecular and metabolic mediators of cholesterol and plasma lipid homeostasis are discussed in relation to how they are modulated by curcumin or statins. Overall, curcumin influences the same mediators of plasma lipid alteration as statins do. Almost all the pathways through which cholesterol trafficking takes place are affected by these agents. These include gastrointestinal absorption of dietary cholesterol, hepatocellular removal of plasma cholesterol, the mediators of reverse cholesterol transport and removal of cholesterol from peripheral tissues. Moreover, the reactive oxygen species (ROS) scavenging potential of curcumin limits the risk of lipid peroxidation that triggers inflammatory responses causing cardiovascular diseases (CVD) and atherosclerosis. Taken together, curcumin could be used as a safe and well‐tolerated adjunct to statins to control hyperlipidaemia more effectively than statins alone. This article is protected by copyright. All rights reserved

  December 24, 2016   doi: 10.1002/jcp.25756   open full text
• Diverse Functions and Signal Transduction of the Exocyst Complex in Tumor Cells.
  Toshiaki Tanaka, Kaoru Goto, Mitsuyoshi Iino.
  Journal of Cellular Physiology. December 22, 2016
  The exocyst complex is a large conserved hetero‐oligomeric complex that consists of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 subunits. It has been implicated in the targeting of vesicles for regulated exocytosis in various cell types, and is also important for targeted exocytosis of post‐Golgi transport vesicles to the plasma membrane. The exocyst complex is essential for membrane growth, secretion, and function during exocytosis and endocytosis. Moreover, the individual components of the complex are thought to act on specific biological processes, such as cytokinesis, ciliogenesis, apoptosis, autophagy, and epithelial‐mesenchymal transition (EMT). As a result, recent studies suggest that the exocyst complex may be involved in several diseases such as kidney disease, neuropathogenesis, diabetes, and cancer. In this review, we focus on the diverse functions and cellular signaling pathways of the exocyst complex in various tumors. J. Cell. Physiol. 232: 939–957, 2017. © 2016 Wiley Periodicals, Inc. Exo70 interacts strongly with the Sec10–Sec15 subcomplex and Exo84 interacts with the Sec10–Sec15–Exo70 subcomplex. Sec3, Sec5, Sec6, and Sec8 form a tight quaternary subcomplex and Sec8 interacts with Sec10. Each component of the exocyst complex contributes to cancer progression and survival in diverse ways such as break of cell–cell contact, EMT, cell cycle progression, DNA repair, migration, autophagy, and anti‐apoptosis.

  December 22, 2016   doi: 10.1002/jcp.25619   open full text
• Differential Expression of miR‐4520a Associated With Pyrin Mutations in Familial Mediterranean Fever (FMF).
  Helen Latsoudis, Mir‐Farzin Mashreghi, Joachim R. Grün, Hyun‐Dong Chang, Bruno Stuhlmüller, Argyro Repa, Irini Gergiannaki, Eleni Kabouraki, George S. Vlachos, Thomas Häupl, Andreas Radbruch, Prodromos Sidiropoulos, Kimon Doukoumetzidis, Dimitris Kardassis, Timothy B. Niewold, Dimitrios T. Boumpas, George N. Goulielmos.
  Journal of Cellular Physiology. December 20, 2016
  Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self‐limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR‐4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR‐4520a. Although the relative expression levels of miR‐4520a were variable among FMF patients, the statistical expression of miR‐4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR‐4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR‐4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR‐4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326–1336, 2017. © 2016 Wiley Periodicals, Inc. Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute and self‐limiting attacks of fever. In the present study we presented for the first time results supporting a role of deregulated autophagy in the pathogenesis of FMF.

  December 20, 2016   doi: 10.1002/jcp.25602   open full text
• Overexpression of Gremlin1 in Mesenchymal Stem Cells Improves Hindlimb Ischemia in Mice by Enhancing Cell Survival.
  Qiuling Xiang, Dongxi Hong, Yan Liao, Yong Cao, Muyun Liu, Jun Pang, Junjie Zhou, Guang Wang, Renhao Yang, Maosheng Wang, Andy Peng Xiang.
  Journal of Cellular Physiology. December 20, 2016
  Mesenchymal stem cells (MSCs) are a promising cell resource for the treatment of ischemic diseases, partially through paracrine effects. One of the major obstacles of MSC treatment is the poor survival rate and low efficiency of transplanted stem cells due to ischemic or inflammatory environments. Gremlin1 (GREM1), a regulator of growth, differentiation and development, has been identified as a novel proangiogenic factor. However, the role and mechanism of GREM1 in MSCs remains unclear. Therefore, we assessed the putative beneficial effects of GREM1 on MSC‐based therapy for hindlimb ischemia. The lentiviral vector, EF1a‐GREM1, was constructed using the Multisite Gateway System and used to transduce MSCs. In vitro studies demonstrated increased survival of GREM1‐MSCs exposed to H2O2, which is consistent with the activation of caspase‐3. Conditional medium from GREM1‐MSCs (GREM1‐MSC‐CM) increased the anti‐apoptotic effects of human umbilical vein endothelial cells (HUVECs), and this effect was attenuated by treatment with the PI3K/Akt pathway inhibitor LY294002. MSCs modified with GREM1 could significantly increase blood perfusion of the ischemic hindlimb in vivo in a mouse model, which was correlated to improved MSC survival. This study demonstrates that overexpression of GREM1 in MSCs have greater therapeutic effects against ischemia compared with wild‐type MSCs by enhancing the survival of MSCs and ECs, which may provide new tools for studies investigating the treatment of ischemic diseases. J. Cell. Physiol. 232: 996–1007, 2017. © 2016 Wiley Periodicals, Inc. This study demonstrates that overexpression of GREM1 in MSCs have greater therapeutic effects against ischemia compared with wild‐type MSCs by enhancing the survival of MSCs and ECs, which may provide new tools for studies investigating the treatment of ischemic diseases.

  December 20, 2016   doi: 10.1002/jcp.25578   open full text
• Supernatants of Adipocytes From Obese Versus Normal Weight Women and Breast Cancer Cells: In Vitro Impact on Angiogenesis.
  Lauriane Bougaret, Laetitia Delort, Hermine Billard, Charlotte Lequeux, Nicolas Goncalves‐Mendes, Ali Mojallal, Odile Damour, Marie‐Paule Vasson, Florence Caldefie‐Chezet.
  Journal of Cellular Physiology. December 20, 2016
  Breast cancer is correlated with a higher risk of metastasis in obese postmenopausal women. Adipokines, whose plasma concentrations are modulated in obese subjects and adipocytes surround mammary cells, suggesting that adipocyte secretome affect mammary tumorogenesis. We hypothesize that mature adipocyte secretions from obese women conditioned or not by breast neoplasic cells, increase changes on the angiogenesis stages. Supernatants of human mature adipocytes, differentiated from stem cells of either adipose tissue of normal weight (MA20) or obese (MA30) women or obtained from co‐cultures between MA20 and MA30 and breast cancer cell line MCF‐7, were collected. The impact of these supernatants was investigated on proliferation, migration, and tube formation by endothelial cells (HUVEC). MA20 and MA30 showed a preservation of their “metabolic memory” (increase of Leptin, ObR, VEGF, CYP19A1, and a decrease of Adiponectin expression in MA30 compared to MA20). Supernatants from obese‐adipocytes increased HUVEC proliferation, migration, and sprouting like with supernatants obtained from co‐cultures of MA/MCF‐7 regardless the women's BMI. Additional analyses such as the use of neutralizing antibodies, analysis of supernatants (Milliplex®) and variations in gene expression (qRT‐PCR), strongly suggest an implication of IL‐6, or a synergistic action among adipokines, probably associated with that of VEGF or IL‐6. As a conclusion, supernatants from co‐cultures of MA30 and MCF‐7 cells increase proliferation, migration, and sprouting of HUVEC cells. These results provide insights into the interaction between adipocytes and epithelial cancer cells, particularly in case of obesity. The identification of synergistic action of adipokines would therefore be a great interest in developing preventive strategies. J. Cell. Physiol. 232: 1808–1816, 2017. © 2016 Wiley Periodicals, Inc. We characterized the impact of adipokines from obese or normal weight women on different angiogenesis stages (proliferation, migration, and tube formation of endothelial cells) in order to determine the influence of obesity on this process and the impact of adipocytes on breast tumor cells.

  December 20, 2016   doi: 10.1002/jcp.25701   open full text
• Acute Ethanol Increases IGF‐I‐Induced Phosphorylation of ERKs by Enhancing Recruitment of p52‐Shc to the Grb2/Shc Complex.
  Matthew Dean, Adam Lassak, Anna Wilk, Adriana Zapata, Luis Marrero, Patricia Molina, Krzysztof Reiss.
  Journal of Cellular Physiology. December 19, 2016
  Ethanol plays a detrimental role in the development of the brain. Multiple studies have shown that ethanol inhibits insulin‐like growth factor I receptor (IGF‐IR) function. Because the IGF‐IR contributes to brain development by supporting neural growth, survival, and differentiation, we sought to determine the molecular mechanism(s) involved in ethanol's effects on this membrane‐associated tyrosine kinase. Using multiple neuronal cell types, we performed Western blot, immunoprecipitation, and GST‐pulldowns following acute (1–24 h) or chronic (3 weeks) treatment with ethanol. Surprisingly, exposure of multiple neuronal cell types to acute (up to 24 h) ethanol (50 mM) enhanced IGF‐I‐induced phosphorylation of extracellular regulated kinases (ERKs), without affecting IGF‐IR tyrosine phosphorylation itself, or Akt phosphorylation. This acute increase in ERKs phosphorylation was followed by the expected inhibition of the IGF‐IR signaling following 3‐week ethanol exposure. We then expressed a GFP‐tagged IGF‐IR construct in PC12 cells and used them to perform fluorescence recovery after photobleaching (FRAP) analysis. Using these fluorescently labeled cells, we determined that 50 mM ethanol decreased the half‐time of the IGF‐IR‐associated FRAP, which implied that cell membrane‐associated signaling events could be affected. Indeed, co‐immunoprecipitation and GST‐pulldown studies demonstrated that the acute ethanol exposure increased the recruitment of p52‐Shc to the Grb2‐Shc complex, which is known to engage the Ras‐Raf‐ERKs pathway following IGF‐1 stimulation. These experiments indicate that even a short and low‐dose exposure to ethanol may dysregulate function of the receptor, which plays a critical role in brain development. J. Cell. Physiol. 232: 1275–1286, 2017. © 2016 Wiley Periodicals, Inc. In the current study, we show a novel effect of ethanol on the IGF‐I receptor (IGF‐IR) signaling, and provided a possible mechanism through which this occurs. We determine that ethanol enhances mobility of the IGF‐IR molecules within the cell membrane, and that this stimulatory effect enhances formation of the Grb2‐Shc signaling complex leading to the transient increase of the signal toward extracellular regulated kinases (ERKs). Because of the critical role of the IGF‐IR signaling in neuronal growth and survival, and the detrimental effects of alcohol on CNS development, we believe that our new data will help in better understanding of the fetal alcohol syndrome.

  December 19, 2016   doi: 10.1002/jcp.25586   open full text
• Association Between Aerobic Exercise and Rosiglitazone Avoided the NAFLD and Liver Inflammation Exacerbated in PPAR‐α Knockout Mice.
  Helena A.P. Batatinha, Edson A. Lima, Alexandre A.S. Teixeira, Camila O. Souza, Luana A. Biondo, Loreana S. Silveira, Fabio S. Lira, José C. Rosa Neto.
  Journal of Cellular Physiology. November 30, 2016
  Nonalcoholic fatty liver disease (NAFLD) is one of the main liver diseases today, and may progress to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Some studies have shown the beneficial effects of aerobic exercise on reversing NAFLD. To verify whether chronic aerobic exercise improves the insulin resistance, liver inflammation, and steatohepatitis caused by a high fat diet (HF) and whether PPARα is involved in these actions. C57BL6 wild type (WT) and PPAR‐α knockout (KO) mice were fed with a standard diet (SD) or HF during 12 weeks; the HF mice were trained on a treadmill during the last 8 weeks. Serum glucose and insulin tolerances, serum levels of aspartate aminotransferase, hepatic content of triacylglycerol, cytokines, gene expression, and protein expression were evaluated in all animals. Chronic exposure to HF diet increased triacylglycerol accumulation in the liver, leading to NAFLD, increased aminotransferase in the serum, increased peripheral insulin resistance, and higher adiposity index. Exercise reduced all these parameters in both animal genotypes. The liver lipid accumulation was not associated with inflammation; trained KO mice, however, presented a huge inflammatory response that was probably caused by a decrease in PPAR‐γ expression. We conclude that exercise improved the damage caused by a HF independently of PPARα, apparently by a peripheral fatty acid oxidation in the skeletal muscle. We also found that the absence of PPARα together with exercise leads to a decrease in PPAR‐γ and a huge inflammatory response. J. Cell. Physiol. 232: 1008–1019, 2017. © 2016 Wiley Periodicals, Inc. Exercise training improved lipids toxicity in the liver; PPAR‐alpha KO developed liver inflammation after training protocol Roziglitazone reduced liver inflammation independent of PPAR‐alpha.

  November 30, 2016   doi: 10.1002/jcp.25440   open full text
• PPARα Antagonist AA452 Triggers Metabolic Reprogramming and Increases Sensitivity to Radiation Therapy in Human Glioblastoma Primary Cells.
  Elisabetta Benedetti, Michele d'Angelo, Alessandra Ammazzalorso, Giovanni Luca Gravina, Chiara Laezza, Andrea Antonosante, Gloria Panella, Benedetta Cinque, Loredana Cristiano, Anne Chloè Dhez, Carlo Astarita, Renato Galzio, Maria Grazia Cifone, Rodolfo Ippoliti, Rosa Amoroso, Ernesto Di Cesare, Antonio Giordano, Annamaria Cimini.
  Journal of Cellular Physiology. November 30, 2016
  Glioblastoma (GB) is the most common cancer in the brain and with an increasing incidence. Despite major advances in the field, there is no curative therapy for GB to date. Many solid tumors, including GB, experienced metabolic reprogramming in order to sustain uncontrolled proliferation, hypoxic conditions, and angiogenesis. PPARs, member of the steroid hormone receptor superfamily, are particularly involved in the control of energetic metabolism, particularly lipid metabolism, which has been reported deregulated in gliomas. PPARα was previously indicated by us as a potential therapeutic target for this neoplasm, due to the malignancy grade dependency of its expression, being particularly abundant in GB. In this work, we used a new PPARα antagonist on patient‐derived GB primary cells, with particular focus on the effects on lipid metabolism and response to radiotherapy. The results obtained demonstrated that blocking PPARα results in cell death induction, increase of radiosensitivity, and decrease of migration. Therefore, AA452 is proposed as a new adjuvant for the gold standard therapies for GB, opening the possibility for preclinical and clinical trials for this class of compounds. J. Cell. Physiol. 232: 1458–1466, 2017. © 2016 Wiley Periodicals, Inc. A new compound, an inhibitor of PPARalpha trancriptional activity, is reported, able to increase radiation sensitivity of glioblastoma cells.

  November 30, 2016   doi: 10.1002/jcp.25648   open full text
• Inhibitory Effects of Quercetin on Progression of Human Choriocarcinoma Cells Are Mediated Through PI3K/AKT and MAPK Signal Transduction Cascades.
  Whasun Lim, Changwon Yang, Sunwoo Park, Fuller W. Bazer, Gwonhwa Song.
  Journal of Cellular Physiology. November 30, 2016
  As a major dietary flavonol, quercetin mitigates proliferation and progression of cancer due to its anti‐angiogenic, anti‐inflammatory, anti‐oxidant, and apoptotic biological effects on cells. Although its apoptotic effects have been reported for various cancers, little is known of the functional role of quercetin in gestational choriocarcinoma. Results of the present study indicated that quercetin reduced proliferation and induced cell death in two choriocarcinoma cell lines, JAR and JEG3 cells, with an increase in the sub‐G1 phase of the cell cycle. In addition, quercetin induced mitochondrial dysfunction significantly reduced mitochondrial membrane potential (MMP) and increased production of reactive oxygen species (ROS) in both JAR and JEG3 cells. Further, quercetin inhibited phosphorylation of AKT, P70S6K and S6 proteins whereas, it increased phosphorylation of ERK1/2, P38, JNK and P90RSK proteins in JAR and JEG3 cells. The decrease in viability of choriocarcinoma cells treated with quercetin was confirmed by using combinations of quercetin and pharmacological inhibitors of the PI3K and MAPK signaling pathways. Classical chemotherapeutic agents, cisplatin (a platinum‐based drug) and paclitaxel (a taxene‐based drug), inhibited proliferation of JAR and JEG3 cells, and when combined with quercetin, the antiproliferative effects of cisplatin and paclitaxel were enhanced for both choriocarcinoma cell lines. Collectively, these results suggest that quercetin prevents development of choriocarcinoma and may be a valuable therapeutic agent for treatment of choriocarcinoma through its regulation of PI3K and MAPK signal transduction pathways. J. Cell. Physiol. 232: 1428–1440, 2017. © 2016 Wiley Periodicals, Inc. Quercetin prevents development of choriocarcinoma and may be a valuable therapeutic agent for treatment of choriocarcinoma through its regulation of PI3K/AKT and MAPK signaling pathways and intrinsic apoptotic pathways.

  November 30, 2016   doi: 10.1002/jcp.25637   open full text
• TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell Culture Model of the Corneal Epithelium.
  Jacqueline Martínez‐Rendón, Erika Sánchez‐Guzmán, Angélica Rueda, James González, Rosario Gulias‐Cañizo, Guillermo Aquino‐Jarquín, Federico Castro‐Muñozledo, Refugio García‐Villegas.
  Journal of Cellular Physiology. November 30, 2016
  TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca2+ imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN‐1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin‐4 in cell–cell contacts. Epidermal Growth Factor (EGF) up‐regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF‐activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4‐TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794–1807, 2017. © 2016 Wiley Periodicals, Inc. TRPV4 channel activity is necessary for the correct establishment of tight Junctions (TJ) in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. Thus, TRPV4 function is necessary for the terminal differentiation of corneal epithelia.

  November 30, 2016   doi: 10.1002/jcp.25698   open full text
• S100B + A1 CELISA: A Novel Potency Assay and Screening Tool for Redifferentiation Stimuli of Human Articular Chondrocytes.
  Jose Diaz‐Romero, Sibylle Kürsener, Sandro Kohl, Dobrila Nesic.
  Journal of Cellular Physiology. November 30, 2016
  During monolayer expansion, a necessary step in autologous chondrocyte implantation, human articular chondrocytes (HAC) dedifferentiate and lose their capacity to produce stable hyaline cartilage. Determining HAC potency and learning how to trigger their redifferentiation would improve cell‐based cartilage regeneration therapies. We previously identified S100B and S100A1 proteins as markers of HAC redifferentiation potential. Here, we aimed to: (i) demonstrate a correlation between S100B + A1‐positive HAC in monolayer culture and their neochondrogenesis capacity in pellet culture; (ii) develop an S100B + A1 cell‐based ELISA, and (iii) prove that S100B + A1 induction in HAC increases their chondrogenic capacity. Expression patterns of S100A1 and S100B were investigated in HAC during dedifferentiation (monolayer) or redifferentiation (pellet or high‐osmolarity/BMP4 treatment in monolayer) using qRT‐PCR, immunocytochemistry, or immunohistochemistry. A cell‐based ELISA (CELISA) was developed as a 96‐well microplate multiplex assay to measure S100B + A1 (chondrogenesis), alkaline phosphatase (hypertrophy), and DNA amount (normalization), and applied to HAC, bone marrow‐derived mesenchymal stem cells and the chondrocytic cell line ATDC5. The direct correlation between the percentage of S100B + A1‐positive HAC in monolayer and their neochondrogenesis in pellets validates S100B + A1 as a marker of chondrogenic potency. The S100B + A1‐CELISA accurately determines HAC differentiation status, allows identification of chondrogenic stimuli, and permits the simultaneous monitoring of the undesirable hypertrophic phenotype. This novel assay offers a high‐throughput, comprehensive and versatile approach for measuring cell chondrogenic potency and for identifying redifferentiation factors/conditions. HAC improved neochondrogenesis in pellets—induced with high‐osmolarity and BMP4 treatment in monolayer—suggests that cell instruction prior to implantation may improve cartilage repair. J. Cell. Physiol. 232: 1559–1570, 2017. © 2016 Wiley Periodicals, Inc. The novel S100B + A1‐CELISA offers a high‐throughput, comprehensive and versatile approach for measuring cell chondrogenic potency and identifying redifferentiation factors/conditions to improve cartilage repair. Using a combination of high‐osmolarity medium and BMP4 treatment uncovered with this assay, we demonstrate that the induction of chondrocyte redifferentiation in monolayer improves their chondrogenic potential in pellets.

  November 30, 2016   doi: 10.1002/jcp.25682   open full text
• A Real‐World Multicentre Retrospective Study of Paclitaxel‐Bevacizumab and Maintenance Therapy as First‐Line for HER2‐Negative Metastatic Breast Cancer.
  Teresa Gamucci, Lucia Mentuccia, Clara Natoli, Isabella Sperduti, Alessandra Cassano, Andrea Michelotti, Luigi Di Lauro, Domenico Sergi, Alessandra Fabi, Maria G. Sarobba, Paolo Marchetti, Maddalena Barba, Emanuela Magnolfi, Marcello Maugeri‐Saccà, Ernesto Rossi, Valentina Sini, Antonino Grassadonia, Domenica Pellegrini, Antonino Astone, Cecilia Nisticò, Franco Angelini, Angela Vaccaro, Arianna Pellegrino, Claudia De Angelis, Michela Palleschi, Luca Moscetti, Ilaria Bertolini, Simonetta Buglioni, Antonio Giordano, Laura Pizzuti, Patrizia Vici.
  Journal of Cellular Physiology. November 30, 2016
  Bevacizumab in combination with taxanes in HER2‐negative metastatic breast cancer (MBC) patients has shown improved progression‐free survival (PFS), despite the lack of clear overall survival (OS) benefit. We performed a retrospective analysis to evaluate the impact of paclitaxel‐bevacizumab and of maintenance therapy with bevacizumab (BM) and endocrine therapy (ET) in the real‐world practice. We identified 314 HER2‐negative MBC patients treated in 12 cancer centers. Overall, the median PFS and OS were 14 and 40 months, respectively. Among the 254 patients potentially eligible for BM, 183 received BM after paclitaxel discontinuation until progression/toxicity. PFS and OS were improved in patients who had received BM in comparison with those potentially eligible but who did not receive BM (P< 0.0001 and P = 0.001, respectively). Results were confirmed when adjusting for propensity score. Among the 216 hormone‐receptor positive patients eligible for BM, a more favorable PFS and OS were observed when maintenance ET was administered (P < 0.0001). Multivariate analysis showed that PS, BM, number of disease sites and maintenance ET were related to PFS, while response and maintenance ET were related to OS. In hormone‐receptor positive patients, BM produced a significant PFS and a trend towards OS benefit only in absence of maintenance ET (P = 0.0007 and P = 0.06, respectively). In the triple‐negative subgroup, we observed a trend towards a better OS for patients who received BM (P = 0.06), without differences in PFS (P = 0.21). Our results confirmed the efficacy of first‐line paclitaxel‐bevacizumab in real‐world practice; both BM and maintenance ET significantly improved PFS and OS compared to no maintenance therapies. J. Cell. Physiol. 232: 1571–1578, 2017. © 2016 Wiley Periodicals, Inc. We performed a retrospective analysis to evaluate the impact of paclitaxel‐bevacizumab and of maintenance therapy with bevacizumab (BM) and endocrine therapy (ET) in the real‐world practice. Our results confirmed the efficacy of first‐line paclitaxel‐bevacizumab in real‐world practice; both BM and maintenance ET significantly improved PFS and OS compared to no maintenance therapies.

  November 30, 2016   doi: 10.1002/jcp.25685   open full text
• Oligodendrocyte Progenitor Cells Directly Utilize Lactate for Promoting Cell Cycling and Differentiation.
  Yoshinori Ichihara, Toru Doi, Youngjae Ryu, Motoshi Nagao, Yasuhiro Sawada, Toru Ogata.
  Journal of Cellular Physiology. November 30, 2016
  Oligodendrocyte progenitor cells (OPCs) undergo marked morphological changes to become mature oligodendrocytes, but the metabolic resources for this process have not been fully elucidated. Although lactate, a metabolic derivative of glycogen, has been reported to be consumed in oligodendrocytes as a metabolite, and to ameliorate hypomyelination induced by low glucose conditions, it is not clear about the direct contribution of lactate to cell cycling and differentiation of OPCs, and the source of lactate for remyelination. Therefore, we evaluated the effect of 1,4‐dideoxy‐1,4‐imino‐d‐arabinitol (DAB), an inhibitor of the glycogen catabolic enzyme glycogen phosphorylase, in a mouse cuprizone model. Cuprizone induced demyelination in the corpus callosum and remyelination occurred after cuprizone treatment ceased. This remyelination was inhibited by the administration of DAB. To further examine whether lactate affects proliferation or differentiation of OPCs, we cultured mouse primary OPC‐rich cells and analyzed the effect of lactate. Lactate rescued the slowed cell cycling induced by 0.4 mM glucose, as assessed by the BrdU‐positive cell ratio. Lactate also promoted OPC differentiation detected by monitoring the mature oligodendrocyte marker myelin basic protein, in the presence of both 36.6 mM and 0.4 mM glucose. Furthermore, these lactate‐mediated effects were suppressed by the reported monocarboxylate transporter inhibitor, α‐cyano‐4‐hydroxy‐cinnamate. These results suggest that lactate directly promotes the cell cycling rate and differentiation of OPCs, and that glycogen, one of the sources of lactate, contributes to remyelination in vivo. J. Cell. Physiol. 232: 986–995, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. Glycogen phosphorylase inhibitor suppresses remyelination in a cuprizone model. Lactate ameliorates low glucose‐induced slow cell–cell cycling and promotes differentiation of cell in OPC‐rich cultures. Monocarboxylase transport inhibition suppresses lactate effects in OPC‐rich cells.

  November 30, 2016   doi: 10.1002/jcp.25690   open full text
• CD93 as a Potential Target in Neovascular Age‐Related Macular Degeneration.
  Gian Marco Tosi, Elena Caldi, Barbara Parolini, Paolo Toti, Giovanni Neri, Federica Nardi, Claudio Traversi, Gabriele Cevenini, Davide Marigliani, Elisabetta Nuti, Tommaso Bacci, Federico Galvagni, Maurizio Orlandini.
  Journal of Cellular Physiology. November 30, 2016
  In patients with age‐related macular degeneration (AMD), choroidal neovascularization is the major cause of severe visual loss. In these patients, the persistence of neovascular growth despite vascular endothelial growth factor‐A blockage needs the discovery of new endothelial cell targets. The glycoprotein CD93, highly expressed in activated endothelial cells, has been recently involved in the regulation of the angiogenic process both as transmembrane and soluble protein. Choroidal neovascular membranes from patients affected by AMD were examined by immunofluorescence using anti‐CD93 and anti‐von Willebrand factor antibodies. Blood vessels within intraocular and extraocular neoplasias were used as controls for CD93 expression. All choroidal neovascular membranes displayed strong CD93 staining in the von Willebrand factor‐positive endothelial cells, consistently with the analyses showing a high colocalization coefficient in the blood vessels. Intraocular and extraocular tumor vessels showed similar results, whereas the normal choroid displayed blood vessels with only faint CD93 staining. Additionally, the concentration of soluble CD93 was determined in the aqueous humor of patients affected by naïve neovascular AMD by enzyme‐linked immunosorbent assays. Age‐matched cataract patients served as controls. Soluble CD93 was significantly increased in the aqueous humor of naïve neovascular AMD patients and tended to decrease after treatment with an antiangiogenic drug. In conclusion, both transmembrane and soluble CD93 are overexpressed in patients with neovascular AMD, indicating that CD93 may represent a potential new antiangiogenic target in the treatment of choroidal neovascularization. J. Cell. Physiol. 232: 1767–1773, 2017. © 2016 Wiley Periodicals, Inc. In patients with age‐related macular degeneration (AMD), the formation of new blood vessels in the choroid, which is the major cause of severe visual loss, persists even after therapy. In AMD patients, both in the endothelial cells of choroidal neovascular membranes and in the aqueous humor, we found high levels of the glycoprotein CD93, which has been recently involved in the enhancement of tumor vascular function. The presence of CD93 in AMD patients unveils a potential new target for choroidal neovascularization treatment.

  November 30, 2016   doi: 10.1002/jcp.25689   open full text
• E‐Cigarette Vapor Induces an Apoptotic Response in Human Gingival Epithelial Cells Through the Caspase‐3 Pathway.
  Mahmoud Rouabhia, Hyun Jin Park, Abdelhabib Semlali, Andrew Zakrzewski, Witold Chmielewski, Jamila Chakir.
  Journal of Cellular Physiology. November 30, 2016
  Electronic cigarettes represent an increasingly significant proportion of today's consumable tobacco products. E‐cigarettes contain several chemicals which may promote oral diseases. The aim of this study was to investigate the effect of e‐cigarette vapor on human gingival epithelial cells. Results show that e‐cigarette vapor altered the morphology of cells from small cuboidal form to large undefined shapes. Both single and multiple exposures to e‐cigarette vapor led to a bulky morphology with large faint nuclei and an enlarged cytoplasm. E‐cigarette vapor also increased L‐lactate dehydrogenase (LDH) activity in the targeted cells. This activity was greater with repeated exposures. Furthermore, e‐cigarette vapor increased apoptotic/necrotic epithelial cell percentages compared to that observed in the control. Epithelial cell apoptosis was confirmed by TUNEL assay showing that exposure to e‐cigarette vapor increased apoptotic cell numbers, particularly after two and three exposures. This negative effect involved the caspase‐3 pathway, the activity of which was greater with repeated exposure and which decreased following the use of caspase‐3 inhibitor. The adverse effects of e‐cigarette vapor on gingival epithelial cells may lead to dysregulated gingival cell function and result in oral disease. J. Cell. Physiol. 232: 1539–1547, 2017. © 2016 Wiley Periodicals, Inc. E‐cigarette vapor mediated gingival epithelial cell activity by promoting apoptosis which took place through an apoptosis pathway involving caspase‐3. The effects of e‐cigarette vapor on gingival epithelial cells may compromise epithelial tissue, resulting in periodontal disease development and potentiating inflammation resulting from the tissue damage.

  November 30, 2016   doi: 10.1002/jcp.25677   open full text
• Low Shear Stress Attenuates COX‐2 Expression Induced by Resistin in Human Osteoarthritic Chondrocytes.
  Yu‐Ping Su, Cheng‐Nan Chen, Hsin‐I Chang, Kuo‐Chin Huang, Chin‐Chang Cheng, Fang‐Yao Chiu, Ko‐Chao Lee, Chun‐Min Lo, Shun‐Fu Chang.
  Journal of Cellular Physiology. November 20, 2016
  Low shear stress has been proposed to play a reparative role in modulating cartilage homeostasis. Recently, epidemiological studies have found a positive correlation between the resistin level in serum and synovial fluid and osteoarthritis (OA) severity in patients. However, the effect of moderate shear stress on the catabolic stimulation of resistin in OA chondrocytes remains unclear. Hence, this study was to investigate whether low shear stress could regulate resistin‐induced catabolic cyclooxygenase (COX)‐2 expression in human OA chondrocytes and the underlying mechanism. Human OA chondrocytes and SW1353 chondrosarcoma cells were used in this study. Two modes of low shear stress (2 dyn/cm2), pre‐shear and post‐shear, were applied to the chondrocytes. A specific activator and siRNAs were used to investigate the mechanism of low shear stress‐regulated COX‐2 expression of resistin induction. We found that human OA chondrocytes exposed to different modes of low shear stress elicit an opposite effect on resistin‐induced COX‐2 expression: pre‐shear for a short duration attenuates the resistin effect by inhibiting the transcription factor nuclear factor (NF)‐κB‐p65 subunit and the cAMP response element binding protein; however, post‐shear over a longer duration enhances the resistin effect by activating only the NF‐κB‐p65 subunit. Moreover, our results demonstrated that the regulation of both shear modes in resistin‐stimulated COX‐2 expression occurs through increasing AMP‐activated protein kinase activation and then sirtuin 1 expression. This study elucidates the detailed mechanism of low shear stress regulating the resistin‐induced catabolic COX‐2 expression and indicates a possible reparative role of moderate shear force in resistin‐stimulated OA development. J. Cell. Physiol. 232: 1448–1457, 2017. © 2016 Wiley Periodicals, Inc. This study elucidates the detailed mechanism of low shear stress regulating the resistin‐induced catabolic COX‐2 expression and indicates a possible reparative role of moderate shear force in resistin‐stimulated OA development.

  November 20, 2016   doi: 10.1002/jcp.25644   open full text
• Calcium Signaling During Meiotic Cell Cycle Regulation and Apoptosis in Mammalian Oocytes.
  Meenakshi Tiwari, Shilpa Prasad, Tulsidas G. Shrivastav, Shail K. Chaube.
  Journal of Cellular Physiology. November 20, 2016
  Calcium (Ca++) is one of the major signal molecules that regulate various aspects of cell functions including cell cycle progression, arrest, and apoptosis in wide variety of cells. This review summarizes current knowledge on the differential roles of Ca++ in meiotic cell cycle resumption, arrest, and apoptosis in mammalian oocytes. Release of Ca++ from internal stores and/or Ca++ influx from extracellular medium causes moderate increase of intracellular Ca++ ([Ca++]i) level and reactive oxygen species (ROS). Increase of Ca++ as well as ROS levels under physiological range trigger maturation promoting factor (MPF) destabilization, thereby meiotic resumption from diplotene as well as metaphase‐II (M‐II) arrest in oocytes. A sustained increase of [Ca++]i level beyond physiological range induces generation of ROS sufficient enough to cause oxidative stress (OS) in aging oocytes. The increased [Ca++]i triggers Fas ligand‐mediated oocyte apoptosis. Further, OS triggers mitochondria‐mediated oocyte apoptosis in several mammalian species. Thus, Ca++ exerts differential roles on oocyte physiology depending upon its intracellular concentration. A moderate increase of [Ca++]i as well as ROS mediate spontaneous resumption of meiosis from diplotene as well as M‐II arrest, while their high levels cause meiotic cell cycle arrest and apoptosis by operating both mitochondria‐ as well as Fas ligand‐mediated apoptotic pathways. Indeed, Ca++ regulates cellular physiology by modulating meiotic cell cycle and apoptosis in mammalian oocytes. J. Cell. Physiol. 232: 976–981, 2017. © 2016 Wiley Periodicals, Inc. A moderate increase of [Ca++]i level under physiological range induces generation of ROS that result in decrease of cAMP level and increase of CaMKII activity. A decrease of intraoocyte cAMP level and increase of CaMKII activity directly and/or indirectly triggers MPF destabilization thereby meiotic resumption from diplotene as well as M‐II arrest. The high level of [Ca++]i beyond physiological range triggers Fas ligand‐mediated apoptosis and induces OS. The increased OS triggers oocyte apoptosis through mitochondria‐mediated pathway. Indeed, high level of [Ca++]i and ROS‐mediated meiotic cell cycle arrest and apoptosis could be interlinked in mammalian oocytes.

  November 20, 2016   doi: 10.1002/jcp.25670   open full text
• Notch Signaling Pathway Regulates Angiogenesis via Endothelial Cell in 3D Co‐Culture Model.
  Dan Zhao, Changyue Xue, Shiyu Lin, Sirong Shi, Qianshun Li, Mengting Liu, Xiaoxiao Cai, Yunfeng Lin.
  Journal of Cellular Physiology. November 20, 2016
  This study aimed to investigate the role of Notch signaling pathway for angiogenesis in a three‐dimensional (3D) collagen gel model with co‐culture of adipose‐derived stromal cells (ASCs) and endothelial cells (ECs). A 3D collagen gel model was established in vitro by implanting both ASCs from green fluorescent protein‐labeled mouse and ECs from red fluorescent protein‐labeled mouse, and the phenomena of angiogenesis with Notch signaling inducer Jagged1, inhibitor DAPT and PBS, respectively were observed by confocal laser scanning microscopy. Semi‐quantitative PCR and immunofluorescent staining were conducted to detect expressions of angiogenesis‐related genes and proteins. Angiogenesis in the co‐culture gels was promoted by Jagged1 treatment while attenuated by DAPT treatment, compared to control group. In co‐culture system of ASCs and ECs, the gene expressions of VEGFA, VEGFB, Notch1, Notch2, Hes1, Hey1, VEGFR1,and the protein expression of VEGFA, VEGFB, Notch1, Hes1, Hey1 were increased by Jagged1 treatment and decreased by DAPT treatment in ECs. And the result of VEGFR3 was the opposite. However, the same results did not appear completely in ASCs. These results revealed the VEGFA/B‐Notch1/2‐Hes1/Hey1‐ VEGFR1/3 signal axis played an important role in angiogenesis when ASCs and ECs were co‐cultured in a 3D collagen gel model. J. Cell. Physiol. 232: 1548–1558, 2017. © 2016 Wiley Periodicals, Inc. VEGFA/B‐Notch1/2‐Hes1/Hey1‐ VEGFR1/3 signal axis played an important role in angiogenesis when ASCs and ECs were co‐cultured in a 3D collagen gel model.

  November 20, 2016   doi: 10.1002/jcp.25681   open full text
• Wnt3A Induces GSK‐3β Phosphorylation and β‐Catenin Accumulation Through RhoA/ROCK.
  Jae‐Gyu Kim, Myoung‐Ju Kim, Won‐Ji Choi, Mi‐Young Moon, Hee‐Jun Kim, Jae‐Yong Lee, Jaebong Kim, Sung‐Chan Kim, Seung Goo Kang, Goo‐Young Seo, Pyeung‐Hyeun Kim, Jae‐Bong Park.
  Journal of Cellular Physiology. November 20, 2016
  In canonical pathway, Wnt3A has been known to stabilize β‐catenin through the dissociation between β‐catenin and glycogen synthase kinase‐3β (GSK‐3β) that suppresses the phosphorylation and degradation of β‐catenin. In non‐canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross‐talk between canonical and non‐canonical pathways by Wnt signaling; however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK‐3β and accumulation of β‐catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh‐RhoA and Tat‐C3 abolished both the phosphorylation of GSK‐3β and accumulation of β‐catenin. Y27632, an inhibitor of Rho‐associated coiled coil kinase (ROCK) and si‐ROCK inhibited both GSK‐3β phosphorylation and β‐catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK‐3β in vitro. In addition, Wnt3A‐induced cell proliferation and migration, which were inhibited by Tat‐C3 and Y27632. Taken together, we propose the cross‐talk between canonical and non‐canonical signaling pathways of Wnt3A, which induces GSK‐3β phosphorylation and β‐catenin accumulation through RhoA and ROCK activation. J. Cell. Physiol. 232: 1104–1113, 2017. © 2016 Wiley Periodicals, Inc. Wnt3A activates RhoA and ROCK, which induces GSK‐3beta phosphorylation and beta‐catenin accumulation. RhoA and ROCK are involved in the regulation of cell proliferation and migration upon Wnt3A.

  November 20, 2016   doi: 10.1002/jcp.25572   open full text
• ADP‐Induced Ca2+ Signaling and Proliferation of Rat Ventricular Myofibroblasts Depend on Phospholipase C‐Linked TRP Channels Activation Within Lipid Rafts.
  Mariana Certal, Adriana Vinhas, Aurora Barros‐Barbosa, Fátima Ferreirinha, Maria Adelina Costa, Paulo Correia‐de‐Sá.
  Journal of Cellular Physiology. November 10, 2016
  Nucleotides released during heart injury affect myocardium electrophysiology and remodeling through P2 purinoceptors activation in cardiac myofibroblasts. ATP and UTP endorse [Ca2+]i accumulation and growth of DDR‐2/α‐SMA‐expressing myofibroblasts from adult rat ventricles via P2Y4 and P2Y2 receptors activation, respectively. Ventricular myofibroblasts also express ADP‐sensitive P2Y1, P2Y12, and P2Y13 receptors as demonstrated by immunofluorescence confocal microscopy and western blot analysis, but little information exists on ADP effects in these cells. ADP (0.003–3 mM) and its stable analogue, ADPßS (100 μM), caused fast [Ca2+]i transients originated from thapsigargin‐sensitive internal stores, which partially declined to a plateau sustained by capacitative Ca2+ entry through transient receptor potential (TRP) channels inhibited by 2‐APB (50 μM) and flufenamic acid (100 μM). Hydrophobic interactions between Gq/11‐coupled P2Y purinoceptors and TRP channels were suggested by prevention of the ADP‐induced [Ca2+]i plateau following PIP2 depletion with LiCl (10 mM) and cholesterol removal from lipid rafts with methyl‐ß‐cyclodextrin (2 mM). ADP [Ca2+]i transients were insensitive to P2Y1, P2Y12, and P2Y13 receptor antagonists, MRS2179 (10μM), AR‐C66096 (0.1 μM), and MRS2211 (10μM), respectively, but were attenuated by suramin and reactive blue‐2 (100 μM) which also blocked P2Y4 receptors activation by UTP. Cardiac myofibroblasts growth and type I collagen production were favored upon activation of MRS2179‐sensitive P2Y1 receptors with ADP or ADPßS (30 μM). In conclusion, ADP exerts a dual role on ventricular myofibroblasts: [Ca2+]i transients are mediated by fast‐desensitizing P2Y4 receptors, whereas the pro‐fibrotic effect of ADP involves the P2Y1 receptor activation. Data also show that ADP‐induced capacitative Ca2+ influx depends on phospholipase C‐linked TRP channels opening in lipid raft microdomains. J. Cell. Physiol. 232: 1511–1526, 2017. © 2016 Wiley Periodicals, Inc. The P2Y4R mainly mediates ADP‐induced [Ca2+]i rise in rat ventricular myofibroblasts; ADP‐induced fast [Ca2+]i rises depend on Ca2+ recruitment from internal stores; the late [Ca2+]i plateau depends on capacitative Ca2+ entry via TRP channels; PLC‐activating P2Y4 receptors and TRP channels are assembled in lipid rafts; ADP promotes cell growth and collagen production predominantly via P2Y1 activation.

  November 10, 2016   doi: 10.1002/jcp.25656   open full text
• Peripheral Blood Mononuclear Cells Infiltration Downregulates Decidual FAAH Activity in an LPS‐Induced Embryo Resorption Model.
  Manuel Luis Wolfson, Julieta Aisemberg, Fernando Correa, Ana María Franchi.
  Journal of Cellular Physiology. November 10, 2016
  Maternal infections with gram‐negative bacteria are associated with miscarriage and are one of the most common complications during pregnancy. Previous studies from our group have shown that lipopolysaccharide (LPS)‐activated infiltrating peripheral blood mononuclear cells (PBMC) into decidual tissue plays an important role in the establishment of a local inflammatory process that results in embryo cytotoxicity and early embryo resorption. Moreover, we have also shown that an increased endocannabinoid tone mediates LPS‐induced deleterious effects during early pregnancy loss. Here, we sought to investigate whether the infiltrating PBMC modulates the decidual endocannabinoid tone and the molecular mechanisms involved. PBMC isolated from 7‐day pregnant mice subjected to different treatments were co‐cultured in a transwell system with decidual tissue from control 7‐day pregnant mice. Decidual fatty acid amide hydrolase (FAAH) activity was measured by radioconvertion, total decidual protein nitration by Western blot (WB), and decidual FAAH nitration by immunoprecipitation followed by WB. We found that co‐culture of PBMC obtained from LPS‐treated mice increased the level of nitration of decidual FAAH, which resulted in a negative modulation of decidual FAAH activity. Interestingly, co‐treatment with progesterone or aminoguanidine prevented this effect. We found that LPS‐treated PBMC release high amounts of nitric oxide (NO) which causes tyrosine nitration of decidual FAAH, diminishing its enzymatic activity. Inactivation of FAAH, the main degrading enzyme of anandamide and similar endocannabinoids, could lead to an increased decidual endocannabinoid tone with embryotoxic effects. J. Cell. Physiol. 232: 1441–1447, 2017. © 2016 Wiley Periodicals, Inc. PBMC release a soluble factor which modulates the decidual FAAH activity. Peroxynitrite participates in LPS‐treated PBMC decidual FAAH activity modulation. PBMC from LPS‐treated mice increased the nitration levels of decidual FAAH which resulted in a decreased enzymatic activity.

  November 10, 2016   doi: 10.1002/jcp.25640   open full text
• p120‐Catenin Is Required for Dietary Calcium Suppression of Oral Carcinogenesis in Mice.
  Zhongjian Xie, Yuan Yuan, Yi Jiang, Chandrama Shrestha, Ying Chen, Liyan Liao, Shangli Ji, Xiaoge Deng, Eryuan Liao, Daniel D. Bikle.
  Journal of Cellular Physiology. November 10, 2016
  Previous studies have shown that dietary calcium suppresses oral carcinogenesis, but the mechanism is unclear. p120‐catenin (p120) is a cytoplasmic protein closely associated with E‐cadherin to form the E‐cadherin–β‐catenin complex and may function as a tumor suppressor in the oral epithelium. To determine whether p120 is involved in the mechanism by which dietary calcium suppresses oral carcinogenesis, The normal, low, or high calcium diet was fed control mice (designated as floxed p120 mice) or mice in which p120 was specifically deleted in the oral squamous epithelium during the adult stage (designated as p120cKO mice). All mice were exposed to a low dose of oral cancer carcinogen 4‐nitroquinoline 1‐oxide and rates of oral squamous cell carcinoma (OSCC) and proliferation and differentiation in the cancerous and non‐cancerous oral epithelium of these mice were examined. The results showed that the low calcium diet increased rates of OSCC and proliferation of the non‐cancerous oral epithelium and decreased differentiation of the non‐cancerous oral epithelium, but had no effect on cancerous oral epithelium. In contrast, the high calcium diet had opposite effects. However, the effect of the dietary calcium on the rates of OSCC, proliferation, and differentiation of the non‐cancerous epithelium were not seen in p120cKO mice. Based on these results, we conclude that p120 is required for dietary calcium suppression of oral carcinogenesis and oral epithelial proliferation and dietary calcium induction of oral epithelial differentiation. J. Cell. Physiol. 232: 1360–1367, 2017. © 2016 Wiley Periodicals, Inc. The aim of the present study was to determine whether p120‐catenin plays a role in dietary calcium‐induced oral carcinogenesis. The results showed that knockout of p120‐catenin blocked the inhibitory effect of dietary calcium on tumor rates and proliferation and the stimulatory effect of dietary calcium on tumor differentiation in the oral epithelium. These data suggest that p120‐catenin plays a critical role in dietary calcium‐suppressed carcinogenesis and dietary calcium regulation of proliferation, differentiation. Our findings illustrate a mechanism by which dietary calcium suppresses oral carcinogenesis will help in the development of better preventive and therapeutic strategies for oral cancers.

  November 10, 2016   doi: 10.1002/jcp.25620   open full text
• Functional Roles of Eph A‐Ephrin A1 System in Endometrial Luminal Epithelial Cells During Early Pregnancy.
  Whasun Lim, Hyocheol Bae, Fuller W. Bazer, Gwonhwa Song.
  Journal of Cellular Physiology. November 10, 2016
  Eph and ephrin regulate diverse biological events such as proliferation, adhesion, migration, and angiogenesis through cell‐to‐cell interactions. However, little is known of their functional role and mechanisms of action in uterine endometrial cells. In the present study, we demonstrated the effects of the Eph and ephrin on interactions between blastocysts and endometrial luminal epithelial (pLE) cells in the pig that is regarded as an excellent biomedical animal model for research on the peri‐implantation period of pregnancy. Results of this study indicated that among eight members of the Eph A family, expression of Eph A1, A2, A4, and A7 was strongly detected in endometrial epithelial cells during early pregnancy. Of these, for identification of signal transduction pathways induced by ephrin A1, a major ligand for Eph A, cell proliferation assays, and immunofluorescence and cell cycle regulation were analyzed following treatment of pLE cells with ephrin A1. Ephrin A1 stimulated proliferation of pLE cells as evidenced by abundant PCNA expression and an increase in the G2/M phase. Western blot analysis showed that ephrin A1 activated PI3K and MAPK signaling proteins in a time‐dependent manner. Moreover, phosphorylation of AKT, ERK1/2, P38, and JNK proteins were suppressed by their inhibitors wortmannin, U0126, SB203580, and SP600125, respectively. Also, phosphor‐AKT was reduced by ERK1/2 and P38 inhibitors. Ephrin A1‐induced proliferation and migration of pLE cells was also blocked by those inhibitors. Collectively, these results suggest that ephrin A1 enhances interactions between porcine blastocysts and endometrial luminal epithelial cells by activating PI3K and MAPK signal transduction pathways. J. Cell. Physiol. 232: 1527–1538, 2017. © 2016 Wiley Periodicals, Inc. Eph A‐ephrin A1 system plays an important role in uterine receptivity and improvement of litter size through PI3K/AKT and MAPK signal transduction pathways during the peri‐implantation period of pregnancy.

  November 10, 2016   doi: 10.1002/jcp.25659   open full text
• Extracellular Matrix and Colorectal Cancer: How Surrounding Microenvironment Affects Cancer Cell Behavior?
  Sara Crotti, Martina Piccoli, Flavio Rizzolio, Antonio Giordano, Donato Nitti, Marco Agostini.
  Journal of Cellular Physiology. November 10, 2016
  Colorectal cancer (CRC) whit more than a million of new cases per year is one of the most common registered cancers worldwide with few treatment options especially for advanced and metastatic patients.The tumor microenvironment is composed by extracellular matrix (ECM), cells, and interstitial fluids. Among all these constituents, in the last years an increased interest around the ECM and its potential role in cancer tumorigenesis is arisen. During cancer progression the ECM structure and composition became disorganized, allowing cellular transformation and metastasis. Up to now, the focus has mainly been on the characterization of CRC microenvironment analyzing separately structural ECM components or cell secretome modifications. A more extensive view that interconnects these aspects should be addressed. In this review, biochemical (secretome) and biomechanical (structure and architecture) changes of tumor microenvironment will be discussed, giving suggestions on how these changes can affect cancer cell behavior. J. Cell. Physiol. 232: 967–975, 2017. © 2016 Wiley Periodicals, Inc. In this review we discuss how changes in one of the major components of tumoral niche, the extracellular matrix (ECM), allow disease initiation, progression and aggressiveness. The ECM remodeling process in colorectal cancer is presented here considering the alterations occurring in the architecture/structure and in the secretome composition, giving suggestions of new interesting markers of aggressiveness. A comprehensive view of the interactions between tumor cells and ECM should be further investigate in the future to provide new and helpful markers of patients’ response to therapy.

  November 10, 2016   doi: 10.1002/jcp.25658   open full text
• Valproic Acid and Lithium Meditate Anti‐Inflammatory Effects by Differentially Modulating Dendritic Cell Differentiation and Function.
  Sy‐Jye Leu, Yi‐Yuan Yang, Hsing‐Cheng Liu, Chieh‐Yu Cheng, Yu‐Chen Wu, Ming‐Chyi Huang, Yuen‐Lun Lee, Chi‐Ching Chen, Winston W. Shen, Ko‐Jiunn Liu.
  Journal of Cellular Physiology. November 10, 2016
  Valproic acid (VPA), with inhibition activity mainly toward histone deacetylase (HDAC) and Glycogen Synthase Kinase (GSK)‐3, and lithium, with inhibition activity mainly toward GSK‐3, are both prescribed in clinical as mood‐stabilizers and anticonvulsants for the control of bipolar disorder. This study aims to compare the immuno‐modulation activities of VPA and lithium, especially on the differentiation and functions of dendritic cells (DC). Our data show that treatment with VPA or lithium effectively alleviated the severity of collagen‐induced arthritis triggered by LPS in mice. Both agents reduced the serum level of IL‐6 and IL‐10 after LPS challenge in mice. VPA and lithium both induce significant down‐regulation of group I CD1 expression and secretion of IL‐6 during differentiation of human monocyte‐derived immature DC, while they differ in the induction of CD83 and CD86 expression, secretion of IL‐8, IL‐10, and TNF‐α. Upon stimulation of immature DC with LPS, VPA, and lithium both reduced the secretion of IL‐6 and TNF‐α. However, only lithium significantly increased the production of IL‐10, while VPA increased the production of IL‐8 but substantially reduce the secretion of IL‐10 and IL‐23. Treatment with VPA resulted in a reduced capacity of LPS‐stimulated DC to promote the differentiation of T helper 17 cells that are critical in the promotion of inflammatory responses. Taken together, our results suggest that VPA and lithium may differentially modulate inflammation through regulating the capacity of DC to mediate distinct T cell responses, and they may provide a complementary immunomodulatory effects for the treatment of inflammation‐related diseases. J. Cell. Physiol. 232: 1176–1186, 2017. © 2016 Wiley Periodicals, Inc. Upon stimulation of immature DC with LPS, VPA, and lithium both reduced the secretion of IL‐6 and TNF‐α. However, only lithium significantly increased the production of IL‐10, while VPA increased the production of IL‐8 but substantially reduce the secretion of IL‐10 and IL‐23. Treatment with VPA resulted in a reduced capacity of LPS‐stimulated DC to promote the differentiation of T helper 17 cells. VPA and lithium may differentially modulate inflammation through regulating the capacity of DC to mediate distinct T cell responses, and they may provide a complementary immunomodulatory effects for the treatment of inflammation‐related diseases.

  November 10, 2016   doi: 10.1002/jcp.25604   open full text
• Src Family Kinase Links Insulin Signaling to Short Term Regulation of Na,K‐ATPase in Nonpigmented Ciliary Epithelium.
  Mohammad Shahidullah, Amritlal Mandal, Nicholas A. Delamere.
  Journal of Cellular Physiology. November 10, 2016
  Insulin has been shown to elicit changes of Na,K‐ATPase activity in various tissues. Na,K‐ATPase in the nonpigmented ciliary epithelium (NPE) plays a role in aqueous humor secretion and changes of Na,K‐ATPase activity impact the driving force. Because we detect a change of NPE Na,K‐ATPase activity in response to insulin, studies were carried out to examine the response mechanism. Ouabain‐sensitive rubidium (Rb) uptake by cultured NPE cells, measured as a functional index of Na,K‐ATPase‐mediated inward potassium transport, was found to increase in cells exposed for 5 min to insulin. The maximally effective concentration was 100 nM. An intrinsic increase of Na,K‐ATPase activity evident as a >2‐fold increase in the rate of ouabain‐sensitive ATP hydrolysis in homogenates obtained from cells exposed to 100 nM insulin for 5 min was also observed. Insulin‐treated cells exhibited Akt, Src family kinase (SFK), ERK1/2, and p38 activation, all of which were prevented by a pI3 kinase inhibitor LY294002. The Rb uptake and Na,K‐ATPase activity response to insulin both were abolished by PP2, an SFK inhibitor which also prevented p38 and ERK1/2 but not Akt activation. The Akt inhibitor MK‐2206 did not change the Na,K‐ATPase response to insulin. The findings suggest insulin activates pI3K‐dependent Akt and SFK signaling pathways that are separate. ERK1/2 and p38 activation is secondary to and dependent on SFK activation. The increase of Na,K‐ATPase activity is dependent on activation of the SFK pathway. The findings are consistent with previous studies that indicate a link between Na,K‐ATPase activity and SFK signaling. J. Cell. Physiol. 232: 1489–1500, 2017. © 2016 Wiley Periodicals, Inc. Studies were conducted to examine the insulin response in ocular nonpigmented ciliary epithelium. Insulin elicited a pronounced short term increase in Na,K‐ATPase activity and a complex signaling response that involved Akt (protein kinase B), Src family kinase (SFK), ERK1/2, and p38 MAPK all of which were dependent on PI3K activation. ERK1/2 and p38 MAPK activation were secondary to and dependent on SFK activation. The insulin‐dependent increase in Na,K‐ATPase activity was linked to SFK activation. In contrast, Akt activation has no discernable effect on Na,K‐ATPase activity.

  November 10, 2016   doi: 10.1002/jcp.25654   open full text
• Neuroprotection and Blood–Brain Barrier Restoration by Salubrinal After a Cortical Stab Injury.
  M. Asunción Barreda‐Manso, Natalia Yanguas‐Casás, Manuel Nieto‐Sampedro, Lorenzo Romero‐Ramírez.
  Journal of Cellular Physiology. November 10, 2016
  Following a central nervous system (CNS) injury, restoration of the blood‐brain barrier (BBB) integrity is essential for recovering homeostasis. When this process is delayed or impeded, blood substances and cells enter the CNS parenchyma, initiating an additional inflammatory process that extends the initial injury and causes so‐called secondary neuronal loss. Astrocytes and profibrotic mesenchymal cells react to the injury and migrate to the lesion site, creating a new glia limitans that restores the BBB. This process is beneficial for the resolution of the inflammation, neuronal survival, and the initiation of the healing process. Salubrinal is a small molecule with neuroprotective properties in different animal models of stroke and trauma to the CNS. Here, we show that salubrinal increased neuronal survival in the neighbourhood of a cerebral cortex stab injury. Moreover, salubrinal reduced cortical blood leakage into the parenchyma of injured animals compared with injured controls. Adjacent to the site of injury, salubrinal induced immunoreactivity for platelet‐derived growth factor subunit B (PDGF‐B), a specific mitogenic factor for mesenchymal cells. This effect might be responsible for the increased immunoreactivity for fibronectin and the decreased activation of microglia and macrophages in injured mice treated with salubrinal, compared with injured controls. The immunoreactivity for PDGF‐B colocalized with neuronal nuclei (NeuN), suggesting that cortical neurons in the proximity of the injury were the main source of PDGF‐B. Our results suggest that after an injury, neurons play an important role in both, the healing process and the restoration of the BBB integrity. J. Cell. Physiol. 232: 1501–1510, 2017. © 2016 Wiley Periodicals, Inc. Salubrinal is neuroprotective in an animal model of cortical stab injury and facilitated the restoration of the BBB integrity, by inducing the neuronal production of PDGF‐B. Our results suggest that neurons in the proximity of the lesion have a previously underrated, major role in the restoration of the BBB.

  November 10, 2016   doi: 10.1002/jcp.25655   open full text
• The Emerging Role of TRAF7 in Tumor Development.
  Tiziana Zotti, Ivan Scudiero, Pasquale Vito, Romania Stilo.
  Journal of Cellular Physiology. November 03, 2016
  The seven members of the tumor necrosis factor receptor (TNF‐R)‐associated factor (TRAF) family of intracellular proteins were originally discovered and characterized as signaling adaptor molecules coupled to the cytoplasmic regions of receptors of the TNF‐R superfamily. Functionally, TRAFs act both as a scaffold and/or enzymatic proteins to regulate activation of mitogen‐activated protein kinases (MAPKs) and transcription factors of nuclear factor‐κB family (NF‐κB). Given the wide variety of stimuli intracellularly conveyed by TRAF proteins, they are physiologically involved in multiple biological processes, including embryonic development, tissue homeostasis and regulation of innate and adaptive immune responses. In the last few years, it has become increasingly evident the involvement of TRAF7, the last member of the TRAF family to be discovered, in the genesis and progression of several human cancers, placing TRAF7 in the spotlight as a novel tumor suppressor protein. In this paper, we review and discuss the literature recently produced on this subject. This article is protected by copyright. All rights reserved

  November 03, 2016   doi: 10.1002/jcp.25676   open full text
• Novel Instruments for the Implementation of Electrochemotherapy Protocols: From Bench Side to Veterinary Clinic.
  Enrico P. Spugnini, Stefano Fais, Tommaso Azzarito, Alfonso Baldi.
  Journal of Cellular Physiology. October 27, 2016
  Electrochemotherapy (ECT) is a medical strategy that allows an increased efficacy of chemotherapy agents after the application of permeabilizing electric pulses having appropriate characteristics (form, voltage, frequency). In the past 10 years, the clinical efficacy of this therapeutic approach in several spontaneous models of tumors in animals has been shown. Moreover, some of the molecular and cellular mechanisms responsible for this phenomenon have been elucidated. Our group has been deeply involved in the development of new ECT protocols for companion animals, implementing the use of the technique as first line treatment, and evaluating different chemotherapy agents in laboratory animals as well as pets. This article summarizes the most important advances in veterinary ECT, including the development of novel equipment, therapeutic protocols, and their translation to humans. J. Cell. Physiol. 232: 490–495, 2017. © 2016 Wiley Periodicals, Inc. Electrochemotherapy (ECT) is a medical strategy that allows an increased efficacy of chemotherapy agents after the application of permeabilizing electric pulses having appropriate characteristics (form, voltage, frequency). Our group has been deeply involved in the development of new ECT protocols for companion animals, implementing the use of the technique as first line treatment, and evaluating different chemotherapy agents in laboratory animals as well as pets. This article summarizes the most important advances in veterinary ECT, including the development of novel equipment, therapeutic protocols, and their translation to humans.

  October 27, 2016   doi: 10.1002/jcp.25505   open full text
• Pathophysiology of the Desmo‐Adhesome.
  Antonio Celentano, Michele Davide Mignogna, Michael McCullough, Nicola Cirillo.
  Journal of Cellular Physiology. October 27, 2016
  Advances in our understanding of desmosomal diseases have provided a clear demonstration of the key role played by desmosomes in tissue and organ physiology, highlighting the importance of their dynamic and finely regulated structure. In this context, non‐desmosomal regulatory molecules have acquired increasing relevance in the study of this organelle resulting in extending the desmosomal interactome, named the “desmo‐adhesome.” Spatiotemporal changes in the expression and regulation of the desmo‐adhesome underlie a number of genetic, infectious, autoimmune, and malignant conditions. The aim of the present article was to examine the structural and functional relationship of the desmosome, by providing a comprehensive, yet focused overview of the constituents targeted in human disease. The inclusion of the novel regulatory network in the desmo‐adhesome pathophysiology opens new avenues to a deeper understanding of desmosomal diseases, potentially unveiling pathogenic mechanisms waiting to be explored. J. Cell. Physiol. 232: 496–505, 2017. © 2016 Wiley Periodicals, Inc. The desmosome has been the subject of intense research over the last 20 years. While several reviews have recently been published on this topic, none has investigated the desmosomal interactome as a whole. This review is unique in that it not only takes into account the diseases that are linked to primary desmosomal components but, also, includes the potential role of accessory molecules in epithelial disease and cancer.

  October 27, 2016   doi: 10.1002/jcp.25515   open full text
• Role of microRNAs on the Regulation of Mitochondrial Biogenesis and Insulin Signaling in Skeletal Muscle.
  Tanes I. Lima, Hygor N. Araujo, Eveline S. Menezes, Carlos H. Sponton, Michel B. Araújo, Lucas H.M. Bomfim, André L. Queiroz, Madla A. Passos, Thais Amaral e Sousa, Sandro M. Hirabara, Amanda R. Martins, Helena C.L.B. Sampaio, Alice Rodrigues, Rui Curi, Everardo M. Carneiro, Antônio C. Boschero, Leonardo R. Silveira.
  Journal of Cellular Physiology. October 26, 2016
  Mitochondria play a critical role in several cellular processes and cellular homeostasis. Mitochondrion dysfunction has been correlated with numerous metabolic diseases such as obesity and type 2 diabetes. MicroRNAs are non‐coding RNAs that have emerged as key regulators of cell metabolism. The microRNAs act as central regulators of metabolic gene networks by leading to the degradation of their target messenger RNA or repression of protein translation. In addition, vesicular and non‐vesicular circulating miRNAs exhibit a potential role as mediators of the cross‐talk between the skeletal muscle and other tissues/organs. In this review, we will focus on the emerging knowledge of miRNAs controlling mitochondrial function and insulin signaling in skeletal muscle cells. J. Cell. Physiol. 232: 958–966, 2017. © 2016 Wiley Periodicals, Inc. Mitochondrion dysfunction in skeletal muscle has been correlated with innumerous metabolic diseases. MicroRNAs have emerged as key regulators of cell metabolism. In addition, vesicular and non‐vesicular circulating miRNAs exhibit potential role as mediators of the cross‐talking between skeletal muscle and other tissues controlling mitochondrial function and insulin signaling.

  October 26, 2016   doi: 10.1002/jcp.25645   open full text
• Increased Chondrogenic Potential of Mesenchymal Cells From Adipose Tissue Versus Bone Marrow‐Derived Cells in Osteoarthritic In Vitro Models.
  Stefania Pagani, Veronica Borsari, Francesca Veronesi, Andrea Ferrari, Simona Cepollaro, Paola Torricelli, Giuseppe Filardo, Milena Fini.
  Journal of Cellular Physiology. October 26, 2016
  Primarily, to compare the behavior of human mesenchymal stem cells (MSCs) derived from bone marrow (hBMSCs) and adipose tissue (hADSCs) in an osteoarthritic (OA) microenvironment; secondly, to investigate the reaction of these cell types in two alternative in vitro culture systems, obtained by using TNFα and/or IL1β as inflammation mediators, or by using synovial fluid harvested by OA patients (OSF) to simulate the complex inflamed knee microenvironment. 3D micromass cultures of hBMSCs or hADSCs were grown in chondrogenic medium (CTR), in the presence of TNFα and/or IL1β, or synovial fluid from OA patients. After 1 month of culture, the chondrogenic differentiation of micromasses was evaluated by gene expression, matrix composition, and organization. Both hMSCs types formed mature micromasses in CTR, but a better response of hADSCs to the inflammatory environment was documented by micromass area and Bern score evaluations. The addition of OSF elicited a milder reaction than with TNFα and/or IL1β by both cell types, probably due to the presence of both catabolic and protective factors. In particular, SOX9 and ACAN gene expression and GAG synthesis were more abundant in hADSCs than hBMSCs when cultured in OSF. The expression of MMP1 was increased for both hMSCs in inflammatory conditions, but in particular by hBMSCs. hADSCs showed an increased chondrogenic potential in inflammatory culture systems, suggesting a better response of hADSCs in the OA environment, thus underlining the importance of appropriate in vitro models to study MSCs and potential advantages of using these cells for future clinical applications. J. Cell. Physiol. 232: 1478–1488, 2017. © 2016 Wiley Periodicals, Inc. Primary objective of this paper was to compare the behavior of human mesenchymal stem cells (MSCs) derived from bone marrow (hBMSCs) and adipose tissue (hADSCs) in an osteoarthritic (OA) microenvironment; secondly, to investigate the reaction of these cell types in two alternative in vitro culture systems, obtained by using TNFα and/or IL1β as inflammation mediators, or by using synovial fluid harvested by OA patients (OSF) to simulate the complex inflamed knee microenvironment.

  October 26, 2016   doi: 10.1002/jcp.25651   open full text
• Essential Role of CFTR in PKA‐Dependent Phosphorylation, Alkalinization, and Hyperpolarization During Human Sperm Capacitation.
  Lis C. Puga Molina, Nicolás A. Pinto, Paulina Torres Rodríguez, Ana Romarowski, Alberto Vicens Sanchez, Pablo E. Visconti, Alberto Darszon, Claudia L. Treviño, Mariano G. Buffone.
  Journal of Cellular Physiology. October 26, 2016
  Mammalian sperm require to spend a limited period of time in the female reproductive tract to become competent to fertilize in a process called capacitation. It is well established that HCO3− is essential for capacitation because it activates the atypical soluble adenylate cyclase ADCY10 leading to cAMP production, and promotes alkalinization of cytoplasm, and membrane hyperpolarization. However, how HCO3− is transported into the sperm is not well understood. There is evidence that CFTR activity is involved in the human sperm capacitation but how this channel is integrated in the complex signaling cascades associated with this process remains largely unknown. In the present work, we have analyzed the extent to which CFTR regulates different events in human sperm capacitation. We observed that inhibition of CFTR affects HCO3−‐entrance dependent events resulting in lower PKA activity. CFTR inhibition also affected cAMP/PKA‐downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility, and acrosome reaction. In addition, we demonstrated for the first time, that CFTR and PKA activity are essential for the regulation of intracellular pH, and membrane potential in human sperm. Addition of permeable cAMP partially recovered all the PKA‐dependent events altered in the presence of inh‐172 which is consistent with a role of CFTR upstream of PKA activation. J. Cell. Physiol. 232: 1404–1414, 2017. © 2016 Wiley Periodicals, Inc. We observed that inhibition of CFTR affects HCO3‐entrance dependent events resulting in lower PKA activity.

  October 26, 2016   doi: 10.1002/jcp.25634   open full text
• Interfering of the Reelin/ApoER2/PSD95 Signaling Axis Reactivates Dendritogenesis of Mature Hippocampal Neurons.
  Estibaliz Ampuero, Nur Jury, Steffen Härtel, María‐Paz Marzolo, Brigitte van Zundert.
  Journal of Cellular Physiology. October 25, 2016
  Reelin, an extracellular glycoprotein secreted in embryonic and adult brain, participates in neuronal migration and neuronal plasticity. Extensive evidence shows that reelin via activation of the ApoER2 and VLDLR receptors promotes dendrite and spine formation during early development. Further evidence suggests that reelin signaling is needed to maintain a stable architecture in mature neurons, but, direct evidence is lacking. During activity‐dependent maturation of the neuronal circuitry, the synaptic protein PSD95 is inserted into the postsynaptic membrane to induce structural refinement and stability of spines and dendrites. Given that ApoER2 interacts with PSD95, we tested if reelin signaling interference in adult neurons reactivates the dendritic architecture. Unlike findings in developing cultures, the presently obtained in vitro and in vivo data show, for the first time, that reelin signaling interference robustly increase dendritogenesis and reduce spine density in mature hippocampal neurons. In particular, the expression of a mutant ApoER2 form (ApoER2‐tailless), which is unable to interact with PSD95 and hence cannot transduce reelin signaling, resulted in robust dendritogenesis in mature hippocampal neurons in vitro. These results indicate that reelin/ApoER2/PSD95 signaling is important for neuronal structure maintenance in mature neurons. Mechanistically, obtained immunofluorescent data indicate that reelin signaling impairment reduced synaptic PSD95 levels, consequently leading to synaptic re‐insertion of NR2B‐NMDARs. Our findings underscore the importance of reelin in maintaining adult network stability and reveal a new mode for reactivating dendritogenesis in neurological disorders where dendritic arbor complexity is limited, such as in depression, Alzheimer's disease, and stroke. J. Cell. Physiol. 232: 1187–1199, 2017. © 2016 Wiley Periodicals, Inc. In mature neurons the reelin interference reactivates the dendritogenesis in the adults reelin signaling maintains adult network stability.

  October 25, 2016   doi: 10.1002/jcp.25605   open full text
• Heat Stress Modulates Both Anabolic and Catabolic Signaling Pathways Preventing Dexamethasone‐Induced Muscle Atrophy In Vitro.
  Wakako Tsuchida, Masahiro Iwata, Takayuki Akimoto, Shingo Matsuo, Yuji Asai, Shigeyuki Suzuki.
  Journal of Cellular Physiology. October 19, 2016
  It is generally recognized that synthetic glucocorticoids induce skeletal muscle weakness, and endogenous glucocorticoid levels increase in patients with muscle atrophy. It is reported that heat stress attenuates glucocorticoid‐induced muscle atrophy; however, the mechanisms involved are unknown. Therefore, we examined the mechanisms underlying the effects of heat stress against glucocorticoid‐induced muscle atrophy using C2C12 myotubes in vitro, focusing on expression of key molecules and signaling pathways involved in regulating protein synthesis and degradation. The synthetic glucocorticoid dexamethasone decreased myotube diameter and protein content, and heat stress prevented the morphological and biochemical glucocorticoid effects. Heat stress also attenuated increases in mRNAs of regulated in development and DNA damage responses 1 (REDD1) and Kruppel‐like factor 15 (KLF15). Heat stress recovered the dexamethasone‐induced inhibition of PI3K/Akt signaling. These data suggest that changes in anabolic and catabolic signals are involved in heat stress‐induced protection against glucocorticoid‐induced muscle atrophy. These results have a potentially broad clinical impact because elevated glucocorticoid levels are implicated in a wide range of diseases associated with muscle wasting. J. Cell. Physiol. 232: 650–664, 2017. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc. Heat stress attenuated increases in REDD1 and KLF15, two genes directly targeted by glucocorticoids that decrease protein synthesis by inhibiting mTORC1. Heat stress also normalized dexamethasone‐induced increases in key factors promoting protein degradation through the ubiquitin‐proteasome system, including KLF15.

  October 19, 2016   doi: 10.1002/jcp.25609   open full text
• Short‐ and Long‐Term Hindlimb Immobilization and Reloading: Profile of Epigenetic Events in Gastrocnemius.
  Alba Chacon‐Cabrera, Joaquim Gea, Esther Barreiro.
  Journal of Cellular Physiology. October 19, 2016
  Skeletal muscle dysfunction and atrophy are characteristic features accompanying chronic conditions. Epigenetic events regulate muscle mass and function maintenance. We hypothesized that the pattern of epigenetic events (muscle‐enriched microRNAs and histone acetylation) and acetylation of transcription factors known to signal muscle wasting may differ between early‐ and late‐time points in skeletal muscles of mice exposed to hindlimb immobilization (I) and recovery following I. Body and muscle weights, grip strength, muscle‐enriched microRNAs, histone deacetylases (HDACs), acetylation of proteins, histones, and transcription factors (TF), myogenic TF factors, and muscle phenotype were assessed in gastrocnemius of mice exposed to periods (1, 2, 3, 7, 15, and 30 days, I groups) of hindlimb immobilization, and in those exposed to reloading for different periods of time (1, 3, 7, 15, and 30 days, R groups) following 7‐day immobilization. Compared to non‐immobilized controls, muscle weight, limb strength, microRNAs, especially miR‐486, SIRT1 levels, and slow‐ and fast‐twitch cross‐sectional areas were decreased in mice of I groups, whereas Pax7 and acetylated FoxO1 and FoxO3 levels were increased. Muscle reloading following splint removal improved muscle mass loss, strength, and fiber atrophy, by increasing microRNAs, particularly miR‐486, and SIRT1 content, while decreasing acetylated FoxO1 and FoxO3 levels. In this mouse model of disuse muscle atrophy, muscle‐enriched microRNAs, especially miR‐486, through Pax7 regulation delayed muscle cell differentiation following unloading of gastrocnemius muscle. Acetylation of FoxO1 and 3 seemed to drive muscle mass loss and atrophy, while deacetylation of these factors through SIRT1 would enable the muscle fibers to regenerate. J. Cell. Physiol. 232: 1415–1427, 2017. © 2016 Wiley Periodicals, Inc. In this mouse model of disuse muscle atrophy, muscle‐enriched microRNAs, especially miR‐486, through Pax7 regulation delayed muscle cell differentiation following unloading of gastrocnemius muscle. Acetylation of FoxO1 and 3 seemed to drive muscle mass loss and atrophy, while deacetylation of these factors through SIRT1 would enable the muscle fibers to regenerate.

  October 19, 2016   doi: 10.1002/jcp.25635   open full text
• Osteoclast Fusion: Time‐Lapse Reveals Involvement of CD47 and Syncytin‐1 at Different Stages of Nuclearity.
  Anaïs Marie Julie Møller, Jean‐Marie Delaissé, Kent Søe.
  Journal of Cellular Physiology. October 19, 2016
  Investigations addressing the molecular keys of osteoclast fusion are primarily based on end‐point analyses. No matter if investigations are performed in vivo or in vitro the impact of a given factor is predominantly analyzed by counting the number of multi‐nucleated cells, the number of nuclei per multinucleated cell or TRAcP activity. But end‐point analyses do not show how the fusion came about. This would not be a problem if fusion of osteoclasts was a random process and occurred by the same molecular mechanism from beginning to end. However, we and others have in the recent period published data suggesting that fusion partners may specifically select each other and that heterogeneity between the partners seems to play a role. Therefore, we set out to directly test the hypothesis that fusion factors have a heterogenic involvement at different stages of nuclearity. Therefore, we have analyzed individual fusion events using time‐lapse and antagonists of CD47 and syncytin‐1. All time‐lapse recordings have been studied by two independent observers. A total of 1808 fusion events were analyzed. The present study shows that CD47 and syncytin‐1 have different roles in osteoclast fusion depending on the nuclearity of fusion partners. While CD47 promotes cell fusions involving mono‐nucleated pre‐osteoclasts, syncytin‐1 promotes fusion of two multi‐nucleated osteoclasts, but also reduces the number of fusions between mono‐nucleated pre‐osteoclasts. Furthermore, CD47 seems to mediate fusion mostly through broad contact surfaces between the partners’ cell membrane while syncytin‐1 mediate fusion through phagocytic‐cup like structure. J. Cell. Physiol. 232: 1396–1403, 2017. © 2016 Wiley Periodicals, Inc. Time‐lapse recording has allowed us to categorize cell fusion events according to the number of nuclei of the fusion partners. We show here that factors commonly believed to be critical for fusion, are in fact only involved in some categories of fusion. This points to the existence of distinct molecular mechanisms of cell fusion depending on the degree of nuclearity.

  October 19, 2016   doi: 10.1002/jcp.25633   open full text
• MicroRNA‐494 Activation Suppresses Bone Marrow Stromal Cell‐Mediated Drug Resistance in Acute Myeloid Leukemia Cells.
  Chen Tian, Guoguang Zheng, Hongqing Zhuang, Xubin Li, Dongzhi Hu, Lei Zhu, Tengteng Wang, Mingjian James You, Yizhuo Zhang.
  Journal of Cellular Physiology. October 19, 2016
  Acute myeloid leukemia (AML) is not sensitive to chemotherapy partially because of the protection of AML cells by mesenchymal stromal cells (MSCs). Our previous studies found that MSCs protected AML cells from apoptosis through the c‐Myc‐dependent pathway. However, the mechanism by which MSCs regulate c‐Myc in AML cells is still unknown. To elucidate the mechanism, we performed microRNA array analysis of AML cell lines and validated by TaqMan realtime PCR. The results showed that the expression of microRNA‐494 (miR‐494) in AML cells after coculture with MSCs was downregulated. Reporter gene analysis confirmed miR‐494 as one of the regulators of c‐Myc. In the coculture system, activation of miR‐494 in AML cells suppressed proliferation and induced apoptosis of AML cells in vitro. After addition of mitoxantrone to the coculture system, the proliferation of AML cells with miR‐494 activation was suppressed more than that of control cells. After subcutaneous injection of AML cell lines in combination with MSC, tumor growth was suppressed in mice injected with miR‐494‐overexpressing AML cells. The rate of tumor formation was even lower after mitoxantrone treatment in the miR‐494 overexpressing group. Moreover, miR‐494 activation resulted in a decrease of leukemic cell counts in peripheral blood (PB) and bone marrow, and prolonged survival in mice injected with miR‐494‐overexpressing AML cellls and MSCs compared to the control mice. Our results indicate that miR‐494 suppresses drug resistance in AML cells by downregulating c‐Myc through interaction with MSCs and that miR‐494 therefore is a potential therapeutic target. J. Cell. Physiol. 232: 1387–1395, 2017. © 2016 Wiley Periodicals, Inc. miR‐494 suppresses drug resistance in AML cells by downregulating c‐Myc through interaction with MSCs and that miR‐494 therefore is a potential therapeutic target.

  October 19, 2016   doi: 10.1002/jcp.25628   open full text
• Increased Amyloid Precursor Protein and Tau Expression Manifests as Key Secondary Cell Death in Chronic Traumatic Brain Injury.
  Sandra A. Acosta, Naoki Tajiri, Paul R. Sanberg, Yuji Kaneko, Cesar V. Borlongan.
  Journal of Cellular Physiology. October 19, 2016
  In testing the hypothesis of Alzheimer's disease (AD)‐like pathology in late stage traumatic brain injury (TBI), we evaluated AD pathological markers in late stage TBI model. Sprague–Dawley male rats were subjected to moderate controlled cortical impact (CCI) injury, and 6 months later euthanized and brain tissues harvested. Results from H&E staining revealed significant 33% and 10% reduction in the ipsilateral and contralateral hippocampal CA3 interneurons, increased MHCII‐activated inflammatory cells in many gray matter (8–20‐fold increase) and white matter (6–30‐fold increased) regions of both the ipsilateral and contralateral hemispheres, decreased cell cycle regulating protein marker by 1.6‐ and 1‐fold in the SVZ and a 2.3‐ and 1.5‐fold reductions in the ipsilateral and contralateral dentate gyrus, diminution of immature neuronal marker by two‐ and onefold in both the ipsilateral and contralateral SVZ and dentate gyrus, and amplified amyloid precursor protein (APP) distribution volumes in white matter including corpus callosum, fornix, and internal capsule (4–38‐fold increase), as well as in the cortical gray matter, such as the striatum hilus, SVZ, and dentate gyrus (6–40‐fold increase) in TBI animals compared to controls (P's < 0.001). Surrogate AD‐like phenotypic markers revealed a significant accumulation of phosphorylated tau (AT8) and oligomeric tau (T22) within the neuronal cell bodies in ipsilateral and contralateral cortex, and dentate gyrus relative to sham control, further supporting the rampant neurodegenerative pathology in TBI secondary cell death. These findings indicate that AD‐like pathological features may prove to be valuable markers and therapeutic targets for late stage TBI. J. Cell. Physiol. 232: 665–677, 2017. © 2016 Wiley Periodicals, Inc. In testing the hypothesis of Alzheimer's disease (AD)‐like pathology in late stage traumatic brain injury (TBI), we evaluated AD pathological markers in late stage TBI model. We provided evidence that neuronal cell loss, inflammation, decreased cell proliferation and impaired neurogenesis, occurred in discreet neurostructures exhibiting abnormal intra‐neuronal APP expressions, indicating an extensive AD‐like neuropathology in advanced TBI. Moreover, surrogate AD‐like phenotypic markers revealed a significant accumulation of phosphorylated tau (AT8) and oligomeric tau (T22) within the neuronal cell bodies in ipsilateral and contralateral cortex, and dentate gyrus relative to sham control, further supporting the rampant neurodegenerative pathology in TBI secondary cell death. These findings indicate that AD‐like pathological features may prove to be valuable markers and therapeutic targets for late stage TBI.

  October 19, 2016   doi: 10.1002/jcp.25629   open full text
• Indispensable Role of Ion Channels and Transporters in the Auditory System.
  Rahul Mittal, Mayank Aranke, Luca H. Debs, Desiree Nguyen, Amit P. Patel, M'hamed Grati, Jeenu Mittal, Denise Yan, Prem Chapagain, Adrien A. Eshraghi, Xue Zhong Liu.
  Journal of Cellular Physiology. October 17, 2016
  Ear is a complex system where appropriate ionic composition is essential for maintaining the tissue homeostasis and hearing function. Ion transporters and channels present in the auditory system plays a crucial role in maintaining proper ionic composition in the ear. The extracellular fluid, called endolymph, found in the cochlea of the mammalian inner ear is particularly unique due to its electrochemical properties. At an endocochlear potential of about +80 mV, signaling initiated by acoustic stimuli at the level of the hair cells is dependent on the unusually high potassium (K+) concentration of endolymph. There are ion channels and transporters that exists in the ear to ensure that K+ is continually being cycled into the stria media endolymph. This review is focused on the discussion of the molecular and genetic basis of previously and newly recognized ion channels and transporters that support sensory hair cell excitation based on recent knock‐in and knock‐out studies of these channels. This article also addresses the molecular and genetic defects and the pathophysiology behind Meniere's disease as well as how the dysregulation of these ion transporters can result in severe defects in hearing or even deafness. Understanding the role of ion channels and transporters in the auditory system will facilitate in designing effective treatment modalities against ear disorders including Meniere's disease and hearing loss. J. Cell. Physiol. 232: 743–758, 2017. © 2016 Wiley Periodicals, Inc. We review the role of ion channels and transporters in the auditory system and summarize recent advancements in understanding the role of ion channels using genetic and molecular tools. Dysregulation of ion channels and transporters causes ear disorders including Meniere's disease and hearing loss. SLC22 family of ion transporters plays a crucial role in maintaining the integrity of stria vascularis and preventing age‐related hearing loss.

  October 17, 2016   doi: 10.1002/jcp.25631   open full text
• Early Growth Response 3 (Egr3) Contributes a Maintenance of C2C12 Myoblast Proliferation.
  Mitsutoshi Kurosaka, Yuji Ogura, Toshiya Funabashi, Tatsuo Akema.
  Journal of Cellular Physiology. October 12, 2016
  Satellite cell proliferation is a crucially important process for adult myogenesis. However, its regulatory mechanisms remain unknown. Early growth response 3 (Egr3) is a zinc‐finger transcription factor that regulates different cellular functions. Reportedly, Egr3 interacts with multiple signaling molecules that are also known to regulate satellite cell proliferation. Therefore, it is possible that Egr3 is involved in satellite cell proliferation. Results of this study have demonstrated that Egr3 transcript levels are upregulated in regenerating mouse skeletal muscle after cardiotoxin injury. Using C2C12 myoblast culture (a model of activated satellite cells), results show that inhibition of Egr3 by shRNA impairs the myoblast proliferation rate. Results also show reduction of NF‐кB transcriptional activity in Egr3‐inhibited cells. Inhibition of Egr3 is associated with an increase in annexin V+ cell fraction and apoptotic protein activity including caspase‐3 and caspase‐7, and Poly‐ADP ribose polymerase. By contrast, the reduction of cellular proliferation by inhibition of Egr3 was partially recovered by treatment of pan‐caspase inhibitor Z‐VAD‐FMK. Collectively, these results suggest that Egr3 is involved in myoblast proliferation by interaction with survival signaling. J. Cell. Physiol. 232: 1114–1122, 2017. © 2016 Wiley Periodicals, Inc. Egr3 maintains myoblast proliferation by stabilizing basal NF‐κB activity. However, the inhibition of Egr3 results in activation of apoptotic signaling by suppressing NF‐κB activity. The activation of apoptotic signaling is associated with proliferation defect in myoblast.

  October 12, 2016   doi: 10.1002/jcp.25574   open full text
• Gene and MicroRNA Expression Are Predictive of Tumor Response in Rectal Adenocarcinoma Patients Treated With Preoperative Chemoradiotherapy.
  Caterina Millino, Isacco Maretto, Beniamina Pacchioni, Maura Digito, Antonino De Paoli, Vincenzo Canzonieri, Edoardo D'Angelo, Marco Agostini, Flavio Rizzolio, Antonio Giordano, Andrea Barina, Senthilkumar Rajendran, Giovanni Esposito, Gerolamo Lanfranchi, Donato Nitti, Salvatore Pucciarelli.
  Journal of Cellular Physiology. October 07, 2016
  Preoperative chemoradiotherapy (pCRT) followed by surgery is the standard treatment for locally advanced rectal cancer (LARC). However, tumor response to pCRT is not uniform, and there are no effective predictive methods. This study investigated whether specific gene and miRNA expression are associated with tumor response to pCRT. Tissue biopsies were obtained from patients before pCRT and resection. Gene and miRNA expression were analyzed using a one‐color microarray technique that compares signatures between responders (R) and non‐responders (NR), as measured based on tumor regression grade. Two groups composed of 38 “exploration cohort” and 21 “validation cohort” LARC patients were considered for a total of 32 NR and 27 R patients. In the first cohort, using SAM Two Class analysis, 256 genes and 29 miRNAs that were differentially expressed between the NR and R patients were identified. The anti‐correlation analysis showed that the same 8 miRNA interacted with different networks of transcripts. The miR‐630 appeared only with the NR patients and was anti‐correlated with a single transcript: RAB5B. After PAM, the following eight transcripts were strong predictors of tumor response: TMEM188, ITGA2, NRG, TRAM1, BCL2L13, MYO1B, KLF7, and GTSE1. Using this gene set, an unsupervised cluster analysis was applied to the validation cohort and correctly assigned the patients to the NR or R group with 85.7% accuracy, 90% sensitivity, and 82% specificity. All three parameters reached 100% when both cohorts were considered together. In conclusion, gene and miRNA expression profiles may be helpful for predicting response to pCRT in LARC patients. J. Cell. Physiol. 232: 426–435, 2017. © 2016 Wiley Periodicals, Inc. Preoperative chemoradiotherapy (pCRT) followed by surgery is the standard treatment for locally advanced rectal cancer. This study investigated whether specific gene and miRNA expression are associated with tumor response to pCRT.

  October 07, 2016   doi: 10.1002/jcp.25441   open full text
• Pulsed Electromagnetic Field Exposure Reduces Hypoxia and Inflammation Damage in Neuron‐Like and Microglial Cells.
  Fabrizio Vincenzi, Annalisa Ravani, Silvia Pasquini, Stefania Merighi, Stefania Gessi, Stefania Setti, Ruggero Cadossi, Pier Andrea Borea, Katia Varani.
  Journal of Cellular Physiology. October 07, 2016
  In the present study, the effect of low‐frequency, low‐energy pulsed electromagnetic fields (PEMFs) has been investigated by using different cell lines derived from neuron‐like cells and microglial cells. In particular, the primary aim was to evaluate the effect of PEMF exposure in inflammation‐ and hypoxia‐induced injury in two different neuronal cell models, the human neuroblastoma‐derived SH‐SY5Y cells and rat pheochromocytoma PC12 cells and in N9 microglial cells. In neuron‐like cells, live/dead and apoptosis assays were performed in hypoxia conditions from 2 to 48 h. Interestingly, PEMF exposure counteracted hypoxia damage significantly reducing cell death and apoptosis. In the same cell lines, PEMFs inhibited the activation of the hypoxia‐inducible factor 1α (HIF‐1α), the master transcriptional regulator of cellular response to hypoxia. The effect of PEMF exposure on reactive oxygen species (ROS) production in both neuron‐like and microglial cells was investigated considering their key role in ischemic injury. PEMFs significantly decreased hypoxia‐induced ROS generation in PC12, SH‐SY5Y, and N9 cells after 24 or 48 h of incubation. Moreover, PEMFs were able to reduce some of the most well‐known pro‐inflammatory cytokines such as tumor necrosis factor–α (TNF‐α), interleukin (IL)‐1β, IL‐6, and IL‐8 release in N9 microglial cells stimulated with different concentrations of LPS for 24 or 48 h of incubation time. These results show a protective effect of PEMFs on hypoxia damage in neuron‐like cells and an anti‐inflammatory effect in microglial cells suggesting that PEMFs could represent a potential therapeutic approach in cerebral ischemic conditions. J. Cell. Physiol. 232: 1200–1208, 2017. © 2016 Wiley Periodicals, Inc. In the present study, the effect of low‐frequency, low‐energy pulsed electromagnetic fields (PEMFs) has been investigated by using different cell lines derived from neuron‐like cells and microglial cells. PEMF exposure counteracted hypoxia damage significantly reducing cell death and apoptosis, reduced HIF‐1α, ROS, and proinflammatory cytokines. PEMFs could represent a potential therapeutic approach in cerebral ischemic conditions.

  October 07, 2016   doi: 10.1002/jcp.25606   open full text
• Inflammatory Events and Oxidant Production in the Diaphragm, Gastrocnemius, and Blood of Rats Exposed to Chronic Intermittent Hypoxia: Therapeutic Strategies.
  Marisol Domínguez‐Álvarez, Joaquim Gea, Esther Barreiro.
  Journal of Cellular Physiology. September 30, 2016
  We hypothesized that inflammatory events and reactive oxygen species (ROS) production may be differentially expressed in respiratory and limb muscles, and blood of a chronic intermittent hypoxia (CIH) experimental model and that antioxidants and TNF‐alpha blockade may influence those events. In blood, diaphragm, and gastrocnemius of rats non‐invasively exposed to CIH (10% hypoxia, 2 h/day, 14 consecutive days) with/without concomitant treatment with either anti‐TNF‐alpha antibody (infliximab) or N‐acetyl cysteine (NAC), inflammatory cytokines, superoxide anion production, muscle structural abnormalities, and fiber‐type composition were assessed. Compared to non‐exposed controls, in CIH‐exposed rats, body weight gain was reduced, TNF‐alpha, IL‐1beta, IL‐6, and interferon‐gamma levels were increased in diaphragm, TNF‐alpha, and IL‐1 beta plasma levels were greater, systemic and muscle superoxide anion production was higher, diaphragm and gastrocnemius inflammatory cells and internal nuclei were higher, and muscle fiber‐type and morphometry remained unmodified. CIH rats treated with infliximab further increased TNF‐alpha, IL‐1beta, IL‐6, and interferon‐gamma diaphragm levels, whereas NAC induced a reduction only in TNF‐alpha and IL‐1beta levels in diaphragm and plasma. Infliximab and NAC elicited a significant decline in superoxide anion production in diaphragm, gastrocnemius, and plasma, while inducing a further increase in inflammatory cells and internal nuclei in both muscles. Proinflammatory cytokines are differentially expressed in respiratory and limb muscles and plasma of CIH‐exposed rats, while superoxide anion production increased in both muscle types and blood. Infliximab and NAC exerted different effects. These findings may help understand the biology underlying CIH in skeletal muscles and blood of patients with chronic respiratory diseases. J. Cell. Physiol. 232: 1165–1175, 2017. © 2016 Wiley Periodicals, Inc. Proinflammatory cytokines are differentially expressed in respiratory and limb muscles and plasma of CIH‐exposed rats, while superoxide anion production increased in both muscle types and blood. Infliximab and NAC exerted different effects. These findings may help understand the biology underlying CIH in skeletal muscles and blood of patients with chronic respiratory diseases.

  September 30, 2016   doi: 10.1002/jcp.25600   open full text
• MED28 Regulates Epithelial–Mesenchymal Transition Through NFκB in Human Breast Cancer Cells.
  Chun‐Yin Huang, Nien‐Tsu Hsieh, Chun‐I Li, Yu‐Ting Weng, Hsiao‐Sheng Liu, Ming‐Fen Lee.
  Journal of Cellular Physiology. September 30, 2016
  MED28, a mammalian Mediator subunit, was found highly expressed in several types of malignancy, including breast cancer. Recently, we have identified a role of MED28 in regulating both cell growth and migration in human breast cancer cells. In epithelium‐derived solid tumor, migration and invasion are preceded by the progression of epithelial–mesenchymal transition (EMT) which calls for downregulation of epithelial markers as well as upregulation of mesenchymal markers, among other features. The objective of this study was to investigate a putative role of MED28 in the progression of EMT in human breast cancer cells. In fibroblast‐like MDA‐MB‐231 cells, suppression of MED28 attenuated the mesenchymal morphology, concomitantly with a reduction of several mesenchymal biomarkers and Snail, a transcriptional repressor of E‐cadherin. The suppression effect was also accompanied by downregulation of p‐NFκB/p65. However, overexpression of MED28 exhibited in an opposite manner. In epithelial MCF7 cells, administration of Adriamycin®, an experimental EMT induction system, led to a mesenchyme‐like appearance correlated with increased expression of MED28, p‐p65, and Snail, and a reciprocal change of epithelial and mesenchymal markers. Furthermore, suppression of MED28 attenuated the experimental EMT effect and restored the original expression status of E‐cadherin and MMP9 in MCF7 cells. Our data indicate that MED28 modulates the development of EMT through NFκB in human breast cancer cells, further reinforcing the significance of MED28 in the progression of breast cancer on top of its role in cell growth and migration. J. Cell. Physiol. 232: 1337–1345, 2017. © 2016 Wiley Periodicals, Inc. MED28 regulates the expression of epithelial markers and mesenchymal markers through NFκB, thereby modulating the development of epithelial–mesenchymal transition. These findings further reinforce the significance of MED28 in the progression of breast cancer on top of its role in cell growth and migration.

  September 30, 2016   doi: 10.1002/jcp.25610   open full text
• Hypoglycemia Enhances Epithelial‐Mesenchymal Transition and Invasiveness, and Restrains the Warburg Phenotype, in Hypoxic HeLa Cell Cultures and Microspheroids.
  Álvaro Marín‐Hernández, Juan Carlos Gallardo‐Pérez, Ileana Hernández‐Reséndiz, Isis Del Mazo‐Monsalvo, Diana Xochiquetzal Robledo‐Cadena, Rafael Moreno‐Sánchez, Sara Rodríguez‐Enríquez.
  Journal of Cellular Physiology. September 30, 2016
  The accelerated growth of solid tumors leads to episodes of both hypoxia and hypoglycemia (HH) affecting their intermediary metabolism, signal transduction, and transcriptional activity. A previous study showed that normoxia (20% O2) plus 24 h hypoglycemia (2.5 mM glucose) increased glycolytic flux whereas oxidative phosphorylation (OxPhos) was unchanged versus normoglycemia in HeLa cells. However, the simultaneous effect of HH on energy metabolism has not been yet examined. Therefore, the effect of hypoxia (0.1–1% O2) plus hypoglycemia on the energy metabolism of HeLa cells was analyzed by evaluating protein content and activity, along with fluxes of both glycolysis and OxPhos. Under hypoxia, in which cell growth ceased and OxPhos enzyme activities, ΔΨm and flux were depressed, hypoglycemia did not stimulate glycolytic flux despite increasing H‐RAS, p‐AMPK, GLUT1, GLUT3, and HKI levels, and further decreasing mitochondrial enzyme content. The impaired mitochondrial function in HH cells correlated with mitophagy activation. The depressed OxPhos and unchanged glycolysis pattern was also observed in quiescent cells from mature multicellular tumor spheroids, suggesting that these inner cell layers are similarly subjected to HH. The principal ATP supplier was glycolysis for HH 2D monolayer and 3D quiescent spheroid cells. Accordingly, the glycolytic inhibitors iodoacetate and gossypol were more effective than mitochondrial inhibitors in decreasing HH‐cancer cell viability. Under HH, stem cell‐, angiogenic‐, and EMT‐biomarkers, as well as glycoprotein‐P content and invasiveness, were also enhanced. These observations indicate that HH cancer cells develop an attenuated Warburg and pronounced EMT‐ and invasive‐phenotype. J. Cell. Physiol. 232: 1346–1359, 2017. © 2016 Wiley Periodicals, Inc. Hypoglycemia enhances (i) epithelial‐mesenchymal transition, (ii) invasiveness, and (iii) restrains the Warburg phenotype in hypoxic HeLa cells.

  September 30, 2016   doi: 10.1002/jcp.25617   open full text
• PACAP and VIP Inhibit HIF‐1α‐Mediated VEGF Expression in a Model of Diabetic Macular Edema.
  Grazia Maugeri, Agata Grazia D'Amico, Salvatore Saccone, Concetta Federico, Sebastiano Cavallaro, Velia D'Agata.
  Journal of Cellular Physiology. September 30, 2016
  Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) exert a protective role against retinal injuries, including diabetic macular edema (DME). The macular damage is induced by hyperglycemia, which damages vessels supplying blood to the retina and induces hypoxia. The microenvironmental changes stimulate the expression of hypoxia‐inducible factors (HIFs), which promote the choroidal endothelial cell transmigration across the retinal pigmented epithelium (RPE) into neurosensory retina, where they proliferate into new vessels under stimulation of the vascular endothelial growth factor (VEGF). In the present study, we have investigated whether PACAP and VIP prevent retinal damage by modulating the expression of HIFs, VEGF, and its receptors. In accord to our hypothesis, we have shown that both peptides are able to significantly reduce HIF‐1α and increase HIF‐3α expression in ARPE‐19 cells exposed to hyperglycemic/hypoxic insult. This effect is also related to a reduction of VEGF and its receptors expression. Moreover, both peptides also reduce the activation of p38 mitogen‐activated protein kinase (MAPK), a pro‐apoptotic signaling pathway, which is activated by VEGFR‐1 and 2 receptors. In conclusion, our study has further elucidated the protective role performed by PACAP and VIP, against the harmful combined effect of hyperglycemia/hypoxia characterizing the DME microenvironment. J. Cell. Physiol. 232: 1209–1215, 2017. © 2016 Wiley Periodicals, Inc. PACAP and VIP prevent retinal damage by modulating the expression of HIFs, VEGF, and its receptors; PACAP and VIP act against the harmful combined effect of hyperglycemia/hypoxia.

  September 30, 2016   doi: 10.1002/jcp.25616   open full text
• Differences in Activation of HIV‐1 Replication by Superinfection With HIV‐1 and HIV‐2 in U1 Cells.
  Xue Wang, Bing Sun, Christelle Mbondji, Santanu Biswas, Jiangqin Zhao, Indira Hewlett.
  Journal of Cellular Physiology. September 30, 2016
  Macrophages contribute to HIV‐1 pathogenesis by forming a viral reservoir that serve as a viral source for the infection of CD4 T cells. The relationship between HIV‐1 latent infection and superinfection in macrophages has not been well studied. Using susceptible U1 cells chronically infected with HIV‐1, we studied the effects of HIV superinfection on latency and differences in superinfection with HIV‐1 and HIV‐2 in macrophages. We found that HIV‐1 (MN) superinfection displayed increased HIV‐1 replication in a time‐dependent manner; while cells infected with HIV‐2 (Rod) initially showed increased HIV‐1 replication, followed by a decrease in HIV‐1 RNA production. HIV‐1 superinfection upregulated/activated NF‐ĸB, NFAT, AP‐1, SP‐1, and MAPK Erk through expression/activation of molecules, CD4, CD3, TCRβ, Zap‐70, PLCγ1, and PKCΘ in T cell receptor‐related signaling pathways; while HIV‐2 superinfection initially increased expression/activation of these molecules followed by decreased protein expression/activation. HIV superinfection initially downregulated HDAC1 and upregulated acetyl‐histone H3 and histone H3 (K4), while HIV‐2 superinfection demonstrated an increase in HDAC1 and a decrease in acetyl‐histone H3 and histone H3 (K4) relative to HIV‐1 superinfection. U1 cells superinfected with HIV‐1 or HIV‐2 showed differential expression of proteins, IL‐2, PARP‐1, YB‐1, and LysRS. These findings indicate that superinfection with HIV‐1 or HIV‐2 has different effects on reactivation of HIV‐1 replication. HIV‐1 superinfection with high load of viral replication may result in high levels of cytotoxicity relative to HIV‐2 superinfection. Cells infected with HIV‐2 showed lower level of HIV‐1 replication, suggesting that co‐infection with HIV‐2 may result in slower progression toward AIDS. J. Cell. Physiol. 232: 1746–1753, 2017. © 2016 Wiley Periodicals, Inc.

  September 30, 2016   doi: 10.1002/jcp.25614   open full text
• Risk Differences Between Prediabetes And Diabetes According To Breast Cancer Molecular Subtypes.
  A. Crispo, L.S.A. Augustin, M. Grimaldi, F. Nocerino, A. Giudice, E. Cavalcanti, M. Di Bonito, G. Botti, M. De Laurentiis, M. Rinaldo, E. Esposito, G. Riccardi, A. Amore, M. Libra, G. Ciliberto, D.J.A. Jenkins, M. Montella.
  Journal of Cellular Physiology. September 30, 2016
  Hyperglycemia and hyperinsulinemia may play a role in breast carcinogenesis and prediabetes and diabetes have been associated with increased breast cancer (BC) risk. However, whether BC molecular subtypes may modify these associations is less clear. We therefore investigated these associations in all cases and by BC molecular subtypes among women living in Southern Italy. Cases were 557 patients with non‐metastatic incident BC and controls were 592 outpatients enrolled during the same period as cases and in the same hospital for skin‐related non‐malignant conditions. Adjusted multivariate logistic regression models were built to assess the risks of developing BC in the presence of prediabetes or diabetes. The analyses were repeated by strata of BC molecular subtypes: Luminal A, Luminal B, HER2+, and Triple Negative (TN). Prediabetes and diabetes were significantly associated with higher BC incidence after controlling for known risk factors (OR = 1.94, 95% CI 1.32–2.87 and OR = 2.46, 95% CI 1.38–4.37, respectively). Similar results were seen in Luminal A and B while in the TN subtype only prediabetes was associated with BC (OR = 2.43, 95% CI 1.11–5.32). Among HER2+ patients, only diabetes was significantly associated with BC risk (OR = 3.04, 95% CI 1.24–7.47). Furthermore, when postmenopausal HER2+ was split into hormone receptor positive versus negative, the association with diabetes remained significant only in the former (OR = 5.13, 95% CI 1.53–17.22). These results suggest that prediabetes and diabetes are strongly associated with BC incidence and that these metabolic conditions may be more relevant in the presence of breast cancer molecular subtypes with positive hormone receptors. J. Cell. Physiol. 232: 1144–1150, 2017. © 2016 Wiley Periodicals, Inc.

  September 30, 2016   doi: 10.1002/jcp.25579   open full text
• Hyperexcitability of Mesencephalic Trigeminal Neurons and Reorganization of Ion Channel Expression in a Rett Syndrome Model.
  Max F. Oginsky, Ningren Cui, Weiwei Zhong, Christopher M. Johnson, Chun Jiang.
  Journal of Cellular Physiology. September 30, 2016
  People with Rett syndrome (RTT) have defects in motor function also seen in Mecp2‐null mice. Motor function depends on not only central motor commands but also sensory feedback that is vulnerable to changes in excitability of propriosensory neurons. Here we report evidence for hyperexcitability of mesencephalic trigeminal (Me5) neurons in Mecp2‐null mice and a novel cellular mechanism for lowering its impact. In in vitro brain slices, the Me5 neurons in both Mecp2−/Y male and symptomatic Mecp2+/− female mice were overly excitable showing increased firing activity in comparison to their wild‐type (WT) male and asymptomatic counterparts. In Mecp2−/Y males, Me5 neurons showed a reduced firing threshold. Consistently, the steady‐state activation of voltage‐gated Na+ currents (INa) displayed a hyperpolarizing shift in the Mecp2‐null neurons with no change in the INa density. This seems to be due to NaV1.1, SCN1B and SCN4B overexpression and NaV1.2 and SCN3B under‐expression. In contrast to the hyperexcitability, the sag potential and postinhibitory rebound (PIR) were reduced in Mecp2‐null mice. In voltage‐clamp, the IH density was deficient by ∼33%, and the steady‐state half‐activation had a depolarizing shift of ∼10 mV in the Mecp2‐null mice. Quantitative PCR analysis indicated that HCN2 was decreased, HCN1 was upregulated with no change in HCN4 in Mecp2−/Y mice compared to WT. Lastly, blocking IH reduced the firing rate much more in WT than in Mecp2‐null neurons. These data suggest that the Mecp2 defect causes an increase in Me5 neuronal excitability likely attributable to alterations in INa, meanwhile IH is reduced likely altering neuronal excitability as well. J. Cell. Physiol. 232: 1151–1164, 2017. © 2016 Wiley Periodicals, Inc. Mesencephalic trigeminal V neurons are hyperexcitable in Mecp2‐null mice. Our data suggest that the Mecp2 defect that causes an increase in Me5 neuronal excitability is likely attributable to alterations in INa, meanwhile IH is reduced likely altering neuronal excitability as well. Further, the changes in these currents seem to be due to alterations in channel subunit composition.

  September 30, 2016   doi: 10.1002/jcp.25589   open full text
• Nephroprotective Effects of Metformin in Diabetic Nephropathy.
  Sreenithya Ravindran, Vinitha Kuruvilla, Kerry Wilbur, Shankar Munusamy.
  Journal of Cellular Physiology. September 26, 2016
  Metformin, a well‐known anti‐diabetic agent, is very effective in lowering blood glucose in patients with type 2 diabetes with minimal side‐effects. Metformin is also being recommended in the treatment of obesity and polycystic ovary syndrome. Metformin elicits its therapeutic effects mainly via activation of AMP‐activated kinase (AMPK) pathway. Renal cells under hyperglycemic or proteinuric conditions exhibit inactivation of cell defense mechanisms such as AMPK and autophagy, and activation of pathologic pathways such as mammalian target of rapamycin (mTOR), endoplasmic reticulum (ER) stress, epithelial‐to‐mesenchymal transition (EMT), oxidative stress, and hypoxia. As these pathologic pathways are intertwined with AMPK signaling, the potential benefits of metformin therapy in patients with type 2 diabetes would extend beyond its anti‐hyperglycemic effects. However, since metformin is eliminated unchanged through the kidneys and some studies have shown the incidence of lactic acidosis with its use during severe renal dysfunction, the use of metformin was contraindicated in patients with renal disease until recently. With more studies indicating the relatively low incidence of lactic acidosis and revealing the additional benefits with metformin therapy, the US FDA has now approved metformin to be administered in patients with established renal disease based on their renal function. The purpose of this review is to highlight the various mechanisms by which metformin protects renal cells that have lost its functionality in a diabetic or non‐diabetic setting and to enlighten the advantages and therapeutic potential of metformin as a nephroprotectant for patients with diabetic nephropathy and other non‐diabetic forms of chronic kidney disease. J. Cell. Physiol. 232: 731–742, 2017. © 2016 Wiley Periodicals, Inc. Metformin, a popular anti‐diabetic drug and an AMPK activator, modulates various signaling pathways altered in diabetic nephropathy and exhibits nephroprotective properties in various animal models of diabetes. Additional studies in humans are needed to ascertain its therapeutic potential as a nephroprotectant to treat nephropathy in patients with diabetes.

  September 26, 2016   doi: 10.1002/jcp.25598   open full text
• VIP Family Members Prevent Outer Blood Retinal Barrier Damage in a Model of Diabetic Macular Edema.
  Grazia Maugeri, Agata Grazia D'Amico, Caterina Gagliano, Salvatore Saccone, Concetta Federico, Sebastiano Cavallaro, Velia D'Agata.
  Journal of Cellular Physiology. September 21, 2016
  Diabetic macular edema (DME), characterized by an increase of thickness in the eye macular area, is due to breakdown of the blood‐retinal barrier (BRB). Hypoxia plays a key role in the progression of this pathology by activating the hypoxia‐inducible factors. In the last years, various studies have put their attention on the role of pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in retinal dysfunction. However, until now, no study has investigated their protective role against the harmful combined effect of both hyperglycemia and hypoxia on outer BRB. Therefore, in the present study, we have analyzed the role of these peptides on permeability, restoration of tight junctions expression and inhibition of hyperglycemia/hypoxia‐induced apoptosis, in an experimental in vitro model of outer BRB. Our results have demonstrated that the peptides' treatment have restored the integrity of outer BRB induced by cell exposure to hyperglycemia/hypoxia. Their effect is mediated through the activation of phosphoinositide 3 kinase (PI3K)/Akt and mammalian mitogen activated protein kinase/Erk kinase (MAPK/ERK) signaling pathways. In conclusion, our study further clarifies the mechanism through which PACAP and VIP perform the beneficial effect on retinal damage induced by hyperglycemic/hypoxic insult, responsible of DME progression. J. Cell. Physiol. 232: 1079–1085, 2017. © 2016 Wiley Periodicals, Inc. PACAP and VIP restore the integrity of outer BRB exposed to hyperglycemia/hypoxia. PACAP and VIP perform their beneficial effect on retinal damage through activation of PI3K/Akt and MAPK/ERK signaling cascades.

  September 21, 2016   doi: 10.1002/jcp.25510   open full text
• Effects of Conjugated Linoleic Acid Associated With Endurance Exercise on Muscle Fibres and Peroxisome Proliferator‐Activated Receptor γ Coactivator 1 α Isoforms.
  Rosario Barone, Claudia Sangiorgi, Antonella Marino Gammazza, Daniela D'Amico, Monica Salerno, Francesco Cappello, Cristoforo Pomara, Giovanni Zummo, Felicia Farina, Valentina Di Felice, Filippo Macaluso.
  Journal of Cellular Physiology. September 21, 2016
  Conjugated linoleic acid (CLA) has been reported to improve muscle hypertrophy, steroidogenesis, physical activity, and endurance capacity in mice, although the molecular mechanisms of its actions are not completely understood. The aim of the present study was to identify whether CLA alters the expression of any of the peroxisome proliferator‐activated receptor γ coactivator 1α (PGC1α) isoforms, and to evaluate the possible existence of fibre‐type‐specific hypertrophy in the gastrocnemius and plantaris muscles. Mice were randomly assigned to one of four groups: placebo sedentary, CLA sedentary, placebo trained, or CLA trained. The CLA groups were gavaged with 35 μl per day of Tonalin® FFA 80 food supplement containing CLA throughout the 6‐week experimental period, whereas the placebo groups were gavaged with 35 μl sunflower oil each day. Each administered dose of CLA corresponded to approximately 0.7 g/kg or 0.5%, of the dietary daily intake. Trained groups ran 5 days per week on a Rota‐Rod for 6 weeks at increasing speeds and durations. Mice were sacrificed by cervical dislocation and hind limb posterior muscle groups were dissected and used for histological and molecular analyses. Endurance training stimulated mitochondrial biogenesis by PGC1α isoforms (tot, α1, α2, and α3) but CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. In the plantaris muscle, CLA supplementation induced a fibre‐type‐specific hypertrophy of type IIx muscle fibres, which was associated with increased capillary density and was different from the fibre‐type‐specific hypertrophy induced by endurance exercise (of types I and IIb muscle fibres). J. Cell. Physiol. 232: 1086–1094, 2017. © 2016 Wiley Periodicals, Inc. CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. CLA supplementation induced a fibre‐type‐specific hypertrophy of type IIx muscle fibres.

  September 21, 2016   doi: 10.1002/jcp.25511   open full text
• Endothelial STAT3 Activation Increases Vascular Leakage Through Downregulating Tight Junction Proteins: Implications for Diabetic Retinopathy.
  Jang‐Hyuk Yun, Sung Wook Park, Kyung‐Jin Kim, Jong‐Sup Bae, Eun Hui Lee, Sun Ha Paek, Seung U. Kim, Sangkyu Ye, Jeong‐Hun Kim, Chung‐Hyun Cho.
  Journal of Cellular Physiology. September 21, 2016
  Vascular inflammation is characteristic feature of diabetic retinopathy. In diabetic retina, a variety of the pro‐inflammatory cytokines are elevated and involved in endothelial dysfunction. STAT3 transcription factor has been implicated in mediating cytokine signaling during vascular inflammation. However, whether and how STAT3 is involved in the direct regulation of the endothelial permeability is currently undefined. Our studies revealed that IL‐6‐induced STAT3 activation increases retinal endothelial permeability and vascular leakage in retinas of mice through the reduced expression of the tight junction proteins ZO‐1 and occludin. In a co‐culture model with microglia and endothelial cells under a high glucose condition, the microglia‐derived IL‐6 induced STAT3 activation in the retinal endothelial cells, leading to increasing endothelial permeability. In addition, IL‐6‐induced STAT3 activation was independent of ROS generation in the retinal endothelial cells. Moreover, we demonstrated that STAT3 activation downregulates the ZO‐1 and occludin levels and increases the endothelial permeability through the induction of VEGF production in retinal endothelial cells. These results suggest the potential importance of IL‐6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy. J. Cell. Physiol. 232: 1123–1134, 2017. © 2016 Wiley Periodicals, Inc. Vascular inflammation is characteristic feature of diabetic retinopathy. We demonstrated that STAT3 in retinal endothelial cells plays a causative role in downregulating ZO‐1 and occludin levels and increasing the endothelial permeability through VEGF production in retinal endothelial cells. These results suggest the potential importance of IL‐6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy.

  September 21, 2016   doi: 10.1002/jcp.25575   open full text
• HMGB1 Inhibits Apoptosis Following MI and Induces Autophagy via mTORC1 Inhibition.
  Eleonora Foglio, Giovanni Puddighinu, Antonia Germani, Matteo A. Russo, Federica Limana.
  Journal of Cellular Physiology. September 21, 2016
  Exogenous High Mobility Group Box‐1 protein (HMGB1) has been reported to protect the infarcted heart but the underlying mechanism is quite complex. In particular, its effect on ischemic cardiomyocytes has been poorly investigated. Aim of the present study was to verify whether and how autophagy and apoptosis were involved in HMGB1‐induced heart repair following myocardial infarction (MI). HMGB1 (200 ng) or denatured HMGB1 were injected in the peri‐infarcted region of mouse hearts following acute MI. Three days after treatment, an upregulation of autophagy was detected in infarcted HMGB1 treated hearts compared to controls. Specifically, HMGB1 induced autophagy by significantly upregulating the protein expression of LC3, Beclin‐1, and Atg7 in the border zone. To gain further insights into the molecular mechanism of HMGB1‐mediated autophagy, WB analysis were performed in cardiomyocytes isolated from 3 days infarcted hearts in the presence and in the absence of HMGB1 treatment. Results showed that upregulation of autophagy by HMGB1 treatment was potentially related to activation of AMP‐activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1). Accordingly, in these hearts, phospho‐Akt signaling pathway was inhibited. The induction of autophagy was accompanied by reduced cardiomyocyte apoptotic rate and decreased expression levels of Bax/Bcl‐2 and active caspase‐3 in the border zone of 3 days infarcted mice following HMGB1 treatment. We report the first in vivo evidence that HMGB1 treatment in a murine model of acute MI might induce cardiomyocyte survival through attenuation of apoptosis and AMP‐activated protein kinase‐dependent autophagy. J. Cell. Physiol. 232: 1135–1143, 2017. © 2016 Wiley Periodicals, Inc. Exogenous High Mobility Group Box‐1 protein (HMGB1) has been reported to protect the infarcted heart but the underlying mechanism is quite complex; in particular, its effect on ischemic cardiomyocytes has been poorly investigated. HMGB1 (200 ng) or denatured HMGB1 were injected in the peri‐infarcted region of mouse hearts following acute MI. We report the first in vivo evidence that HMGB1 treatment in a murine model of acute MI might induce cardiomyocyte survival through attenuation of apoptosis and AMP‐activated protein kinase‐dependent autophagy.

  September 21, 2016   doi: 10.1002/jcp.25576   open full text
• Functional Effects of Cigarette Smoke‐Induced Changes in Airway Smooth Muscle Mitochondrial Morphology.
  Bharathi Aravamudan, Michael Thompson, Gary C. Sieck, Robert Vassallo, Christina M. Pabelick, Y. S. Prakash.
  Journal of Cellular Physiology. September 21, 2016
  Long‐term exposure to cigarette smoke (CS) triggers airway hyperreactivity and remodeling, effects that involve airway smooth muscle (ASM). We previously showed that CS destabilizes the networked morphology of mitochondria in human ASM by regulating the expression of mitochondrial fission and fusion proteins via multiple signaling mechanisms. Emerging data link regulation of mitochondrial morphology to cellular structure and function. We hypothesized that CS‐induced changes in ASM mitochondrial morphology detrimentally affect mitochondrial function, leading to CS effects on contractility and remodeling. Here, ASM cells were exposed to 1% cigarette smoke extract (CSE) for 48 h to alter mitochondrial fission/fusion, or by inhibiting the fission protein Drp1 or the fusion protein Mfn2. Mitochondrial function was assessed via changes in bioenergetics or altered rates of proliferation and apoptosis. Our results indicate that both exposure to CS and inhibition of mitochondrial fission/fusion proteins affect mitochondrial function (i.e., energy metabolism, proliferation, and apoptosis) in ASM cells. In vivo, the airways in mice chronically exposed to CS are thickened and fibrotic, and the expression of proteins involved in mitochondrial function is dramatically altered in the ASM of these mice. We conclude that CS‐induced changes in mitochondrial morphology (fission/fusion balance) correlate with mitochondrial function, and thus may control ASM proliferation, which plays a central role in airway health. J. Cell. Physiol. 232: 1053–1068, 2017. © 2016 Wiley Periodicals, Inc. Long‐term exposure to cigarette smoke (CS) triggers airway hyperreactivity and remodeling, effects that involve airway smooth muscle (ASM). Previous studies show that CS destabilizes the networked morphology of mitochondria in human ASM. We hypothesized that CS‐induced changes in ASM mitochondrial morphology detrimentally affect mitochondrial function, leading to CS effects on contractility and remodeling.

  September 21, 2016   doi: 10.1002/jcp.25508   open full text
• Store‐Operated Ca2+ Entry in Oocytes Modulate the Dynamics of IP3‐Dependent Ca2+ Release From Oscillatory to Tonic.
  Raphaël Courjaret, Maya Dib, Khaled Machaca.
  Journal of Cellular Physiology. September 07, 2016
  Ca2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal, and amplitude features. Here, we investigate the role of store‐operated Ca2+ entry (SOCE) in regulating the temporal dynamics of Ca2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca2+ release leads to Ca2+ store depletion, this triggers the activation of SOCE that translates into a low‐amplitude tonic Ca2+ signal. SOCE has also been implicated in fueling Ca2+ oscillations when activated at low levels. Here, we show that sustained SOCE activation in the presence of IP3 to gate IP3 receptors (IP3R) results in a pump‐leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca2+ oscillations and produces a tonic Ca2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca2+ entry through SOCE ER‐plasma membrane (PM) junctions, Ca2+ uptake into the ER, followed by release through open IP3Rs at distant sites, a process we refer to as “Ca2+ teleporting.” Therefore, sustained SOCE activation in the presence of an IP3‐dependent “leak” pathway at the ER membrane results in a switch from oscillatory to tonic Ca2+ signaling. J. Cell. Physiol. 232: 1095–1103, 2017. © 2016 Wiley Periodicals, Inc. Calcium influx through SOCE inhibits calcium oscillations and favors a tonic mode of signaling. This is achieved through focal calcium entry at SOCE clusters, followed by calcium uptake into the ER through SERCA and re‐release through open IP3 Receptors. A process referred to as “calcium teleporting.”

  September 07, 2016   doi: 10.1002/jcp.25513   open full text
• MiR‐145 Regulates Lipogenesis in Goat Mammary Cells Via Targeting INSIG1 and Epigenetic Regulation of Lipid‐Related Genes.
  Hui Wang, Huaiping Shi, Jun Luo, Yongqing Yi, Dawei Yao, Xueying Zhang, Gongzhen Ma, Juan J. Loor.
  Journal of Cellular Physiology. September 07, 2016
  MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression at the post‐transcriptional level to cause translational repression or degradation of targets. The profiles of miRNAs across stages of lactation in small ruminant species such as dairy goats is unknown. A small RNA library was constructed using tissue samples from mammary gland of Saanen dairy goats harvested at mid‐lactation followed by sequencing via Solexa technology. A total of 796 conserved miRNAs, 263 new miRNAs, and 821 pre‐miRNAs were uncovered. After comparative analyses of our sequence data with published mammary gland transcriptome data across different stages of lactation, a total of 37 miRNAs (including miR‐145) had significant differences in expression over the lactation cycle. Further studies revealed that miR‐145 regulates metabolism of fatty acids in goat mammary gland epithelial cells (GMEC). Compared with nonlactating mammary tissue, lactating mammary gland had a marked increase in expression of miR‐145. Overexpression of miR‐145 increased transcription of genes associated with milk fat synthesis resulting in greater fat droplet formation, triacylglycerol accumulation, and proportion of unsaturated fatty acids. In contrast, silencing of miR‐145 impaired fatty acid synthesis. Inhibition of miR‐145 increased methylation levels of fatty acid synthase (FASN), stearoyl‐CoA desaturase 1 (SCD1), peroxisome proliferator‐activated receptor gamma (PPARG), and sterol regulatory element binding transcription factor 1 (SREBF1). Luciferase reporter assays confirmed that insulin induced gene 1 (INSIG1) is a direct target of miR‐145. These findings underscore the need for further studies to evaluate the potential for targeting miR‐145 for improving beneficial milk components in ruminant milk. J. Cell. Physiol. 232: 1030–1040, 2017. © 2016 Wiley Periodicals, Inc. Mammary lipogenesis is partly regulated by epigenetic mechanisms through miR‐145 and INSIG1.

  September 07, 2016   doi: 10.1002/jcp.25499   open full text
• Hypoxia Modulates the Swelling‐Activated Cl Current in Human Glioblastoma Cells: Role in Volume Regulation and Cell Survival.
  Luigi Sforna, Marta Cenciarini, Silvia Belia, Antonio Michelucci, Mauro Pessia, Fabio Franciolini, Luigi Catacuzzeno.
  Journal of Cellular Physiology. August 26, 2016
  The malignancy of glioblastoma multiforme (GBM), the most common human brain tumor, correlates with the presence of hypoxic areas, but the underlying mechanisms are unclear. GBM cells express abundant Cl channels whose activity supports cell volume and membrane potential changes, ultimately leading to cell proliferation, migration, and escaping death. In non‐tumor tissues Cl channels are modulated by hypoxia, which prompted us to verify whether hypoxia would also modulate Cl channels in GBM cells. Our results show that in GBM cell lines, acute application of a hypoxic solution activates a Cl current displaying the biophysical and pharmacological features of the swelling‐activated Cl current (ICl,swell). We also found that acute hypoxia increased the cell volume by about 20%, and a 30% hypertonic solution partially inhibited the hypoxia‐activated Cl current, suggesting that cell swelling and the activation of the Cl current are sequential events. Notably, the hypoxia‐induced cell swelling was followed by a regulatory volume decrease (RVD) mediated mainly by ICl,swell. Since, a hypoxia‐induced prolonged cell swelling is usually regarded as a death insult, we hypothesized that the hypoxia‐activated Cl current could limit cell swelling and prevent necrotic death of GBM cells under hypoxic conditions. In accordance, we found that the ICl,swell inhibitor DCPIB hampered the RVD process, and more importantly it sensibly increased the hypoxia‐induced necrotic death in these cells. Taken together, these results suggest that Cl channels are strongly involved in the survival of GBM cells in a hypoxic environment, and may thus represent a new therapeutic target for this malignant tumor. J. Cell. Physiol. 232: 91–100, 2017. © 2016 Wiley Periodicals, Inc.

  August 26, 2016   doi: 10.1002/jcp.25393   open full text
• Extracellular Proteasomes Are Deficient in 19S Subunits as Revealed by iTRAQ Quantitative Proteomics.
  Anna S. Tsimokha, Julia J. Zaykova, Andrew Bottrill, Nikolai A. Barlev.
  Journal of Cellular Physiology. August 16, 2016
  Proteasome‐mediated proteolysis is critical for regulation of vast majority of cellular processes. In addition to their well‐documented functions in the nucleus and cytoplasm proteasomes have also been found in extracellular space. The origin and functions of these proteasomes, dubbed as circulating/plasmatic or extracellular proteasomes, are unclear. To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ‐based quantitative proteomics (iTRAQ LC/MS‐MS). Our analysis of purified from K562 cells or conditioned medium intact proteasome complexes led to an identification and quantification of 114 proteins, out of which 19 were 26S proteasome proteins (all subunits of the 20S proteasome and a small number of the 19S regulatory particle proteins), and 3 belonged to the ubiquitin system. Sixty‐two of proteasome interacting proteins (PIPs) were differentially represented in CP versus EP, with folds difference ranging from 1.5 to 4.8. The bioinformatics analysis revealed that functionally most of EP‐PIPs were associated with protein biosynthesis and, unlike CP‐PIPs, were under represented by chaperon/ATP‐binding proteins. Identities of some of the proteasome proteins and PIPs were verified by Western blotting. Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes. Specifically, the EP prep contained only three 19S subunits versus at least 18 in the CP one, suggesting that the extracellular proteasomes are deficient in 19S complexes, which may imply that they have special biological functions. J. Cell. Physiol. 232: 842–851, 2017. © 2016 Wiley Periodicals, Inc. To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ‐based quantitative proteomics (iTRAQ LC/MS‐MS). Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes, which implies that the two species of proteasomes may have different biological functions.

  August 16, 2016   doi: 10.1002/jcp.25492   open full text
• Uric Acid Amplifies Aβ Amyloid Effects Involved in the Cognitive Dysfunction/Dementia: Evidences From an Experimental Model In Vitro.
  Giovambattista Desideri, Roberta Gentile, Andrea Antonosante, Elisabetta Benedetti, Davide Grassi, Loredana Cristiano, Antonello Manocchio, Sara Selli, Rodolfo Ippoliti, Claudio Ferri, Claudio Borghi, Antonio Giordano, Annamaria Cimini.
  Journal of Cellular Physiology. August 16, 2016
  There is still a considerable debate concerning whether uric acid is neuroprotective or neurotoxic agent. To clarify this topic, we tested the effects of uric acid on neuronal cells biology by using differentiated SHSY5Y neuroblastoma cells incubated with amyloid β to reproduce an in vitro model of Alzheimer's disease. The incubation of cells with uric acid at the dose of 40 µM or higher significantly reduced cell viability and potentiated the proapoptotic effect of amyloid β. Finally, uric acid enhanced the generation of 4‐hydroxynonenal and the expression of PPARβ/δ promoted by amyloid β, indicating a prooxidant effects. In conclusion, uric acid could exert a detrimental influence on neuronal biology being this influence further potentiated by the concomitant exposure to neurotoxic stimuli. This effect is evident for uric acid concentrations close to those achievable in cerebrospinal fluid in presence of mild hyperuricemia thus suggesting a potential role of uric acid in pathophysiology of cognitive dysfunction. These effects are influenced by the concentrations of uric acid and by the presence of favoring conditions that commonly occur in neurodegenerative disorders and well as in the aging brain, including increased oxidative stress and exposure to amyloid β. J. Cell. Physiol. 232: 1069–1078, 2017. © 2016 Wiley Periodicals, Inc. The study suggests that uric acid could exert detrimental effects on brain structure and function by directly influencing the viability of neuronal cells and their ability to establish synaptic connections.

  August 16, 2016   doi: 10.1002/jcp.25509   open full text
• Chondrocyte Morphology in Stiff and Soft Agarose Gels and the Influence of Fetal Calf Serum.
  Asima Karim, Andrew C. Hall.
  Journal of Cellular Physiology. August 16, 2016
  Changes to chondrocyte volume/morphology may have deleterious effects on extracellular matrix (ECM) metabolism potentially leading to cartilage deterioration and osteoarthritis (OA). The factors controlling chondrocyte properties are poorly understood, however, pericellular matrix (PCM) weakening may be involved. We have studied the density, volume, morphology, and clustering of cultured bovine articular chondrocytes within stiff (2% w/v) and soft (0.2% w/v) three‐dimensional agarose gels. Gels with encapsulated chondrocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM; fetal calf serum (FCS) 1–10%;380 mOsm) for up to 7 days. Chondrocytes were fluorescently labeled after 1, 3, and 7 days with 5‐chloromethylfluorescein‐diacetate (CMFDA) and propidium iodide (PI) or 1,5‐bis{[2‐(di‐methylamino)ethyl]amino}‐4,8‐dihydroxyanthracene‐9,10‐dione (DRAQ5) to identify cytoplasmic space or DNA and imaged by confocal laser scanning microscopy (CLSM). Chondrocyte density, volume, morphology, and clustering were quantified using Volocity™ software. In stiff gels after 7 d with 10% FCS, chondrocyte density remained unaffected and morphology was relatively normal with occasional cytoplasmic processes. However, in soft gels by day 1, chondrocyte volume increased (P = 0.0058) and by day 7, density increased (P = 0.0080), along with the percentage of chondrocytes of abnormal morphology (P < 0.0001) and enhanced clustering (P < 0.05), compared to stiff gels. FCS exacerbated changes to density (P < 0.01), abnormal morphology (P < 0.001) and clustering (P < 0.01) compared to lower concentrations at the same gel strength. Reduced gel stiffness and/or increased FCS concentrations promoted chondrocyte proliferation and clustering, increased cell volume, and stimulated abnormal morphology, producing similar changes to those occurring in OA. The increased penetration of factors in FCS into soft gels may be important in the development of these abnormal chondrocyte properties. J. Cell. Physiol. 232: 1041–1052, 2017. © 2016 Wiley Periodicals, Inc. Changes to chondrocyte properties occur during the process of osteoarthritis but their role in this disorder is poorly understood. We described a three‐dimensional culture model using stiff and soft agarose and quantified chondrocyte properties of density, morphology, volume, and clustering. Some of the changes we observed are very similar to those that occur in OA and the results suggested that the penetration of serum factors in the culture medium could be responsible.

  August 16, 2016   doi: 10.1002/jcp.25507   open full text
• Ascorbic Acid Induces Necrosis in Human Laryngeal Squamous Cell Carcinoma via ROS, PKC, and Calcium Signaling.
  Min‐Woo Baek, Heui‐Seung Cho, Sun‐Hun Kim, Won‐Jae Kim, Ji‐Yeon Jung.
  Journal of Cellular Physiology. August 16, 2016
  Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N‐acetylcysteine blocked ascorbic acid‐induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid‐induced necrotic cell death was inhibited by Ro‐31‐8425 (PKC inhibitor) and BAPTA‐AM (cytosolic calcium‐selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro‐31‐8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid‐treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 232: 417–425, 2017. © 2016 Wiley Periodicals, Inc. Ascorbic acid induced necrotic cell death in human laryngeal squamous cell carcinoma through ROS, PKC, and calcium signaling pathway.

  August 16, 2016   doi: 10.1002/jcp.25438   open full text
• 3‐Methylcholanthrene/Aryl‐Hydrocarbon Receptor‐Mediated Hypertension Through eNOS Inactivation.
  Chih‐Cheng Chang, Yung‐Ho Hsu, Hsiu‐Chu Chou, Yuan‐Chii G. Lee, Shu‐Hui Juan.
  Journal of Cellular Physiology. August 10, 2016
  Endothelial nitric oxide synthase (eNOS) modulates vascular blood pressure and is predominantly expressed in endothelial cells and activated through the protein kinase B (Akt/PKB)‐dependent pathway. We previously reported that 3‐methylcholanthrene (3MC) activates the aryl hydrocarbon receptor (AhR) and reduces PI3K/Akt phosphorylation. This study investigated the mechanism underlying the downregulatory effects of 3‐MC on nitric oxide (NO) production occurring through the AhR/RhoA/Akt‐mediated mechanism. The mechanism underlying the effects of 3‐MC on eNOS activity and blood pressure was examined in vitro and in vivo through genetic and pharmacological approaches. Results indicated that 3‐MC modified heat shock protein 90 (HSP90), caveolin‐1, dynein, and eNOS mRNA and protein expression through the AhR/RhoA‐dependent mechanism in mouse cerebral vascular endothelial cells (MCVECs) and that 3‐MC reduced eNOS phosphorylation through the AhR/RhoA‐mediated inactivation of Akt1. The upregulation of dynein expression was associated with decreased eNOS dimer formation (eNOS dimer; an activated form of the enzyme). Coimmunoprecipitation assay results indicated that 3‐MC significantly reduced the interaction between eNOS and its regulatory proteins, including Akt1 and HSP90, but increased the interaction between eNOS and caveolin‐1. Immunofluorescence and Western blot analysis revealed that 3‐MC reduced the amount of membrane‐bound activated eNOS, and a modified Griess assay revealed that 3‐MC concomitantly reduced NO production. However, simvastatin reduced 3‐MC‐mediated murine hypertension. Our study results indicate that AhR, RhoA, and eNOS have major roles in blood pressure regulation. Statin intervention might provide a potential therapeutic approach for reducing hypertension caused by 3‐MC. J. Cell. Physiol. 232: 1020–1029, 2017. © 2016 Wiley Periodicals, Inc. Our study is the first to report that the AhR ligand 3‐MC inactivated eNOS associated with downregulated HSP90 and upregulated caveolin‐1 and dynein expression through AhR/RhoA‐mediated PI3K/Akt inactivation, leading to hypertension in mice. The blockade of AhR and RhoA by using siAhR, DNRhoA, or statins reversed the inhibitory effects of 3‐MC on eNOS by correcting the alterations of eNOS regulatory proteins.

  August 10, 2016   doi: 10.1002/jcp.25497   open full text
• Differentiation of Keratinocytes Modulates Skin HPA Analog.
  Justyna M. Wierzbicka, Michał A. Żmijewski, Jakub Antoniewicz, Michal Sobjanek, Andrzej T. Slominski.
  Journal of Cellular Physiology. August 10, 2016
  It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus–pituitary–adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1‐3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R, and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from days 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. J. Cell. Physiol. 232: 154–166, 2017. © 2016 Wiley Periodicals, Inc. It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus–pituitary–adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. Here, we are showing for the first time preferential expression of HPA axis elements by populations of keratinocytes on different stage of differentiation that resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis.

  August 10, 2016   doi: 10.1002/jcp.25400   open full text
• Tissue Inhibitor of Metalloproteinase 1 Influences Vascular Adaptations to Chronic Alterations in Blood Flow.
  Erin R. Mandel, Cassandra Uchida, Emmanuel Nwadozi, Armin Makki, Tara L. Haas.
  Journal of Cellular Physiology. July 28, 2016
  Remodeling of the skeletal muscle microvasculature involves the coordinated actions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). We hypothesized that the loss of TIMP1 would enhance both ischemia and flow‐induced vascular remodeling by increasing MMP activity. TIMP1 deficient (Timp1−/−) and wild‐type (WT) C57BL/6 mice underwent unilateral femoral artery (FA) ligation or were treated with prazosin, an alpha‐1 adrenergic receptor antagonist, in order to investigate vascular remodeling to altered flow. Under basal conditions, Timp1−/− mice had reduced microvascular content as compared to WT mice. Furthermore, vascular remodeling was impaired in Timp1−/− mice. Timp1−/− mice displayed reduced blood flow recovery in response to FA ligation and no arteriogenic response to prazosin treatment. Timp1−/− mice failed to undergo angiogenesis in response to ischemia or prazosin, despite maintaining the capacity to increase VEGF‐A and eNOS mRNA. Vascular permeability was increased in muscles of Timp1−/− mice in response to both prazosin treatment and FA ligation, but this was not accompanied by greater MMP activity. This study highlights a previously undescribed integral role for TIMP1 in both vascular network maturation and adaptations to ischemia or alterations in flow. J. Cell. Physiol. 232: 831–841, 2017. © 2016 Wiley Periodicals, Inc. Remodeling of the skeletal muscle microvasculature involves the coordinated actions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). This study highlights a previously undescribed integral role for TIMP1 in vascular network maturation and in adaptations to altered blood flow.

  July 28, 2016   doi: 10.1002/jcp.25491   open full text
• Coumestrol Inhibits Proliferation and Migration of Prostate Cancer Cells by Regulating AKT, ERK1/2, and JNK MAPK Cell Signaling Cascades.
  Whasun Lim, Muhah Jeong, Fuller W. Bazer, Gwonhwa Song.
  Journal of Cellular Physiology. July 28, 2016
  Coumestrol is the one of the major phytoestrogens which is abundant in soybeans, legumes, brussel sprouts, and spinach. The beneficial effects of coumestrol are well known in various biological processes including; neuroprotective effects on the nervous system, function of the female reproductive system, anti‐bacterial properties, and anti‐cancer effects. Although the anti‐tumor activity of coumestrol has been demonstrated for ovarian, breast, lung, and cervical cancers, little is known of its effects on prostate cancer. Therefore, in the present study, we investigated the chemotherapeutic effects of coumestrol on two prostate cancer cell lines, PC3 and LNCaP. Our results showed that coumestrol decreased proliferation and migration and induced apoptosis in both PC3 and LNCaP cells. Moreover, effects of coumestrol on cell signaling pathways were investigated and it increased phosphorylation of ERK1/2, JNK, P90RSK, and P53 proteins in a dose‐ and time‐dependent manner whereas phosphorylation of AKT was reduced by coumestrol under the same conditions for culture of PC3 and LNCaP cells. In addition, mitochondrial dysfunction was induced by coumestrol as evidenced by a significant loss of mitochondrial membrane potential. Furthermore, cleavage of caspase‐3 and caspase‐9, the apoptotic proteins associated with mitochondria, also changed in response to coumestrol. Coumestrol also caused mitochondrial dysfunction resulting in an increase in ROS production in PC3 and LNCaP cells. These results suggest that coumestrol can inhibit progression of prostate cancer and may be a novel chemotherapeutic agent for treatment of prostate cancer via effects mediated via the PI3K/AKT and ERK1/2 and JNK MAPK cell signaling pathways. J. Cell. Physiol. 232: 862–871, 2017. © 2016 Wiley Periodicals, Inc. Our results indicated that coumestrol can inhibit progression of prostate cancer cells by affecting their viability and mitochondrial functions, and may be a novel chemotherapeutic agent for treatment of prostate cancer via effects mediated via the PI3K/AKT and ERK1/2 and JNK MAPK cell signal transduction cascades.

  July 28, 2016   doi: 10.1002/jcp.25494   open full text
• Nestin Expressed by Pre‐Existing Cardiomyocytes Recapitulated in Part an Embryonic Phenotype; Suppressive Role of p38 MAPK.
  Marc‐Andre Meus, Vanessa Hertig, Louis Villeneuve, Jean‐Francois Jasmin, Angelino Calderone.
  Journal of Cellular Physiology. July 27, 2016
  Nestin(+)‐cardiomyocytes were identified in the ischemically damaged human/rodent heart, albeit the cellular source, and signaling events implicated in the appearance of the intermediate filament protein remained undefined. Expression of the enhanced green fluorescent protein (EGFP) driven by the second intron of the nestin gene identified a subpopulation of EGFP/nestin(+) cells that differentiated to a vascular phenotype in the peri‐infarct/infarct region of post‐MI mice albeit the transgene was not detected in nestin(+)‐cardiomyocytes. α‐MHC‐driven expression of the reporter mCherry was detected in troponin‐T(+)‐ and nestin(+)‐cardiomyocytes in the peri‐infarct/infarct region of post‐MI mice. However, the cell cycle re‐entry of nestin/mCherry(+)‐cardiomyocytes was not observed. Nestin staining was identified in a paucity of neonatal rat ventricular cardiomyocytes (NNVM). Exposure to phorbol 12,13‐dibutyrate (PDBu) induced NNVM hypertrophy but did not promote nestin expression or Brdu incorporation. PDBu treatment of NNVMs phosphorylated p38 MAPK and HSP27 and HSP27 phosphorylation was abrogated by the p38 MAPK inhibitor SB203580. PDBu/SB203580 co‐treatment significantly increased the percentage of NNVMs that expressed nestin and incorporated Brdu. In the heart of embryonic 10.5 day mice, nestin immunoreactivity was observed in cycling troponin‐T(+)‐cardiomyocytes. Nestin was also detected in embryonic rat ventricular cardiomyocytes and depletion of the intermediate filament protein attenuated cell cycle re‐entry. Thus, nestin expressed by pre‐existing cardiomyocytes following ischemic damage recapitulated in part an embryonic trait and may provide the requisite phenotype to initiate cell cycle re‐entry. However, the overt activation of the p38 MAPK pathway post‐MI may in part limit the appearance and inhibit the cell cycle re‐entry of nestin(+)‐cardiomyocytes. J. Cell. Physiol. 232: 1717–1727, 2017. © 2016 Wiley Periodicals, Inc. Nestin was expressed by pre‐existing cardiomyocytes following ischemic damage to the adult heart and recapitulated in part an embryonic phenotype. However, nestin‐expressing cardiomyocytes were unable to re‐enter the cell cycle following ischemic damage because of the suppressive effect of the p38 MAPK pathway.

  July 27, 2016   doi: 10.1002/jcp.25496   open full text
• Late Endosomal Recycling of Open MHC‐I Conformers.
  Hana Mahmutefendić, Gordana Blagojević Zagorac, Kristina Grabušić, Ljerka Karleuša, Senka Maćešić, Frank Momburg, Pero Lučin.
  Journal of Cellular Physiology. July 27, 2016
  With an increasing number of endosomal cargo molecules studied, it is becoming clear that endocytic routes are diverse, and the cell uses more pathways to adjust expression of cell surface proteins. Intracellular itinerary of integral membrane proteins that avoid the early endosomal recycling route is not enough studied. Therefore, we studied endocytic trafficking of empty Ld (eLd) molecules, an open form of murine MHC‐I allele, in fibroblast‐like cells. Pulse labeling of cell surface eLd with mAbs and internalization kinetics suggest two steps of endosomal recycling: rapid and late. The same kinetics was also observed for human open MHC‐I conformers. Kinetic modeling, using in‐house developed software for multicompartment analysis, colocalization studies and established protocols for enriched labeling of the late endosomal (LE) pool of eLd demonstrated that the late step of recycling occurs from an LE compartment. Although the majority of eLd distributed into pre‐degradative multivesicular bodies (MVBs), these LE subsets were not a source for eLd recycling. The LE recycling of eLd did not require Rab7 membrane domains, as demonstrated by Rab7‐silencing, but required vectorial LE motility, suggesting that LE recycling occurs from dynamic tubulovesicular LE domains prior segregation of eLd in MVBs. Thus, our study indicates that LE system should not be simply considered as a feeder for loading of the degradative tract of the cell but also as a feeder for loading of the plasma membrane and thereby contribute to the maintenance of homeostasis of plasma membrane proteins. J. Cell. Physiol. 232: 872–887, 2017. © 2016 Wiley Periodicals, Inc. Study of endosomal trafficking of open MHC‐I conformers reveals that endosomal recycling may occur from late endosomes, suggesting that the late endosomal system should not be simply considered as a feeder for loading of the degradative tract of the cell.

  July 27, 2016   doi: 10.1002/jcp.25495   open full text
• Tyrosine Residues Regulate Multiple Nuclear Functions of P54nrb.
  Ahn R. Lee, Wayne Hung, Ning Xie, Liangliang Liu, Leye He, Xuesen Dong.
  Journal of Cellular Physiology. July 27, 2016
  The non‐POU‐domain‐containing octamer binding protein (NONO; also known as p54nrb) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although tyrosine phosphorylation has been proposed to account for the multi‐functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. To investigate the tyrosine phosphorylation status of p54nrb, we performed site‐directed mutagenesis on the five tyrosine residues of p54nrb, replacing the tyrosine residues with phenylalanine or alanine, and immunoblotted for tyrosine phosphorylation. We then preceded with luciferase reporter assays, RNA splicing minigene assays, co‐immunoprecipitation, and confocal microscopy to study the function of p54nrb tyrosine residues on transcription, RNA splicing, protein–protein interaction, and cellular localization. We found that p54nrb was not phosphorylated at tyrosine residues. Rather, it has non‐specific binding affinity to anti‐phosphotyrosine antibodies. However, replacement of tyrosine with phenylalanine altered p54nrb activities in transcription co‐repression and RNA splicing in gene context‐dependent fashions by means of differential regulation of p54nrb protein association with i