The urea channel UT-A1 and the water channel aquaporin-2 (AQP2) mediate vasopressin-regulated transport in the renal inner medullary collecting duct (IMCD). To identify the proteins that interact with UT-A1 and AQP2 in native rat IMCD cells, we carried out chemical cross-linking followed by detergent solubilization, immunoprecipitation, and LC-MS/MS analysis of the immunoprecipitated material. The analyses revealed 133 UT-A1-interacting proteins and 139 AQP2-interacting proteins, each identified in multiple replicates. 53 proteins that were present in both the UT-A1 and AQP2 interactomes can be considered as mediators of housekeeping interactions, likely common to all plasma membrane proteins. Among proteins unique to the UT-A1 list were those involved in post-translational modifications: phosphorylation (protein kinases Cdc42bpb, Phkb, Camk2d and Mtor), ubiquitylation/deubiquitylation (Uba1, Usp9x), and neddylation (Nae1 and Uba3). Among the proteins unique to the AQP2 list were several Rab proteins (Rab1a, Rab2a, Rab5b, Rab5c, Rab7a, Rab11a, Rab11b, Rab14, Rab17) involved in membrane trafficking. UT-A1 was found to interact with UT-A3, although quantitative proteomics revealed that most UT-A1 molecules in the cell are not bound to UT-A3. In vitro incubation of UT-A1 peptides with the protein kinases identified in the UT-A1 interactome revealed that all except Mtor were capable of phosphorylating known sites in UT-A1. Overall, the UT-A1 and AQP2 interactomes provide a snapshot of a dynamic process in which UT-A1 and AQP2 are produced in the rough endoplasmic reticulum, processed through the Golgi apparatus, delivered to endosomes that move into and out of the plasma membrane, and are regulated in the plasma membrane.
The mitochondrial network in muscle is controlled by the opposing processes of mitochondrial biogenesis and mitophagy. The coactivator PGC-1α regulates biogenesis, while the transcription of mitophagy-related genes is controlled by transcription factor EB (TFEB). PGC-1α activation is induced with exercise, however the effect of exercise on TFEB is not fully known. We investigated the interplay between PGC-1α and TFEB on mitochondria in response to acute contractile activity in C2C12 myotubes, and following exercise in WT and PGC-1α KO mice. TFEB nuclear localization was increased by 1.6-fold following 2 hours of acute myotube contractile activity. TFEB transcription and LC3 localization to mitochondria were also simultaneously increased by 2-3-fold. Viral overexpression of TFEB increased PGC-1α and COXIV gene expression. In WT mice, TFEB translocation to the nucleus increased 2.4-fold in response to acute exercise, while TFEB transcription, assessed through the electroporation of a TFEB promoter construct, was elevated by 4-fold. These exercise effects were dependent on the presence of PGC-1α. Our data suggest that acute exercise provokes TFEB expression and activation both in vitro and in vivo, in a PGC-1α-dependent manner. Our results indicate that TFEB, along with PGC-1α, are important regulators of mitochondrial biogenesis in muscle as a result of exercise.
Spatiotemporal changes in cytosolic Ca2+ concentration ([Ca2+]c) trigger a number of physiological functions in smooth muscle cells (SMCs). We previously imaged Ca2+-induced Ca2+ release following membrane depolarization as local Ca2+ transients, Ca2+ hotspots, in subplasmalemmal regions. In this study, the physiological significance of mitochondria on local Ca2+ signaling was examined. Cytosolic and mitochondrial Ca2+ images following depolarization or action potentials were recorded in single SMCs from the guinea pig urinary bladder using a fast-scanning confocal fluorescent microscope. Depolarization- and action potential-induced [Ca2+]c transients occurred at several discrete sites in subplasmalemmal regions, peaked within 30 ms, and then spread throughout the whole-cell. In contrast, Ca2+ concentration in the mitochondria matrix ([Ca2+]m) increased after a delay of ~50 ms from the start of depolarization, and then peaked within 500 ms. Following repolarization, [Ca2+]c returned to the resting level with a half decay time of ~500 ms, while [Ca2+]m recovered more slowly (~1.5 s). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, a mitochondrial uncoupler, abolished depolarization-induced [Ca2+]m elevations and slowed [Ca2+]c changes. Importantly, short depolarization-induced changes in [Ca2+]m and transmembrane potential in mitochondria coupled to Ca2+ hotspots were significantly larger than those in other mitochondria. Total internal reflection fluorescence imaging revealed that a subset of mitochondria closely localized with ryanodine receptors and voltage-dependent Ca2+ channels. These results indicate that particular mitochondria are functionally coupled to ion channels and sarcoplasmic reticulum fragments within the local Ca2+ microdomain, and thus, strongly contribute to [Ca2+]c regulation in SMCs.
(Pro)renin receptor (PRR), a new component of renin-angiotensin system (RAS), has emerged as a new regulator of collecting duct function. The present study was designed to investigate the role of PRR in high salt-induced apoptosis in cultured mouse inner medullary collecting duct cells, mIMCD-K2 cells. Exposure to high NaCl at 550 mOsm/kg H2O increased PRR protein abundance so did mannitol, sodium gluconate or choline chloride. This was accompanied with upregulation of phosphorylated NF-B p65 protein abundance. NF-B inhibition with either QNZ, CAPE, or siRNA-mediated silencing of NF-B p65 attenuated high NaCl-induced PRR upregulation. Exposure to high salt for 24 hours induced apoptosis as assessed by immunoblotting analysis of cleaved caspase-3 and flow cytometry analysis of the number of apoptotic cells. High NaCl-induced apoptosis was attenuated by a PRR decoy inhibitor PRO20 or siRNA-mediated silencing of NF-B p65. In conclusion, PRR expression is induced by high NaCl through NF-B and contributes to cell apoptosis under this circumstance.
Air pollution stimulates airway epithelial secretion through a cholinergic reflex that is unaffected in cystic fibrosis (CF), yet a strong correlation is observed between passive smoke exposure in the home and impaired lung function in CF children. Our aim was to study the effects of low smoke concentrations on CFTR function in vitro. Cigarette smoke extract stimulated robust anion secretion that was transient, mediated by cystic fibrosis transmembrane conductance regulator (CFTR), and dependent on cAMP-dependent protein kinase activation. Secretion was initiated by reactive oxygen species (ROS) and mediated by at least two distinct pathways; autocrine activation of EP4 prostanoid receptors, and stimulation of Ca2+ store-operated cAMP signaling. The response was absent in cells expressing the most common disease-causing mutant F508del-CFTR. In addition to the initial secretion, prolonged exposure of non-CF bronchial epithelial cells to low levels of smoke also caused a gradual decline in CFTR functional expression. F508del-CFTR channels that had been rescued by the CF drug combination VX-809 (lumacaftor) + VX-770 (ivacaftor) were more sensitive to this downregulation than wild-type CFTR. The results suggest that CFTR-mediated secretion during acute cigarette smoke exposure initially protects the airway epithelium while prolonged exposure reduces CFTR functional expression and reduces the efficacy of CF drugs.
Sclerostin (SCL) has emerged as an important regulator of bone mass. We have shown that SCL can act by targeting late osteoblasts/osteocytes to inhibit bone mineralisation and to upregulate osteocyte expression of catabolic factors, resulting in osteocytic osteolysis. Here we sought to examine the effect of exogenous sclerostin on osteocytes in trabecular bone mechanically loaded ex vivo. Bovine trabecular bone cores, with bone marrow removed, were inserted into individual chambers and subjected to daily episodes of dynamic loading. Cores were perfused with either osteogenic media alone or media containing human recombinant sclerostin (rhSCL) (50 ng/ml). Loaded control bone increased in apparent stiffness over time compared to unloaded bone, and this was abrogated in the presence of rhSCL. Loaded bone showed an increase in calcein uptake as a surrogate of mineral accretion, compared to unloaded bone, in which this was substantially inhibited by rhSCL treatment. Sclerostin treatment induced a significant increase in the ionised calcium concentration in the perfusate and the release of β-CTX at several time points, an increased mean osteocyte lacunar size, indicative of osteocytic osteolysis and the expression of catabolism-related genes. Human primary osteocyte-like cultures treated with rhSCL also released β-CTX from their matrix. These results suggest that osteocytes contribute directly to bone mineral accretion, and to the mechanical properties of bone. Moreover, it appears that sclerostin, acting on osteocytes, can negate this effect by modulating the dimensions of the lacunocanalicular porosity and the composition of the peri-osteocyte matrix.
As an alpha-chemokine receptor specific for stromal-derived-factor-1 (SDF-1, also called CXCL12), CXCR4 plays a vital role in chemotactically attracting lymphocytes during inflammation. CXCR4 also regulates HIV infection due to its role as one of the chemokine co-receptors for HIV entry into CD4+ T cells. Chemokine receptors and their signaling pathways have been shown to be regulated by the process of ubiquitination, a post-translational modification, guided by ubiquitin E3 ligases, which covalently links ubiquitin chains to lysine residues within target substrates. Here we describe a novel mechanism regulating CXCR4 protein levels and subsequent CXCR4/CXCL12 signaling pathway through the ubiquitination and degradation of the receptor in response to ligand stimulation. We identify that an uncharacterized RING finger ubiquitin E3 ligase, RNF113A, directly ubiquitinates CXCR4 in cells, leading to CXCR4 degradation, and therefore disrupts the signaling cascade. We determined that the K331 residue within CXCR4 is essential for RNF113A-mediated ubiquitin conjugation. Overexpression of RNF113A significantly reduces CXCL12-induced kinase activation in HeLa cells, whereas RNF113A knockdown enhances CXCL12-induced downstream signaling. Further, RNF113A expression and silencing directly affects cell motility in a wound healing assay. These results suggest that RNF113A plays an important role in CXCR4 signaling through the ubiquitination and degradation of CXCR4. This mechanistic study might provide new understanding of HIV immunity and neutrophils activation and motility regulated by CXCR4.
Endothelial colony forming cells (ECFCs) were proved to take part in post-natal vasculogenesis and injury repair. The angiogenic properties of ECFCs could be influenced by various cytokines, chemokines, and growth factors. Erythropoietin (EPO) is a promising cytokine participating in angiogenesis. However, the mechanisms for EPO's proangiogenic effect still remain largely elusive. Here, we investigated the role of AMP-activated protein kinase (AMPK)-Krüppel-like factor 2 (KLF2) signaling pathway in the proangiogenic effect of EPO in ECFCs. Human ECFCs were isolated from cord blood and cultured. EPO significantly enhanced the migration and tube formation capacities of ECFCs and markedly increased the expression of endothelial markers and vascular endothelial growth factor (VEGF). Further, EPO caused the phosphorylation of AMPK and endothelial nitric oxide synthase (eNOS), in which process KLF2 was also up-regulated on both mRNA and protein levels. The up-regulation of KLF2 was blocked by inhibiting AMPK with Compound C or Ad-AMPK-DN, a recombinant adenovirus which encoded a dominant negative mutant of AMPK. Furthermore, knockdown of KLF2 showed no effect on AMPK but abolished the EPO-enhanced migration and tube formation capacities of ECFCs. Of note, knockdown of KLF2 also diminished the EPO-induced expression of endothelial markers and VEGF; overexpression of KLF2 promoted the expression of endothelial markers and VEGF and enhanced the migration and tube formation capacities of ECFCs. These data suggest that up-regulation of KLF2 by AMPK plays an essential role in EPO-induced angiogenesis.
Type II nephronophthisis (NPHP2) is an autosomal recessive renal cystic disorder characterized by mutations in the inversin gene. Humans and mice with mutations in inversin have enlarged cystic kidneys that may be due to fluid accumulation resulting from altered ion transport. To address this, transepithelial ion transport was measured in shRNA mediated inversin-depleted mouse cortical collecting duct (mCCD) cells. Loss of inversin decreased the basal ion flux in mCCD cells compared to controls. Depletion of inversin decreased vasopressin-induced Na+ absorption, but did not alter Cl- secretion by mCCD cells. Addition of amiloride, a specific blocker of the epithelial sodium channel (ENaC), abolished basal ion transport in both inversin knockdown and control cells indicating ENaC involvement. Transcript levels of ENaCβ subunit were reduced in inversin-knockdown cells consistent with decreased ENaC activity. Furthermore, Nedd4l (neural precursor cell expressed, developmentally downregulated 4 like), an upstream negative regulator of ENaC was evaluated. The relative amount of the phosphorylated, inactive Nedd4l was decreased in inversin-depleted cells consistent with decreased ENaC activity. The protein levels of Sgk1 (serum and glucocorticoid-inducible kinase) that phosphorylates Nedd4l remain unchanged although the transcript levels were increased in inversin-depleted cells. Interestingly, mRNA and protein levels of Crtc2 (Creb-regulated transcription coactivator) kinase, a positive regulator of Sgk1 were decreased in inversin-depleted cells. Together these results suggest that loss of inversin decreases Na+ transport via ENaC, mediated in part by transcriptional and post-translational regulation of Crtc2/Sgk1/Nedd4l axis as a contributory mechanism for enlarged kidneys in NPHP2.
Two prescient 1953 publications set the stage for elucidating a novel endocrine system: Schatzmann's report that cardiotonic steroids (CTSs) are all Na+ pump inhibitors, and Szent-Gyorgi's suggestion that there is an endogenous "missing screw" in heart failure that CTSs like digoxin may replace. In 1977 I postulated that an endogenous Na+ pump inhibitor acts as a natriuretic hormone and simultaneously elevates blood pressure (BP) in salt-dependent hypertension. This hypothesis was based on the idea that excess renal salt retention promoted the secretion of a CTS-like hormone that inhibits renal Na+ pumps and salt reabsorption. The hormone also inhibits arterial Na+ pumps, elevates myocyte Na+ and promotes Na/Ca exchanger-mediated Ca2+ gain. This enhances vasoconstriction and arterial tone - the hallmark of hypertension. Here I describe how those ideas led to the discovery that the CTS-like hormone is endogenous ouabain (EO), a key factor in the pathogenesis of hypertension and heart failure. Seminal observations that underlie the still-emerging picture of the EO-Na+ pump endocrine system in the physiology and pathophysiology of multiple organ systems are summarized. Milestones include: 1. Cloning the Na+ pump isoforms and physiological studies of mutated pumps in mice; 2. Discovery that Na+ pumps are also EO-triggered signaling molecules; 3. Demonstration that ouabain, but not digoxin, is hypertensinogenic; 4. Elucidation of EO's roles in kidney development and cardiovascular and renal physiology and pathophysiology; 4. Discovery of 'brain ouabain', a component of a novel hypothalamic neuromodulatory pathway; 5. Finding that EO and its brain receptors modulate behavior and learning.
The muscle types present with variable fatigue tolerance, in part due to the myosin isoform expressed. However, the critical steps that define 'fatigability' in vivo of fast vs slow myosin isoforms, at the molecular level, are not yet fully understood. We examined the modulation of the ATP-induced myosin sub-fragment 1 (S1) dissociation from pyrene-actin by inorganic phosphate (Pi), pH and temperature using a specially modified stopped-flow system that allowed fast kinetics measurements at physiological temperature. We contrasted the properties of rabbit psoas (fast) and bovine masseter (slow) myosins (obtained from samples collected from New Zealand rabbits and from a licensed abattoir, respectively, according to institutional and national ethics permits). To identify ATP cycling biochemical intermediates, we assessed ATP binding to a pre-equilibrated mixture of actomyosin and variable [ADP], pH (pH 7 vs pH 6.2) and Pi (zero, 15 or 30 added mM Pi) in a range of temperatures (5 to 45°C). Temperature and pH variations had little, if any, effect on the ADP dissociation constant (KADP) for fast S1 but for slow S1 KADP was weakened with increasing temperature or low pH. In the absence of ADP, the dissociation constant for phosphate (KPi) was weakened with increasing temperature for fast S1. In the presence of ADP, myosin type differences were revealed at the apparent phosphate affinity, depending on pH and temperature. Overall, the newly revealed kinetic differences between myosin types could help explain the in vivo observed muscle type functional differences at rest and during fatigue.
The interconversion between epithelial and mesenchymal is proposed to be a key machinery responsible for metastasis-related deaths. Likewise, cancer stem cells (CSCs) have been proposed to be a key driver of tumor metastasis. However, the linkage between the two events and their control mechanisms are unclear. We used a 3D tumor spheroid assay and other CSC-indicating assays to investigate the role of E-cadherin in CSC regulation and its association to epithelial to mesenchymal transition in lung cancer cells. Ectopic overexpression and knockdown of E-cadherin were found to promote and retard, respectively, the formation of tumor spheroids in vitro, but had opposite effects on tumor formation and metastasis in vivo in a xenograft mouse model. We explored the discrepancy between the in vitro and in vivo results and demonstrated for the first time the requirement of E-cadherin as a major survival pathway under detachment conditions. Downregulation of E-cadherin increased the stemness of lung cancer cells, but had an adverse effect on their survival, particularly on non-CSCs. Such downregulation also promoted anoikis resistance and invasiveness of lung cancer cells. These results suggest that anoikis assay could be used as an alternative assay for in vitro assessment of CSCs that involve dysregulated adhesion proteins. Our data also suggest that agents that restore E-cadherin expression may be used as therapeutic agents for metastatic cancers.
Kv1.3 channels are involved in the switch to proliferation of normally quiescent cells, being implicated in the control of cell cycle in many different cell types and in many different ways. They modulate membrane potential controlling K+ fluxes, sense changes in potential and interact with many signaling molecules through their intracellular domains. From a mechanistic point of view, we can describe the role of Kv1.3 channels in proliferation with at least three different models. In the "membrane potential model", membrane hyperpolarization resulting from Kv1.3 activation provides the driving force for Ca2+ influx required to activate Ca2+-dependent transcription. This model explains most of the data obtained from several cells from the immune system. In the "voltage sensor model" Kv1.3 channels serve mainly as sensors that transduce electrical signals into biochemical cascades, independently of their effect on membrane potential. Kv1.3-dependent proliferation of vascular smooth muscle cells (VSMCs) could fit this model. Finally, in the "channelosome balance model", the master switch determining proliferation may be related to the control of the Kv1.3 to Kv1.5 ratio, as described in glial cells and also in VSMCs. Since the three mechanisms cannot function independently, these models are obviously not exclusive. Nevertheless, they could be exploited differentially in different cells and tissues. This large functional flexibility of Kv1.3 channels surely gives a new perspective on their functions beyond their elementary role as ion channels, although a conclusive picture of the mechanisms involved in Kv1.3 signaling to proliferation is yet to be reached.
A considerable amount of the thiamin generated by gut microbiota exists in the form of thiamin pyrophosphate (TPP). We have previously shown that human colonocytes possess an efficient carrier-mediated uptake process for TPP that involves the SLC44A4 system and this uptake process is adaptively-regulated by prevailing extracellular TPP level. Little is known about the molecular mechanisms that mediate this adaptive regulation. We addressed this issue using human-derived colonic epithelial NCM460 cells and mouse colonoids as models. Maintaining NCM460 cells in the presence of high level of TPP (1 mM) for short (2 days) and long (9 days) periods was found to lead to a significant reduction in 3H-TPP uptake compared to cells maintained in its absence. Short-term exposure showed no changes in level of expression of SLC44A4 protein in total cell homogenate (although there was a decreased expression in the membrane fraction), mRNA and promoter activity. However, a significant reduction in the level of expression of the SLC44A4 protein, mRNA and promoter activity was observed upon long-term maintenance with the substrate. Similar changes in Slc44a4 mRNA expression were observed when mouse colonoids were maintained with TPP for short- and long-terms. Expression of the transcription factors ELF3 and CREB-1, (which drive the SLC44A4 promoter), following long-term exposure was unchanged; but their binding affinity to the promoter was decreased and specific histone modifications were also observed. These studies demonstrate that, depending on the period of exposure, different mechanisms are involved in the adaptive regulation of colonic TPP uptake by extracellular substrate level.
Proteinuria is a characteristic of chronic kidney disease (CKD) and also a causative factor that promotes the disease progression in part via activation of intrarenal renin-angiotensin system (RAS). (Pro) renin receptor (PRR), a newly discovered component of the RAS, binds renin and (pro) renin to promote angiotensin I generation. The present study was performed to test the role of soluble PRR (sPRR) in albumin overload-induced responses in cultured human renal proximal tubular cell line HK-2 cells. Bovine serum albumin (BSA) treatment for 24 h at 20 mg/ml induced renin activity and inflammation, both of which were attenuated by a PRR decoy inhibitor PRO20. BSA treatment induced a more than 5-fold increase in medium sPRR due to enhanced cleavage of PRR. Surprisingly, this cleavage event was unaffected by inhibition of furin or ADAM19. Screening for a novel cleavage enzyme led to the identification of site-1 protease (S1P). Inhibition of S1P with PF-429242 or siRNA remarkably suppressed BSA-induced sPRR production, renin activity, and inflammatory response. Administration of a recombinant sPRR, termed sPRR-His, reversed the effects of S1P inhibition. In HK-2 cells overexpressing PRR, mutagenesis of the S1P but not furin cleavage site reduced sPRR levels. Together these results suggest that PRR mediates albumin-induced cellular responses through S1P-derived sPRR.
Red blood cell (RBC)-derived adenosine triphosphate (ATP) has been proposed as an integral component in the regulation of oxygen supply to skeletal muscle. In ex vivo settings RBCs have been shown to release ATP in response to a number of stimuli, including stimulation of adrenergic receptors. Further evidence suggested that ATP release from RBCs was dependent on activation of adenylate cyclase (AC)/ cyclic adenosine monophosphate (cAMP)-dependent pathways and involved the Pannexin 1 (Panx1) channel. Here we show that RBCs express Panx1, and confirm its absence in Panx1 knockout (-/-) RBCs. However, Panx1-/- mice lack any decrease in exercise performance, challenging the assumptions that Panx1 plays an essential role in increased blood perfusion to exercising skeletal muscle and therefore in ATP release from RBCs. We therefore tested the role of Panx1 in ATP release from RBCs ex vivo in RBC suspensions. We found that stimulation with hypotonic potassium gluconate buffer resulted in a significant increase in ATP in the supernatant, but this was highly correlated with RBC lysis. Next, we treated RBCs with a stable cAMP analog, which did not induce ATP release from wild-type (WT) or Panx1-/- mice. Similarly, multiple pharmacological treatments activating AC in RBCs increased intracellular cAMP levels (as measured via mass spectrometry), but did not induce ATP release. The data presented here question the importance of Panx1 for exercise performance and dispute the general assumption that ATP release from RBCs via Panx1 is regulated via cAMP.
Transient muscle paralysis engendered by a single injection of botulinum toxin A (BTxA) rapidly induces profound focal bone resorption within the medullary cavity of adjacent bones. While initially conceived as a model of mechanical disuse, osteoclastic resorption in this model is disproportionately severe compared to the modest gait defect that is created. Preliminary studies of bone marrow following muscle paralysis suggested acute upregulation of inflammatory cytokines, including TNF and IL-1. We therefore hypothesized that BTxA-induced muscle paralysis would rapidly alter the inflammatory microenvironment and the osteoclastic potential of bone marrow. We tested this hypothesis by defining the time course of inflammatory cell infiltration, osteoinflammatory cytokine expression, and alteration in osteoclastogenic potential in the tibia bone marrow following transient muscle paralysis of the calf muscles. Our findings identified inflammatory cell infiltration within 24 hours of muscle paralysis. By 72 hours, osteoclast fusion and pro-osteoclastic inflammatory gene expression were upregulated in tibia bone marrow. These alterations coincided with bone marrow becoming permissive to the formation of osteoclasts of greater size and greater nuclei numbers. Taken together, our data are consistent with the thesis that transient calf muscle paralysis induces acute inflammation within the marrow of the adjacent tibia and that these alterations are temporally consistent with a role in mediating muscle paralysis-induced bone resorption.
We are interested in understanding mechanisms that govern the protective role of exercise against lipid-induced insulin resistance, a key driver of type 2 diabetes. In this context, cell culture models provide a level of abstraction that aid in our understanding of cellular physiology. Here we describe the development of an in vitro myotube contraction system that provides this protective effect, and which we have harnessed to investigate lipid-induced insulin resistance. C2C12 myocytes were differentiated into contractile myotubes. A custom manufactured platinum electrode system and pulse stimulator, with polarity switching, provided an electrical pulse stimulus (EPS) (1Hz, 6ms pulsewidth, 1.5V/mm, 16 hours). Contractility was assessed by optical flow flied spot noise mapping and inhibited by application of ammonium acetate. Following EPS, myotubes were challenged with 0.5 mM palmitate for 4 hours. Cells were then treated with or without insulin for glucose uptake (30 mins), secondary insulin signaling activation (10 mins), and phosphoinositide 3-kinase-α (PI3Kα) activity (5 mins). Prolonged EPS increased non-insulin stimulated glucose uptake (83%, P=0.002), Akt (Thr308) phosphorylation (P=0.005), and IRS-1 associated PI3Kα activity (P=0.048). Palmitate reduced insulin specific action on glucose uptake (-49%, P<0.001) and inhibited insulin stimulated Akt phosphorylation (P=0.049) and whole cell PI3Kα activity (P=0.009). The inhibitory effects of palmitate were completely absent with EPS pretreatment at the levels of glucose uptake, insulin responsiveness, Akt phosphorylation, and whole cell PI3Kα activity. This model suggests that muscle contraction alone is a sufficient stimulus to protect against lipid-induced insulin resistance as evidenced by changes in proximal canonical insulin-signaling pathway.
Rabbit corpus cavernosum smooth muscle (RCCSM) cells express channels that produce Ca2+-activated Cl- (IClCa) current, but low sensitivity to conventional antagonists have made its role in tone generation difficult to evaluate. We have re-examined this question using two new generation IClCa blockers, T16Ainh-A01 and CaCCinh-A01. Isolated RCCSM cells were studied using the perforated patch method. Current-voltage protocols revealed both L-type Ca2+ current and IClCa. T16Ainh-A01 and CaCCinh-A01 (10 M) reduced IClCa by ~85%, while 30 M abolished it. L-type Ca2+ current was unaffected by 10 M CaCCinh-A01, but was reduced by 50% at 30 M CaCCinh-A01, 46% at 10 M T16Ainh-A01 and 78% at 30 M T16Ainh-A01. Both drugs reduced spontaneous isometric tension in RCCSM strips, by 60-70% at 10 M and >90% at 30 M. Phenylephrine (PE)-enhanced tension was also reduced (ED50 = 3 μM, CaCCinh-A01; 14 μM, T16Ainh-A01). CaCCinh-A01 10 M had little effect on 60 mM KCl contractures, though they were reduced by 30 M CaCCinh-A01 and T16Ainh-A01 (10 M & 30 M) consistent with their effects on L-type Ca2+ current. Both drugs also reversed the stimulatory effect of PE on intracellular Ca2+ waves, studied with laser scanning confocal microscopy in isolated RCCSM cells. Although both drugs were effective blockers of IClCa, the effect of T16Ainh-A01 on L-type Ca2+ current preclude its use for evaluating the role of IClCa in tone generation. However, 10 μM CaCCinh-A01 selectively blocked IClCa vs L-type Ca2+ current and reduced spontaneous and PE-induced tone, suggesting that IClCa is important in maintaining penile detumescence.
Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate TRPC6, and the subsequent increase in intracellular Ca2+ leads to TRPC5 activation. The goal of this study is to elucidate the steps in the pathway between TRPC6 activation and TRPC5 externalization. Following TRPC6 activation by lysoPC, extracellular regulated kinase (ERK) is phosphorylated. This leads to phosphorylation of p47phox and subsequent NADPH oxidase activation with increased production of reactive oxygen species. ERK activation requires TRPC6 opening and influx of Ca2+ as evidenced by the failure of lysoPC to induce ERK phosphorylation in TRPC6-/- endothelial cells. ERK siRNA blocks the lysoPC-induced activation of NADPH oxidase, demonstrating that ERK activation is upstream of NADPH oxidase. The reactive oxygen species produced by NADPH oxidase promote myosin light chain kinase (MLCK) activation with phosphorylation of MLC and TRPC5 externalization. Downregulation of ERK, NADPH oxidase, or MLCK with the relevant siRNA prevents TRPC5 externalization. Blocking MLCK activation prevents the prolonged rise in intracellular calcium levels and preserves endothelial migration in the presence of lysoPC.
Muscle stretch activation (SA) is critical for optimal cardiac and insect indirect flight muscle (IFM) power generation. The SA mechanism has been investigated for decades with many theories proposed, but none proven. One reason for the slow progress could be that multiple SA mechanisms may have evolved in multiple species or muscle types. Laboratories studying IFM SA in the same or different species have reported differing SA functional properties which would, if true, suggest divergent mechanisms. However, these conflicting results might be due to different experimental methodologies. Thus, we directly compared SA characteristics of IFMs from two SA model systems, Drosophila and Lethocerus, using two different fiber bathing solutions. Compared to Drosophila IFM, Lethocerus IFM isometric tension is 10 or 17-fold higher and SA tension was 5 or 10-fold higher, depending on the bathing solution. However, the rate of SA tension generation was 9-fold faster for Drosophila IFM. The inverse differences between rate and tension in the two species causes maximum power output to be similar, where Drosophila power is optimized in the bathing solution that favors faster muscle kinetics and Lethocerus in the solution that favors greater tension generation. We found that isometric tension and SA tension increased with calcium concentration for both species in both solutions, reaching a maximum plateau around pCa 5.0. Our results favor a similar mechanism for both species, perhaps involving a troponin complex that does not fully calcium activate the thin filament thus leaving room for further tension generation by SA.
Mammary epithelial cells are regulated by steroid hormones, growth factors, and even microRNAs. MiR-15b has been found to regulate lipid metabolism in adipocytes; however its effects on lipid metabolism in mammary epithelial cells, the cells of lipid synthesis and secretion, are as yet unknown. The main purpose of this investigation was to explore the effect of miR-15b on lipid metabolism in mammary epithelial cells, along with the underlying mechanisms. MiR-15b was overexpressed or inhibited by miRNA mimics or inhibitors; subsequently lipid formation in mammary epithelial cells, and proteins related to lipid metabolism, were investigated. Through overexpression or inhibition of miR-15b expression, the current investigation found that miR-15b down-regulates lipid metabolism in mammary epithelial cells and is expressed differentially at various stages of mouse and goat mammary gland development. Inhibition of miR-15b expression increased lipid content in mammary epithelial cells through elevation of the lipid synthesis enzyme FASN, and overexpression of miR-15b reduced lipid content in mammary epithelial cells with decreasing levels of FASN. Moreover, the steroid hormones estradiol and progesterone decreased miR-15b expression with a subsequent increase in lipid formation in mammary epithelial cells. The expression of miR-15b was lower during lactation and negatively correlated with lipid synthesis proteins, which suggests that it may be involved in lipid synthesis and milk production. MiR-15b might be a useful target for altering lipid production and milk yield.
Long before the molecular identity of the Na+-dependent K+-Cl- cotransporters was uncovered in the mid-nineties, a Na+-independent K+-Cl- cotransport system was also known to exist. It was initially observed in sheep and goat red blood cells where it was shown to be ouabain-insensitive and to increase in the presence of N-ethylmaleimide (NEM). After it was established between the early and mid-nineties, the expressed sequence tag (EST) databank was found to include a sequence that was highly homologous to those of the Na+-dependent K+-Cl- cotransporters. This sequence was eventually found to code for the Na+-independent K+-Cl- cotransport function that was described in red blood cells several years before. It was termed KCC1 and led to the discovery of three isoforms called KCC2, KCC3 and KCC4. Since then, it has become obvious that each one of these isoforms exhibits unique patterns of distribution and fulfill distinct physiological roles. Among them, KCC3 has been the subject of great attention in view of its important role in the nervous system and its association with a rare hereditary sensorimotor neuropathy (called Andermann syndrome) that affects many individuals in Québec province (Canada). It was also found to play important roles in the cardiovascular system, the organ of Corti and circulating blood cells. As will be seen in this review, however, there are still a number of uncertainties regarding the transport properties, structural organization and regulation of KCC3. The same is true regarding the mechanisms by which KCC3 accomplishes its numerous functions in animal cells.
Oxidized low density lipoprotein (oxLDL) accumulates early in atherosclerotic lesions and plays an important role in the progressive formation of atherosclerotic plaques. Endothelial derived microparticles (EMPs) form a heterogeneous population of < 1μm particles that shed from endothelial membranes upon activation. While EMPs are shown to be involved in atherosclerotic pathophysiology and progression, there is no report regarding to the relationship between oxLDL and EMPs. In this study we aim to determine the influence of oxLDL on endothelial microparticle release and the subsequent regulation of the endothelial activation. EMPs were collected from the medium of human umbilical vein endothelial cells (HUVECs) treated with oxLDL or PBS as control. We find that oxLDL increases the release of EMPs containing intercellular adhesion molecule 1 (ICAM-1), but not vascular cell adhesion molecule 1 (VCAM-1). Confocal microscopy analysis further demonstrates that these EMPs interact with endothelial cells and increase the expression of ICAM-1 in HUVECs. The fact that injecting oxLDL induced EMPs via the tail vein of ICR mice augments ICAM-1 expression on aortic endothelial cells confirms our results in vivo. Finally, oxLDL induced EMPs from HUVECs increase the adhesion of monocytes to endothelial cells as determined by the adhesion assay. Our study suggests that oxLDL may augment the release of EMPs harboring increased levels of ICAM-1 that can be transferred to endothelial cells elsewhere. This leads to increased monocyte recruitment in other regions where oxLDL accumulation was initially more limited. EMPs may therefore serve as the mediator that propagates oxLDL induced endothelial inflammation.
Oocyte meiotic spindles are associated with spindle-enriched mRNAs, phosphorylated ribosome protein S6, and phosphorylated variants of the key translational regulator EIF4EBP1, consistent with translational control of localized mRNAs by EIF4EBP1 in facilitating spindle formation and stability. Using specific kinase inhibitors, we determined which kinases regulate phosphorylation status of EIF4EBP1 associated with meiotic spindles in mouse oocytes, and effects of kinase inhibition on chromosome congression and spindle formation. Neither ATM nor mTOR inhibition significantly affected phosphorylation status of spindle-associated EIF4EBP1 at the phosphorylation sites examined. Spindle-associated phospho-EIF4EBP1, spindle formation, and chromosome congression were strongly disrupted by PLK1 inhibition at both MI and MII. In addition, direct inhibition of EIF4EBP1 via 4EGI led to spindle defects at MI, indicating a direct role for EIF4EBP1 phosphorylation in meiotic spindle formation. PLK1 also regulated microtubule dynamics throughout the ooplasm, indicating likely coordination between spindle dynamics and broader ooplasm cytoskeletal dynamics. Because diverse upstream signaling pathways converge on PLK1, these results implicate PLK1 as a major regulatory nexus coupling endogenous and exogenous signals via EIF4EBP1 to the regulation of spindle formation and stability.
Glucose constitutes the major source of energy of mammalian brains. Glucose uptake at the blood-brain barrier (BBB) occurs through a facilitated glucose transport, through glucose transporter 1 (GLUT1), although other isoforms have been described at the BBB. Mutations in GLUT1 is associated with the GLUT1 deficiency syndrome (GLUT1DS), yet none of the current in vitro models of the human BBB maybe suited for modelling such disorder. In this study, we investigated the expression of glucose transporters and glucose diffusion across the human BBB using brain microvascular endothelial cells (BMECs) derived from healthy patient-derived induced pluripotent stem cells (iPSCs). We investigated the expression of different glucose transporters at the BBB using immunocytochemistry and flow cytometry and measured glucose uptake and diffusion across BMEC monolayers obtained from two iPSC lines and from hCMEC/D3 cells. BMECs monolayers showed expression of several glucose transporters, in particular GLUT1, GLUT3 and GLUT4. Diffusion of glucose across the monolayers were mediated via a saturable transcellular mechanism and partially inhibited by pharmacological inhibitors. Taken together, our study suggests the presence of several glucose transporters isoforms at the human BBB and demonstrate the feasibility of modelling glucose across the BBB using patient-derived stem cells.
We examined the roles played by gap junctions (GJs) and the GJ channel protein connexin43 (Cx43) in arginine vasopressin (AVP)-induced vasoconstriction after hemorrhagic shock, and their relationship to Rho kinase (ROCK) and protein kinase C (PKC). The results showed AVP induced an endothelium-independent contraction in rat superior mesenteric arteries (SMAs). Blocking the GJs significantly decreased the contractile response of SMAs and vascular smooth muscle cells (VSMCs) to AVP after shock and hypoxia. The selective Cx43-mimetic peptide inhibited the vascular contractile effect of AVP after shock and hypoxia. AVP restored hypoxia-induced decrease of Cx43 phosphorylation at Ser262 and gap-junctional communication in VSMCs. Activation of RhoA with U46619 increased the contractile effect of AVP. This effect was antagonized by the ROCK inhibitor Y27632 and the Cx43-mimetic peptide. In contrast, neither an agonist nor an inhibitor of PKC had significant effects on AVP-induced contraction after hemorrhagic shock. In addition, silencing of Cx43 with siRNA blocked the AVP-induced increase of ROCK activity in hypoxic VSMCs. In conclusion, AVP-mediated vascular contractile effects are endothelium and myoendothelial gap junctions independent. Gap junctions between VSMCs, gap-junctional communication and Cx43 phosphorylation at Ser262 play important roles in the vascular effects of AVP. RhoA/ROCK, but not PKC, is involved in this process.
IL-6 and LIF, members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mTORC1-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of gp130 receptor and downstream signaling pathways, including PI3K-Akt-mTORC1 and STAT3-SOCS3, were investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knock-down and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6 or LIF induced SOCS3 negatively regulates the activation of myotube protein synthesis.
Pigment epithelium derived factor (PEDF) is an endogenous inhibitor of angiogenesis. Although various ocular cell types including retinal endothelial cells (EC) produce PEDF, we know very little about cell autonomous effects of PEDF in these cell types. Here we determined how PEDF expression affects retinal EC proangiogenic properties. Retinal EC were prepared from wild type (PEDF+/+) and PEDF-deficient (PEDF-/-) mice. The identity of EC was confirmed by staining for specific markers including VE-cadherin, CD31, and B4-lectin. Retinal EC also expressed VEGF-R1 and endoglin, as well as ICAM-1, ICAM-2, and VCAM-1. PEDF-/- retinal EC were more proliferative, less apoptotic when challenged with H2O2, less migratory, and less adherent compared with PEDF+/+ EC. These changes could be associated, at least in part, with increased levels of tenascin C, fibronectin, thrombospondin-1 and collagen IV, and lower amounts of osteopontin. PEDF-/- EC also exhibited alterations in expression of a number of integrins including α2, αv, β1, β8, and αvβ3, and cell-cell adhesion molecules including CD31, ZO-1, and occludin. These observations correlated with attenuation of capillary morphogenesis and increased levels of oxidative stress in PEDF-/- EC. PEDF -/- EC also produced lower levels of VEGF compared with PEDF+/+ cells. Thus, PEDF deficiency has a significant impact on retinal EC adhesion and migration, perhaps through altered production of ECM and junctional proteins in response to increased oxidative stress affecting their proangiogenic activity.
Both zinc (Zn2+) and reactive oxygen species (ROS) have been shown to accumulate during hypoxic-ischemic stress and play important roles in pathological processes. Here we studied Zn2+ and ROS accumulations by employing fluorescent probes in HeLa cells to further the understanding of cause and effect relationship of these two important cellular signaling during chemical-ischemia, stimulated by oxygen and glucose deprivation (OGD). We observed two Zn2+ rises that were divided into four phases in the course of 30 minutes OGD. The first Zn2+ rise was a transient, which was followed by a latent phase during which Zn2+ levels recovered, however remained above a basal level. The final phase was the second Zn2+ rise that reached a sustained plateau called Zn2+ overload. Zn2+ rises were not observed when were treated by TPEN (a Zn2+ chelator) or thapsigargin (depleting Zn2+ from intracellular stores), indicating that Zn2+ originated from intracellular stores. Damaging mitochondria with FCCP significantly reduced the second Zn2+ rise, indicating that the mitochondrial Zn2+ accumulation contributes to Zn2+ overload. We also detected two OGD-induced ROS rises. Two Zn2+ rises preceded two ROS rises. Removal of Zn2+ reduced or delayed OGD- and FCCP-induced ROS generation, indicating that Zn2+ contributes to mitochondrial ROS generation. There was a Zn2+-induced increase in functional component of NADPH oxidase, p47phox, thus suggesting that NADPH oxidase may mediate Zn2+-induced ROS. We suggest a new mechanism of crosstalk between Zn2+ and mitochondrial ROS through positive feedback processes that eventually causes excessive free Zn2+ and ROS accumulations during the course of ischemic-stress.
MicroRNAs (miRNAs) can regulate proliferative status of pulmonary artery smooth muscle cells (PASMCs), which is a core factor modulating pulmonary vascular remodeling diseases, such as atherosclerosis and pulmonary arterial hypertension (PAH). Our previous work has shown that miR-4632, a rarely reported miRNA, is significantly downregulated in Platelet-Derived Growth Factor (PDGF)BB-stimulated human pulmonary artery smooth muscle cells (HPASMCs), yet its cell function and the underlying molecular mechanisms remain to be elucidated. Here, we find that miR-4632 is highly expressed in HPASMCs and its expression significantly decreased in response to different stimuli. Functional studies revealed that miR-4632 inhibited proliferation and promoted apoptosis of HPASMCs, but had no effects on cell contraction and migration. Furthermore, the cJUN was identified as a direct target gene of miR-4632, while knockdown of cJUN was necessary for miR-4632-mediated HPASMCs proliferation and apoptosis. In addition, the downregulation of miR-4632 by PDGFBB was found to associate with histone deacetylation through the activation of PDGFR/PI3K/HDAC4 signaling. Finally, the expression of miR-4632 was reduced in serum of patients with PAH. Overall, our results suggest that miR-4632 plays an important role in regulating HPASMCs proliferation and apoptosis by suppression of cJUN, providing a novel therapeutic miRNA candidate for the treatment of pulmonary vascular remodeling diseases. It also implies that serum miR-4632 has the potential to serve as a circulating biomarker for PAH diagnosis.
Aberrant expression of microRNAs (miRs) contributes to diabetic renal complications including renal hypertrophy and matrix protein accumulation. Reduced expression of PTEN by hyperglycemia contributes to these processes. We considered involvement of microRNA in the downregulation of PTEN. In the renal cortex of type 1 diabetic mice, we detected increased expression of miR-214 in association with decreased levels of PTEN and, enhanced Akt phosphorylation and fibronectin expression. Mesangial and proximal tubular epithelial cells exposed to high glucose showed augmented expression of miR-214. Mutagenesis studies using 3'UTR of PTEN in a reporter construct revealed PTEN as a direct target of miR-214, which controls its expression in both these cells. Overexpression of miR-214 decreased the levels of PTEN and increased Akt activity similar to high glucose and lead to phosphorylation of its substrates GSK3β, PRAS40 and tuberin. In contrast, quenching of miR-214 inhibited high glucose-induced Akt activation and its substrate phosphorylation; these changes were reversed by siRNAs against PTEN. Importantly, respective expression of miR-214 or anti-miR-214 increased or decreased the mTORC1 activity induced by high glucose. Furthermore, mTORC1 activity was controlled by miR-214-targeted PTEN via Akt activation. In addition, neutralization of high glucose-stimulated miR-214 expression significantly inhibited cell hypertrophy and expression of the matrix protein fibronectin. Finally, the anti-miR-214-induced inhibition of these processes was reversed by the expression of constitutively active Akt kinase and hyperactive mTORC1. These results uncover a significant role of miR-214 in the activation of mTORC1 that contributes to high glucose-induced mesangial and proximal tubular cell hypertrophy and fibronectin expression.
Cell-cell adhesion molecules play key roles in maintaining quiescence or promoting activation of various stem cells in their niche. Muscle stem cells called satellite cells (SC) are critical for skeletal muscle regeneration after injury but little is known about the role of adhesion molecules in regulating the behavior of these stem cells. Vascular cell adhesion molecule-1 (VCAM-1) is a cell-cell adhesion protein expressed on quiescent and activated SC whose function is unknown in this context. We deleted Vcam1 from SC using an inducible Cre recombinase in young mice. In the injured niche, Vcam1-/- SC underwent premature lineage progression to a more differentiated state as well as apoptosis leading to a transient delay in myofiber growth during regeneration. Apoptosis was also increased in Vcam1-/- SC in vitro concomitant with decreased levels of p-Akt, a pro-survival signal activated by VCAM-1 signaling in other cell types. During muscle regeneration, we observed an influx of immune cells expressing 4 integrin, a component of the major, high affinity VCAM-1 ligand, α4β1 integrin. Furthermore, 4 integrin mRNA and protein were induced in SC 2 days after injury. These results suggest that SC interact with other SC as well as immune cells through α4β1 integrin in the injured niche to promote expansion of SC. In the uninjured niche, multiple cell types also expressed 4 integrin. However, only basal fusion of Vcam1-/- SC with myofibers was decreased contributing to decreased myofiber growth. These studies define differential roles for VCAM-1 in SC depending on the state of their niche.
Connexin-based therapeutics have shown the potential for therapeutic efficacy in improving wound healing. Our previous work demonstrated that the connexin43 (Cx43)-mimetic peptide JM2 reduced the acute inflammatory response to a submuscular implant model by inhibiting purinergic signaling. Given the prospective application in improving tissue engineered construct tolerance that these results indicated, we sought to determine the mechanism of action for JM2 in the present study. Using confocal microscopy, a gap-FRAP cell communication assay, and an ethidium bromide uptake assay of hemichannel function we found that the peptide reduced cell-surface Cx43 levels, Cx43 gap junction (GJ) size, GJ communication, and hemichannel activity. JM2 is based on the sequence of the Cx43 microtubule binding domain, and microtubules have a confirmed role in intracellular trafficking of Cx43 vesicles. Therefore, we tested the effect of JM2 on Cx43-microtubule interaction and microtubule polymerization. We found that JM2 enhanced Cx43-microtubule interaction, and that microtubule polymerization was significantly enhanced. Taken together, these data suggest that JM2 inhibits trafficking of Cx43 to the cell surface by promoting irrelevant microtubule polymerization, and thereby reduces the number of hemichannels in the plasma membrane available to participate in pro-inflammatory purinergic signaling. Importantly, this work indicates that JM2 may have therapeutic value in the treatment of proliferative diseases such as cancer. We conclude that the targeted action of JM2 on Cx43 channels may improve the tolerance of implanted tissue engineered constructs against the innate inflammatory response.
Oxidized modifications of LDL (oxLDL) are known to play a major role in endothelial dysfunction and the development of atherosclerosis. In this study, we provide comparative analysis of the effects of two major types of oxLDL, copper oxidized LDL (Cu2+-oxLDL) and Lipoxygenase oxidized LDL (LPO-oxLDL) on proliferation of Human Aortic Endothelial Cells (HAECs). We show that Cu2+-oxLDL enhances HAECs proliferation in a dose and oxidation-dependent manner. Similarly, LPO-oxLDL also has a positive effect on HAECs proliferation. Mechanistically, both Cu2+-oxLDL and LPO-oxLDL enhance HAECs proliferation via the same pathway: Rho activation, Akt phosphorylation and a decrease in the expression of Cyclin-dependent kinase inhibitor 1B (p27kip1). Specifically, both, Cu2+-oxLDL or LPO-oxLDL significantly increase Akt phosphorylation whereas inhibiting Akt with a specific inhibitor MK2206 blocks oxLDL-induced increase in the proliferation of HAECs. Furthermore, blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibits oxLDL-induced Akt phosphorylation and cell proliferation induced by both Cu2+- and LPO-oxLDL. In contrast, blocking Rac1 with NSC23766 inhibitor has no effect neither on oxLDL-induced Akt phosphorylation or cell proliferation of HAECs. Further evidence shows that oxLDL-induced Rho/Akt pathway leads to downregulation of p27kip1, a well-known negative regulator of cell proliferation. Notably, we also show that preloading cells with cholesterol prevents oxLDL-induced Akt phosphorylation and its proliferative effect. These observations contribute to our understanding of oxLDL-induced proliferative effect in endothelial cells and provide new evidence for the differential effects of oxLDL and cholesterol on endothelial function.
Olfactory receptor neurons isolated from the newt maintain a high activity of the ciliary beat. A cilium of neuron is so unique that only little is known about regulatory factors for its beat frequency. We examined the olfactory receptor neuron immersed in various extracellular media under the video-enhanced differential interference contrast (DIC) microscope. The activation of voltage-gated Ca2+ channels by K+-depolarization or by application of Ca2+ to membrane-permeabilized olfactory cells did not affect the ciliary movement, suggesting that a Ca2+ influx through the cell membrane has no direct effect on the movement. However, when an extracellular medium contained NaCl or sucrose at concentrations only 30% higher than the normal levels, the ciliary movement was greatly and reversibly suppressed. In contrast, a hypotonic solution of such a solute did not change the ciliary movement. The hypertonic solutions had no effect when applied to permeabilized cells. Suction of the cell membrane with a patch pipette easily suppressed the ciliary movement in an isotonic medium. An application of a positive pressure inside the cell through the same patch pipette eliminated the suppressive effect. From these findings, we concluded that the hypertonic stress suppressed the ciliary movement not by disabling the motor proteins, microtubules or their associates in the cilia, but rather by modifying the chemical environment for the motor proteins. The ciliary motility of the olfactory receptor cell is directly sensitive to the external environment: namely, the air or water on the nasal epithelium depending on life styles of the animal.
White adipose tissue (WAT) has a critical role in lipid handling. Previous work demonstrated that SCD1 is an important regulator of WAT fatty acid (FA) composition; however, its influence on the various interconnected pathways influencing WAT lipid handling remains unclear. Our objective was to investigate the role of SCD1 on WAT lipid handling using Scd1 knock-out (KO) mice and SCD1-inhibited 3T3-L1 adipocytes by measuring gene, protein, and metabolite markers related to FA re-esterification, glyceroneogenesis, and lipolysis. Triacylglycerol (TAG) content was higher in inguinal WAT (iWAT) from KO mice compared to wild-type (WT), but significantly lower in epididymal WAT (eWAT). The SCD1 desaturation index was decreased in both WAT depots in KO mice. FA re-esterification, as measured with a NEFA:glycerol ratio, was reduced in both WAT depots in KO mice, as well as SCD1-inhibited 3T3-L1 adipocytes. Pck1, Atgl, and Hsl gene expression were reduced in both WAT depots of KO mice, while Pck2 and Pdk4 gene expression showed depot-specific regulation. Pck1, Atgl, and Hsl gene expression were reduced, and PEPCK protein content ablated, in SCD1-inhibited adipocytes. Our data provides evidence that SCD1 has a broad impact on WAT lipid handling by altering TAG composition in a depot-specific manner, reducing FA re-esterification, and regulating markers of lipolysis and glyceroneogenesis.
Background/Aim: Interleukin 22 (IL-22) is a Th17 cell hepatoprotective cytokine that is undergoing clinical trials to treat patients with alcoholic hepatitis (AH). Lipopolysaccharide (LPS) activation of macrophage is implicated in hepatocyte cell death and pathogenesis of AH. The role of IL-22 production from macrophage, its regulation by LPS and effects on alcohol induced hepatocyte cell death are unexplored and were examined in this study. Methods/Results: Low levels of IL-22 mRNA/protein were detected in macrophage but were significantly upregulated by 6.5 fold in response to the tissue reparative cytokine IL-10. Conversely, LPS significantly decreased IL-22 mRNA levels in a temporal and concentration dependent manner with a maximum reduction of 5-fold. Since LPS downregulation of IL-22 mRNA levels was rescued in the presence of a pharmacologic inhibitor of c-jun N-terminal kinase (JNK) and by JNK knockdown. Next, we explored if macrophage-derived IL-22 regulated ethanol induced hepatocyte death. Conditioned media from IL-10 stimulated macrophage attenuated ethanol induced hepatocyte caspase-3/7 activity and apoptosis as assessed by fluorimetric assay and TUNEL staining, respectively. This effect was diminished in conditioned media from macrophages with IL-22 knockdown. Cytokine analysis in sera samples of patients with AH revealed that IL-22 levels were significantly elevated compared with healthy controls and heavy drinking controls implying a state of IL-22 resistance in human AH. Conclusions: Macrophage-derived IL-22 protects hepatocytes from ethanol induced cell death. IL-22 downregulation is a new regulatory target of LPS in the pathogenesis of AH. -
Oxidative stress impacts normal cellular function leading to the pathogenesis of various diseases including pulmonary illnesses. Protein arginine methyltransferase 4 (PRMT4) is critical for normal lung alveolar epithelial cell development; however, the regulation of PRMT4 within such pulmonary diseases has yet to be elucidated. Using biochemical approaches, we uncovered that peroxide (H2O2) treatment decreases PRMT4 protein stability in murine lung epithelial (MLE12) cells to impede cell migration. Protein kinase glycogen synthase kinase 3β (GSK-3β) interacts with PRMT4 and catalyzes PRMT4 T132 phosphorylation that protects PRMT4 from ubiquitin proteasomal degradation. H2O2 down-regulates GSK-3β to reduce PRMT4 at protein level. PRMT4 promotes cell migration and H2O2 degrades PRMT4 to inhibit lung epithelial cell migration. These observations demonstrate that oxidative stress destabilizes PRMT4 via GSK-3β signaling to impede lung epithelial cell migration that may hinder lung repair and regeneration process.
The human riboflavin (RF) transporter-3 (hRFVT-3; product of the SLC52A3 gene) plays an essential role in the intestinal RF absorption process and is expressed exclusively at the apical membrane domain of polarized enterocytes. Previous studies have characterized different physiological/biological aspects of this transporter, but nothing is known about the glycosylation status of the hRFVT-3 protein and role of this modification in its physiology/biology. Additionally, little is known about the residues in the hRFVT-3 protein that interact with the ligand, RF. We addressed these issues using appropriate biochemical/molecular approaches, a protein-docking model, and established intestinal/renal epithelial cells. Our results showed that the hRFVT-3 protein is glycosylated and that glycosylation is important for its function. Mutating the predicted N-glycosylation sites at Asn94 and Asn168 led to a significant decrease in RF uptake; it also led to a marked intracellular (in the endoplasmic reticulum, ER) retention of the mutated proteins as shown by live-cell confocal imaging studies. The protein-docking model used in this study has identified a number of putative substrate-interacting sites: Ser16, Ile20, Trp24, Phe142, Thr314 and Asn315. Mutating these potential interacting sites was indeed found to lead to a significant inhibition in RF uptake and to intracellular (ER) retention of the mutated proteins (except for the Phe142 mutant). These results demonstrate that the hRFVT-3 protein is glycosylated and this glycosylation is important for its function and cell surface expression. This study also identified a number of residues in the hRFVT-3 polypeptide that are important for its function/cell surface expression.
Cells are exposed to various intrinsic and extrinsic stresses in both physiological and pathological conditions. To adapt to those conditions, cells have evolved various mechanisms to cope with the disturbances in protein demand, largely through the Unfolded Protein Response (UPR) in the Endoplasmic Reticulum (ER), but also through the Integrated Stress Response (ISR). Both responses initiate downstream signaling to transcription factors that, in turn, trigger adaptive programs and/or in the case of prolonged stress, cell death mechanisms. Recently, non-coding RNAs, including microRNA and long non-coding RNA, have emerged as key players in the stress responses. These non-coding RNAs act as both regulators and effectors of the UPR, and fine-tune the output of the stress signaling pathways. Although much is known about the UPR and the cross-talk that exists between pathways, the contribution of small non-coding RNA has not been fully assessed. Herein we bring together and review the current known functions of non-coding RNA in regulating adaptive pathways in both physiological and pathophysiological conditions, illustrating how they operate within the known UPR functions and contribute to diverse cellular outcomes.
The carotid body (CB) chemoreflex maintains blood PO2 and PCO2/H+ homeostasis and displays sensory plasticity during exposure to chronic hypoxia. Purinergic signaling via P1 and P2 receptors plays a pivotal role in shaping the afferent discharge at the sensory synapse containing catecholaminergic chemoreceptor (type I) cells, glial-like type II cells, and sensory (petrosal) nerve endings. However, little is known about the family of ectonucleotidases that control synaptic nucleotide levels. Using quantitative PCR (QPCR), we first compared expression levels of ectonucleoside triphosphate diphosphohydrolases (NTPDases1,2,3,5,6) and ecto-5'-nucleotidase (E5'Nt/CD73) mRNAs in juvenile rat CB versus brain, petrosal ganglia, sympathetic (superior cervical) ganglia, and a sympathoadrenal chromaffin (MAH) cell line. In whole CB extracts, QPCR revealed a high relative expression of surface-located members NTPDase1,2 and E5'Nt/CD73, compared to low NTPDase3 expression. Immunofluorescence staining of CB sections or dissociated CB cultures localized NTPDase2,3 and E5'Nt/CD73 protein to the periphery of type I clusters, and in association with sensory nerve fibers and/or isolated type II cells. Interestingly, in CBs obtained from rats reared under chronic hypobaric hypoxia (~60 kPa, equivalent to 4,300m) for 5-7 days, in addition to the expected upregulation of tyrosine hydroxylase and VEGF mRNAs, there was a significant upregulation of NTPDase3 and E5'Nt/CD73 mRNA, but a downregulation of NTPDase1 and NTPDase2 relative to normoxic controls. We conclude that NTPDase1,2,3 and E5'Nt/CD73 are the predominant surface-located ectonucleotidases in the rat CB, and suggest that their differential regulation during chronic hypoxia may contribute to CB plasticity via control of synaptic ATP, ADP and adenosine pools.
Angiopoietin-2 (Ang2) contributes to vascular hyporeactivity after hemorrhagic shock and hypoxia through upregulation of inducible nitric oxide synthase (iNOS), in a vascular endothelial cell (VEC)-specific and Ang2/Tie2 receptor-dependent manner. While iNOS is primarily expressed in vascular smooth muscle cells (VSMCs), the mechanisms of signal transfer from VECs to VSMCs are unknown. A double-sided co-culture model with VECs and VSMCs from Sprague Dawley rats was used to investigate the role of myoendothelial gap junctions (MEGJs), the connexin (Cx) isoforms involved and other relevant mechanisms. After hypoxia, VSMCs treated with exogenous Ang2 showed increased iNOS expression and hyporeactivity, as well as MEGJ formation and communication. These Ang2 effects were suppressed by the MEGJ inhibitor, 18α-glycyrrhetic acid (18-GA), Tie2 siRNA or Cx43 siRNA. Reagents antagonizing cAMP or protein kinase A (PKA) in VECs inhibited Cx43 expression in MEGJs, decreasing MEGJ formation and associated communication, after hypoxia following Ang2 treatment. The increased cAMP levels in VSMCs and transfer of Alexa Fluor 488-labeled cAMP from VECs to VSMCs, after hypoxia following Ang2 treatment, was antagonized by Cx43 siRNA. A cAMP antagonist added to VECs or VSMCs inhibited both increased iNOS expression and hyporeactivity in VSMCs subjected to hypoxia following Ang2 treatment. Based on these findings, we propose that Cx43 was the Cx isoform involved in MEGJ-mediated VEC-dependent regulation of Ang2, which induces iNOS protein expression and vascular hyporeactivity after hypoxia. Cx43 was upregulated by cAMP and PKA, permitting cAMP transfer between cells.
Muscle Ankyrin Repeat Proteins (MARPs) are a family of titin-associated stress response molecules and putative transducers of stretch-induced signalling in skeletal muscle. In cardiac muscle, Cardiac Ankyrin Repeat Protein (CARP) and Diabetes-Related Ankyrin Repeat Protein (DARP) reportedly redistribute from binding sites on titin to the nucleus following prolonged stretch. However, it is unclear whether Ankyrin repeat-domain protein2 (Ankrd2) shows comparable stretch-induced redistribution to the nucleus. We measured in rested human skeletal muscle 1) the absolute amount of MARPs, and 2) the distribution of Ankrd2 and DARP in both single fibers and whole muscle preparations. In absolute amounts, Ankrd2 is the most abundant MARP in human skeletal muscle, there being ~3.1 µmol.kg-1, much greater than DARP and CARP (~0.11 and ~0.02 µmol.kg-1, respectively). All DARP was found to be tightly bound at cytoskeletal (or possibly nuclear) sites. In contrast, ~70% of the total Ankrd2 is freely diffusible in the cytosol (including virtually all the phosphorylated p-Ankrd2-Ser99 form), ~15% is bound to non-nuclear membranes and ~15% bound at cytoskeletal sites, likely at the N2A-region of titin. These data are not consistent with the proposal that Ankrd2 per se, or p-Ankrd2-Ser99, mediate stretch-induced signalling in skeletal muscle, dissociating from titin and translocating to the nucleus, because majority of these forms of Ankrd2 are already free in the cytosol. It will be necessary to show that the titin-associated Ankrd2 is modified by stretch in some as yet unidentified way, distinct from the diffusible pool, if it is to act as a stretch-sensitive signalling molecule.
Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5-10 µM) increases excitability in ~60 % of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms which have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels (Ih) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analogue or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability.
Overexpression of sarcolipin (SLN), a regulator of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), stimulates calcineurin signaling to enhance skeletal muscle oxidative capacity. Some studies have shown that calcineurin may also control skeletal muscle mass and remodeling in response to functional overload and unload stimuli by increasing myofiber size and the proportion of slow fibers. To examine whether SLN might mediate these adaptive responses we performed soleus and gastrocnemius tenotomy in wild-type (WT) and Sln¬-null (Sln-/-) mice and examined the overloaded plantaris and unloaded/tenotomized soleus muscles. In the WT overloaded plantaris, we observed ectopic expression of SLN, myofiber hypertrophy, increased fiber number, and a fast-to-slow fiber type shift, which were associated with increased calcineurin signalling (NFAT dephosphorylation and increased stabilin-2 protein content) and reduced SERCA activity. In the WT tenotomized soleus, we observed a 14-fold increase in SLN protein, myofiber atrophy, decreased fiber number, and a slow-to-fast fiber type shift, which were also associated with increased calcineurin signalling and reduced SERCA activity. Genetic deletion of Sln altered these physiological outcomes, with the overloaded plantaris myofibers failing to hypertrophy, increase in fiber number, and transition towards the slow fiber type, while the unloaded soleus muscles exhibited greater myofiber atrophy, reductions in fiber number, and an accelerated slow-to-fast fiber type shift. In both the Sln-/- overloaded and unloaded muscles, these findings were associated with elevated SERCA activity and blunted calcineurin signaling. Thus, SLN plays an important role in adaptive muscle remodeling potentially through calcineurin stimulation, which could have important implications for other muscle diseases and conditions.
Objective Major depression is an important clinical factor in ventricular arrhythmia. Patients diagnosed with major depression overexpress N-methyl-d-aspartate receptors (NMDARs). Previous studies found that chronic NMDAR activation increases susceptibility to ventricular arrhythmias. We aimed to explore the mechanisms by which NMDAR activation may increase susceptibility to ventricular arrhythmias. Methods Male rats were randomly assigned to either normal environments as control (CTL) group or 4weeks of chronic mild stress(CMS) to produce a major depression disorder (MDD) model group. After 4 weeks of CMS, depression-like behaviors were measured in both groups. Varying doses (1-100μM) of NMDA and 10μM NMDA antagonist (MK-801) were perfused through ventricular myocytes isolated from MDD rats to measure the L-type calcium current (ICa-L) and transient outward potassium current(Ito). Structural remodeling was assessed using serial histopathology including Masson's trichrome dye. Electrophysiological characteristics were evaluated using Langendorff perfusion. Results Depression-like behaviors were observed in MDD rats. MDD rats showed longer action potential durations (APD) at 90% repolarization and higher susceptibility to ventricular arrhythmias than CTL rats. MDD rats showed lower ICa-L and Ito current densities than CTL rats. Additionally, NMDA reduced both currents in a concentration-dependent manner, whereas no significant impact on the currents when perfused with MK-801. MDD rats exhibited significantly more fibrosis areas in heart tissue and reduced expression of Kv4.2, Kv4.3, and Cav1.2. Conclusions We observed that acute NMDAR activation led to down-regulation of potassium and L-type calcium currents in a rat model of depression, which may be the mechanism underlying ventricular arrhythmia promotion by depression.
In utero hypoxia influences the structure and function of most fetal arteries, including those of the developing cerebral circulation. Whereas the signals that initiate this hypoxic remodeling remain uncertain, these appear to be distinct from the mechanisms that maintain the remodeled vascular state. The present study explores the hypothesis that chronic hypoxia elicits sustained changes in fetal cerebrovascular reactivity to endothelin-1 (ET-1), a potent vascular contractant and mitogen. In fetal lambs, chronic hypoxia (3820 m altitude for the last 110 days of gestation) had no significant effect on plasma ET-1 levels or ETA receptor density in cerebral arteries but enhanced contractile responses to ET-1 in an ETA-dependent manner. In organ culture (24h), 10 nM ET-1 increased medial thicknesses less in hypoxic than in normoxic arteries, and these increases were ablated by inhibition of PKC (chelerythrine) in both normoxic and hypoxic arteries, but were attenuated by inhibition of CaMKII (KN93) and p38 (SB203580) in normoxic but not hypoxic arteries. As indicated by Ki-67 immunostaining, ET-1 increased medial thicknesses via hypertrophy. Measurements of colocalization between MLCK and SM A revealed that organ culture with ET-1 also promoted contractile dedifferentiation in normoxic, but not hypoxic, arteries through mechanisms attenuated by inhibitors of PKC, CaMKII, and p38. These results support the hypothesis that chronic hypoxia elicits sustained changes in fetal cerebrovascular reactivity to endothelin-1 (ET-1) through pathways dependent upon PKC, CaMKII, and p38 that cause increased ET-1-mediated contractility, decreased ET-1-mediated smooth muscle hypertrophy, and a depressed ability of ET-1 to promote contractile dedifferentiation.
Muscle contraction is commonly associated with the cross-bridge and sliding filament theories, which have received strong support from experiments conducted over the years in different laboratories. However, there are studies that cannot be readily explained by the theories, showing (i) a plateau of the force-length relation extended beyond optimal filament overlap, and forces produced at long sarcomere lengths that are higher than those predicted by the sliding filament theory, (ii) passive forces at long sarcomere lengths that can be modulated by activation and Ca2+, which changes the force-length relation, and (iii) an unexplained high force produced during and after stretch of activated muscle fibers. Some of these studies even propose "new theories of contraction" to explain discrepant results. While some of these observations deserve evaluation, many of these studies present data that lack a rigorous control, and experiments that cannot be repeated in other laboratories. This article reviews these issues, looking into studies that have used intact and permeabilized fibers, myofibrils, isolated sarcomeres and half-sarcomeres. A common mechanism associated with sarcomere and half-sarcomere length non-uniformities, and a Ca2+-induced increase in the stiffness of titin is proposed to explain observations that derive from repeatable studies.
Nanomolar free calcium enhances oxidative phosphorylation. However, the effects over a broad concentration range, at different respiratory states, or on specific energy substrates are less clear. We examined the action of varying [Ca2+] over respiratory states ranging 4 to 3 on skeletal muscle mitochondrial respiration, potential, ATP production, and H2O2 production using ADP recycling to clamp external [ADP]. 450 nM calcium enhanced respiration in mitochondria energized by the complex I substrates, glutamate/malate (but not succinate) at [ADP] of 4-256 µM, but more substantially at intermediate respiratory states and not all at state 4. Using varied [Ca2+], we found that the stimulatory effects on respiration and ATP production were most prominent at nanomolar concentrations, but inhibitory at 10 µM or higher. ATP production decreased more than respiration at 10 µM calcium. However, potential continued to increase up to 10 µM; suggesting a calcium-induced inability to utilize potential for phosphorylation independent of opening of the mitochondrial permeability transition pore (MTP). This effect of 10 µM calcium was confirmed by direct determination of ATP production over a range of potential created by differing substrate concentrations. Consistent with past reports, nanomolar [Ca2+] had a stimulatory effect on utilization of potential for phosphorylation. Increasing [Ca2+] was positively and continuously associated with H2O2 production. In summary, the stimulatory effect of calcium on mitochondrial function is substrate dependent and most prominent over intermediate respiratory states. Calcium stimulates or inhibits utilization of potential for phosphorylation dependent on concentration with inhibition at higher concentration independent of MTP opening.
FXYD5 is a Na+-K+-ATPase regulator, expressed in a variety of normal epithelia. In parallel, it has been found to be associated with several types of cancer and effect lethal outcome by promoting metastasis. However, the molecular mechanism underlying FXYD5 mediated invasion, was has not yet been identified. In this study, using in vivo 4T1 murine breast cancer model, we found that FXYD5 specific shRNA significantly inhibited lung cancer metastasis, without having a substantial effect on primary tumor growth. Our study reveals that FXYD5 participates in multiple stages of metastatic development and exhibits more than one mode of E-cadherin regulation. We provide the first evidence that FXYD5 related morphological changes are mediated through its interaction with Na+-K+-ATPase. Experiments in cultured 4T1 cells have indicated that FXYD5 expression significantly correlates with down-regulation of the β1 isoform of the pump. This behavior could have implications on both transcellular interactions and intracellular events. Further studies suggest that differential localization of the adaptor protein Annexin A2 in FXYD5 expressing cells may correlate with MMP-9 secretion and adhesion changes in 4T1 WT cells.
Dopamine decreases Na-K ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA alpha1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA alpha1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells dopamine decreases NHERF-1 association with NKA alpha1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension (SHRs and aged F344 rats). To test this hypothesis, we isolated and cultured renal PTCs from 22-mo old F344 rats and their controls, normotensive 4-mo old F344 rats, and spontaneously hypertensive rats (SHR) and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K+ transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.
CALHM1 and its C. elegans (ce) homolog, CLHM-1, belong to a new family of physiologically important ion channels that are regulated by voltage and extracellular Ca2+ (Ca2+o), but lack a canonical voltage-sensing domain. Consequently, the intrinsic voltage-dependent gating mechanisms for CALHM channels are unknown. Here, we performed voltage-clamp experiments on ceCLHM-1 chimeric, deletion, insertion and point mutants to assess the role of the N-terminus (NT) in CALHM channel gating. Analyses of chimeric channels in which the ceCLHM-1 and human (h)CALHM1 N-termini were interchanged showed that the hCALHM1 NT destabilized channel-closed states, whereas the ceCLHM-1 NT had a stabilizing effect. In the absence of Ca2+o, deletion of up to eight amino acids from the ceCLHM-1 NT caused a hyperpolarizing shift in the conductance-voltage relationship with little effect on voltage-dependent slope. However, deletion of nine or more amino acids decreased voltage dependence and induced a residual conductance at hyperpolarized voltages. Insertion of amino acids into the N-terminal helix also decreased voltage dependence, but did not prevent channel closure. Mutation of ceCLHM-1 valine 9 and glutamine 13 altered half-maximal activation and voltage dependence in 0-Ca2+, respectively. In 2 mM Ca2+o ceCLHM-1 N-terminal deletion and point mutant channels closed completely at hyperpolarized voltages with apparent affinity for Ca2+o indistinguishable from wild-type ceCLHM-1, although the ceCLHM-1 valine 9 mutant exhibited an altered conductance-voltage relationship and kinetics. We conclude that the NT plays critical roles modulating voltage dependence and stabilizing the closed states of CALHM channels.
Parathyroid hormone (PTH), a pleiotropic hormone that maintains mineral homeostasis, is also essential for controlling pH balance and ion transport across renal and intestinal epithelia. Optimization of luminal pH is important for absorption of trace elements, e.g., calcium and phosphorus. We have previously demonstrated that PTH rapidly stimulated electrogenic HCO3- secretion in intestinal epithelial-like Caco-2 monolayer, but the underlying cellular mechanism, contributions of other ions, particularly Cl- and K+, and long-lasting responses are not completely understood. Herein, PTH and forskolin were confirmed to induce anion secretion, which peaked within 1-3 min (early phase), followed by an abrupt decay and plateau that lasted for 60 min (late phase). In both early and late phases, apical membrane capacitance was increased with a decrease in basolateral capacitance after PTH or forskolin exposure. PTH also induced a transient increase in apical conductance with a long-lasting decrease in basolateral conductance. Anion secretion in both phases was reduced under HCO3--free and/or Cl--free conditions, or after exposure to carbonic anhydrase inhibitor (acetazolamide), CFTR inhibitor (CFTRinh-172), Na+/H+ exchanger (NHE)-3 inhibitor (tenapanor), or K+ channel inhibitors (BaCl2, clotrimazole, and TRAM-34; basolateral side), the latter of which suggested that PTH action was dependent on basolateral K+ recycling. Furthermore, early- and late-phase responses to PTH were diminished by inhibitors of PI3K (wortmannin and LY-294002) and PKA (PKI 14-22). In conclusion, PTH requires NHE3 and basolateral K+ channels to induce HCO3- and Cl- secretion, thus explaining how PTH regulated luminal pH balance and pH-dependent absorption of trace minerals.
We previously demonstrated a role for the myristoylated alanine-rich C kinase substrate (MARCKS) to serve as an adaptor protein in the anionic phospholipid phosphate-dependent regulation of the epithelial sodium channel (ENaC). Both MARCKS and ENaC are regulated by proteolysis. Calpains are a family of ubiquitously expressed intracellular Ca2+-dependent cysteine proteases involved in signal transduction. Here we examine the role of calpain-2 in regulating MARCKS and ENaC in cultured renal epithelial cells and in the mouse kidney. Using recombinant fusion proteins we show MARCKS, but not the ENaC subunits are a substrate of calpain-2 in the presence of Ca2+. Pharmacological inhibition of calpain-2 alters MARCKS protein expression in light density sucrose gradient fractions from cell lysates of mouse cortical collecting duct cells. Calpain-dependent cleaved products of MARCKS are detectable in cultured renal cells. Ca2+ mobilization and calpain-2 inhibition decreases the association between ENaC and MARCKS. The inhibition of calpain-2 reduces ENaC activity as demonstrated by single-channel patch clamp recordings and transepithelial current measurements. These results suggest calpain-2 proteolysis of MARCKS promotes its interaction with lipids and ENaC at the plasma membrane to allow for the PIP2-dependent regulation of ENaC activity in the kidney.
Estrogen plays important roles in regulation of bone formation. Chloride channels in the ClC family are expressed in osteoblasts and are associated with bone physiology and pathology, but the relationship between chloride channels and estrogen is not clear. In this study, the action of estrogen on chloride channels was investigated in osteoblastic MC3T3-E1 cells. Results showed that 17β-estradiol could activate a current which reversed at a potential close to Cl– equilibrium potential with the anion selectivity I- >Br->Cl->gluconate and was inhibited by the chloride channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and knockdown of ClC-3 chloride channel expression. Extracellular application of membrane-impermeable 17β-estradiol-albumin conjugates activated a similar current. The estrogen-activated chloride current could be inhibited by the estrogen receptor antagonist fulvestrant (ICI 182780). The selective ERα agonist, but not ERβ agonist activated a chloride current similar to that induced by 17β-estradiol. Silencing ERα expression prevented activation of estrogen-induced currents. The G protein-coupled estrogen receptor (GPR30) agonist G-1 could not activate chloride channels and the GPR30 antagonist G-15 failed to inhibit estrogen-activated currents. Immunofluorescence and co-immunoprecipitation experiments demonstrated that ClC-3 chloride channels and ERα were co-localized and closely related in cells. Estrogen promoted translocation of ClC-3 and ERα to the cell membrane from the nucleus. In conclusion, it is founded that chloride channels can be activated by estrogen via the ERα receptors located in the cell membrane and suggests that the ClC-3 chloride channel may be one of the action targets of estrogen in regulation of osteoblastic activities.
The key role of the primary cilium in developmental processes is illustrated by ciliopathies resulting from genetic defects of its components. Ciliopathies include a large variety of dysmorphic syndromes that share in common the presence of multiple kidney cysts. These observations suggest that primary cilia may control morphogenetic processes in the developing kidney. In this study, we assessed the role of primary cilium in branching tubulogenesis and/or lumen development using kidney collecting duct-derived mCCDN21 cells that display spontaneous tubulogenic properties when grown in collagen/matrigel matrix. Tubulogenesis and branching were not altered when cilium body growth was inhibited by Kif3A or Ift88 silencing. In agreement with the absence of morphogenetic effect, proliferation and wound-healing assay revealed that neither cell proliferation nor migration were altered by cilium body disruption. The absence of cilium following Kif3A or Ift88 silencing in mCCDN21 cells did not alter the initial stages of tubular lumen generation while lumen maturation and enlargement were delayed. This delay in tubular lumen maturation was not observed after Pkd1 knockdown in mCCDN21 cells. The delayed lumen maturation was explained neither by defective secretion or increased reabsorption of luminal fluid. Our results indicate that primary cilia do not control early morphogenetic processes in renal epithelium. Rather primary cilia modulate tubular lumen maturation and enlargement resulting from luminal fluid accumulation in tubular structures derived from collecting duct cells.
We investigated the effects of S1P3 deficiency on the age-related atrophy, decline of force and regenerative capacity of soleus muscle from 23-month-old male mice. During ageing, in old wild type mice, soleus mass and muscle fiber cross-sectional area (CSA) were reduced by about 26% and 24%, respectively, compared to the adult muscle. By contrast, the mass and fiber CSA of old S1P3-null soleus were comparable to the adult muscle. Moreover, in wild type soleus twitch and tetanic tensions diminished from adult to old age. A slowing of contractile properties was also observed in old wild type soleus. In S1P3-null soleus neither force nor the contractile properties changed during ageing. We also evaluated the regenerative capacity of old S1P3-null soleus by stimulating muscle regeneration through myotoxic injury. After 10 days of regeneration, the mean fiber CSA of old wild type soleus was significantly smaller (- 28%) compared to that of the adult regenerated muscle. On the contrary, the mean fiber CSA of regenerated old S1P3-null soleus was similar to that of the adult muscle. We conclude that in the absence of S1P3 soleus muscle is protected from the decrease of muscle mass and force as well as the attenuation of regenerative capacity, all typical characteristics of ageing.
Aeroallergens produced by Alternaria alternata can elicit life-threatening exacerbations of asthma in patients sensitized to this fungus. In this study, the effect of Alternaria on ion transport mechanisms underlying mucociliary clearance and airway epithelial barrier function was investigated in human airway epithelial cells. Apical exposure to Alternaria induced an increase in anion secretion that was inhibited by blockers of CFTR and Ca2+-activated Cl- channels. Stimulation of anion secretion was dependent on Ca2+ uptake from the apical solution. Alternaria exposure also produced an increase in reactive oxygen species (ROS) that was blocked by pretreatment with the oxidant scavenger glutathione (GSH). GSH and the NADPH oxidase inhibitor/complex 1 electron transport inhibitor diphenylene iodonium chloride (DPI) blocked ATP release and the increase in intracellular [Ca2+] evoked by Alternaria. Alternaria also decreased transepithelial resistance and a portion of this affect was dependent on the increase in ROS. However, the Alternaria-induced increase in unidirectional dextran (mw = 4000 Da) flux across the epithelium could not be accounted for by increased oxidative stress. These results support the conclusion that oxidative stress induced by Alternaria was responsible for regulating Ca2+-dependent anion secretion and tight junction electrical resistance that would be expected to affect mucociliary clearance.
Mesenchymal stem cells (MSCs) have the potential to treat various tissue damages, but the very limited number of cells that migrate to the damaged region strongly restricts their therapeutic applications. Full understanding of mechanisms regulating MSC migration will help to improve their migration ability and therapeutic effects. Increasing evidence shows that microRNAs play important roles in the regulation of MSC migration. In the present study, we reported that miR-9-5p was upregulated in HGF-treated MSCs and in MSCs with high migration ability. Overexpression of miR-9-5p promoted MSC migration, whereas inhibition of endogenous miR-9-5p decreased MSC migration. To elucidate the underlying mechanism, we screened the target genes of miR-9-5p, and reported for the first time that CK1α and GSK3β, two inhibitors of β-catenin signaling pathway, were direct targets of miR-9-5p in MSCs and that overexpression of miR-9-5p upregulated β-catenin signaling pathway. In line with these data, inhibition of β-catenin signaling pathway by FH535 decreased the miR-9-5p-promoted migration of MSCs, while activation of β-catenin signaling pathway by LiCl rescued the impaired migration of MSCs triggered by miR-9-5p inhibitor. Further, the formation and distribution of focal adhesions as well as the reorganization of F-actin were affected by the expression of miR-9-5p. Collectively, these results demonstrate that miR-9-5p promotes MSC migration by upregulating β-catenin signaling pathway, shedding light on the optimization of MSCs for cell replacement therapy through manipulating the expression level of miR-9-5p.
Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl- channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a 4-amino acid PKA activation site, RGET691 of hClC-2 was investigated using WT and mutant (AGET, RGEA and AGAA) hClC-2 expressed in 293EBNA cells as well as involvement of PKA, [cAMP]i, EP2 or EP4 receptor agonist activity. All fatty acids (lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid and elaidic acid) caused significant rightward shifts in concentration-dependent Cl- current activation (increasing EC50s) with mutant compared to WT hClC-2 channels, without changing time- and voltage-dependence, I-V rectification or methadone inhibition of the channel. As with lubiprostone, cobiprostone activation of hClC-2 occurred with PKA inhibitor (myristoylated protein kinase inhibitor) present or when using double PKA activation site (RRAA655/RGEA691) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP]i in hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells cobiprostone increased hClC-2 Cl- currents without increasing [cAMP]i, while PGE2 and forskolin-IBMX increased both. Fatty acids were not agonists of EP2 or EP4 receptors. L-161,982, a supposed EP4 selective inhibitor, had no effect on lubiprostone activated hClC-2 Cl- currents, but significantly decreased T84 cell barrier function measured by TER and fluorescent dextran transepithelial movement. The present findings show that RGET691 of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP]i and EP2 or EP4 receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2.
Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) and mitochondria play a critical role in growth factor-induced switch from a quiescent to an angiogenic phenotype in endothelial cells (ECs). However, how highly diffusible ROS produced from different sources can coordinate to stimulate VEGF signaling and drive the angiogenic process remains unknown. Using the cytosol- and mitochondria-targeted redox-sensitive RoGFP biosensors with real-time imaging, here we show that VEGF stimulation in human ECs rapidly increases cytosolic RoGFP oxidation within 1 min, followed by mitochondrial RoGFP oxidation within 5 min, which continues at least for 60 min. Silencing of Nox4 or Nox2, or overexpression of mitochondria-targeted catalase significantly inhibits VEGF-induced tyrosine phosphorylation of VEGF receptor type2 (VEGFR2-pY), EC migration and proliferation at the similar extent. Exogenous H2O2 or overexpression of Nox4 that produces H2O2, increases mitochondrial ROS (mtROS), which is prevented by Nox2 siRNA, suggesting that Nox2 senses Nox4-derived H2O2 to promote mtROS production. Mechanistically, H2O2 increases S36 phosphorylation of p66Shc, a key mtROS regulator, which is inhibited by siNox2, but not by siNox4. Moreover, Nox2 or Nox4 knockdown, or overexpression of S36 phosphorylation-defective mutant p66Shc(S36A) inhibits VEGF-induced mtROS, VEGFR2-pY, EC migration and proliferation. In summary, Nox4-derived H2O2 in part activates Nox2 to increase mtROS via pSer36-p66Shc, thereby enhancing VEGFR2 signaling and angiogenesis in ECs. This may represent a novel feed-forward mechanism of ROS-induced ROS release orchestrated by the Nox4/Nox2/pSer36-p66Shc/mtROS axis, which drives sustained activation of angiogenesis signaling program.
Platelet adhesion, activation and aggregation are essential for primary haemostasis, but are also critically involved in the development of acute arterial thrombotic occlusion. Stimulation of the collagen receptor glyocoprotein VI (GPVI) leads to PLC-dependent IP3 production with subsequent platelet activation, due to increased intracellular calcium concentration ([Ca2+]i). Although it has been described that tricyclic antidepressants potentially impair platelet activation, nothing is hitherto known about potential effects of the tricyclic antidepressant doxepin on platelet Ca2+ signaling and thrombus formation. As shown in the present study doxepin significantly diminished the stimulatory effect of GPVI agonist collagen-related peptide (CRP) on intracellular Ca2+ release as well as subsequent extracellular Ca2+ influx. Doxepin was partially effective by impairment of CRP-dependent inositol triphosphate (IP3) production. Moreover, doxepin abrogated CRP-induced platelet degranulation, integrin αIIbβ3 activation and aggregation. Finally, doxepin markedly blunted in vitro platelet adhesion to collagen and thrombus formation under high arterial shear rates (1700-sec). In conclusion, doxepin is a powerful inhibitor of GPVI-dependent platelet Ca2+ signaling, platelet activation and thrombus formation.
Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.
Melamine causes renal tubular cell injury through inflammation, fibrosis and apoptosis. While melamine effects the rise in intracellular Ca2+ concentration ([Ca2+]i), reactive oxygen species (ROS) production, and pro-apoptotic pathway activation, the mechanism of upstream Ca2+ signaling is unknown. Since melamine has some structural similarities with L-amino acids, which endogenously activates Ca2+-sensing receptors (CSR), we examined the effect of melamine on CSR-induced Ca2+ signaling and apoptotic cell death. We show here that melamine activates CSR, causing a sustained Ca2+ entry in renal epithelial cell line, LLC-PK1. Moreover, such CSR stimulation resulted a rise in [Ca2+]i, leading to enhanced ROS production. Furthermore, melamine-induced elevated [Ca2+]i and ROS production caused a dose dependent increase in apoptotic (by DAPI staining, DNA laddering, Annexin V assay) and necrotic (propidium iodide staining) cell death. Upon examining the downstream mechanism, we found that transforming growth factor β1 (TGFβ1), which increases extracellular matrix genes and pro-apoptotic signaling were also up-regulated at lower doses of melamine, which could be due to an early event inducing apoptosis. Additionally, cells exposed to melamine displayed a rise in phospho-extracellular signal-regulated kinase (pERK) activation and lactate dehydrogenase (LDH) release resulting in cytotoxicity. These results offer a novel insight into the molecular mechanisms by which melamine exerts its effect on CSR, causing a sustained elevation of [Ca2+]i, leading to ROS generation, fibronectin production, pro-apoptotic pathway activation and renal cell damage. Together, these results thus suggest that melamine-induced apoptosis and/or necrosis may subsequently results in acute kidney injury (AKI) and promote kidney stone formation.
Cells respond to environmental stress in multiple ways. In the germ line, heat shock and nutritive stress trigger the assembly of large ribonucleoprotein (RNP) granules via liquid-liquid phase separation (LLPS). The RNP granules are hypothesized to maintain the quality of oocytes during stress. The goal of this study was to investigate the cellular response to glucose in the germ line and determine if it is an osmotic stress response. We found that exposure to 500 mM glucose induces the assembly of RNP granules in the germ line within one hour. Interestingly, the RNP granules are maintained for up to three hours; however, they dissociate after longer periods of stress. The RNP granules include P body and stress granule proteins, suggesting shared functions. Based on several lines of evidence, the germ line response to glucose largely appears to be an osmotic stress response, thus identifying osmotic stress as a trigger of LLPS. Although RNP granules are not maintained beyond three hours of osmotic stress, the quality of oocytes does not appear to decrease after longer periods of stress, suggesting a secondary adaptation in the germ line. We used an indirect marker of glycerol and observed high levels after five and twenty hours of glucose exposure. Moreover, in gpdh-1;gpdh-2 germ lines glycerol levels are reduced concomitant with RNP granules being maintained for an extended period. We speculate that increased glycerol levels may function as a secondary osmoregulatory adaptive response in the germ line, following a primary response of RNP granule assembly.
Proximal tubule (PT) dysfunction, including tubular proteinuria, is a significant complication in young sickle cell disease (SCD) that can eventually lead to chronic kidney disease. Hemoglobin (Hb) dimers released from red blood cells upon hemolysis are filtered into the kidney and internalized by megalin/cubilin receptors into PT cells. The PT is especially sensitive to heme toxicity, and tubular dysfunction in SCD is thought to result from prolonged exposure to filtered Hb. Here we show that concentrations of Hb predicted to enter the tubule lumen during hemolytic crisis competitively inhibit the uptake of another megalin/cubilin ligand (albumin) by proximal tubule (PT) cells. These effects were independent of heme reduction state. The Glu7Val mutant of Hb that causes SCD was equally effective at inhibiting albumin uptake compared with wild type Hb. Addition of the Hb scavenger haptoglobin (Hpt) restored albumin uptake in the presence of Hb, suggesting that Hpt binding to the Hb αβ dimer-dimer interface interferes with Hb binding to megalin/cubilin. BLAST searches and structural modeling analyses revealed regions of similarity between Hb and albumin that map to this region and may represent sites of Hb interaction with megalin/cubilin. Our studies suggest that impaired endocytosis of megalin/cubilin ligands, rather than heme toxicity, may be the cause of tubular proteinuria in SCD patients. Additionally, loss of these filtered proteins into the urine may contribute to the extra-renal pathogenesis of SCD.
Calcific aortic valve disease (CAVD) is a leading cardiovascular disorder in the elderly. Diseased aortic valves are characterized by sclerosis (fibrosis) and nodular calcification. Sclerosis, an early pathological change, is caused by aortic valve interstitial cell (AVIC) proliferation and over-production of extracellular matrix (ECM) proteins. However, the mechanism of aortic valve sclerosis remains unclear. Recently, we observed that diseased human aortic valves over-express growth factor neurotrophin 3 (NT3). In the present study, we tested the hypothesis that NT3 is a pro-fibrogenic factor to human AVICs. Methods and Results: AVICs isolated from normal human aortic valves were cultured in M199 growth medium and treated with recombinant human NT3 (0.10 µg/ml). An exposure to NT3 induced AVIC proliferation, up-regulated the production of collagen, MMP-9 and augmented collagen deposition. These changes were abolished by inhibition of the Trk receptors. NT3 induced Akt phosphorylation and increased cyclin D1 protein levels in a Trk receptor-dependent fashion. Inhibition of Akt abrogated the effect of NT3 on cyclin D1 production. Further, inhibition of either Akt or cyclin D1 suppressed NT3-induced cellular proliferation, MMP-9 and collagen production, as well as collagen deposition. Conclusions: NT3 up-regulates cellular proliferation, ECM protein production and collagen deposition in human AVICs. It exerts these effects through the Trk-Akt-cyclin D1 cascade. Thus, NT3 is a pro-fibrogenic mediator in human aortic valve, and over-production of NT3 by aortic valve tissue may contribute to the mechanism of valvular sclerosis.
Adrenal gland is a crucial endocrine gland, and the most important function is to synthesize and secrete catecholamines (CATs) which play crucial roles in balancing homeostasis and the responding to stress. microRNA-375 (miR-375) has been detected to highly express in the adrenal, however its role and underlying mechanism are currently unclear. Herein, our results showed that miR-375 was specifically localized to the rat adrenal medulla chromaffin cells, and miR-375 expressing level decreased, when the rats were exposed to stress. The further functional studies demonstrated that the inhibition of endogenous miR-375 induced the secretion of CATs in primary rat medulla chromaffin cells and in PC12 cells, and over-expression of miR-375 resulted in decline of the CATs secretion. Furthermore, the results showed that miR-375 negatively regulated tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) and mediated adrenomedullary CATs biosynthesis. Sp1(a transcriptional activator of TH and DBH) was involved in mediating the regulation of TH and DBH as miR-375 direct target gene. These novel findings suggest that miR-375 acts as a potent negative mediator in regulating the synthesis and secretion of CATs in the adrenal medulla during the maintenance of homeostasis under stress.
Autophagy is a conserved cellular process for degrading aggregate proteins and dysfunctional organelle. It is still debatable if autophagy and mitophagy (a specific process of autophagy of mitochondria) play important roles in myogenic differentiation and functional regeneration of skeletal muscle. We tested the hypothesis that autophagy is critical for functional regeneration of skeletal muscle. We first observed time-dependent increases (3-6 fold) of autophagy-related proteins (Atgs), including Ulk1, Beclin1 and LC3, along with reduced p62 expression during C2C12 differentiation, suggesting increased autophagy capacity and flux during myogenic differentiation. We then used cardiotoxin (CTX) or ischemia-reperfusion (IR) to induce muscle injury and regeneration and observed increases in Atgs between days 2-7 in adult skeletal muscle followed by increased autophagy flux after day 7. Since Ulk1 has been shown to be essential for mitophagy, we asked if Ulk1 is critical for functional regeneration in skeletal muscle. We subjected skeletal muscle-specific Ulk1 knockout mice (MKO) to CTX or IR. MKO mice had significantly impaired recovery of muscle strength and mitochondrial protein content post-CTX or IR. Imaging analysis showed that MKO mice have significantly attenuated recovery of mitochondrial network at 7 and 14 days post-CTX. These findings suggest that increased autophagy protein and flux occur during muscle regeneration, and Ulk1-mediated mitophagy is critical for recovery for mitochondrial network and hence functional regeneration.
The choroid plexus epithelium is a secretory epithelium par excellence. However, this is perhaps not the most prominent reason for the massive interest in this modest-sized tissue residing inside the brain ventricles. Most likely, the dominant reason for extensive studies of the choroid plexus is the identification of this epithelium as the source of the majority of intraventricular cerebrospinal fluid. This finding has direct relevance for studies of diseases and conditions with deranged central fluid volume or ionic balance. While the concept is supported by the vast majority of literature, the implication of the choroid plexus in secretion of the cerebrospinal fluid was recently challenged once again. Three newer and promising areas of current choroid plexus related investigations are: 1) the choroid plexus epithelium as the source of mediators necessary for the central nervous system development, 2) the choroid plexus as a route for microorganisms and immune cells into the central nervous system, and 3) the choroid plexus as a potential route for drug delivery into the central nervous system bypassing the blood brain barrier. Thus, the purpose of this review is to highlight current active areas of research in the choroid plexus physiology and a few matters of continuous controversy.
Mutations in the gene that encodes the principal L-Carnitine transporter, OCTN2, can lead to a reduced intracellular L-Carnitine pool and the disease Primary Carnitine Deficiency. L-Carnitine supplementation is used therapeutically to increase intracellular L-Carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake we hypothesised that AMPK activating compounds and insulin would increase L-Carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase L-Carnitine uptake at 100nM. However, L-Carnitine uptake was modestly increased at a dose of 150nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10mM, 5mM, 1mM, 0.5mM), A23187 (10μM)], inhibit mitochondrial function [Sodium Azide (75μM), Rotenone (1μM), Berberine (100μM), DNP (500μM)] or directly activate AMPK [AICAR (250μM)] were assessed for their ability to regulate L-Carnitine uptake. All compounds tested significantly inhibited L-Carnitine uptake. Inhibition by caffeine was not dantrolene (10μM) sensitive. Saturation curve analysis suggested that caffeine did not competitively inhibit L-Carnitine transport. However, the AMPK inhibitor Compound C (10μM) partially rescued the effect of caffeine suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits L-Carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role.
Artificially generated pancreatic β-cells from pluripotent stem cells are expected for cell replacement therapy for type 1 diabetes. Several strategies are adopted to direct pluripotent stem cells toward pancreatic differentiation. However, a standard differentiation method for clinical application has not been established. It is important to develop more effective and safer methods for generating pancreatic β-cells without toxic or mutagenic chemicals. In the present study, we screened several endogenous factors involved in organ development to identify the factor, which induced the efficiency of pancreatic differentiation and found that treatment with erythropoietin (EPO) facilitated the differentiation of mouse embryonic stem cells (ESCs) into definitive endoderm. At an early stage of differentiation, EPO treatment significantly increased Sox17 gene expression, as a marker of the definitive endoderm. Contrary to the canonical function of EPO, it did not affect the levels of phosphorylated JAK2 and STAT5, but stimulated the phosphorylation of ERK1/2 and AKT. The MEK inhibitor U0126 significantly inhibited EPO-induced Sox17 expression. The differentiation of ESCs into definitive endoderm is an important step for the differentiation into pancreatic and other endodermal lineages. This study suggests a possible role of EPO in embryonic endodermal development and a new agent for directing the differentiation into endodermal lineages like pancreatic β-cells.
The α7β1 integrin is concentrated at the costameres of skeletal muscle and provides a critical link between the actin cytoskeleton and laminin in the basement membrane. We previously have demonstrated that expression of the α7BX2 integrin subunit (MCK:α7BX2) preserves muscle integrity and enhances myofiber cross sectional area following eccentric exercise. The purpose of this study was to utilize gene expression profiling to reveal potential mechanisms by which the α7BX2 integrin contributes to improvements in muscle growth post-exercise. A microarray analysis was performed using RNA extracted from skeletal muscle of wild type or transgenic mice, under sedentary conditions and 3 hrs following an acute bout of downhill running. Genes with FDR p-values below the cutoff of 0.05 (n=73) were found to be regulated by either exercise or transgene expression. KEGG pathway analysis detected upregulation of genes involved in ER protein processing with integrin overexpression. Targeted analyses verified increased transcription of Rpl13a, Nosip, Ang, Scl7a5, Gys1, Ndrg2, Hspa5, and Hsp40 as a result of integrin overexpression, alone or in combination with exercise (P<0.05). A significant increase in HSPa5 and a decrease in CHOP protein was detected in transgenic muscle (P < 0.05). In vitro knockdown experiments verified integrin-mediated regulation of Scl7a5. The results from this study suggest that the α7β1 integrin initiates transcription of genes that allow for protection from stress, including activation of a beneficial unfolded protein response, and modulation of protein synthesis, both which may contribute to positive adaptations in skeletal muscle as a result of engagement in eccentric exercise.
Reactive oxygen species (ROS) play a profound role in cardiorespiratory function under normal physiological conditions and disease states. ROS can influence neuronal activity by altering various ion channels and transporters. Within the nucleus tractus solitarii (nTS), a vital brainstem area for cardiorespiratory control, hydrogen peroxide (H2O2) induces sustained hyperexcitability following an initial depression of neuronal activity. The mechanism(s) associated with the delayed hyperexcitability are unknown. Here we evaluate the effect(s) of H2O2 on cytosolic Ca++ (via Fura-2 imaging) and voltage-dependent calcium currents in dissociated rat nTS neurons. H2O2 perfusion (200 µM; 1 min) induced a delayed, slow, and moderate increase (~27%) in intracellular Ca++ ([Ca++]i). The H2O2-mediated increase in [Ca++]i prevailed during Thapsigargin, excluding the endoplasmic reticulum as a Ca++ source. The effect, however, was abolished by removal of extracellular Ca++ or the addition of cadmium to the bath solution, suggesting voltage-gated Ca++ channels (VGCCs) as targets for H2O2 modulation. Recording of the total voltage-dependent Ca++ current confirmed H2O2 enhanced Ca++ entry. Blocking VGCC L-, N-, and P/Q-subtype decreased the number of cells and their calcium currents that respond to H2O2. The number of responder cells to H2O2 also decreased in the presence of Dithiothreitol, suggesting the actions of H2O2 were dependent on sulfhydryl-oxidation. In summary, here, we have shown that H2O2 increases [Ca++]i and their Ca++ currents, which is dependent on multiple VGCCs likely by oxidation of sulfhydryl groups. These processes presumably contribute to the previously observed delayed hyperexcitability of nTS neurons in in vitro brainstem slices.
Mitochondria are comprised of both nuclear- and mitochondrially-encoded proteins requiring precise stoichiometry for their integration into functional complexes. The augmented protein synthesis associated with mitochondrial biogenesis results in the accumulation of unfolded proteins, thus triggering cellular stress. As such, the unfolded protein responses emanating from the endoplasmic reticulum (UPRER) or the mitochondrion (UPRMT) are triggered to ensure correct protein handling. Whether this response is necessary for mitochondrial adaptations is unknown. Two models of mitochondrial biogenesis were used: muscle differentiation and chronic contractile activity (CCA) in murine muscle cells. After 4 days of differentiation, our findings depict selective activation of the UPRMT in which chaperones decreased, however Sirt3 and UPRER markers were elevated. To delineate the role of ER stress in mitochondrial adaptations, the ER stress inhibitor TUDCA was administered. Surprisingly, mitochondrial markers COX-I, COX-IV, and PGC-1α protein levels were augmented up to 1.5-fold above that of vehicle-treated cells. Similar results were obtained in myotubes undergoing CCA in which biogenesis was enhanced by ~2-3-fold, along with elevated UPRMT markers Sirt3 and CPN10. To verify whether the findings were attributable to the terminal UPRER branch directed by the transcription factor CHOP, cells were transfected with CHOP siRNA. Basally, COX-I levels increased (~20%) and COX-IV decreased (~30%), suggesting that CHOP influences mitochondrial composition. This effect was fully restored by CCA. Therefore, our results suggest that mitochondrial biogenesis is independent of the terminal UPRER. Under basal conditions CHOP is required for the maintenance of mitochondrial composition, but not for differentiation- or CCA-induced mitochondrial biogenesis.
Marked loss of skeletal muscle mass occurs under various conditions of disuse, but the molecular and cellular mechanisms leading to atrophy are not completely understood. We investigate early molecular events which might play a role in skeletal muscle remodeling during mechanical unloading (disuse). The effects of acute (6 - 12 h) hindlimb suspension on the soleus muscles from adult rats were examined. The integrity of plasma membrane lipid rafts was tested utilizing cholera toxin B subunit, or fluorescent sterols. In addition, resting intracellular Ca2+ level was analyzed. Acute disuse disturbed the plasma membrane lipid-ordered phase throughout the sarcolemma and was more pronounced in junctional membrane regions. Ouabain (1 µM), which specifically inhibits the Na,K-ATPase α2 isozyme in rodent skeletal muscles, produced similar lipid rafts changes in control muscles, but was ineffective in suspended muscles, which show an initial loss of α2 Na,K-ATPase activity. Lipid rafts were able to recover with cholesterol supplementation, suggesting that disturbance results from cholesterol loss. Repetitive nerve stimulation also restores lipid rafts, specifically in junctional sarcolemma region. Disuse locally lowered the resting intracellular Ca2+ concentration only near the neuromuscular junction of muscle fibers. Our results provide the evidence to suggest that the ordering of lipid rafts strongly depends on motor nerve input and may involve interactions with the α2 Na,K-ATPase. Lipid rafts disturbance, accompanied by intracellular Ca2+ dysregulation are among the earliest remodeling events induced by skeletal muscle disuse.
Scleroderma is a multisystem fibroproliferative disease with no effective medical treatment. Myofibroblasts are critical to the fibrogenic tissue repair process in the skin and many internal organs. Emerging data support a role for both matrix stiffness, and transforming growth factor β1 (TGFβ1), in myofibroblast differentiation. Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive ion channel activated by both mechanical and biochemical stimuli. The objective of this study was to determine the role of TRPV4 in TGFβ1- and matrix stiffness-induced differentiation of dermal fibroblasts. We found that TRPV4 channels are expressed and functional in both human (HDF) and mouse (MDF) dermal fibroblasts. TRPV4 activity (agonist-induced Ca2+ influx) was induced by both matrix stiffness and TGFβ1 in dermal fibroblasts. TGFβ1 induced expression of TRPV4 proteins in a dose-dependent manner. Genetic ablation or pharmacologic antagonism of TRPV4 channel abrogated Ca2+ influx and both TGFβ1-induced and matrix stiffness-induced myofibroblast differentiation as assessed by i) α-smooth muscle actin expression/incorporation into stress fibers, ii) generation of polymerized actin, and iii) expression of collagen-1. We found that TRPV4 inhibition abrogated TGFβ1-induced activation of AKT but not of Smad2/3, suggesting that the mechanism by which profibrotic TGFβ1 signaling in dermal fibroblasts is modified by TRPV4 may be through non-Smad pathways. Altogether, these data identify a novel reciprocal functional link between TRPV4 activation and TGFβ1 signals regulating dermal myofibroblast differentiation. These findings suggest that therapeutic inhibition of TRPV4 activity may provide a targeted approach to the treatment of scleroderma.
Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as Type 2 Diabetes and atherosclerosis) has shifted the focus from high-fat high-cholesterol containing western type diet (WD)-induced changes in gut microbiota per se to release of gut bacteria-derived products (e.g., LPS) into circulation due to intestinal barrier dysfunction as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. We demonstrated earlier that oral supplementation with curcumin attenuates WD-induced development of Type-2 diabetes and atherosclerosis. Poor bioavailability of curcumin has precluded the establishment of a causal relationship between oral supplementation and it's in vivo effects. We hypothesized that curcumin attenuates WD-induced chronic inflammation and associated metabolic diseases by modulating the function of intestinal epithelial cells (IECs) and the intestinal barrier function. The objective of the present study was to delineate the underlying mechanisms. Human IEC lines, Caco-2 and HT-29 were used for these studies and modulation of direct as well as indirect effects of LPS on intracellular signaling as well as tight junctions were examined. Pre-treatment with Curcumin significantly attenuated LPS-induced secretion of master cytokine IL-1 from IECs and macrophages. Furthermore, curcumin also reduced IL1-induced activation of p-38 MAPK in IECs and subsequent increase in expression of Myosin Light Chain Kinase involved in the phosphorylation of tight junction proteins and ensuing disruption of their normal arrangement. The major site of action of curcumin is, therefore, likely the IECs and the intestinal barrier and by reducing intestinal barrier dysfunction, curcumin modulates chronic inflammatory diseases despite poor bioavailability.
Silicon (Si) has long been known to play a major physiological and structural role in certain organisms, including diatoms, sponges, and many higher plants, leading to the recent identification of multiple proteins responsible for Si transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding about the biochemical pathways that enable Si homeostasis. Here we report the identification of a mammalian efflux Si transporter, namely Slc34a2 (also termed NaPiIIb) a known sodium-phosphate co-transporter, which was upregulated in rat kidney following chronic dietary Si deprivation. Normal rat renal epithelium demonstrated punctate expression of Slc34a2 and when the protein was heterologously expressed in Xenopus laevis oocytes, Si efflux activity (i.e. movement of Si out of cells) was induced and was quantitatively similar to that induced by the known plant Si transporter OsLsi2 in the same expression system. Interestingly, Si efflux appeared saturable over time, but it did not vary as a function of extracellular HPO42- or Na+ concentration, suggesting that Slc34a2 harbors a functionally independent transport site for Si operating in the reverse direction to the site for phosphate. Indeed, in rats with dietary Si depletion-induced upregulation of transporter expression, there was increased urinary phosphate excretion. This is the first evidence of an active Si transport protein in mammals and points towards an important role for Si in vertebrates and explains interactions between dietary phosphate and silicon.
Mechanochemical signal transduction occurs when mechanical forces, such as fluid shear stress, are converted into biochemical responses within the cell. The molecular mechanisms by which endothelial cells (ECs) sense/transduce shear stress into biological signals, including the nature of the mechanosensor, are still unclear. G proteins and G protein-coupled receptors (GPCRs) have been postulated independently to mediate mechanotransduction. In this study, we used in situ proximity ligation assay (PLA) to investigate the role of a specific GPCR/Gαq/11 pair in EC shear stress-induced mechanotransduction. We demonstrated that S1P stimulation causes a rapid dissociation at 0.5 min of Gαq/11 from its receptor, S1P3, followed by an increased association within 2 min of GRK2 and β-arrestin 1/2 with S1P3 in human coronary artery ECs, which are consistent with GPCR/Gαq/11 activation and receptor desensitization/internalization. The G protein activator, AlF4-, resulted in increased dissociation of Gαq/11 from S1P3, but no increase in association between S1P3 and either GRK2 or β-arrestin 1/2. The G protein inhibitor, GDP-β-S, and the S1P3 antagonist, VPC23019, both prevented S1P-induced activation. Shear stress also caused the rapid activation within 7 sec of S1P3/Gαq/11. There were no increased associations between S1P3 and GRK2 or S1P3 and β-arrestin 1/2 until 5 min. GDP-β-S, but not VPC23019, prevented dissociation of Gαq/11 from S1P3 in response to shear stress. Shear stress did not induce rapid dephosphorylation of β-arrestin 1 nor rapid internalization of S1P3, indicating no GPCR activation. These findings suggest that Gαq/11 participates in the sensing/transducing of shear stress independent of GPCR activation in ECs.
Upon activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42 and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. While RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP)VI-specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.
Little is known regarding the role of the Suppressor of Cytokine Signaling (SOCS) in the control of cytokine signaling in cardiomyocytes. We investigated the consequences of SOCS2 ablation for Leukemia Inhibitory Factor (LIF)-induced enhancement of intracellular Ca2+ ([Ca2+]i) transient by performing experiments with cardiomyocytes from SOCS2-knockout (ko) mice. Similar levels of SOCS3 transcripts were seen in cardiomyocytes from wild-type and SOCS2-ko, while SOCS1 mRNA was reduced in SOCS2-ko. Immunoprecipitation experiments showed increased SOCS3 association with gp130 receptor in SOCS2-ko myocytes. Measurements of Ca2+ in wild-type myocytes exposed to LIF showed a significant increase in the magnitude of the Ca2+ transient. These changes were absent in LIF-treated SOCS2-ko cells. LIF activation of ERK and STAT3 was observed in both wild-type and SOCS2-ko cells, indicating that in SOCS2-ko LIF receptors were functional, despite the lack of effect in the Ca2+ transient. In wild-type cells, LIF-induced increase in [Ca2+]i and phospholamban (PLN)(Thr17) phosphorylation was inhibited by KN93, indicating a role for CaMKII in LIF-induced Ca2+ raise. LIF-induced phosphorylation of PLN(Thr17) was abrogated in SOCS2-ko myocytes. In wild-type cardiomyocytes, LIF treatment increased L-type Ca2+ current (ICa,L), a key activator of CaMKII in response to LIF. Conversely, SOCS2-ko myocytes failed to activate ICa,L in response to LIF, providing a rational for the lack of LIF effect on Ca2+ transient. Our data show that absence of SOCS2 turns cardiomyocytes unresponsive to LIF-induced [Ca2+] raise, indicating that endogenous levels of SOCS2 are crucial for full activation of LIF signaling in the heart.
Highly tumorigenic cancer stem cells (CSCs) residing in most cancers are responsible for cancer progression and treatment failure. Zinc is an element regulator of several cell functions; however, its role in regulation of stem cell program in lung cancer has not been demonstrated. The present study reveals for the first time that zinc can suppress stem cell properties of lung cancer cells. Such findings were proved in different lung cancer cell lines (H460, H23, and H292) and it was found that CSC markers (CD133 and ALDH1A1), stem cell-associated transcription factors (Oct4, Nanog, and Sox-2), and the ability to form tumor spheroid were dramatically suppressed by zinc treatments. Zinc was found to activate Protein Kinase C-alpha (PKCα) that further phosphorylated and mediated β-catenin degradation through the ubiquitin-proteasomal pathway. Zinc was found to increase the β-catenin-ubiquitin complex which can be inhibited by a specific PKC inhibitor, bisindolylmaleimide I. Using specific ROS detection and antioxidants, we have demonstrated that superoxide anions generated by zinc are a key up-stream mechanism for PKCα activation leading to the subsequent suppression of stem cell features of lung cancer. Zinc increased cellular superoxide anions and the addition of superoxide anion scavenger prevented the activation of PKCα and β-catenin degradation. These findings indicate a novel role for zinc regulation in the PKCα/β-catenin pathway and explain an important mechanism for controlling of stem cell program in lung cancer cells.
The β1- and β2-adrenergic signaling systems play different roles in the functioning of cardiac cells. Experimental data shows that the activation of β1-adrenergic signaling system produces significant inotropic, lusitropic, and chronotropic effects in the heart, while the effects of the β2-adrenergic signaling system is less apparent. In this paper, a comprehensive compartmentalized experimentally-based mathematical model of the combined β1- and β2-adrenergic signaling systems in mouse ventricular myocytes is developed to simulate the experimental findings and make testable predictions of the behavior of the cardiac cells under different physiological conditions. Simulations describe the dynamics of major signaling molecules in different subcellular compartments; kinetics and magnitudes of phosphorylation of ion channels, transporters, and Ca2+ handling proteins; modifications of action potential shape and duration; and [Ca2+]i and [Na+]i dynamics upon stimulation of β1- and β2-adrenergic receptors (β1- and β2-ARs). The model reveals physiological conditions when β2-ARs do not produce significant physiological effects and when their effects can be measured experimentally. Simulations demonstrated that stimulation of β2-ARs with isoproterenol caused a marked increase in the magnitude of the L-type Ca2+ current, [Ca2+]i transient, and phosphorylation of phospholamban only upon additional application of pertussis toxin or inhibition of phosphodiesterases of type 3 and 4. The model also made testable predictions of the changes in magnitudes of [Ca2+]i and [Na+]i fluxes, the rate of decay of [Na+]i concentration upon both combined and separate stimulation of β1- and β2-ARs, and the contribution of phosphorylation of PKA targets to the changes in the action potential and [Ca2+]i transient.
Connexins (Cxs) are a group of integral membrane proteins which can form gap junctions between adjacent cells. Recently, it was reported that Cx43 is expressed not only in the plasma membrane, but also in the inner mitochondrial membrane, and that it regulates mitochondrial functions. Cx40 is predominantly expressed in vascular endothelial cells (ECs) and plays an important role in the electrical propagation between ECs and endothelial/smooth muscle cells. However, it is unknown whether Cx40 is expressed in the mitochondria and what the role of mitochondrial Cx40 is in endothelial functions. We observed in coronary ECs that Cx40 protein was expressed in the mitochondria, as determined by Western blot and immunofluorescence studies. We found that mouse coronary ECs (MCECs) isolated from Cx40 knockout (Cx40 KO) mice exhibited significantly lower resting mitochondrial calcium concentration ([Ca2+]mito) than MCECs from wild-type (Wt) mice. After increasing the cytosolic Ca2+ concentration ([Ca2+]cyto) using cyclopiazonic acid, calcium uptake into the mitochondria was significantly attenuated in MCECs from Cx40 KO mice compared to Wt MCECs. There was no difference in the resting [Ca2+]cyto and store-operated calcium entry in MCECs from Wt and Cx40 KO mice. We also detected a significant decrease in the concentration of mitochondrial reactive oxygen species (ROS) in Cx40 KO MCECs. Cx40 overexpression in ECs significantly increased resting [Ca2+]mito level and calcium uptake by mitochondria in response to increased [Ca2+]cyto and augmented mitochondrial ROS production. These data suggest that mitochondrial Cx40 contributes to the regulation of mitochondrial calcium homeostasis.
Communication between vascular smooth muscle cells (VSMCs) is dependent on gap junctions and is regulated by the Na,K-ATPase. The Na,K-ATPase is therefore important for synchronized VSMCs oscillatory activity, i.e. vasomotion. The signaling between the Na,K-ATPase and gap junctions is unknown. We tested here the hypothesis that that this signaling involves cSrc kinase. Intercellular communication was assessed by membrane capacitance measurements of electrically coupled VSMCs. Vasomotion in isometric myograph, input resistance and synchronized [Ca2+]i transients were used as readout for intercellular coupling in rat mesenteric small arteries in vitro. Phosphorylation of cSrc kinase and connexin43 (Cx43) were semi-quantified by Western blotting. Micromole concentration of ouabain reduced the amplitude of norepinephrine-induced vasomotion and desynchronized Ca2+ transients in VSMC in the arterial wall. Ouabain also increased input resistance in the arterial wall. These effects of ouabain were antagonized by inhibition of tyrosine phosphorylation with genistein, PP2 and by an inhibitor of the Na,K-ATPase-dependent cSrc activation, pNaKtide. Moreover, inhibition of cSrc phosphorylation increased vasomotion amplitude, and decreased the resistance between cells in the vascular wall. Ouabain inhibited the electrical coupling between A7r5 cells, but pNaKtide restored the electrical coupling. Ouabain increased cSrc autophosphorylation of tyrosine 418 (Y418) required for full catalytic activity while pNaKtide antagonized it. This cSrc activation was associated with Cx43 phosphorylation of tyrosine 265 (Y265). Our findings demonstrate that Na,K-ATPase regulates intercellular communication in the vascular wall via cSrc-dependent Cx43 tyrosine phosphorylation.
The substituted cysteine accessibility method (SCAM) is widely used to study the structure and function of channels, receptors and transporters. In its usual application, a cysteine residue is introduced into a protein which lacks native cysteines following which the accessibility of the residue to the aqueous compartment is assessed. Implicit, and generally assumed, is that if the cysteine-substituted residue is not available to react with sulfhydryl reagents it is not exposed to the extracellular compartment or within the aqueous translocation pathway. We demonstrate here, in a Hela-derived cell line, that some cysteine-substituted residues of the proton-coupled folate transporter (PCFT, SLC46A) that are inaccessible to 2-((biotinoyl)amino)ethyl methanethiosulfonate are glutathionylated by biotinylated glutathione ethyl ester in the absence of an oxidizing agent. Intramolecular disulfide formation involving cysteine-substituted residues was also identified in some instances. These post-translational modifications limit the accessibility of the cysteine residues to sulfhydryl reagents and can have a profound impact on the interpretation of SCAM but may not alter function. When a post-translationally modified residue is used as a reference extracellular control, the high level of exposure required for detection on Western blot results in erroneous detection of otherwise inaccessible intracellular cysteine-substituted residues. The data indicate that in the application of SCAM, when a cysteine-substituted residue does not appear to be accessible to sulfhydryl-reactive reagents, the possibility of a post-translational modification should be excluded. The data explain the discrepancies in the assessment, and confirm the localization, of the first intracellular loop of PCFT.
Caveolins (Cavs) are ~20 kDa scaffolding proteins that assemble as oligomeric complexes in lipid raft domains to form caveolae, flask-shaped plasma membrane (PM) invaginations. Caveolae ("little caves") require lipid-lipid, protein-lipid, and protein-protein interactions that can modulate the localization, conformational stability, ligand affinity, effector specificity, and other functions of proteins that are partners of Cavs. Cavs are assembled into small oligomers in the endoplasmic reticulum (ER), transported to the Golgi for assembly with cholesterol and other oligomers, and then exported to the PM as an intact coat complex. At the PM, cavins, ~50 kDa adapter proteins, oligomerize into an outer coat complex that remodels the membrane into caveolae. The structure of caveolae protects their contents (i.e., lipids and proteins) from degradation. Cellular changes, including signal transduction effects, can destabilize caveolae and produce cavin dissociation, restructuring of Cav oligomers, ubquitination, internalization, and degradation. In this review, we provide a perspective of the life cycle (biogenesis, degradation), composition, and physiologic roles of Cavs and caveolae and identify unanswered questions regarding the roles of Cavs and cavins in caveolae and in regulating cell physiology.
In the healthy lung the opportunistic pathogen, P. aeruginosa, is rapidly eliminated by mucociliary clearance, a process that is dependent on the activity of the CFTR anion channel that, in concert with a number of other transport proteins, regulates the volume and composition of the periciliary surface liquid. This fluid layer is essential to enable cilia to clear pathogens from the lungs. However, in cystic fibrosis (CF), mutations in the CFTR gene reduce Cl- and HCO3- secretion, thereby decreasing periciliary surface liquid volume and mucociliary clearance of bacteria. In CF this leads to persistent infection with the opportunistic pathogen, P. aeruginosa, which is the cause of reduced lung function and death in ~95% of CF patients. Others and we have conducted studies to elucidate the effects of P. aeruginosa on wild type and Phe508del-CFTR Cl- secretion as well as on the host immune response. These studies have demonstrated that Cif (CFTR Inhibitory Factor), a virulence factor secreted by P. aeruginosa, is associated with reduced lung function in CF, induces the ubiquitination and degradation of wt-CFTR as well as TAP1, which plays a key role in viral and bacterial antigen presentation, and inhibits the generation of host proresolving lipids. Cif also enhances the degradation of Phe508del-CFTR that has been rescued by ORKAMBI, a drug approved for CF patients homozygous for the PheF508del-CFTR mutation, thereby reducing drug efficacy. This review is based on the Hans Ussing Distinguished Lecture at the 2016 Experimental Biology Meeting given by the author.
Activating transcription factor 6 (ATF6) is an important factor in the endoplasmic reticulum (ER) stress signaling pathway. The aim of this study was to assess the role of ATF6 in mouse granulosa cells with respect to apoptosis, the cell cycle, and steroid hormone production, as well as several key genes related to follicular development, via RNA interference, immunohistochemical staining, real-time quantitative polymerase chain reaction, western blotting, flow cytometry, TUNEL assay, and ELISA. Immunohistochemical staining revealed that ATF6 was extensively distributed in the granulosa cells of various ovarian follicles and oocytes in adult female mice. FSH or LH treatment significantly increased ATF6 protein levels .Flow cytometry and TUNEL assays analysis indicated that ATF6 depletion decreased apoptosis and arrested the S-phase of cell cycle. Consistent with these results, p53, Caspase-3, Bax, Chop, CyclinA1, CyclinB1, and CyclinD2 mRNA expression decreased, whereas Bcl-2 and Grp78 mRNA expression increased. Interestingly, ATF6 knock-down obviously increased progesterone and estradiol production. Cyp1b1 mRNA levels were down-regulated, whereas Cyp11a1, Star and Cyp19a1 mRNA levels were up-regulated, in keeping with the changes in steroid hormones. Furthermore, ATF6 disruption remarkably increased Igfbp4 expression and decreased Has2, Ptgs2 and Ptgfr expression. But after treating with tunicamycin (Tm), the levels of Has2, Ptgs2 and Ptgfr increased relatively whereas Igfbp4 expression decreased. Collectively, these results imply that ATF6 may regulate apoptosis, the cell cycle, steroid hormone synthesis, and other modulators related to folliculogenesis, which may indirectly involve in the development, ovulation, and atresia of ovarian follicles by affecting the physiological function of granulosa cells.
Peptide growth factors stimulate cellular responses through activation of their trans-membrane receptors. Multiple intracellular signaling cascades are engaged following growth factor - receptor binding, leading to short- and long-term biological effects. Each receptor-activated signaling pathway does not act in isolation, but rather interacts at different levels with other pathways to shape signaling networks that are distinctive for each growth factor. To gain insights into the specifics of growth factor-regulated interactions among different signaling cascades, we developed a HeLa cell line stably expressing fluorescent live-cell imaging reporters that are readouts for two major growth factor-stimulated pathways, Ras - Raf - Mek - Erk and PI3-kinase - Akt. Incubation of cells with EGF resulted in rapid, robust, and sustained Erk signaling but shorter-term activation of Akt. In contrast, HGF induced sustained Akt signaling, but weak and short-lived Erk activity, and IGF-I stimulated strong long-term Akt responses, but negligible Erk signaling. To address potential interactions between signaling pathways, we employed specific small molecule inhibitors. In cells incubated with EGF or PDGF-AA, Raf activation and the subsequent stimulation of Erk reduced Akt signaling, while Mek inhibition, which blocked Erk activation, enhanced Akt, and turned transient effects into sustained responses. Our results reveal that individual growth factors initiate signaling cascades that vary markedly in strength and duration, and demonstrate in living cells the dramatic effects of crosstalk from Raf and Mek to PI3-kinase and Akt. Our data further indicate how specific growth factors can encode distinct cellular behaviors by promoting complex interactions among signaling pathways.
c-Jun is an AP-1 transcription factor and implicated in many aspects of cellular functions, but its exact role in the regulation of early intestinal epithelial restitution after injury remains largely unknown. Phospholipase C-1 (PLC1) catalyzes hydrolysis of phosphatidylinositol 4,5 biphosphate into the second messenger diacylglycerol and inositol 1,4,5 triphosphate, and coordinates Ca2+ stores mobilization, and regulates cell migration and proliferation in response to stress. Here we reported that c-Jun up-regulates PLC1 expression and enhances PLC1-induced Ca2+ signaling, thus promoting intestinal epithelial restitution after wounding. Ectopically expressed c-Jun increased PLC1 expression at the transcription level and this stimulation is mediated by directly interacting with AP-1- and C/EBP-binding sites that are located at proximal region of the PLC1 promoter. Increased levels of PLC1 by c-Jun elevated cytosolic free Ca2+ concentration and stimulated intestinal epithelial cell migration over the denuded area after wounding. The c-Jun-mediated PLC1/Ca2+ signal also plays an important role in polyamine-induced cell migration after wounding, since increased c-Jun rescued Ca2+ influx and cell migration in polyamine-deficient cells. These findings indicate that c-Jun induces PLC1 expression transcriptionally and enhances rapid epithelial restitution after injury by activating Ca2+ signal.
An increase in oxidative stress is suggested to be the main cause in Doxorubicin (Dox) -induced cardiotoxicity. However, there is now evidence that activation of inducible nitric oxide synthase (iNOS) and nitrosative stress are also involved. The role of Vitamin C (Vit C) in the regulation of nitric oxide synthase (NOS) and reduction of nitrosative stress in Dox-induced cardiotoxicity is unknown. The present study investigated the effects of Vit C in the mitigation of Dox-induced changes in the levels of nitric oxide (NO), NOS activity, protein expression of NOS isoforms and nitrosative stress as well as cytokines TNFα and IL-10 in isolated cardiomyocytes. Cardiomyocytes isolated from adult Sprague Dawley rats were segregated into four groups: i) control; ii) Vit C (25 µM); iii) Dox (10 µM); and iv) Vit C + Dox. Dox caused significant increase in the generation of superoxide radical (O2-.), peroxynitrite and NO and these effects of Dox were blunted by Vit C. Dox increased the expression of iNOS and altered protein expression as well as activation of endothelial NOS (eNOS). These changes were prevented by Vit C. Dox-induced increase in the ratio of monomeric/dimeric eNOS, promoting the production of O2-., which was prevented by Vit C by increasing the stability of dimeric form of eNOS. Vit C protected against Dox-induced increase in TNFα as well as a reduction in IL-10. These results suggest that Vit C provides cardioprotection by reducing oxidative/nitrosative stress and inflammation via a modulation of Dox-induced increase in the NO levels and NOS activity.
Elevation of blood triglycerides, primarily as triglyceride-rich lipoproteins (TGRL), has been linked to cerebrovascular inflammation, vascular dementia, and Alzheimer's disease (AD). Brain microvascular endothelial cells and astrocytes, two cell components of the neurovascular unit, participate in controlling blood-brain barrier (BBB) permeability and regulating neurovascular unit homeostasis. Our studies showed that infusion of high physiological concentrations of TGRL lipolysis products (TGRL + lipoprotein lipase) activate and injure brain endothelial cells and transiently increases the BBB transfer coefficient (Ki=permeability x surface area/volume) in vivo. However, little is known about how blood lipids affect astrocyte lipid accumulation and inflammation. To address this, we first demonstrated TGRL lipolysis products increased lipid droplet formation in cultured normal human astrocytes. We then evaluated the transcriptional pathways activated in astrocytes by TGRL lipolysis products and found up-regulated stress and inflammatory-related genes including activating transcription factor 3 (ATF3), macrophage inflammatory protein-3α (MIP-3α), growth differentiation factor-15 (GDF15), and prostaglandin-endoperoxide synthase 2 (COX2). TGRL lipolysis products also activated the JNK/cJUN/ATF3 pathway, induced ER stress protein CHOP, and the NFB pathway, while increasing secretion of MIP-3α, GDF15, and IL-8. Thus, our results demonstrate TGRL lipolysis products increase the BBB transfer coefficient (Ki), induce astrocyte lipid droplet formation, activate cell stress pathways, and induce secretion of inflammatory cytokines. Our observations are consistent with evidence for lipid-induced neurovascular injury and inflammation, and we, therefore, speculate that lipid-induced astrocyte injury could play a role in cognitive decline.
Background: Calcific aortic valve disease (CAVD) is a chronic inflammatory condition and the inflammatory responses of aortic valve interstitial cells (AVICs) play a critical role in the disease progression. Double-stranded RNA (dsRNA) released from damaged or stressed cells is pro-inflammatory and may contribute to the mechanism of chronic inflammation observed in diseased aortic valves. The objective of this study is to determine the effect of dsRNA on AVIC inflammatory responses and the underlying mechanism. Methods and results: AVICs from normal human aortic valves were stimulated with polyinosinic-polycytidylic acid [poly(I:C)], a mimic of dsRNA. Poly(I:C) increased the production of interleukin(IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1). Poly(I:C) also induced robust activation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) and nuclear factor-kappaB (NF-B). Knockdown of Toll-like receptor (TLR) 3 or Toll-interleukin 1 receptor domain-containing adapter-inducing interferon-β (TRIF) suppressed ERK1/2 and NF-B p65 phosphorylation, and reduced inflammatory mediator production induced by poly(I:C). Inhibition of NF-B, not ERK1/2, reduced inflammatory mediator production in AVICs exposed to poly(I:C). Interestingly, inhibition of NF-B by prevention of p50 migration failed to suppress inflammatory mediator production. NF-B p65 intranuclear translocation induced by TLR4 agonist was reduced by inhibition of p50 migration, however, poly(I:C)-induced p65 translocation was not although the p65/p50 heterodimer is present in AVICs. Conclusion: Poly(I:C) up-regulates the production of multiple inflammatory mediators through the TLR3-TRIF-NF-B pathway in human AVICs. The NF-B activated by dsRNA appears not the canonical p65/p50 heterodimers.
Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter, SMVT. While different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. While no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2 as well as mutating the putative CK2 phosphorylation site (Thr78Ala) in the SMVT protein led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time, that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter, and that these effects likely involve a CK2 - mediated pathway.
Severely injured burn patients receive multiple blood transfusions for anemia of critical illness despite the adverse consequences. One limiting factor to consider alternate treatment strategies is the lack of a reliable test platform to study molecular mechanisms of impaired erythropoiesis. This study illustrates how conditions resulting in high catecholamine microenvironment such as burns can instigate myelo-erythroid reprioritization influenced by beta-adrenergic stimulation leading to anemia. In mouse model of scald burn injury we observed, along with a 3-fold increase in bone marrow LSKs (linneg Sca1+cKit+), the myeloid shift is accompanied with a significant reduction in megakaryocyte erythrocyte progenitors (MEPs). Beta-blocker administration (propranolol) for six days post burn not only reduced the number of LSKs and MafB+ cells in multi potent progenitors but also influenced myelo-erythroid bifurcation by increasing the MEPs and reducing the granulocyte monocyte progenitors (GMPs) in the bone marrow of burn mice. Furthermore, similar results were observed in burn patients' PBMC derived ex-vivo culture system demonstrating that commitment stage of erythropoiesis is impaired in burn patients and intervention with propranolol (non-selective beta 1,2-adrenergic blocker) increases MEPs. Also, MafB+ cells that were significantly increased following standard burn care could be mitigated when propranolol was administered to burn patients establishing the mechanistic regulation of erythroid commitment by myeloid regulatory transcription factor MafB. Overall, results demonstrate that beta- adrenergic blockers following burn injury can redirect the hematopoietic commitment toward erythroid lineage by lowering MafB expression in multi potent progenitors and be of potential therapeutic value to increase erythropoietin responsiveness in burn patients.
The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier (oBRB) and is the prime target of early age-related macular degeneration (AMD). C-reactive protein (CRP), a serum biomarker for chronic inflammation and AMD, presents two different isoforms, monomeric (mCRP) and pentameric (pCRP) that may have a different effect on inflammation and barrier function in the RPE. The results reported in this study suggest that mCRP but not pCRP impairs RPE functionality by increasing paracellular permeability and disrupting the tight junction proteins ZO-1 and occludin in RPE cells. Additionally, we evaluated the effect of drugs commonly used in the clinical setting over the mCRP-induced barrier dysfunction. We found that corticosteroids (methylprednisolone) and anti-VEGF agents (bevacizumab) prevented mCRP-induced ARPE-19 barrier disruption and IL-8 production. Furthermore, bevacizumab was also able to revert mCRP-induced IL-8 increase after mCRP stimulation. In conclusion, the presence of mCRP within retinal tissue may lead to disruption of the oBRB, effect that may be modified in the presence of corticosteroids or anti-VEGF drugs.
Over the last several years, converging lines of evidence have indicated that miR-206 plays a pivotal role in promoting muscle differentiation and regeneration, thereby potentially impacting positively on the progression of neuromuscular disorders including Duchenne muscular dystrophy (DMD). Despite several studies showing the regulatory function of miR-206 on target mRNAs in skeletal muscle cells, the effects of overexpression of miR-206 in dystrophic muscles remain to be established. Here, we found that miR-206 overexpression in mdx mouse muscles simultaneously targets multiple mRNAs and proteins implicated in satellite cell differentiation, muscle regeneration, and at the neuromuscular junction. Overexpression of miR-206 also increased the levels of several muscle-specific mRNAs/proteins while enhancing utrophin A expression at the sarcolemma. Finally, we also observed that the increase expression of miR-206 in dystrophin-deficient mouse muscle decreased the production of pro-inflammatory cytokines and infiltration of macrophages. Taken together, our results show that miR-206 acts as a pleiotropic regulator that targets multiple key mRNAs and proteins expected to provide beneficial adaptations in dystrophic muscle, thus highlighting its therapeutic potential for DMD.
Type IV collagen, a non-fibrillar type, is ubiquitously expressed in the basement membrane around cardiomyocytes. Canstatin, a cleaved product of α2 chain of type IV collagen, is an anti-angiogenic factor. Because it has not been clarified whether canstatin exerts other biological activities in heart, we investigated the effects of canstatin on adult rat cardiac fibroblasts. Cell migration was determined by Boyden chamber assay. Western blotting was performed to detect secretion of matrix metalloproteinase (MMP)-2 and MMP-9 and phosphorylation of extracellular signal-regulated kinase (ERK). Localization of MMP-2 was detected by immunofluorescence staining. Canstatin (250 ng/ml) significantly increased migration, secretion and activity of MMP-2 but not MMP-9. CTTFWGFTLC peptide, an MMPs inhibitor and small interfering RNA (siRNA) against MMP-2 suppressed the canstatin (250 ng/ml, 24 h)-induced migration. Canstatin (250 ng/ml, 30 min) significantly increased phosphorylation of ERK. PD98059, a MEK inhibitor significantly suppressed the canstatin (250 ng/ml, 24 h)-induced migration but not secretion of MMP-2. An increase in MMP-2 expression was observed in cytoplasm of the canstatin (250 ng/ml)-treated cardiac fibroblasts (within 30 min). Canstatin induced actin stress fiber formation, which was inhibited by Y-27632, a Rho-associated kinase inhibitor. Y-27632 also suppressed the canstatin (250 ng/ml, 24 h)-induced MMP-2 secretion. Canstatin (250 ng/ml, 30 min) failed to induce ERK phosphorylation in MMP-2 siRNA-treated cardiac fibroblasts. In conclusion, this study revealed a novel function of canstatin for inducing cell migration of adult rat cardiac fibroblasts at least in part by ERK phosphorylation through MMP-2 secretion possibly via actin cytoskeletal change.
Drosophila melanogaster is widely used as a model system to study innate immunity and signaling pathways related to innate immunity, including the Toll signaling pathway. Although this pathway is well-studied, the precise mechanisms of post-transcriptional regulation of key components of the Toll signaling pathway by microRNAs (miRNAs) remain obscure. In this study we used an in silico strategy in combination with the Gal80ts-Gal4 driver system to identify microRNA-958 (miR-958) as a candidate Toll pathway regulating miRNA in Drosophila. We report that overexpression of miR-958 significantly reduces the expression of Drosomycin, a key antimicrobial peptide involved in Toll signaling and the innate immune response. We further demonstrate in vitro and in vivo that miR-958 targets the Toll and Dif genes, key components of the Toll signaling pathway, to negatively regulate Drosomycin expression. In addition, a miR-958-sponge rescued the expression of Toll and Dif, resulting in increased expression of Drosomycin. These results not only revealed a novel function and modulation pattern of miR-958, but also provided a new insight into the underlying molecular mechanisms of Toll signaling in regulation of innate immunity.
Nitric oxide is generated in skeletal muscle with activity and decreases Ca2+-sensitivity of the contractile apparatus, putatively by S-nitrosylation of an unidentified protein. We investigate the mechanistic basis of this effect and its relationship to the oxidation-induced increase in Ca2+-sensitivity in mammalian fast-twitch (FT) fibers mediated by S-glutathionylation of Cys134 on fast troponin I (TnIf). Force-[Ca2+] characteristics of the contractile apparatus in mechanically-skinned fibers were assessed by direct activation with heavily Ca2+-buffered solutions. Treatment with S-nitrosylating agents, S-nitrosoglutathione (GSNO) or S-nitroso-N-acetyl-penicillamine (SNAP), decreased pCa50 (= log10 [Ca2+] at half maximal activation) by ~-0.07 pCa units in rat and human FT fibers without affecting maximum force, but had no effect on rat and human slow-twitch fibers or toad or chicken FT fibers, which all lack Cys134. The Ca2+-sensitivity decrease was i) fully reversed with dithiothreitol or reduced glutathione, ii) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and iii) irreversibly blocked by low concentration of the alkylating agent, N-ethylmaleimide (NEM). The biotin-switch assay showed that both GSNO and SNAP treatments caused S nitrosylation of TnIf. S-glutathionylation pretreatment blocked the effects of S-nitrosylation on Ca2+-sensitivity, and vice-versa. S-nitrosylation pretreatment prevented NEM from irreversibly blocking S-glutathionylation of TnIf and its effects on Ca2+-sensitivity, and likewise S-glutathionylation pretreatment prevented NEM block of S-nitrosylation. Following substitution of TnIf into rat slow-twitch fibers, S-nitrosylation treatment caused decreased Ca2+-sensitivity. These findings demonstrate that S-nitrosylation and S-glutathionylation exert opposing effects on Ca2+-sensitivity in mammalian FT muscle fibers, mediated by competitive actions on Cys134 of TnIf.
Multi-nucleated muscle fibers are formed by the fusion of myogenic progenitor cells during embryonic and fetal myogenesis. However, the role of prenatally incorporated myonuclei in the skeletal muscle fibers of adult animals is poorly understood. We demonstrated, using the muscle-specific reporter mice, that the prenatal myonuclei remained in the adult soleus muscle, although cardiotoxin injection caused the loss of prenatal myonuclei. Overloading by the tendon transection of synergists failed to induce compensatory hypertrophy in regenerated soleus muscle fibers of adult rats, whereas unloading by tail suspension normally induced the fiber atrophy. Loss of hypertrophying function correlated with the lowered histone acetylation at the transcription start site of Igf1r gene, which was one of the genes that did not respond to the overloading. These parameters were improved by the transplantation of cells harvested from the juvenile soleus muscles of neonatal rats in association with enhanced histone acetylation of Igf1r gene. These results indicated that the presence of prenatal myonuclei was closely related to the status of histone acetylation, which could regulate the responsiveness of muscle fibers to physiological stimuli.
The relationship between iron and β cell dysfunction has long been recognized as individuals with iron overload display an increased incidence of diabetes. This link is usually attributed to the accumulation of excess iron in β cells leading to cellular damage and impaired function. Yet, the molecular mechanism(s) by which human β cells take up iron has not been determined. In the present study, we assessed the contribution of the metal-ion transporters ZIP14, ZIP8, and DMT1 to iron uptake by human β cells. Iron was provided to the cells as non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload and is a major contributor to tissue iron loading. We found that overexpression of ZIP14 and ZIP8, but not DMT1, resulted in increased NTBI uptake by βlox5 cells, a human β cell line. Conversely, siRNA-mediated knockdown of ZIP14, but not ZIP8, resulted in 50% lower NTBI uptake in βlox5 cells. In primary human islets, knockdown of ZIP14 also reduced NTBI uptake by 50%. Immunofluorescence analysis of islets from human pancreatic sections localized ZIP14 and DMT1 nearly exclusively to β cells. Studies in primary human islets suggest that ZIP14 protein levels do not vary with iron status or treatment with IL-1β. Collectively, these observations identify ZIP14 as a major contributor to NTBI uptake by β cells and suggest differential regulation of ZIP14 in primary human islets compared to other cell types such as hepatocytes.
L-arginine (L-Arg) is the substrate for nitric oxide synthase (NOS) to produce nitric oxide (NO), a signaling molecule that is key in cardiovascular physiology and pathology. In cardiac myocytes, L-Arg is incorporated from the circulation through the functioning of system-y+ cationic amino acid transporters. Depletion of L-Arg leads to NOS uncoupling, with O2 rather than L-Arg as terminal electron acceptor, resulting in superoxide formation. The reactive oxygen species (ROS) superoxide (O2•–), combined with NO, may lead to the production of the reactive nitrogen species (RNS) peroxynitrite (ONOO-), which is recognized as a major contributor to myocardial depression. In this study we aimed to determine the levels of external L-Arg that trigger ROS/RNS production in cardiac myocytes. To this goal, we used a two-step experimental design in which acutely-isolated cardiomyocytes were loaded with the dye coelenterazine that greatly increases its fluorescence quantum yield in the presence of ONOO- and O2•–. Cells were then exposed to different concentrations of extracellular L-Arg and changes in fluorescence were followed spectrofluorometrically. It was found that below a threshold value of ~100 µM, decreasing concentrations of L-Arg progressively increased ONOO-/ O2•–-induced fluorescence, an effect that was not mimicked by D-arginine or L-lysine and was fully blocked by the NOS inhibitor L-NAME. These results can be explained by NOS aberrant enzymatic activity and provide an estimate for the levels of circulating L-Arg below which ROS/RNS-mediated harmful effects arise in cardiac muscle.
Aspirin, an anti-inflammatory and anti-thrombotic drug, has become the focus of intense research as a potential anti-cancer agent owing to its ability to reduce tumor proliferation in vitro and to prevent tumorigenesis in patients. Studies have found an anti-cancer effect of aspirin when used in low, anti-platelet doses. However, the mechanism(s) through which low dose aspirin works is poorly understood. In this study we aimed to determine the effect of aspirin on the crosstalk between platelets and cancer cells. For our study we used 2 colon cancer cell lines isolated from the same donor but characterized by different metastatic potential, SW480 (nonmetastatic) and SW620 (metastatic) cancer cells, and a pancreatic cancer cell line, PANC-1 (nonmetastatic). We found that SW480 and PANC-1 cancer cell proliferation was potentiated by human platelets in manner dependent upon the upregulation and activation of the oncoprotein c-MYC. The ability of platelets to upregulate c-MYC and cancer cell proliferation was reversed by an anti-platelet concentration of aspirin. In conclusion, we show for the first time that inhibition of platelets by aspirin can affect their ability to induce cancer cell proliferation through the modulation of the c-MYC oncoprotein.
The Na/K-ATPase α1 polypeptide supports both ion pumping and signaling functions. The Na/K-ATPase α3 polypeptide differs from α1 in both its primary structure and its tissue distribution. Alpha3 expression seems particularly important in neurons, and recent clinical evidence supports a unique role of this isoform in normal brain function. The nature of this specific role of α3 has remained elusive, because the ubiquitous presence of α1 has hindered efforts to characterize α3-specific functions in mammalian cell systems. Using Na/K-ATPase α1 knock-down pig kidney cells (PY-17), we generated the first stable mammalian cell line expressing a ouabain-resistant form of rat Na/K-ATPase α3 in the absence of endogenous α1 detectable by western-blotting. In these cells, Na/K-ATPase α3 formed a functional ion-pumping enzyme and rescued the expression of Na/K-ATPase β1 and caveolin-1 to levels comparable to those observed in PY-17 cells rescued with a rat Na/K-ATPase α1 (AAC-19). The α3-containing enzymes had lower Na+ affinity and lower ouabain-sensitive transport activity than their α1-containing counterparts under basal conditions, but showed a greater capacity to be activated when intracellular Na+ was increased. In contrast to Na/K-ATPase α1, α3 could not regulate Src. Upon exposure to ouabain, Src activation did not occur, yet ERK was activated through Src-independent pathways involving PI3K and PKC. Hence, α3 expression confers signaling and pumping properties that are clearly distinct from that of cells expressing Na/K-ATPase α1.
Alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are the leading causes of cirrhosis and increase the risk of hepatocellular carcinoma and liver-related death. ALD and NAFLD share common pathogenic features extending from isolated steatosis to steatohepatitis, steatofibrosis, which can progress to cirrhosis and hepatocellular carcinoma. The pathophysiological mechanisms of the progression of NAFLD and ALD are complex and still unclear. Important links between the regulation of autophagy (Macroautophagy and Chaperome-mediated autophagy) and chronic liver diseases have been reported. Autophagy may protect against steatosis and progression to steatohepatitis by limiting hepatocyte injury and reducing M1 polarization, as well as promoting liver regeneration. Its role in fibrosis and hepatocarcinogenesis are more complex. It has pro- and anti-fibrogenic properties depending on the hepatic cell type concerned, and beneficial and deleterious effects on hepatocarcinogenesis at initiating and late phases, respectively. This review summarizes the latest advances on the role of autophagy in different stages of fatty liver disease progression, and describes its divergent and cell-specific effects during chronic liver injury.
MicroRNA-125a-5p(miR-125a-5p)could participate in the pathogenesis of vascular diseases. In this study, we investigated the role of miR-125a-5p in oxidized low density lipoprotein (ox-LDL) induced functional changes in human brain microvessel endothelial cells (HBMEC). The reactive oxygen species (ROS) production, nitric oxide (NO) generation, senescence, apoptosis and functions of HBMEC were analyzed. For mechanism study, the epidermal growth factor receptor (EGFR)/ extracellular regulated protein kinases (ERK)/ p38 mitogen-activated protein kinase (p38 MAPK), and phosphatidylinositol-3-kinase (PI3K), serine/threonine kinase (Akt), endothelial nitric oxide synthase (eNOS), and p-Akt were analyzed. Results showed that: 1) MiR-125a-5p expression was reduced in ox-LDL treated HBMEC. 2) Overexpression of miR-125a-5p prevented HBMEC from ox-LDL induced apoptosis, senescence, ROS production, and NO reduction. 3) Overexpression of miR-125a-5p increased HBMEC proliferation, migration and tube formation, while decreased HBMEC adhesion to leukocytes, as well as counteracted the effects of ox-LDL on those functions. 4) The levels of EGFR/ERK/p38 MAPK pathway, PI3K/Akt/eNOS pathway, cleaved caspase 3 and adherent molecular ICAM-1 and VCAM-1 were associated with the effects of ox-LDL on these HBMEC functions. In conclusion, miR-125a-5p could counteract the effects of ox-LDL on various HBMEC functions via regulating the EGFR/ERK/p38 MAPK and PI3K/Akt/eNOS pathways and cleaved caspase 3, ICAM-1 and VCAM-1 expression.
Recent studies have implicated the Hippo pathway and its transcriptional effectors YAP and TAZ as necessary for fibroblast activation and tissue fibrosis. To test the specific and sufficient roles for TAZ in driving autonomous fibroblast activation, we cultured NIH3T3 fibroblasts expressing a doxycycline-inducible nuclear-localized mutant of TAZ (TAZ4SA) in scaffold-free 3D hanging drop spheroids, or on matrices of specified mechanical rigidity. Control NIH3T3 fibroblasts formed spheroids in hanging drop culture that remained stable and neither increased nor decreased in size significantly over 15 days. In contrast, TAZ4SA-transduced fibroblasts grew robustly in spheroid culture, and expressed enhanced levels of genes encoding pro-fibrotic soluble factors CTGF, Et-1 and PAI-1. However, TAZ4SA expression was unable to enhance expression of ECM-encoding genes Col1a1, Col1a2, Col3a1 or Fn1 in spheroid culture. Micromechanical testing indicated that spheroids composed of either control or TAZ4SA expressing cells were highly compliant and indistinguishable in mechanical properties. In fibroblasts cultured on 2D matrices of compliance similar to spheroids, TAZ4SA expression was able to enhance contractile force generation, but was unable to enhance ECM gene expression. In contrast, culture on stiff hydrogels potentiated TAZ4SA enhancement of ECM expression. TAZ4SA enhancement of Col1a1 expression on soft matrices was potentiated by TGF-β1 while on stiff matrices it was abrogated by inhibition of MRTF, demonstrating context dependent crosstalk of TAZ with these pathways. These findings demonstrate sufficiency of TAZ activation for driving fibroblast proliferation, contraction, and soluble pro-fibrotic factor expression, and mechanical context-dependent crosstalk of TAZ with other pathways in regulating Col1a1 expression.
Tissue extracellular matrix provides structural support and creates unique environments for resident cells. However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I expressing cells in all three cell populations increase proportionally in fibrotic muscle indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I expressing cell populations differentially express distinct ECM proteins - fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins and skeletal muscle progenitor cells differentially express genes important for the satellite cell.
Angiotensin-converting enzyme inhibitors and mineralocorticoid receptor (MR) antagonists are FDA approved drugs that inhibit the renin-angiotensin-aldosterone system (RAAS) and are used to treat heart failure. Combined treatment with the angiotensin-converting enzyme inhibitor lisinopril and the non-specific MR antagonist spironolactone surprisingly improves skeletal muscle, in addition to heart function and pathology in a Duchenne muscular dystrophy mouse model. We recently demonstrated that MR is present in all limb and respiratory muscles and functions as a steroid hormone receptor in differentiated normal human skeletal muscle fibers. The goals of the current study were to begin to define cellular and molecular mechanisms mediating the skeletal muscle efficacy of RAAS inhibitor treatment. We also compared molecular changes resulting from RAAS inhibition with those resulting from the current Duchenne muscular dystrophy standard-of-care glucocorticoid treatment. Direct assessment of muscle membrane integrity demonstrated improvement in dystrophic mice treated with lisinopril and spironolactone compared to untreated mice. Short-term treatments of dystrophic mice with specific and non-specific MR antagonists combined with lisinopril led to overlapping gene expression profiles with beneficial regulation of metabolic processes and decreased inflammatory gene expression. Glucocorticoids increased apoptotic, proteolytic and chemokine gene expression that were not changed by RAAS inhibitors in dystrophic mice. Microarray data identified potential genes that may underlie RAAS inhibitor treatment efficacy and the side-effects of glucocorticoids. Direct effects of RAAS inhibitors on membrane integrity also contribute to improved pathology of dystrophic muscles. Together, these data will inform clinical development of MR antagonists for treating skeletal muscles in Duchenne muscular dystrophy.
Stretch activation (SA) is a delayed increase in force that enables high power and efficiency from a cyclically contracting muscle. SA exists in various degrees in almost all muscle types. In Drosophila, the indirect flight muscle (IFM) displays exceptionally high SA force production (FSA), whereas the jump muscle produces only minimal FSA. We previously found that expressing an embryonic (EMB) myosin heavy chain (MHC) isoform in the jump muscle transforms it into a moderately SA muscle type and enables positive cyclical power generation. To investigate if variation in MHC isoforms is sufficient to produce even higher FSA, we substituted the IFM MHC isoform (IFI) into the jump muscle. Surprisingly, we found that IFI only caused a 1.7-fold increase in FSA, less than half the increase previously observed with EMB, and only at a high Pi concentration, 16 mM. This IFI induced FSA is much less than what occurs in IFM, relative to isometric tension, and did not enable positive cyclical power generation by the jump muscle. Both isometric tension and FSA of control fibers decreased with increasing Pi concentration. However, for IFI expressing fibers, only isometric tension decreased. The rate of FSA generation was about 1.5-fold faster for IFI fibers than control fibers, and both rates were Pi dependent. We conclude that MHC isoforms can alter FSA and hence cyclical power generation, but that isoforms can only endow a muscle type with moderate FSA. Highly SA muscle types, such as IFM, likely use a different or additional mechanism.
Healthy expansion of human adipose tissue requires mesenchymal stem cells (hMSC) able to proliferate and differentiate into mature adipocytes. Characterization of those factors that coordinate hMSC-to-adipocyte transition is hence of paramount importance to modulate the adipose tissue expansion. It has been previously reported that the adipogenic program of hMSC can be disrupted by up-regulating caveolar proteins and Polymerase I and Transcript Release Factor (PTRF) is an integral component of caveolae highly expressed in adipose tissue. Here, we hypothesized that the role of PTRF in adipocyte functionality might stem from an effect on hMSC. To test this hypothesis we isolated hMSC from the subcutaneous fat depot. We found an upregulated expression of the PTRF associated with decreased adipogenic potential of hMSC, likely due to the existence of senescent adipocyte precursors. Employing shRNA-based constructs to stably reduce PTRF we were able to restore insulin sensitivity and reduced basal lipolysis and leptin levels in human adipocytes with high levels of PTRF. Additionally, we pinpointed the detrimental effect caused by PTRF on the adipose tissue to the existence of senescent adipocyte precursors unable to proliferate and differentiate into adipocytes. This study provides evidence that impaired adipocyte functionality can be corrected, at least partially, by PTRF down-regulation, and warrants further in vivo research in patients with dysfunctional adipose tissue to prevent metabolic complications.
Angiogenesis is an energy demanding process, however, the role of cellular energy pathways and their regulation by extracellular stimuli, especially extracellular nucleotides, remain largely unexplored. Using metabolic inhibitors of glycolysis (2-deoxyglucose, 2-DG) and oxidative phosphorylation (oligomycin, rotenone, and FCCP) we demonstrate that glycolysis and OXPHOS are both essential for angiogenic responses of vasa vasorum endothelial cell (VVEC). Treatment with P2R agonists, ATP and MeSADP, but not P1 receptor agonist, adenosine, increased glycolytic activity in VVEC (measured by extracellular acidification rate and lactate production). Stimulation of glycolysis was accompanied by increased levels of phospho-phosphofructokinase (PFKB3), hexokinase (HK), and GLUT 1, but not lactate dehydrogenase (LDH). Moreover, extracellular ATP and MeSADP, and to a lesser extent, adenosine increased basal and maximal oxygen consumption rates (OCR) in VVEC. These effects were potentiated when the cells were cultured in 20 mM galactose and 5mM glucose compared to 25 mM glucose. Treatment with P2R agonists decreased phosphorylation of pyruvate dehydrogenase (PHD-E1α) and increased succinate dehydrogenase (SDH), cytochrome oxidase (COX IV), and β subunit of F1F0 ATP synthase expression. In addition, P2R stimulation transiently elevated mitochondrial [Ca2+], implying involvement of mitochondria in VVEC angiogenic activation. We also demonstrated a critical role of PI3K and Akt pathways in lactate production, pyruvate dehydrogenase complex E1α subunit (PHD-E1α) phosphorylation, and the expression of HK, SDH, and GLUT1 in ATP-stimulated VVEC. Together, our findings suggest that purinergic and metabolic regulation of VVEC energy pathways are essential for VV angiogenesis and may contribute to pathologic vascular remodeling in PH.
The multidrug resistance-associated protein 2 (Mrp2) is an ATP-binding cassette transporter that transports a wide variety of organic anions across the apical membrane of epithelial cells. The expression of Mrp2 on the plasma membrane is regulated by protein-protein interactions. CFTR-associated ligand (CAL) interacts with transmembrane proteins via its PDZ domain and reduces their cell surface expression by increasing lysosomal degradation and intracellular retention. Our results showed that CAL is localized at the trans-Golgi network of rat hepatocytes. The expression of CAL is increased, and Mrp2 expression is decreased, in the liver of mice deficient in sodium-hydrogen exchanger regulatory factor-1 (NHERF-1). To determine if CAL interacts with Mrp2 and is involved in the post-transcriptional regulation of Mrp2, we used GST fusion proteins with or without the C-terminal PDZ binding motif of Mrp2 as the bait in GST pull-down assays. We demonstrated that Mrp2 binds to CAL via its C-terminal PDZ-binding motif in GST pull-down assays, an interaction verified by co-immunoprecipitation of these two proteins in co-transfected COS-7 cells. In COS-7 and LLC-PK1 cells transfected with Mrp2 alone, only a mature, high molecular weight band of Mrp2 was detected. However, when cells were co-transfected with Mrp2 and CAL, Mrp2 was expressed as both mature and immature forms. Biotinylation and streptavidin pull-down assays confirmed that CAL dramatically reduces the expression level of total and cell surface Mrp2 in Huh-7 cells. Our findings suggest that CAL interacts with Mrp2 and is a negative regulator of Mrp2 expression.
Hypoxia induces angiogenesis through the VEGF signaling pathway; however, signal propagation of VEGF under hypoxia is not understood fully. In this study, we examined alterations of VEGF signaling under hypoxia and its determinant in endothelial cells. To analyze VEGF signaling during hypoxia, human umbilical vein endothelial cells (HUVECs) were exposed to 3 h hypoxia (1% O2), followed by 3 h reoxygenation or 12 h hypoxia. Hypoxia induced the expression of VEGF mRNA; however, it was not associated with an increase of tube formation by HUVECs. During 3 h hypoxia, VEGF-induced phosphorylation of VEGFR-2 and downstream molecules were significantly inhibited without a change of VEGFR-2 expression, but it was completely restored by reoxygenation. VEGF-mediated VEGFR-2 phosphorylation was associated with a reduction of cellular ATP under hypoxia (65.93 ± 8.32% of normoxia; mean ± s.e.m.; P<0.01). Interestingly, attenuation of VEGFR-2 phosphorylation was restored by the addition of ATP to prepared membranes from 3 h hypoxia-treated cells. In contrast with 3 h hypoxia, exposure of cells to 12 h hypoxia decreased VEGFR-2 expression and VEGF-mediated VEGFR-2 phosphorylation. The magnitude of VEGFR-2 phosphorylation was not fully restored by the addition of ATP to prepared membranes from cells exposed to 12 h hypoxia. These data indicate that ATP is an important determinant of VEGF signaling under hypoxia, and suggest that the activation process of VEGFR-2 was modified by sustained hypoxia. These observations contribute to our understanding of signal alterations of VEGF in endothelial cells under hypoxia.
Dehydrogenase/reductase member 7C (DHRS7C) is a newly identified NAD/NADH-dependent dehydrogenase that is expressed in cardiac and skeletal muscle and localized in the endoplasmic/sarcoplasmic reticulum (ER/SR). However, its functional role in muscle cells remains to be fully elucidated. Here, we investigated the role of DHRS7C by analyzing mouse C2C12 myoblasts deficient in DHRS7C (DHRS7C-KO cells), overexpressing wild-type DHRS7C (DHRS7C-WT cells), or expressing mutant DHRS7C (DHRS7C-Y191F or DHRS7C-K195Q cells, harboring point mutations in the NAD/NADH-dependent dehydrogenase catalytic core domain [YXXXK]). DHRS7C expression was induced as C2C12 myoblasts differentiated into mature myotubes, whereas DHRS7C-KO myotubes exhibited enlarged cellular morphology after differentiation. Notably, both DHRS7C-Y191F and DHRS7C-K195Q cells also showed similar enlarged cellular morphology, suggesting that the NAD/NADH-dependent dehydrogenase catalytic core domain is pivotal for DHRS7C function. In DHRS7C-KO, DHRS7C-Y191F, and DHRS7C-K195Q cells, the resting level of cytosolic Ca2+ and total amount of Ca2+ storage in the ER/SR were significantly higher than those in control C2C12 and DHRS7C-WT cells after differentiation. Additionally, Ca2+ release from the ER/SR induced by thapsigargin and 4-chloro-m-cresol was augmented in these cells and calpain, a calcium-dependent protease, was significantly activated in DHRS7C-KO, DHRS7C-Y191F, and DHRS7C-K195Q myotubes, consistent with the higher resting level of cytosolic Ca2+ concentration and enlarged morphology after differentiation. Furthermore, treatment with a calpain inhibitor abolished the enlarged cellular morphology. Taken together, our findings suggested that DHRS7C maintains intracellular Ca2+ homeostasis involving the ER/SR and that functional loss of DHRS7C leads to Ca2+ overload in the cytosol and ER/SR, resulting in enlarged cellular morphology via calpain activation.
Zn2+ deficiency (ZnD) is comorbid with chronic kidney disease (CKD) and worsens kidney complications. Oxidative stress is implicated in the detrimental effects of ZnD. However, the sources of oxidative stress continue to be identified. Since NADPH oxidases (Nox) are the primary enzymes that contribute to renal reactive oxygen species (ROS) generation, this study's objective was to determine the role of these enzymes in ZnD-induced oxidative stress. We hypothesized that ZnD promotes Nox upregulation resulting in oxidative stress and kidney damage. To test this hypothesis, WT mice were pair-fed a ZnD- or Zn2+ adequate-diet. To further investigate the effects of Zn2+ bioavailability on Nox regulation, mouse tubular epithelial cells (mTEC) were exposed to the Zn2+ chelator N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or vehicle followed by Zn2+ supplementation. The findings show that mice fed a ZnD-diet develop microalbuminuria, electrolyte imbalance and whole kidney hypertrophy. These markers of kidney damage are accompanied by elevated Nox2 expression and H2O2 levels. In mTEC, TPEN-induced ZnD stimulates H2O2 generation. In this in vitro model of ZnD, enhanced H2O2 generation is prevented with Nox inhibition by diphenyleneiodonium. Specifically, TPEN promotes Nox2 expression and activation which are reversed when intracellular Zn2+ levels are restored following Zn2+ supplementation. Finally, Nox2 knock-down by si-RNA prevents TPEN-induced H2O2 generation and cellular hypertrophy in vitro. Taken together, these findings reveal that Nox2 is a Zn2+-regulated enzyme that mediates ZnD-induced oxidative stress and kidney hypertrophy. Understanding the specific mechanisms by which ZnD contributes to kidney damage may have an important impact on the treatment of CKD.
We recently reported that skeletal muscle fibers of obscurin knockout (KO) mice present altered distribution of ankB, disorganization of the sub-sarcolemmal microtubules and reduced localization of dystrophin at costameres. In addition, these mice have impaired running endurance and increased exercise-induced sarcolemmal damage compared to wild-type animals. Here, we report results from a combined approach of physiological, morphological, and structural studies in which we further characterize the skeletal muscles of obscurin KO mice. A detailed examination of exercise performance, using different running protocols, revealed that the reduced endurance of obscurin KO animals on the treadmill depends on exercise intensity and age. Indeed, a mild running protocol did not evidence significant differences between control and obscurin KO mice whereas comparison of running abilities of 2, 6 and 11 month-old mice exercised at exhaustion revealed a progressive, age-dependent reduction of the exercise tolerance in KO mice. Histological analysis indicated that a heavy exercise induced leucocyte infiltration, fibrotic connective tissue deposition and hyper-contractures in the diaphragm of KO mice. On the same line, electron microscopy revealed that in the diaphragm of exercised obscurin KO mice, but not in the hind limb muscles, both M-line and H-zone of sarcomeres appeared wavy and less defined. Altogether, these results suggest that obscurin is required for the maintenance of morphological and ultrastructural integrity of skeletal muscle fibers against damage induced by intense mechanical stress and point to the diaphragm as the skeletal muscle most severely affected in obscurin-deficient mice.
The effects of acute exposure to acidic water on Na+ and Cl- homeostasis, and the mechanisms underlying their compensatory regulation, were investigated in the larval zebrafish Danio rerio. Exposure to acidic water (pH 4.0; control pH 7.6) for 2 h significantly reduced Na+ uptake and whole body Na+ content. Nevertheless, the capacity for Na+ uptake was substantially increased in fish pre-exposed to acidic water but measured in control water. Based on the accumulation of the Na+-selective dye, Sodium GreenTM, two ionocyte sub-types exhibited intracellular Na+ enrichment after pre-exposure to acidic water; H+-ATPase rich (HR) cells which co-express the Na+/H+ exchanger isoform 3b (NHE3b) and a non-HR cell population. In fish experiencing Na+-Cl- cotransporter (NCC) knockdown, we observed no Sodium GreenTM accumulation in the latter cell type, suggesting the non-HR cells were NCC cells. Elimination of NHE3b-expressing HR cells did not prevent the increased Na+ uptake following acid exposure. On the other hand, the increased Na+ uptake was abolished when the acidic water was enriched with Na+ and Cl-, but not with Na+ only, indicating that the elevated Na+ uptake after acid exposure was associated with the compensatory regulation of Cl-. Further examinations demonstrated that acute acid exposure also reduced whole body Cl- levels and increased the capacity for Cl- uptake. Moreover, knockdown of NCC prevented the increased uptake of both Na+ and Cl- after exposure to acidic water. Together, the results of the present study revealed a novel role of NCC in the compensatory regulation of Na+ and Cl- uptake following acute acidosis.
NADPH oxidase (Nox)-derived oxyradicals contribute to atherosclerosis by oxidizing low-density lipoproteins (LDL), leading to their phagocytosis by vascular macrophages. Endocanna-binoids, such as 2-arachidonoylglycerol (2-AG), might be an important link between oxidative stress and atherosclerosis. We hypothesized that 2-AG biosynthesis in macrophages is enhanced following ligation of oxidized LDL by scavenger receptors via a signal transduction pathway in-volving Nox-derived ROS that activates diacylglycerol lipase β (DAGLβ), the 2-AG biosynthetic enzyme. To test this idea, we challenged macrophage cell lines and murine primary macrophages with a xanthine oxidase system or with non-physiologic and physiologic Nox stimulants [phorbol 12-myristate 13-acetate (PMA) and arachidonic acid (AA)]. Each stressor increased cellular su-peroxide levels and enhanced 2-AG biosynthetic activity in a Nox-dependent manner. Levels of cPLA2-dependent AA metabolites (eicosanoids) in primary macrophages were also dependent on Nox-mediated ROS. In addition, 2-AG levels in DAGL-overexpressing COS7 cells were atten-uated by inhibitors of Nox and DAGL. Furthermore, ROS induced by menadione (a redox cy-cling agent) or PMA could be partially attenuated by the cannabinoid 1/2 receptor agonist (WIN 55,212-2). Finally, cells that overexpress Nox2 components (Phox-COS7) synthesized larger amounts of 2-AG as compared to the parental COS7 cells. Together, the results suggest a positive correlation between heightened oxygen radical flux and 2-AG biosynthesis in macrophage cell lines and primary macrophages. Because of the antioxidant and anti-inflammatory effects associ-ated with 2-AG, the increased levels of this bioactive lipid might be an adaptive response to oxi-dative stress. Thus, oxyradical stress may be counteracted by the enhanced endocannabinoid tone.
Intestinal anoxia/reoxygenation (A/R) injury induces loss of barrier function followed by epithelial repair. Myosin light chain kinase (MLCK) has been shown to alter barrier function via regulation of interepithelial tight junctions (TJ), but has not been studied in intestinal A/R injury. We hypothesized that A/R injury would disrupt TJ barrier function via MLCK activation and MLC phosphorylation. Caco-2BBe1 monolayers were subjected to anoxia for 2 hours followed by reoxygenation in 21% O2, after which barrier function was determined by measuring transepithelial resistance (TER) and FITC-dextran flux. TJ proteins and MLCK signaling were assessed by western blotting, real time-PCR, or immunofluorescence microscopy. The role of MLCK was further investigated with select inhibitors (ML-7 and peptide 18) using in vitro and ex vivo models. Following A/R injury, there was a significant increase in paracellular permeability compared to control cells, as determined by TER and dextran fluxes (P<0.05). The TJ protein occludin was internalized during A/R injury and re-localized to the region of the TJ after 4h recovery. MLC phosphorylation was significantly increased by A/R injury (P<0.05) and treatment with the MLCK inhibitor peptide 18 attenuated the increased epithelial monolayer permeability and occludin endocytosis caused by A/R injury. Application of MLCK inhibitors to ischemia-injured porcine ileal mucosa induced significant increases in TER and reduced mucosal-to-serosal fluxes of 3H-labeled mannitol. These data suggest that MLCK-induced occludin endocytosis mediates intestinal epithelial barrier dysfunction during A/R injury. Our results also indicate that MLCK-dependent occludin regulation may be a target for the therapeutic treatment of ischemia/reperfusion injury.
Vascular smooth muscle cell (SMC) migration is an essential step involved in neointimal formation in restenosis and atherosclerosis. Lysophosphatidic acid (LPA) is a bioactive component of oxidized low density lipoprotein and is produced by activated platelets, implying that LPA influences vascular remodeling. Our previous study revealed that matricellular protein CCN1, a prominent extracellular matrix (ECM) protein, mediates LPA-induced SMC migration in vitro. Here we examined the role of CCN1 in LPA-induced neointimal formation. By using LPA infusion of carotid artery in a mouse model, we demonstrated that LPA highly induced CCN1 expression (6~7 fold) in neointimal lesions. Down-regulation of CCN1 expression with the specific CCN1 siRNA in carotid arteries blocked LPA-induced neointimal formation, indicating that CCN1 is essential in LPA-induced neointimal formation. We then used LPA receptor knockout (LPA1-/-, LPA2-/- and LPA3-/-) mice to examine LPA receptor function in CCN1 expression in vivo and in LPA-induced neointimal formation. Our data reveal that LPA1 deficiency, but not LPA2 or LPA3 deficiency, prevents LPA-induced CCN1 expression in vivo in mouse carotid arteries. We also observed that LPA1 deficiency blunted LPA infusion-induced neointimal formation, indicating that LPA1 is the major mediator for LPA-induced vascular remodeling. Our in vivo model of LPA-induced neointimal formation established a key role of the ECM protein CCN1 in mediating LPA-induced neointimal formation. Our data support the notion that the LPA1-CCN1 axis may be the central control for SMC migration and vascular remodeling. CCN1 may serve as an important vascular disease marker and potential target for vascular therapeutic intervention.
Hyperactivated DEG/ENaC channels cause neuronal death mediated by intracellular Ca2+ overload. Mammalian ASIC1a and C. elegans MEC-4(d) neurotoxic channels conduct both Na+ and Ca2+ raising the possibility that direct Ca2+ influx through these channels contributes to the intracellular Ca2+ overload. However, we showed that homologous C. elegans DEG/ENaC channel UNC-8(d) is not Ca2+ permeable yet it is neurotoxic, suggesting that Na+ influx is sufficient to induce cell death. Interestingly, UNC-8(d) shows small currents due to extracellular Ca2+ block in the Xenopus oocytes expression system. Thus, MEC-4(d) and UNC-8(d) differ both in current amplitude and Ca2+ permeability. Given that these two channels show a striking difference in toxicity, we asked what is the contribution of Na+ conductance versus Ca2+ permeability to cell death. To address this question we built a UNC-8/ MEC-4 chimeric channel that retains the calcium permeability of MEC-4 and characterized its properties in Xenopus oocytes. Our data support the hypothesis that for Ca2+ permeable DEG/ENaC channels, both Ca2+ permeability and Na+ conductance contribute to toxicity. However, for Ca2+ impermeable DEG/ENaCs (e.g., UNC-8) our evidence shows that constitutive Na+ conductance is sufficient to induce toxicity and that this effect is enhanced as current amplitude increases. Our work further refines the contribution of different channel properties to cellular toxicity induced by hyperactive DEG/ENaC channels.
MicroRNAs play diverse roles in various physiological processes during Drosophila development. In the present study, we reported that miR-11 regulates pupal size during Drosophila metamorphosis via targeting Ras85D with following evidences: pupal size was increased in the miR-11 deletion mutant; restoration of miR-11 in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant; ectopic expression of miR-11 in brain insulin-producing cells (IPCs) and whole body shows consistent alteration of pupal size; Dilps and Ras85D expressions were negatively regulated by miR-11 in vivo; miR-11 targets Ras85D through directly binding to Ras85D 3'UTR in vitro; removal of one copy of Ras85D in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant. Thus, our current work provides a novel mechanism of pupal size determination by microRNAs during Drosophila melanogaster metamorphosis.
The RNA-binding protein HuR is crucial for normal intestinal mucosal regeneration by modulating the stability and translation of target mRNAs, but the exact mechanism underlying HuR trafficking between the cytoplasm and nucleus remains largely unknown. Here we report a novel function of transcription factor JunD in the regulation of HuR subcellular localization through the control of importin-α1 expression in intestinal epithelial cells (IECs). Ectopically expressed JunD specifically inhibited importin-α1 at the transcription level, and this repression is mediated via interaction with CREB-binding site that was located at the proximal region of importin-α1 promoter. Reduction in the levels of importin-α1 by JunD increased cytoplasmic levels of HuR, although it failed to alter whole-cell HuR levels. Increased levels of endogenous JunD by depleting cellular polyamines also inhibited importin-α1 expression and increased cytoplasmic HuR levels, whereas JunD silencing rescued importin-α1 expression and enhanced HuR nuclear translocation in polyamine-deficient cells. Moreover, importin-α1 silencing protected IECs against apoptosis, which was prevented by HuR silencing. These results indicate that JunD regulates HuR subcellular distribution by down-regulating importin-α1, thus contributing to the maintenance of gut epithelium homeostasis.
Central CO2 chemosensitive neurons in the caudal solitary complex (cSC) are stimulated not only by hypercapnic acidosis (HA), but by hyperoxia as well. While a cellular mechanism for the CO2 response has yet to be isolated, previous data show that a redox-sensitive mechanism underlies neuronal excitability to hyperoxia. However, it remains unknown how changes in pO2 affect the production of reactive oxygen and nitrogen species (RONS) in the cSC that can lead to increased cellular excitability and with larger doses, cellular dysfunction and death. To this end, we used fluorescence microscopy in real time to determine how normobaric hyperoxia increases the production of key RONS in the cSC. Because neurons in the region are CO2 sensitive, we also examined the potential effects of CO2 narcosis, used during euthanasia prior to brain slice harvesting, on RONS production. Our findings show that normobaric hyperoxia (0.4 0.95 ATA O2) increases the fluorescence rates of fluorogenic dyes specific to both superoxide and nitric oxide. Interestingly, different results were seen for superoxide fluorescence when CO2 narcosis was used during euthanasia, suggesting long-lasting changes in superoxide production and/or antioxidant activity subsequent to CO2 narcosis prior to brain slicing. Further research needs to distinguish whether the increased levels of RONS reported here are merely increases in oxidative and nitrosative signaling or, alternatively, evidence of redox and nitrosative stress.
Central CO2 chemoreceptive neurons in the caudal solitary complex (cSC) are stimulated by hyperoxia via a free radical mechanism. Hyperoxia has been shown to increase superoxide and nitric oxide in the cSC, but it remains unknown how changes in pCO2 during hyperoxia affect the production of O2-dependent reactive oxygen and nitrogen species (RONS) downstream that can lead to increased levels of oxidative and nitrosative stress, cellular excitability, and potentially, dysfunction. We used real time fluorescence microscopy in rat brain slices to determine how hyperoxia and hypercapnic acidosis (HA) modulate one another in the production of key RONS, as well as colorimetric assays to measure levels of oxidized and nitrated lipids and proteins. We also examined the effects of CO2 narcosis and hypoxia prior to euthanasia and brain slice harvesting, as these neurons are CO2 sensitive and hypothesized to employ CO2/H+ mechanisms that exacerbate RONS production and potentially oxidative stress. Our findings show that hyperoxia ± HA increases the production of peroxynitrite and its derivatives, while increases in Fenton chemistry are most prominent during hyperoxia + HA. Using CO2 narcosis prior to euthanasia modulates cellular sensitivity to HA post-mortem and enhances the magnitude of the peroxynitrite pathway, but blunts the activity of Fenton chemistry. Overall, hyperoxia and HA do not result in increased production of markers of oxidative and nitrosative stress as expected. We postulate this is due to antioxidant and proteosomal removal of damaged lipids and proteins to maintain cell viability and avoid death during protracted hyperoxia.
Autophagy is a dynamic recycling process responsible for the breakdown of misfolded proteins and damaged organelles, providing nutrients and energy for cellular renovation and homeostasis. Loss of autophagy is associated with cardiovascular diseases. Caveolin-3 (Cav-3), a muscle-specific isoform, is a structural protein within caveolae and is critical to stress adaptation in the heart. Whether Cav-3 plays a role in regulating autophagy to modulate cardiac stress responses remains unknown. In the present study, we used HL-1 cells, a cardiac muscle cell line, with stable Cav-3 knockdown (Cav-3 KD) and Cav-3 over-expression (Cav-3 OE) to study the impact of Cav-3 in regulation of autophagy. We show that traditional stimulators of autophagy (i.e., rapamycin and starvation) result in upregulation of the process in Cav-3 OE cells while Cav-3 KD cells have a blunted response. Cav-3 co-immunoprecipitated with beclin-1 and Atg12, showing an interaction of caveolin with autophagy related proteins. In the heart, autophagy may be a major regulator of protection from ischemic stress. We found that Cav-3 KD cells have a decreased expression of autophagy markers (beclin-1, light chain (LC3-II) after simulated ischemia and I/R when compared to WT whereas OE cells showed increased expression. Moreover, Cav-3 KD cells showed increased cell death and higher level of apoptotic proteins (cleaved caspase-3 and cytochrome c) with suppressed mitochondrial function in response to simulated ischemia and I/R whereas Cav-3 OE cells were protected and had preserved mitochondrial function. Taken together, these results indicate that autophagy regulates adaptation to cardiac stress in a Cav-3 dependent manner.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a difficult diagnosis and prognosis. In this regard, new and more reliable biomarkers for the disease are needed. We propose peripheral blood, and more specifically the Hematopoietic Stem and Progenitor Cells (HSPCs) as potential prognostic biomarkers in the SOD1G93A murine model of ALS. We accurately and serially studied three HSPCs, Hematopoietic Stem Cells (HSCs), Common Lymphoid Progenitors (CLPs) and Common Myeloid Progenitors (CMPs), in both control and SOD1G93A mice along the disease progression by RT-PCR and flow cytometry analysis. We found interesting differences for every HSPCs type in the transgenic mice compared to the control mice at every time point selected, as well as differences along the disease course. The results showed a maintained compensatory increase of HSCs along disease progression. However, the downregulated levels of CLPs and CMPs suggested an exit of these cell populations to the peripheral tissues, probably due to their supporting role to the damaged tissues. In addition, a positive correlation of the percentage of CLPs and CMPs with the longevity was found, as well as a positive correlation of HSCs and CMPs with motor function and weight, thus reinforcing the idea that HSPCs play a relevant role in the longevity of the SOD1G93A mice. Based on these results, both CLPs and CMPs could be considered prognostic biomarkers of longevity in this animal model, opening the door to future studies in human patients for their potential clinical use.
The brush border Cl-oxalate exchanger SLC26A6 plays an essential role in mediating intestinal secretion of oxalate, and is crucial for the maintenance of oxalate homeostasis and the prevention of hyperoxaluria and calcium oxalate nephrolithiasis. Previous in vitro studies have suggested that SLC26A6 is heavily N-glycosylated. N-linked glycosylation is known to critically affect folding, trafficking and function in a wide variety of integral membrane proteins and could therefore potentially have a critical impact on SLC26A6 function and subsequent oxalate homeostasis. Through a series of enzymatic deglycosylation studies we confirmed that endogenously expressed mouse and human SLC26A6 are indeed glycosylated, that the oligosaccharides are principally attached via N-glycosidic linkage, and that there are tissue-specific differences in glycosylation. In vitro cell culture experiments were then used to elucidate the functional significance of the addition of the carbohydrate moieties. Biotinylation studies of SLC26A6 glycosylation mutants indicated that glycosylation is not essential for cell surface delivery of SLC26A6, but suggested that it may affect the efficacy with which it is trafficked and maintained in the plasma membrane. Functional studies of transfected SLC26A6 demonstrated that glycosylation at two sites in the putative second extracellular loop of SLC26A6 are critically important for chloride-dependent oxalate transport, and that enzymatic deglycosylation of SLC26A6 expressed on the plasma membrane of intact cells strongly reduced oxalate transport activity. Taken together, these studies indicated that oxalate transport function of SLC26A6 is critically dependent on glycosylation, and that exoglycosidase mediated deglycosylation of SLC26A6 has the capacity to profoundly modulate SLC26A6 function.
Diabetic cardiomyopathy is associated with metabolic changes including decreased glucose oxidation (Gox) and increased fatty acid oxidation (FAox), which result in cardiac energetic deficiency. Diabetic hyperglycemia is a pathophysiological mechanism that triggers multiple maladaptive phenomena. The mitochondrial calcium uniporter (MCU) is the channel responsible for Ca2+ uptake in mitochondria and free mitochondrial calcium concentration ([Ca2+]m) regulates mitochondrial metabolism. Experiments with cardiac myocytes (CM) exposed to simulated hyperglycemia revealed both reduced [Ca2+]m and MCU protein levels. Therefore, we investigated whether returning [Ca2+]m to normal levels in CM by expressing MCU could lead to normalization of Gox and FAox with no detrimental effects. Mouse neonatal CM were exposed for 72 h to either normal glucose (5.5 mM glucose plus 19.5 mM manitol, NG), high glucose (25 mM glucose, HG), or HG plus adenoviral MCU expression. Gox and FAox, [Ca2+]m, MCU levels, pyruvate dehydrogenase activity (PDH), oxidative stress, mitochondrial membrane potential (m) and apoptosis were assessed. Results showed that [Ca2+]m and MCU protein levels were reduced after 72h of HG. Gox was decreased and FAox was increased in HG along with decreased PDH activity, higher phosphorylated PDH levels and reduced m. MCU expression returned these parameters towards NG levels. Moreover, increased oxidative stress and apoptosis were reduced in HG by MCU expression. Relevantly, we also observed reduced MCU protein levels and [Ca2+]m in hearts from type 1 diabetic mice. Thus, we conclude that HG-induced metabolic alterations can be reverted by restoring MCU levels resulting in return of [Ca2+]m to normal.
The SLC4A11 gene encodes the bicarbonate-transporter-related protein BTR1, which is mutated in syndromes characterized by vision and hearing loss. Signs of these diseases (congenital hereditary endothelial dystrophy (CHED) and Harboyan Syndrome) are evident in mouse models of Slc4a11 disruption. However, the intrinsic activity of Slc4a11 remains controversial, complicating assignment of its (patho)physiological role. Most studies concur that Slc4a11 transports H+ (or the thermodynamically equivalent species OH–) rather than HCO3– but disparities have arisen as to whether the transport is coupled to another species such as Na+ or NH3/NH4+. Here for the first time, we examine the action of mouse Slc4a11 in Xenopus oocytes. We simultaneously monitor changes in intracellular pH, membrane potential and conductance as we alter extracellular pH, revealing the electrical and chemical driving forces that underlie the observed ion fluxes. We find that mSlc4a11 is an ideally selective H+/OH– conductive pathway, the action of which is uncoupled from the cotransport of any other ion. We also find that the activity of mSlc4a11 is independently enhanced by both extracellular and intracellular alkalinization, suggesting OH– as the most likely substrate and providing a novel explanation for the apparent NH3-dependence of Slc4a11-mediated currents reported by others. We suggest that the unique properties of Slc4a11 action underlie its value as a pH regulator in corneal endothelial cells.
The endothelial surface glycocalyx (ESG) is a carbohydrate-rich layer found on the vascular endothelium, serving critical functions in the mechanotransduction of blood flow induced forces. One of the most important protective functions of the ESG is to mediate the production of nitric oxide (NO) in response to blood flow. However, the detailed mechanism underlying ESG's mechanotransduction of the production of NO has not been completely identified. Herein, using the cultured rat brain microvascular endothelial cells (bEnd.3) as a model system, we have implemented a combined atomic force and fluorescence microscopy approach to show that the ESG senses and transduces vertical mechanical stretch to produce NO. This rapid NO production is dependent on the presence of both heparan sulfate (HS) and hyaluronic acid (HA) in ESG, as the removal of HS and/or HA leads to a significant decrease in NO production. Moreover, the production of NO is dependent on the intake of Ca2+ via endothelial Transient Receptor Potential (TRP) channels. Together, our results demonstrate the molecular mechanism of rapid production of NO in response to vertical mechanical stretch.
In the shark rectal gland (SRG), apical chloride secretion is electrically coupled to a basolateral K+ conductance whose type and molecular identity are unknown. We performed studies in the perfused SRG with 17 K+ channel inhibitors to begin this search. Maximal chloride secretion was inhibited by low perfusate pH, quinine, bupivicaine, and anandamide, consistent with the properties of an acid sensitive four transmembrane, two-pore-domain K+ channel (4TM-K2P). Using PCR with degenerate primers to this family, we identified a TASK-1 fragment in shark rectal gland, brain, gill, and kidney. Using 5'and 3' RACE PCR and genomic walking, we cloned the full length shark gene whose open reading frame encodes a protein of 375 amino acids that was 80% identical to the human TASK-1 protein. We expressed shark and human TASK-1 cRNA in Xenopus oocytes and characterized these channels using two electrode voltage clamping (TEVC). Both channels had identical current voltage relationships and a reversal potential of -90 mV. Both were inhibited by quinine, bupivicaine, and acidic pH. We identified this protein in SRG by Western blot and confocal immuno-fluorescent microscopy and detected the protein in SRG and human airway cells. Shark TASK-1 is the major K+ channel coupled to chloride secretion in the SRG, is the oldest 4TM 2P family member identified, and is the first TASK-1 channel identified to play a role in setting the driving force for chloride secretion in epithelia. The detection of this potassium channel in lung tissue has implications for human biology and disease.
Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder caused mostly by disruption of the MECP2 gene. Among several RTT-like mouse models, one of them is a strain of mice that carries an R168X point mutation in Mecp2, and resembles one of the most common RTT-causing mutations in humans. Although several behavioral defects have previously been found in Mecp2R168X/Y mice, alterations in nerve cells remain unknown. Here we compare several behavioral and cellular outcomes between this Mecp2R168X/Y model and a widely used Mecp2Bird/Y mouse model. With lower body weight and shorter lifespan than their wild-type littermates, the Mecp2R168X/Y mice showed impairments of breathing and motor function. Thus we studied brainstem CO2 chemosensitive neurons and propriosensory cells that are associated with these two functions, respectively. Neurons in the locus coeruleus (LC) of both mutant strains showed defects in their intrinsic membrane properties, including changes in action potential morphology and excessive firing activity. Neurons in the mesencephalic trigeminal nucleus (Me5) of both strains displayed a higher firing response to depolarization than their WT littermates, likely due to a lower firing threshold. Because the increased excitability in LC and Me5 neurons tends to impact the excitation-inhibition balances in brainstem neuronal networks as well as their associated functions, it is likely that the defects in the intrinsic membrane properties of these brainstem neurons contribute to the breathing abnormalities and motor dysfunction. Furthermore, our results showing comparable phenotypical outcomes of Mecp2R168X/Y mice with Mecp2Bird/Y mice suggest that both strains are valid animal models for RTT research.
We previously demonstrated that smooth muscle (SM) 22α promotes the migration activity in contractile vascular smooth muscle cells (VSMCs). Based on the varied functions exhibited by SM22α in different VSMC phenotypes, we investigated the effect of SM22α on VSMC migration under pathological conditions. The results demonstrated that SM22α overexpression in synthetic VSMCs inhibited platelet-derived growth factor (PDGF)-BB-induced cell lamellipodium formation and migration, which was different from its action in contractile cells. The results indicated two distinct mechanisms underlying inhibition of lamellipodium formation by SM22α, increased actin dynamic stability and decreased Ras activity via interference with interactions between Ras and guanine nucleotide exchange factor. The former inhibited actin cytoskeleton rearrangement in the cell cortex, while the latter significantly disrupted actin nucleation activation of the Arp2/3 complex. Baicalin, a herb-derived flavonoid compound, inhibited VSMC migration via upregulation of SM22α expression in vitro and in vivo. These data suggest that SM22α regulates lamellipodium formation and cell migration in a phenotype-dependent manner in VSMCs, which may be a new therapeutic target for vascular lesion formation.
Autonomic neural activation of intracellular Ca2+ release in parotid acinar cells induces the secretion of the fluid and protein components of primary saliva critical for maintaining overall oral homeostasis. In the current study, we profiled the role of acidic organelles in shaping the Ca2+ signals of parotid acini using a variety of imaging and pharmacological approaches. Results demonstrate that zymogen granules predominate as an apically polarized population of acidic organelles that contributes to the initial Ca2+ release. Moreover, we provide evidence that indicates a role for the intracellular messenger NAADP in the release of Ca2+ from acidic organelles following elevation of cAMP. Our data are consistent with the "trigger" hypothesis where localized release of Ca2+ sensitizes canonical intracellular Ca2+ channels to enhance signals from the ER. Release from acidic stores may be important for initiating saliva secretion at low levels of stimulation and a potential therapeutic target to augment secretory activity in hypofunctioning salivary glands.
Vitamin C, or ascorbic acid, both tightens the endothelial permeability barrier in basal cells and prevents barrier leak induced by inflammatory agents. Barrier tightening by ascorbate in basal endothelial cells requires nitric oxide derived from activation of nitric oxide synthase. Although ascorbate did not affect cyclic AMP levels in our previous study, there remains a question of whether it might activate downstream cyclic AMP-dependent pathways. In this work, we found in both primary and immortalized cultured endothelial cells that ascorbate tightened the endothelial permeability barrier by about 30%. In human umbilical vein endothelial cells, this occurred at what are likely physiologic intracellular ascorbate concentrations. In so doing, ascorbate decreased measures of oxidative stress and flattened the cells to increase cell-to-cell contacts. Inhibition of downstream cyclic AMP-dependent proteins via protein kinase A did not prevent ascorbate from tightening the endothelial permeability barrier, whereas inhibition of Epac1 did block the ascorbate effect. Although Epac1 was required, its mediator Rap1 was not activated. Further, ascorbate acutely stabilized microtubules during depolymerization induced by colchicine and nocodazole. Over several days in culture, ascorbate also increased the amount of stable acetylated α-tubulin. Microtubule stabilization was further suggested by the finding that ascorbate increased the amount of Epac1 bound to α-tubulin. These results suggest that physiologic ascorbate concentrations tighten the endothelial permeability barrier in unstimulated cells by stabilizing microtubules in a manner downstream of cyclic AMP that might be due both to increasing nitric oxide availability and to scavenging of reactive oxygen or nitrogen species.
Secreted Frizzled-related protein 2 (sFRP2) plays a key role in chronic fibrosis after myocardial infarction and in heart failure. The study was aimed at elucidating the mechanisms through which sFRP2 may regulate the growth and extracellular matrix (ECM) remodeling of adult mouse cardiac fibroblasts (CFs). We found that sFRP2 activates CFs in part through canonical Wnt/β-catenin signaling as evidenced by increased expression of Axin2 and Wnt3a, but not Wnt5a, as well as accumulation of nuclear β-catenin. CFs in response to sFRP2 exhibited robust cell proliferation associated with increased glucose consumption and lactate production, a phenomenon termed "the Warburg effect" in oncology. The coupling between CF expansion and anaerobic glycolysis is marked by upregulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and tissue-nonspecific alkaline phosphatase (TNAP). In conjunction with these phenotypic changes, the CFs accelerated ECM remodeling through upregulating expression of the matrix metalloproteinase 1 (MMP1) and MMP13 genes, two members of the Collagenase subfamily, and enzyme activities of MMP2 and MMP9, two members of the Gelatinase subfamily. Consistent with the induction of multiple MMPs possessing collagenolytic activities, the steady-state level of collagen type I present in CF spent medium was reduced by sFRP2. Analysis of non-CF cell types revealed that the observed multifaceted effects of sFRP2 in growth control, glucose metabolism and ECM regulation are largely restricted to CFs and highly sensitive to Wnt signaling perturbation. The study provides a molecular framework upon which the functional versatility and signaling complexity of sFRP2 in cardiac fibrosis may be better defined.
Ribosome production is an early event during skeletal muscle hypertrophy and precedes muscle protein accretion. Signaling via mTOR is crucial for ribosome production and hypertrophy, however the mechanisms by which it regulates these processes remain to be identified. Herein, we investigated the activation of mTOR signaling in hypertrophying myotubes, and determined that mTOR coordinates various aspects of gene expression important for ribosome production. First, inhibition of translation with cycloheximide had a more potent effect on protein synthesis than Rapamycin indicating that mTOR function during hypertrophy is not on general, but rather specific protein synthesis. Second, blocking Pol II transcription had a similar effect as Rapamycin and unexpectedly, revealed the necessity of Pol II transcription for Pol I transcription, suggesting that mTOR may regulate ribosome production also by controlling Class II genes at the transcription level. Third, Pol I activity is essential for rDNA transcription, and surprisingly, for protein synthesis as selective Pol I inhibition blunted rDNA transcription, protein synthesis and the hypertrophic response of myotubes. Finally, mTOR has nuclear localization in muscle, which is not sensitive to Rapamycin. Inhibition of mTOR signaling by Rapamycin disrupted mTOR-rDNA promoter interaction, and resulted in altered histone marks indicative of repressed transcription and formation of higher order chromatin structure. Thus mTOR signaling appears to regulate muscle hypertrophy by affecting protein synthesis, Class I and II gene expression and chromatin remodeling.
Congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (CHED with progressive sensorineural deafness), and potentially a subset of individuals with late onset Fuchs endothelial corneal dystrophy (FECD) are caused by mutations in the SLC4A11 gene that results in corneal endothelial cell abnormalities. Originally classified as a borate transporter, the function of SLC4A11 as a transport protein remains poorly understood. Elucidating the transport function(s) of SLC4A11 is needed to better understand how its loss results in the aforementioned posterior corneal dystrophic disease processes. qPCR experiments demonstrated that of the three known human N-terminal variants, SLC4A11-C is the major transcript expressed in human corneal endothelium. We studied the expression pattern of the three variants in mammalian HEK-293 cells and demonstrated that the SLC4A11-B and SLC4A11-C variants are plasma membrane proteins whereas SLC4A11-A is localized intracellularly. SLC4A11-B and SLC4A11-C were shown to be multifunctional ion transporters capable of transporting H+ equivalents in both a Na+-independent and Na+-coupled mode. In both transport modes, SLC4A11-C H+ flux was significantly greater than SLC4A11-B. In the presence of ammonia, SLC4A11-B and SLC4A11-C generated inward currents that were comparable in magnitude. Chimera SLC4A11-C-N-terminus-SLC4A11-B experiments demonstrated that the SLC4A11-C N-terminus functions as an auto-activating domain, enhancing Na+-independent and Na+-coupled H+ flux without significantly affecting the electrogenic NH3-H+(n) cotransport mode. All three modes of transport were significantly impaired in the presence of the CHED causing p.R109H (SLC4A11-C numbering) mutation. These complex ion transport properties need to be addressed in the context of corneal endothelial disease processes caused by mutations in SLC4A11.
The Cl– secretion via Ca2+-activated Cl– channel (CaCC) is critical for fluid secretion in exocrine glands like the salivary gland. Also in the mammary gland, it has been hypothesized that CaCC plays an important role in the secretion of Cl– and aqueous phase of milk. However, there has been no evidence for the functional expression of CaCC in native mammary secretory (MS) cells of lactating animals. We therefore assessed the membrane current in the MS cells that were freshly isolated from lactating mice using the whole-cell patch-clamp techniques. In the MS cells, we detected the CaCC current that exhibited the following characters: 1) Ca2+-dependnet activation at the concentrations of submicromolar range, 2) voltage-dependent activation, 3) slow kinetics for activation and deactivation, 4) outward rectification of the steady-state current, 5) anion permeability in the sequence of I– > NO3– > Br– > Cl– >> glutamate, 6) inhibition by Cl– channel blockers (NFA, DIDS, and CaCCinh-A01). These characters of the native CaCC current were similar to reported characters of heterologously expressed TMEM16A. RT-PCR analyses showed the expression of multiple CaCC channels including TMEM16A, Best1, and Best3 in the mammary glands of lactating mice. Immunohistochemical staining revealed the localization of TMEM16A protein at the apical membrane of the MS cells. Collectively, our data strongly suggest that MS cells functionally express CaCC, which is at least partly constituted by TMEM16A. The CaCC such as TMEM16A at the apical membrane of the MS cells may influence the quantity and/or quality of milk.
SLC4A11, a member of the SLC4 family of bicarbonate transporters, is a widely expressed integral membrane protein, abundant in kidney and cornea. Mutations of SLC4A11 cause some cases of the blinding corneal dystrophies, congenital hereditary endothelial dystrophy and Fuchs endothelial corneal dystrophy. These diseases are marked by fluid accumulation in the corneal stroma, secondary to defective fluid reabsorption by the corneal endothelium. The role of SLC4A11 in these corneal dystrophies is not firmly established, as SLC4A11 function remains unclear. To clarify the normal function(s) of SLC4A11, we characterized the protein following expression in the simple, low-background, expression system, X. laevis oocytes. Since plant and fungal SLC4A11 orthologs transport borate, we measured cell swelling associated with accumulation of solute, borate. The plant water/borate transporter, Nip5;1, manifested borate transport, whereas human SLC4A11 did not. SLC4A11 supported osmotically-driven water accumulation that was electroneutral and Na+-independent. Studies in oocytes and HEK293 cells could not detect Na+-coupled HCO3- transport or Cl-/HCO3- exchange by SLC4A11. SLC4A11 mediated electroneutral NH3 transport in oocytes. Voltage-dependent OH- or H+ movement was not measurable in SLC4A11 expressing oocytes, but SLC4A11-expressing HEK293 cells manifested low level cytosolic acidification at baseline. In mammalian cells, but not oocytes, OH-/H+ conductance may arise when SLC4A11 activates another protein or itself is activated by another protein. These data argue against a role of human SLC4A11 in bicarbonate or borate transport. This work provides additional support for water and ammonia transport by SLC4A11. When expressed in oocytes, SLC4A11 transported NH3, not NH3/H+.
Channel activities of skeletal muscle ryanodine receptor (RyR1) are activated by micromolar Ca2+ and inactivated by higher (~1 mM) Ca2+. To gain insight into a mechanism underlying Ca2+-dependent inactivation of RyR1 and its relationship with skeletal muscle diseases, we constructed nine recombinant RyR1 mutants carrying malignant hyperthermia or centronuclear myopathy associated mutations and determined RyR1 channel activities by [3H]ryanodine binding assay. These mutations are localized in or near the RyR1 domains which are responsible for Ca2+-dependent inactivation of RyR1. Four RyR1 mutations (F4732D, G4733E, R4736W and R4736Q) in the cytoplasmic loop between S2 and S3 transmembrane segments (S2-S3 loop) greatly reduced Ca2+-dependent channel inactivation. Activities of these mutant channels were suppressed at 10-100 µM Ca2+, and the suppressions were relieved by 1 mM Mg2+. The Ca2+- and Mg2+-dependent regulation of S2-S3 loop RyR1 mutants are similar to those of the cardiac isoform of RyR (RyR2) rather than wild type RyR1. Two mutations (T4825I and H4832Y) in the S4-S5 cytoplasmic loop increased Ca2+ affinities for channel activation and decreased Ca2+ affinities for inactivation, but impairment of Ca2+-dependent inactivation was not as prominent as those of S2-S3 loop mutants. Three mutations (T4082M, S4113L and N4120Y) in the EF hand domain showed essentially the same Ca2+-dependent channel regulation as that of wild-type RyR1. The results suggest that nine RyR1 mutants associated with skeletal muscle diseases were differently regulated by Ca2+ and Mg2+. Four malignant hyperthermia-associated RyR1 mutations in the S2-S3 loop conferred RyR2-type Ca2+- and Mg2+-dependent channel regulation.
Bile acids are known to initiate intricate signaling events in a variety of tissues, primarily in the liver and gastrointestinal tract. Of the known bile acids, only the dihydroxy species, deoxycholic acid and chenodeoxycholic acid (CDCA), and their conjugates, activate processes that stimulate epithelial Cl- secretion. We have previously published that CDCA acts in a rapid manner to stimulate colonic ion secretion via protein kinase A (PKA)-mediated activation of the dominant Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) (AJP 305:C447-56, 2013); however, PKA signaling did not account for the entire CDCA response. Here we show that in human colonic T84 cells, CDCA's induction of CFTR activity, measured as changes in short-circuit current (Isc), is dependent on epidermal growth factor receptor (EGFR) activation, and does not involve the bile acid receptors TGR5 or FXR. CDCA activation of Cl- secretion does not require Src, mitogen activated protein kinases, or phosphoinositide-3 kinase downstream of EGFR, but does require an increase in cytosolic Ca2+. In addition to PKA signaling, we found that the CDCA response requires a novel involvement of the exchange protein directly activated by cAMP (EPAC). EPAC is a known hub for cAMP and Ca2+ cross talk. Downstream of EPAC, CDCA activates Rap2, and changes in [Ca2+]i were dependent on both EPAC and EGFR activation. This study establishes the complexity of CDCA signaling in the colonic epithelium, and shows the contribution of EGFR, EPAC and Ca2+ in CDCA-induced activation of CFTR-dependent Cl- secretion.
Skeletal muscle mass can increase during hypertrophy or decline dramatically in response to normal or pathological signals that trigger atrophy. Many reports have documented that the number of nuclei within these cells is also plastic. It has been proposed that an as yet to be defined regulatory mechanism functions to maintain a relatively stable relationship between the cytoplasmic volume and nuclear number within the cell, a phenomenon known as the "myonuclear domain hypothesis". While it is accepted that hypertrophy is typically associated with the addition of new nuclei to the muscle fiber from stem cells such as satellite cells, the loss of myonuclei during atrophy has been controversial. The intersegmental muscles (ISMs) from the tobacco hawkmoth Manduca sexta are composed of giant syncytial cells that undergo sequential developmental programs of atrophy and programmed cell death at the end of metamorphosis. Since the ISMs lack satellite cells or regenerative capacity, the tissue is not "contaminated" by these non-muscle nuclei. Consequently, we monitored muscle mass, cross-sectional area, nuclear number, and cellular DNA content during atrophy and the early phases of cell death. Despite a ~75-80% decline in muscle mass and cross-sectional area during the period under investigation, there were no reductions in either nuclear number or DNA content and the myonuclear domain was reduced by about 85%. These data suggest that the myonuclear domain is not an intrinsic property of skeletal muscle and that nuclei persist through atrophy and programmed cell death.
(Pro)renin receptor (PRR) is predominantly expressed in the collecting duct (CD) with unclear functional implication. It is not known whether CD PRR is regulated by high potassium (HK). Here, we aimed to investigate the effect of HK on PRR expression and its role in regulation of aldosterone synthesis and release in the CD. In primary rat inner medullary CD cells, HK augmented PRR expression and soluble PPR (sPRR) release in a time- and dose- dependent manner, which was attenuated by PRR siRNA, eplerenone, and losartan. HK upregulated aldosterone release in parallel with an increase of CYP11B2 (cytochrome P450, family 11, subfamily B, polypeptide 2) protein expression and upregulation of medium renin activity, both of which were attenuated by a PRR antagonist PRO20, PRR siRNA, eplerenone, and losartan. Similarly, prorenin upregulated aldosterone release and CYP11B2 expression, both of which were attenuated by PRR siRNA. Interestingly, a recombinant sPRR (sPRR-His) also stimulated aldosterone release and CYP11B2 expression. Taken together, we conclude that HK enhances a local renin-angiotensin-aldosterone system (RAAS) leading to increased PRR expression which in turn amplifies the response of the RAAS, ultimately contributing to heightened aldosterone release.
It is often assumed that mechanical factors are important for effects of exercise on muscle, but during voluntary training and most experimental conditions the effects could solely be attributed to differences in electrical activity, and direct evidence for a mechano-sensory pathway has been scarce. We here show that in rat muscles stimulated in vivo under deep anesthesia with identical electrical activity patterns isometric contractions induced two-fold more hypertrophy than contractions with 50-60 % of the isometric force. The number of myonuclei and the RNA levels of myogenin and MRF4 were increased with high load, suggesting that activation of satellite cells are mechano-dependent. On the other hand, training induced a major shift in fiber type distribution from type 2b to 2x that was load-independent, indicating that the electrical signaling rather than mechano-signaling controls fiber type. Akt and S6K1 were not significantly differentially activated by load, suggesting that the difference in mechanical factors were not important for activating the Akt/mTOR/S6K1-pathway. The transmembrane molecule syndecan-4 implied in overload hypertrophy in cardiac muscle was not load dependent suggesting that mechano-signaling in skeletal muscle is different.
The regulation of the luminal pH of each organelle is crucial for its function and must be controlled tightly. Nevertheless, it has been assumed that the nuclear pH is regulated by the cytoplasmic proton transporters via the diffusion of H+ across the nuclear pores because of their large diameter. However, it has been demonstrated that ion gradients exist between cytosol and nucleus suggesting that the permeability of ions across the nuclear pores is restricted. V-H+-ATPase is responsible for the creation and maintenance of trans-membrane electrochemical gradient. We hypothesize that V-H+-ATPase located in the nuclear membranes functions as the primary mechanism to regulate nuclear pH and generate H+ gradients across the nuclear envelope. We studied the sub-cellular heterogeneity of H+ concentration in the nucleus and cytosol using ratio imaging microscopy and SNARF-1, a pH indicator, in prostate cells. Our results indicated that there are proton gradients across the nuclear membranes that are generated by V-H+-ATPase located in the outer and inner nuclear membranes. We demonstrated that these gradients are mostly dissipated by inhibiting V-H+-ATPase. Immunoblots and V-H+-ATPase activities corroborated the existence of V-H+-ATPase in the nuclear membranes. This study demonstrates that V-H+-ATPase is functionally expressed in nuclear membranes and is responsible for nuclear H+ gradients that may promote not only the coupled transport of substrates, but also most electrochemically driven events across the nuclear membranes. This study represents a paradigm shift that the nucleus can regulate its own pH micro-environment, providing new insights into nuclear ion homeostasis and signaling.
Binding of angiotensin II (Ang II) to the AT1 receptor (AT1R) in the proximal tubule stimulates NHE3 activity through multiple signaling pathways. However, the effects of Ang II/AT1R-induced Gi activation and subsequent decrease in cAMP accumulation on NHE3 regulation are not well established. We therefore tested the hypothesis that Ang II reduces cAMP/PKA-mediated phosphorylation of NHE3 on serine 552 and, in doing so, stimulates NHE3 activity. Under basal conditions, Ang II stimulated NHE3 activity but did not affect PKA-mediated NHE3 phosphorylation at serine 552 in opossum kidney (OKP) cells. However, in the presence of cAMP-elevating agent forskolin (FSK), Ang II blocked FSK-induced NHE3 inhibition, reduced intracellular cAMP concentrations, lowered PKA activity and prevented the FSK-mediated increase in NHE3 serine 552 phosphorylation. All effects of Ang II were blocked by pretreating OKP cells with the AT1R antagonist losartan, highlighting the contribution of the AT1R/Gi pathway in Ang II-mediated NHE3 upregulation under cAMP-elevating conditions. Accordingly, Gi inhibition by pertussis toxin treatment decreased NHE3 activity both in vitro and in vivo and, more importantly, prevented the stimulatory effect of Ang II on NHE3 activity in rat proximal tubules. Collectively, our results suggest that Ang II counteracts the effects of cAMP/PKA on NHE3 phosphorylation and inhibition by activating the AT1R/Gi pathway. Moreover, these findings support the notion that NHE3 dephosphorylation at serine 552 may represent a key event in the regulation of renal proximal tubule sodium handling by Ang II in the presence of natriuretic hormones that promote cAMP accumulation and transporter phosphorylation.
Mammalian cells of various types exhibit the remarkable ability to adapt to externally-applied mechanical stresses and strains. Because of this adaptation, cells can maintain their endogenous mechanical tension at a preferred (homeostatic) level, which is essential for normal physiological functions of cells and tissues and provides protection against various diseases, including atherosclerosis and cancer. Conventional wisdom is that the cell possesses the ability to maintain tensional homeostasis on its own. Recent findings showed, however, that isolated cells cannot maintain tensional homeostasis. Here we studied the effect of multicellular interactions on tensional homeostasis by measuring traction forces in isolated bovine aortic endothelial cells and in confluent and non-confluent cell clusters of different sizes. We found that in isolated cells the traction field exhibited a highly dynamic and erratic behavior. However, in cell clusters, dynamic fluctuations of the traction field became attenuated with increasing cluster size at a rate which was faster in non-confluent than in confluent clusters. The driving mechanism of attenuation of traction field fluctuations was statistical averaging of the noise and the impeding mechanism was non-uniform stress distribution in the clusters, which resulted from intercellular force transmission known as a "global tug-of-war". These results showed that isolated cells could not maintain tensional homeostasis, which confirmed previous findings, and that tensional homeostasis is a multicellular phenomenon, which was a novel finding.
Although leucine has many positive effects on metabolism in multiple tissues, elevated levels of this amino acid and the other branched-chain amino acids (BCAAs) and their metabolites are implicated in obesity and insulin resistance. While some controversies exist about the direct effect of leucine on insulin action in skeletal muscle, little is known about the direct effect of BCAA metabolites. Here, we first showed that the inhibitory effect of leucine on insulin stimulated glucose transport in L6 myotubes was dampened when other amino acids were present, due in part to a 140% stimulation of basal glucose transport (P < 0.05). Importantly, we also showed that α-ketoisocaproic acid (KIC), an obligatory metabolite of leucine, stimulated mTORC1 signaling but suppressed insulin stimulated glucose transport (-34%, P < 0.05) in an mTORC1-dependent manner. The effect of KIC on insulin-stimulated glucose transport was abrogated in cells depleted of branched-chain aminotransferase 2 (BCAT2), the enzyme that catalyzes the reversible transamination of KIC to leucine. We conclude that although KIC can modulate muscle glucose metabolism, this effect is likely a result of its transamination back to leucine. Therefore, limiting the availability of leucine, rather than those of its metabolites, to skeletal muscle may be more critical in the management of insulin resistance and its sequelae.
Hyperhomocysteinemia (HHcy) has been observed to promote hypertension, but the mechanisms are unclear. Toll-like receptor 4 (TLR-4) is a cellular membrane protein that is ubiquitously expressed in all cell types of the vasculature. TLR-4 activation has been known to promote inflammation that has been associated with pathogenesis of hypertension. In this study, we hypothesize that HHcy induces hypertension by TLR-4 activation that promotes inflammatory cytokine up-regulation (IL-1β, IL-6, TNF-α) and initiation of mitochondria- dependent apoptosis leading to cell death and chronic vascular inflammation. To test the hypothesis, we used C57BL/6J mice (WT); Cystathionine-β-synthase deficient mice (CBS+/-) with genetic mild HHcy; C3H/HeJ (C3H) mice, with TLR-4 mutation and mice with combined genetic HHcy and TLR-4 mutation (CBS+/-/C3H). Ultrasonography of the superior mesenteric artery (SMA) detected an increase in wall-to-lumen ratio, resistive index (RI) and pulsatility index (PI). The tail cuff blood pressure (BP) measurement revealed elevated blood pressure in CBS+/- mice. The RI, PI and wall-to-lumen ratio of the SMA in CBS+/-/C3H mice were similar to the control group and blood pressure was significantly alleviated. The expression of TLR-4, IL-1β, IL-6 and TNF-α was up regulated in the SMA tissue of CBS+/- mice group and reduced in CBS+/-/C3H group. CBS+/- mice exhibited an up regulation of molecules (BAX, caspase-9, caspase-3) involved in mitochondria- mediated cell death pathway that were attenuated in CBS+/-/C3H mice group. We conclude that HHcy promotes TLR-4- driven chronic vascular inflammation and mitochondria- mediated cell death inducing hypertension. TLR-4 mutation attenuates vascular inflammation and cell death that suppresses hypertension.
Oxidative stress and impaired antioxidant defense are believed to be contributors to the cardiovascular aging process. The transcription factor NF-E2 related factor 2 (Nrf2) plays a key role in orchestrating cellular antioxidant defenses and maintaining redox homeostasis. Our previous study showed that Exendin-4, a GLP-1 analogue, alleviates angiotensin II (Ang II)-induced VSMC senescence by inhibiting Rac1 activation via cAMP/PKA. The objective of this study is to investigate if Nrf2 mediates the anti-senescent effect of Exendin-4 in Ang II-induced VSMCs. Here we report that Exendin-4 triggered Nrf2 nuclear translocation, a downstream target of cAMP responsive element-binding protein (CREB) and expressions of antioxidant genes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO-1) in a dose- and time-dependent manner. In addition, knock-down of Nrf2 attenuated the inhibitory effects of Exendin-4 on Ang II-induced superoxidant generation and VSMC senescence. PKA/CREB pathway participated in the upregulations of HO-1 and NQO-1 induced by Exendin-4. Notably, our study revealed that Exendin-4 dose-dependently increased the acetylation of Nrf2 and the the recruitment of transcriptional coactivator CREB binding protein (CBP) to Nrf2. The Exendin-4-induced Nrf2 transactivation was diminished in the presence of CBP siRNA. Microscope imaging of Nrf2 as well as immunoblotting for Nrf2 showed that the Exendin-4-evoked Nrf2 acetylation favored its nuclear retention. Importantly, CBP silencing attenuated the suppressing effects of Exendin-4 on Ang II-induced VSMC senescence and superoxidant production. In conclusion, these results provide a mechanistic insight into how Nrf2 signaling mediates the anti-senescent and anti-oxidative effects induced by Exendin-4 in VSMCs.
SMCTs move several important fuel molecules that are involved in lipid, carbohydrate, and amino acid metabolism, but their regulation has been poorly studied. Insulin controls the translocation of several solutes that are involved in energetic cellular metabolism, including glucose. We studied the effect of insulin on the function of human SMCT1 expressed in Xenopus oocytes. The addition of insulin reduced α-keto-isocaproate (KIC)-dependent 22Na+ uptake by 29%. Consistent with this result, the co-injection of SMCT1 with SGK1 cRNA decreased the KIC-dependent 22Na+ uptake by 34%. The reduction of SMCT1 activity by SGK1 depends on its kinase activity, and it was observed that the co-injection of SMCT1 with S442D-SGK1 (a constitutively active mutant) decreased the KIC-dependent 22Na+ uptake by 50%. In contrast, an SMCT1 co-injection with K127M-SGK1 (an inactive mutant) had no effect on the KIC-dependent Na+ uptake. The decreasing SMCT1 function by insulin or SGK1 was corroborated by measuring [1-14C]-acetate uptake and the electric currents of SMCT1-injected oocytes. Previously, we found that SMCT2/Slc5a12-mRNA, but not SMCT1/Slc5a8-mRNA, is present in zebrafish pancreas (by in situ hybridization); however, SLC5a8 gene silencing was associated with the development of human pancreatic cancer. We confirmed that the mRNA and protein of both transporters were present in rat pancreas using RT-PCR with specific primers, Western blot analysis and immunohistochemistry. Additionally, significant propionate-dependent 22Na+ uptake occurred in pancreatic islets and was reduced by insulin treatment. Our data indicate that human SMCT1 is regulated by insulin and SGK1 and that both SMCTs are present in the mammalian pancreas.
Phosphatidylinositol-4,5-bisphosphate (PIP2) is a membrane phosphoinositide that regulates the activity of many ion channels. Influx of calcium primarily through voltage-gated calcium (CaV) channels promotes insulin secretion in pancreatic β-cells. However, whether CaV channels are regulated by PIP2, as is the case for some noninsulin-secreting cells, is unknown. The purpose of this study was to investigate whether CaV channels are regulated by PIP2 depletion in pancreatic β-cells through activation of a muscarinic pathway induced by oxotremorine methiodide (Oxo-M). CaV channel currents were recorded by the patch clamp technique. The CaV current amplitude was reduced by activation of the muscarinic receptor 1 (M1R) in the absence of kinetic changes. The Oxo-M-induced inhibition exhibited the hallmarks of voltage-independent regulation and did not involve PKC activation. A small fraction of the Oxo-M-induced CaV inhibition was diminished by a high concentration of Ca2+ chelator, whereas ≥ 50% of this inhibition was prevented by diC8-PIP2 dialysis. Localization of PIP2 in the plasma membrane was examined by transfecting INS-1 cells with PH-PLC1, which revealed a close temporal association between PIP2 hydrolysis and CaV channel inhibition. Furthermore, the depletion of PIP2 by a voltage-sensitive phosphatase reduced CaV currents in a way similar to that observed following M1R activation. These results indicate that activation of the M1R pathway inhibits the CaV channel via PIP2 depletion by a Ca2+-dependent mechanism in pancreatic β- and INS-1 cells and thereby support the hypothesis that membrane phospholipids regulate ion channel activity by interacting with ion channels.
Skeletal muscle KATP channel is crucial in preventing fiber damage and contractile dysfunctions, possibly by preventing large damaging ATP depletion. The objective of this study was to investigate changes in energy metabolism during fatigue in wild type and Kir6.2-/- flexor digitorum brevis (FDB), the latter muscle lacking functional KATP channels. Fatigue was elicited with one tetanic contraction every sec. In contrast to wild type, Kir6.2-/- FDB had significantly greater decreases in ATP and total adenylate levels during the last 2 min of the fatigue period. Glycogen depletion was greater in Kir6.2-/- FDB for the first 60 sec but not by the end of the fatigue period while there was no difference in glucose uptake. The total amount of glucosyl units entering glycolysis was the same between wild type and Kir6.2-/- FDB. During the first 60 sec, Kir6.2-/- generated less lactate and more CO2, while in the last 120 sec Kir6.2-/- FDB stopped generating CO2 and produced more lactate. The ATP generated during fatigue from phosphocreatine, glycolysis (lactate) and oxidative phosphorylation (CO2) was 3.3-fold greater in Kir6.2-/- than wild type FDB. Considering that ATP and total adenylate were significantly less in Kir6.2-/- FDB, it is suggested that Kir6.2-/- FDB had a greater energy deficit despite a greater ATP production, which was further supported by the fact that glucose uptake, lactate and CO2 production were greater in Kir6.2-/- FDB during the recovery period. It is thus concluded that a lack of functional KATP channels results in an impairment of energy metabolism.
Pituitary adenylate cyclase activating polypeptide (PACAP) peptides (Adcyap1) signaling at the selective PAC1 receptor (Adcyap1r1) participates in multiple homeostatic and stress-related responses, yet the cellular mechanisms underlying PACAP actions remain to be completely elucidated. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia and as these neurons are readily accessible, this neuronal system is particularly amenable to studying PACAP modulation of ionic conductances. The present study investigated how PACAP activation of MEK/ERK signaling contributed to the peptide-induced increase in cardiac neuron excitability. Treatment with the MEK inhibitor PD 98059 blocked PACAP-stimulated pERK and in parallel, suppressed the increase in cardiac neuron excitability. However, PD 98059 did not blunt the ability of PACAP to enhance two inward ionic currents, one flowing through hyperpolarization-activated nonselective cationic channels (Ih), and a second flowing through low voltage-activated calcium channels (IT), which support the peptide-induced increase in excitability. Thus, a PACAP and MEK/ERK-sensitive, voltage-dependent conductance(s), in addition to Ih and IT, modulate neuronal excitability. Despite prior work implicating PACAP down regulation of KV4.2 in modulation of excitability in other cells, treatment with the KV4.2 current blocker 4-aminopyridine did not replicate the PACAP-induced increase in excitability in cardiac neurons. However, cardiac neurons express the ERK target NaV1.7 channel and treatment with the selective NaV1.7 inhibitor PF-04856264 decreased the PACAP modulation of excitability. From these results, PACAP/PAC1 activation of MEK/ERK signaling may phosphorylate NaV1.7, enhancing sodium currents near the threshold, an action contributing to repetitive firing of the cardiac neurons exposed to PACAP.
Calponin is an actin cytoskeleton-associated protein that regulates motility-based cellular functions. Three isoforms of calponin are present in vertebrates, among which calponin 2 encoded by the Cnn2 gene is expressed in multiple types of cells, including blood cells from the myeloid lineage. Our previous studies demonstrated that macrophages from Cnn2 knockout (KO) mice exhibit increased migration and phagocytosis. Intrigued by an observation that monocytes and macrophages from patients with rheumatoid arthritis had increased calponin 2, we investigated anti-glucose-6-phosphate isomerase serum-induced arthritis in Cnn2-KO mice for the effect of calponin 2 deletion on the pathogenesis and pathology of inflammatory arthritis. The results showed that the development of arthritis was attenuated in systemic Cnn2-KO mice with significantly reduced inflammation and bone erosion than that in age- and stain background-matched C57BL/6 wild type (WT) mice. In vitro differentiation of calponin 2-null mouse bone marrow cells produced fewer osteoclasts with decreased bone resorption. The attenuation of inflammatory arthritis was confirmed in conditional myeloid cell-specific Cnn2-KO mice. The increased phagocytotic activity of calponin 2-null macrophages may facilitate the clearance of autoimmune complexes and the resolution of inflammation, whereas the decreased substrate adhesion may reduce osteoclastogenesis and bone resorption. The data suggest that calponin 2 regulation of cytoskeleton function plays a novel role in the pathogenesis of inflammatory arthritis, implicating a potentially therapeutic target.
Two-pore domain potassium (K2P) channels influence basic cellular parameters such as resting membrane potential, cellular excitability or intracellular Ca2+-concentration [Ca2+]i. While the physiological importance of K2P-channels in dif-ferent organ systems (e.g. heart, CNS or immune system) has become increas-ingly clear over the last decade, their expression profile and functional role in skeletal muscle cells (SkMC) remains largely unknown. The mouse SkMC cell line C2C12, wild type mouse muscle tissue and primary mouse muscle cells (PMM) were analyzed using quantitative PCR, western blotting and immuno-histochemical stainings as well as functional analysis including patch-clamp measurements and Ca2+-imaging. Mouse SkMC express TASK2, TREK1, TREK2 and TRAAK. Except TASK2 all mentioned channels were upregulated in vitro during differentiation from myoblasts to myotubes. TASK2 and TREK1 were also functionally expressed and upregulated in PMMs isolated from mouse muscle tissue. Inhibition of TASK2 and TREK1 during differentiation revealed a morphological impairment of myoblast fusion accompanied by a downregulation of maturation markers. TASK2 and TREK1 blockade led to a decreased K+ out-ward current and a decrease of ACh-dependent Ca2+-influx in C2C12 cells as potential underlying mechanism. K2P-channel expression was also detected in human muscle tissue by immunohistochemistry pointing towards possible rele-vance for human muscle cell maturation and function. In conclusion, our find-ings for the first time demonstrate the functional expression of TASK2 and TREK1 in muscle cells with implications for differentiation processes warranting further investigations in physiologic and pathophysiologic scenarios.
We previously reported that hypoxia augments alpha-adrenergic contraction (HVC) of skeletal arteries in rats. The underlying mechanism may involve hypoxic inhibition of eNOS expressed in skeletal arterial myocytes (14). To further explore the novel role of muscular eNOS in the skeletal artery, we compared HVC in femoral arteries (FAs) from eNOS knockout (KO) mice with that from wild-type (WT) and heterozygote (HZ) mice. Immunohistochemical assays revealed that, in addition to endothelia, eNOS is expressed in the FA medial layer, albeit at a much lower level. However, the medial eNOS signal was not evident in HZ FAs, despite strong expression in the endothelium; similar observations were made in WT carotid arteries (CAs). The amplitude of contraction induced by 1 µM phenylephrine (PhE) was greater in HZ than in WT FAs. Hypoxia (3% PO2) significantly augmented PhE-induced contraction in WT FAs, but not in HZ or KO FAs. No HVC was observed in PhE-pretreated WT CAs. The NOS inhibitor L-NAME (0.1 mM) also augmented PhE contraction in endothelium-denuded WT FAs, but not in WT CAs. Inhibitors specific to nNOS and iNOS did not augment PhE-induced contraction of WT FAs. NOX4 inhibitor (GKT137831, 5 μM) but not NOX2 inhibitor (apocynin, 100 μM) suppressed HVC. Consistent with the role of reactive oxygen species, HVC was also inhibited by pretreatment with tiron or PEG-catalase. Taken together, these data suggest that the eNOS expressed in smooth muscle cells in FAs attenuates alpha-adrenergic vasoconstriction; this suppression is alleviated under hypoxia, which potentiates vasoconstriction in a NOX4/ROS-dependent mechanism.
Resting metabolic rate (RMR) in human shows pronounced individual variations, but the underlying molecular mechanism remains elusive. Cytochrome C oxidase (COX) plays a key role in control of metabolic rate and recent studies of the subunit 4 isoform 2 (COX IV-2) indicate involvement in the cellular response to hypoxia and oxidative stress. We evaluated whether the COX subunit IV isoform composition may explain the pronounced individual variations in resting metabolic rate (RMR). RMR was determined in healthy humans by indirect calorimetry and correlated to levels of COX IV-2 and COX IV-1 in Vastus Lateralis. Over expression and knock down of the COX IV isoforms were performed in primary myotubes followed by evaluation of the cell respiration and production of reactive oxygen species. Here we show that COX IV-2 protein is constitutively expressed in human skeletal muscle and strongly correlated to RMR. Primary human myotubes overexpressing COX IV-2 displayed markedly (>60%) lower respiration, reduced (>50%) cellular H2O2 production, higher resistance towards both oxidative stress and severe hypoxia compared to control cells. These results suggest an important role of isoform COX IV-2 in the control of energy expenditure, hypoxic tolerance and mitochondrial ROS homeostasis in humans.
Defects in the outer blood-retinal barrier have significant impact on the pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin. We recently reported a method for culture of retinal pigment epithelial (RPE) cells from wild type and transgenic mice. The RPE cells are responsible for maintaining the integrity of outer blood-retinal barrier whose dysfunction during diabetes has a significant impact on vision. Here we determined the impact of high glucose on the function of RPE cells. We showed that high glucose conditions resulted in enhanced migration and increased level of oxidative stress in RPE cells, but minimally impacted their rate of proliferation and apoptosis. High glucose also minimally affected the cell-matrix and cell-cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including pigment epithelium derived factor (PEDF) were altered under high glucose conditions. Incubation of RPE cells with the anti-oxidant N-acetylcysteine under high glucose conditions restored normal migration and PEDF expression. These cells also exhibited increased nuclear localization of the antioxidant transcription factor NRF2 and ZO-1, reduced levels of β-catenin and phagocytic activity, and minimal effect on production of vascular endothelial growth factor, inflammatory cytokines, and AKT, MAPK, and SRC signaling pathways. Thus, high glucose conditions promote RPE cell migration through increased oxidative stress and expression of PEDF without a significant effect on the rate of proliferation and apoptosis.
Anoctamin-1 (ANO1) is a Ca2+-activated Cl- channel expressed in many types of cells. Splice variants of ANO1 have been shown to influence the biophysical properties of the conductance. Several new antagonists of ANO1 with relatively high affinity and selectivity have been suggested to have usefulness for experimental and potentially for therapeutic purposes. We investigated the effects of intracellular Ca2+ (100-1000 nM), a concentration range that might be achieved in cells during physiological activation of ANO1 channels, on the block of ANO1 channels expressed in HEK 293 cells. Tests were performed on a variety of naturally occurring splice variants of ANO1 using whole cell and excised patch configurations of the patch clamp technique. Block of ANO1 currents with aminophenylthiazole (T16Ainh-A01) was highly dependent upon [Ca2+]i. Increasing [Ca2+]i reduced the potency of this blocker. Similar Ca2+-dependent effects were also observed with benzbromarone. Experiments on excised, inside-out patches showed that the diminished potency of the blockers caused by intracellular Ca2+ might involve a competitive interaction for a common binding site or repulsion of the blocking drugs by electrostatic forces at the cytoplasmic surface of the channels. The degree of interaction between the channel blockers and [Ca2+]i depended upon the splice variant expressed. These experiments demonstrate that the efficacy of ANO1 antagonists depends upon [Ca2+]i, suggesting a need for caution when using ANO1 blockers to determine the role of ANO1 in physiological functions and in use as therapeutic agents.
M cells are phagocytic intestinal epithelial cells in the follicle-associated epithelium (FAE) of Peyer's patches (PPs) that transport particulate antigens from the gut lumen into the subepithelial dome. Differentiation of M cells from epithelial stem cells in intestinal crypts requires the cytokine RANKL and the transcription factor Spi-B. We used three-dimensional enteroid cultures established with small intestinal crypts from mice as a model system to investigate signaling pathways involved in M cell differentiation and the influence of other cytokines on RANKL-induced M cell differentiation. RANKL addition to enteroids induced expression of multiple M-cell-associated genes by 1 day, including Spib, Ccl9, Tnfaip2, Anxa5 and Marcksl1. The mature M-cell marker Gp2 was strongly induced by 3 days and expressed by 11% of cells in enteroids. The noncanonical NF-B pathway was required for RANKL-induced M cell differentiation in enteroids, as addition of RANKL to enteroids from mice with a null mutation in the Map3k14 gene encoding NF-B-inducing kinase failed to induce M-cell-associated genes. While the cytokine TNF-α alone had little if any effect on expression of M-cell-associated genes, adding TNF-α to RANKL consistently resulted in 3- to 6-fold higher levels of multiple M-cell-associated genes compared to RANKL alone. One contributing mechanism is the rapid induction by TNF-α of Relb and Nfkb2, genes encoding the two subunits of the noncanonical NF-B heterodimer. We conclude that endogenous activators of canonical NF-B signaling present in the GALT microenvironment including TNF-α can play a supportive role in the RANKL-dependent differentiation of M cells in the FAE.
Rationale: Cigarette-smoke (CS) exposure and aging are the leading causes of chronic obstructive pulmonary disease (COPD)-emphysema development, although the molecular mechanism that mediates disease pathogenesis remains poorly understood. Objectives: To investigate the impact of CS-exposure and aging on autophagy, and pathophysiological changes associated with lung aging (senescence) and emphysema progression. Methods: Beas2b cells, C57BL/6 mice and human (GOLD 0-IV) lung tissues were used to determine the central mechanism involved in CS/age-related COPD-emphysema pathogenesis. Results: Beas2b cells and murine lungs exposed to CSE/CS showed a significant (p<0.05) accumulation of poly-ubiquitinated proteins and impaired-autophagy marker, p62, in aggresome-bodies. Moreover, treatment with autophagy-inducing antioxidant drug, cysteamine significantly (p<0.001) decreased CSE/CS-induced aggresome-bodies. We also found a significant (p<0.001) increase in levels of aggresome-bodies in the lungs of smokers and COPD-subjects in comparison to non-smoker controls. Furthermore, the presence and levels of aggresome-bodies statistically correlated with severity of emphysema and alveolar senescence. In addition to CS exposure, lungs from old mice also showed accumulation of aggresome-bodies, suggesting this as a common mechanism to initiate cellular senescence and emphysema. Additionally, Beas2b cells and murine lungs exposed to CSE/CS showed cellular apoptosis and senescence, which were both controlled by cysteamine treatment. In parallel, we evaluated the impact of CS on pulmonary exacerbation, using mice exposed to CS and/or infected with Pseudomonas aeruginosa (Pa), and confirmed cysteamine's potential as an autophagy-inducing antibacterial drug, based on its ability to control CS-induced pulmonary exacerbation (Pa-bacterial counts) and resulting inflammation. Conclusion: CS induced autophagy-impairment accelerates lung aging, COPD-emphysema exacerbations and pathogenesis.
The water-soluble, biotin (vitamin B7), is indispensable for normal human health. The vitamin acts as a co-factor for five carboxylases that are critical for fatty acid, glucose and amino acid metabolism. Biotin deficiency is associated with various diseases, and mice deficient in this vitamin display enhanced inflammation. Previous studies have shown that biotin affects the functions of adaptive immune T and NK cells, but its effect(s) on innate immune cells is not known. Because of that and because vitamins such as vitamins A and D have a profound effect on dendritic cell (DC) function, we investigated the effect of biotin levels on the functions of human monocyte derived DCs. Culture of DCs in a biotin deficient medium (BDM) and subsequent activation with LPS resulted in enhanced secretion of pro-inflammatory cytokines, TNF-α, IL-12p40, IL-23 and IL-1β compared to LPS-activated DCs cultured in biotin sufficient (control) and biotin over-supplemented media. Furthermore, LPS-activated DCs cultured in BDM displayed a significantly higher induction of IFN- and IL-17 indicating Th1/Th17 bias in T cells compared to cells maintained in biotin control or over-supplemented media. Investigations into the mechanisms suggested that impaired activation of AMP kinase in DCs cultured in BDM may be responsible for the observed increase in inflammatory responses. In summary, these results demonstrate for the first time that biotin deficiency enhances the inflammatory responses of DCs. This may therefore be one of the mechanism(s) that mediates the observed inflammation that occurs in biotin deficiency.
Aims: We have previously demonstrated that low-energy extracorporeal cardiac shock wave (SW) therapy improves myocardial ischemia through enhanced myocardial angiogenesis in a porcine model of chronic myocardial ischemia and in patients with refractory angina pectoris. However, the detailed molecular mechanisms for the SW-induced angiogenesis remain unclear. In this study, we thus examined the effects of SW irradiation on intracellular signaling pathways in vitro. Methods and Results: Cultured human umbilical vein endothelial cells (HUVECs) were treated with 800 shots of low-energy SW (1 Hz at an energy level of 0.03 mJ/mm2). The SW therapy significantly up-regulated mRNA expression and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). The SW therapy also enhanced phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and Akt. Furthermore, the SW therapy enhanced phosphorylation of caveolin-1 and the expression of HUTS-4 that represents β1-integrin activity. These results suggest that caveolin-1 and β1-integrin are involved in the SW-induced activation of angiogenic signaling pathways. To further examine the signaling pathways involved in the SW-induced angiogenesis, HUVECs were transfected with siRNA of either β1-integrin or caveolin-1. Knockdown of either caveolin-1 or β1-integrin suppressed the SW-induced phosphorylation of Erk1/2 and Akt and up-regulation of VEGF and eNOS. Knockdown of either caveolin-1 or β1-integrin also suppressed SW-induced enhancement of HUVEC migration in scratch assay. Conclusions: These results suggest that activation of mechanosensors on cell membranes, such as caveolin-1 and β1-integrin, and subsequent phosphorylation of Erk and Akt may play pivotal roles in the SW-induced angiogenesis.
Ubiquitin-proteasome system (UPS) is considered to be the key regulator in protein degradation. Bortezomib (BTZ) is the first proteasome inhibitor that is approved by FDA for the treatment of relapsed multiple myeloma and mantle cell lymphoma. Recently, BTZ has been reported to be effective against the pulmonary vascular characteristic associated with chronic hypoxia- and monocrotaline(MCT)-induced pulmonary hypertension. However, the underlying detailed mechanisms remains poorly understood. We previously confirmed that hypoxia-elevated basal intracellular Ca2+ concentration ([Ca2+]i) and store-operated Ca2+ entry (SOCE) in pulmonary artery smooth muscle cells (PASMCs) involve in the pulmonary vascular remodeling process. In this study, we investigated the effects of BTZ on proliferation as well as [Ca2+]i, especially the process of SOCE, and a list of SOCE-related regulatory proteins such like HIF-1α, BMP4, PPAR, TRPC1 and TRPC6 in rat distal PASMCs. Results showed that: 1) In either hypoxia-induced or MCT-induced rat PH models, BTZ markedly attenuated the elevated right ventricular pressure, right ventricular hypertrophy and pulmonary vascular remodeling; 2) BTZ prevented the process of PH by inhibiting hypoxia-increased cell proliferation, basal [Ca2+]i and SOCE in PASMCs; 3) BTZ also significantly normalized the hypoxia-upregulated HIF-1α, BMP4, TRPC1 and TRPC6 expression and the downregulated PPAR expression in distal pulmonary arteries (PAs) and PASMCs. These results indicated that BTZ exerts its protective role in the development of PH likely by inhibiting the TRPC-SOCE-[Ca2+]i signaling axis in PASMCs.
Blood acid/base regulation by specialized epithelia such as gills and kidney requires the ability to sense blood acid/base status. Here, we developed primary cultures of ray (Urolophus halleri) gill cells to study mechanisms for acid/base sensing without the interference of whole-animal hormonal regulation. Ray gills had abundant base-secreting cells, identified by being rich in V-H+-ATPase (VHA), and also expressed the evolutionarily conserved acid/base sensor soluble adenylyl cyclase (sAC). Exposing cultured cells to extracellular alkalosis (pH 8.0; 40 mM HCO3-) triggered VHA translocation to the cell membrane, similar to previous reports in live animals experiencing blood alkalosis. VHA translocation was dependent on sAC, as it was blocked by the sAC-specific inhibitor KH7. Ray gill base-secreting cells also express transmembrane adenylyl cyclases (tmACs); however, tmAC inhibition by 2'5'-dideoxyadenosine did not prevent alkalosis dependent VHA translocation, and tmAC activation by forskolin reduced the abundance of VHA at the cell membrane. This study demonstrates that sAC is a necessary and sufficient sensor of extracellular alkalosis in ray gill base-secreting cells. In addition, this study indicates that different sources of cAMP differentially modulate cell biology.
The renal filtration barrier is maintained by the renal podocyte, an epithelial postmitotic cell. Immortalized mouse podocyte cell lines - both in the differentiated and undifferentiated state - are widely utilized tools to estimate podocyte injury and cytoskeletal rearrangement processes in vitro. Here, we mapped the cultured podocyte proteome at a depth of more than 8800 proteins and quantified 7240 proteins. Copy numbers of proteins mutated in forms of hereditary nephrotic syndrome or focal segmental glomerulosclerosis (FSGS) were assessed. We found that cultured podocytes express abundant copy numbers of endogenous receptors such as tyrosine kinase membrane receptors, the G-protein coupled receptor (GPCR), NPR3 (ANP receptor) and several poorly characterized GPCRs. The dataset was correlated with deep mapping mRNA sequencing ("mRNAseq") data from the native mouse podocyte, the native mouse podocyte proteome and staining intensities from the human protein atlas. The generated dataset was similar to these previously published resources, but several native and high-abundant podocyte-specific proteins were not identified in the dataset. Notably, this dataset detected general perturbations in proteostatic mechanisms as a dominant alteration during podocyte differentiation, with high proteasome activity in the undifferentiated state and markedly increased expression of lysosomal proteins in the differentiated state. Phosphoproteomics analysis of mouse podocytes at a resolution of more than 3000 sites suggested a preference of phosphorylation of actin-filament associated proteins in the differentiated state. The dataset obtained here provides a resource and provides the means for deep mapping of the native podocyte proteome and phosphoproteome in a similar manner.
5'AMP activated protein kinase is activated as a consequence of lipolysis and has been shown to play a role in regulating adipose tissue mitochondrial content. Conversely, the inhibition of lipolysis has been reported to potentiate the induction of protein kinase A targeted genes involved in the regulation of oxidative metabolism. The purpose of the current study was to address these apparent discrepancies and to more fully examine the relationship between lipolysis, 5'AMP activated protein kinase, and the beta-adrenergic mediated regulation of gene expression. In 3T3-L1 adipocytes the adipose tissue triglyceride lipase inhibitor, ATGListatin, attenuated the Thr-172 phosphorylation of 5'AMP activated protein kinase by a beta 3 adrenergic agonist (CL 316,243) independent of changes in protein kinase A signaling. Similarly, CL 316,243 induced increases in the Thr-172 phosphorylation of 5'AMP activated protein kinase phosphorylation were reduced in adipose tissue from whole body ATGL deficient mice. Despite reductions in the activation of 5'AMP activated protein kinase, the induction of protein kinase A targeted genes was intact, or in some cases increased. Similarly, markers of mitochondrial content and respiration were increased in adipose tissue from ATGL knockout mice independent of changes in the Thr-172 phosphorylation of 5'AMP activated protein kinase phosphorylation. Taken together our data provides evidence that 5'AMP activated protein kinase is not required for the regulation of adipose tissue oxidative capacity in conditions of reduced fatty acid release.
The phenotype conversion of fibroblasts to myofibroblasts plays a key role in the pathogenesis of cardiac fibrosis. Numerous triggers of this conversion process have been identified, including plating of cells on solid substrates, cytokines such as Transforming Growth Factor β, and mechanical stretch, however the underlying mechanisms remain incompletely defined. Recent studies from our laboratory revealed that the transcription factor scleraxis is a key regulator of cardiac fibroblast phenotype and extracellular matrix expression. Here we report that mechanical stretch induces type I collagen expression and morphological changes indicative of cardiac myofibroblast conversion, as well as scleraxis expression via activation of the scleraxis promoter. Scleraxis causes phenotypic changes similar to stretch, and the effect of stretch is attenuated in scleraxis null cells. Scleraxis was also sufficient to up-regulate expression of vinculin and F-actin, to induce stress fiber and focal adhesion formation, and to attenuate both cell migration and proliferation - further evidence of scleraxis-mediated regulation of fibroblast to myofibroblast conversion. Together, these data confirm that scleraxis is sufficient to promote the myofibroblast phenotype, and is a required effector of stretch-mediated conversion. Scleraxis may thus represent a potential target for the development of novel anti-fibrotic therapies aimed at inhibiting myofibroblast formation.
Fibroblasts, the most abundant cells in the heart, contribute to cardiac fibrosis, the substrate for the development of arrythmogenesis, and therefore are potential targets for preventing arrhythmic cardiac remodeling. Chamber-specific difference in the responsiveness of fibroblasts from the atria and ventricles toward cytokine and growth factors has been described in animal models, but it is unclear if similar differences exist in human cardiac fibroblasts and whether drugs affect their proliferation differentially. Using cardiac fibroblasts from humans, differences between atrial and ventricular fibroblasts in serum-induced proliferation, DNA synthesis, cell cycle progression, cyclin gene expression, and their inhibition by simvastatin were determined. The serum-induced proliferation rate of human atrial fibroblasts was more than 3-fold greater than ventricular fibroblasts with faster DNA synthesis and higher mRNA levels of cyclin genes. Simvastatin predominantly decreased the rate of proliferation of atrial fibroblasts, with inhibition of cell cycle progression and an increase in the G0/G1 phase in atrial fibroblasts with higher sensitivity toward inhibition compared to ventricular fibroblasts. DNA synthesis and mRNA levels of cyclin A, D, and E were significantly reduced by simvastatin in atrial but not in ventricular fibroblasts. The inhibitory effect of simvastatin on atrial fibroblasts was abrogated by mevalonic acid (500 µM) that bypasses HMG-CoA reductase inhibition. Chamber-specific differences exist in the human heart with atrial fibroblasts having a higher proliferative capacity and being more sensitive to simvastatin-mediated inhibition through HMG-CoA reductase pathway. This mechanism may be useful in selectively preventing excessive atrial fibrosis without inhibiting adaptive ventricular remodeling during cardiac injury.
The two evolutionarily conserved mammalian lipid kinases Vps34 and PIKfyve are involved in an important physiological relationship, whereby the former produces phosphatidylinositol (PtdIns) 3P that is used as a substrate for PtdIns(3,5)P2 synthesis by the latter. Reduced production of PtdIns(3,5)P2 in proliferating mammalian cells is phenotypically manifested by the formation of multiple translucent cytoplasmic vacuoles, readily rescued upon exogenous delivery of PtdIns(3,5)P2 or overproduction of PIKfyve. Although the aberrant vacuolation phenomenon has been frequently used as a sensitive functional measure of localized PtdIns(3,5)P2 reduction, cellular factors governing the appearance of cytoplasmic vacuoles under PtdIns3P-PtdIns(3,5)P2 loss remain elusive. To gain further mechanistic insight about the vacuolation process following PtdIns(3,5)P2 reduction, in this study we sought for cellular mechanisms required for manifestation of the aberrant endomembrane vacuoles triggered by PIKfyve or Vps34 dysfunction. The latter was achieved by various means such as pharmacological inhibition, gene disruption or dominant-interference in several proliferating mammalian cell types. We report here that inhibition of V-ATPase with bafilomycin A1 as well as inactivation of the GTP-GDP cycle of Rab5a GTPase phenotypically rescued or completely precluded the cytoplasmic vacuolization despite the continued presence of inactivated PIKfyve or Vps34. Together, our work identifies for the first time that factors such as active V-ATPase or functional Rab5a cycle are acting coincidentally with the PtdIns(3,5)P2 reduction in triggering formation of aberrant cytoplasmic vacuoles under PIKfyve or Vps34 dysfunction.
Glucocorticoids strongly influence the mucosal-defense functions performed by the bronchial epithelium and inhaled corticosteroids (ICS) are critical in the treatment of patients with inflammatory airway diseases such as asthma, COPD and cystic fibrosis. A common pathology associated with these diseases is reduced mucociliary clearance, a defense mechanism involving the coordinated transport of salt, water and mucus by the bronchial epithelium, ultimately leading to the retention of pathogens and particles in the airways and to further disease progression. In the present study, we investigated the role of hydrocortisone (HC) in differentiation and development of the ion transport phenotype of normal human bronchial epithelial (NHBE) cells under air-liquid interface (ALI) conditions. NHBE cells differentiated in the absence of HC (HC0) showed significantly less benzamil-sensitive short-circuit current compared to controls as well as a reduced response after stimulation with the selective β2-adrenergic receptor (AR) agonist salbutamol. Apical membrane localization of ENaCα subunits were similarly reduced in HC0 cells compared to controls, supporting a role of HC in the trafficking and density of sodium channels in the plasma membrane. Additionally, glucocorticoid exposure during differentiation regulated the transcription of CFTR and β2-AR mRNAs and appeared to be necessary for the expression of CFTR-dependent anion secretion in response to β2-agonists. HC had no significant effect on surface cell differentiation but did modulate the expression of mucin mRNAs. These findings indicated that glucocorticoids support mucosal defense by regulating critical transport pathways essential for effective mucociliary clearance.
Conjunctival integrity and preservation is indispensable for vision. The self-renewing capacity of conjunctival cells control conjunctival homeostasis and regeneration; however, the source of conjunctival self-renewal and the underlying mechanism is currently unclear. Here, we characterize the biochemical phenotype and proliferative potential of conjunctival epithelial cells in adult mouse by detecting proliferation-related signatures and conducting clonal analysis. Further, we show transcription factor 7-like 2 (T-cell-specific transcription factor 4), a DNA binding protein expressed in multiple types of adult stem cells, is highly correlated with proliferative signatures in basal conjunctival epithelia. Clonal studies demonstrated that Transcription factor 7-like 2 (Tcf7l2) was coexpressed with p63α and proliferating cell nuclear antigen (PCNA) in propagative colonies. Furthermore, Tcf7l2 was actively transcribed concurrently with conjunctival epithelial proliferation in vitro. Collectively, we suggest that Tcf7l2 may involve in maintenance of stem/progenitor cells properties of conjunctival epithelial stem/progenitor cells, and the fornix as the optimal sites to isolate highly proliferative conjunctival epithelial cells in adult mouse.
The primary objective of this study was to determine whether alterations in mitochondria affect recovery of skeletal muscle strength and mitochondrial enzyme activities following myotoxic injury. 3-methyladenine (3-MA) was administered daily to blunt autophagy (15mg/kg) and the creatine analog guanidionpropionic acid (β-GPA) (1% in chow) was administered daily to enhance oxidative capacity. Male C57BL/6 mice were randomly assigned to non-treatment (Con; n=6), 3-MA (n=6), or β-GPA (n=8) groups for 10 wk of treatment. Mice were sacrificed two weeks after myotoxic injury to assess mitochondrial remodeling during regeneration and its association with the recovery of muscle strength. Injured muscles had a greater expression of several autophagy-related proteins (e.g., pUlk1 ~2-4 fold, p<0.049) compared to uninjured muscles indicating a relationship between muscle regeneration/remodeling and autophagy. By 2 weeks post-injury, 3-MA-treated mice had significantly less recovery of strength (18% less, p=0.03) and lower mitochondrial enzyme activity (e.g., citrate synthase (CS): 22% less, p =0.049) compared to Con, suggesting that the autophagy process plays an important role during muscle regeneration. In contrast, muscle regeneration was nearly complete in β-GPA-treated mice, i.e., strength recovered to 93% of baseline vs.78% for Con mice. Remarkably, 14 d was sufficient for a near complete recovery of mitochondrial function in β-GPA-treated mice (e.g., no difference between injured and uninjured in CS activity, p=0.49), indicating a robust mitochondrial remodeling process during muscle regeneration. In conclusion, autophagy is likely activated following muscle injury and appears to play an important role in functional muscle regeneration.
We previously reported that high pathophysiological concentrations of leptin, the adipocyte-secreted peptide, upregulates the expression of a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in vascular smooth muscle cells (VSMC). Moreover, this regulation was found to occur at the level of transcription; however, the underlying molecular mechanisms remain unknown. The goal of the present study was to investigate the specific transcriptional mechanisms that mediate upregulation of TSP-1 expression by leptin. Primary human aortic smooth muscle cell (HASMC) cultures were transiently transfected with different TSP-1 gene (THBS1) promoter linked-luciferase reporter constructs and luciferase activity in response to leptin (100ng/ml) was assessed. We identified a long THBS1 promoter (-1270/+750) fragment with specific leptin response elements that was required for increased TSP-1 transcription by leptin. Promoter analyses, protein/DNA array and gel shift assays demonstrated activation and association of transcription factors, interferon regulatory factor-1 (IRF-1) and cAMP response element-binding protein (CREB), to the distal fragment of the THBS1 promoter in response to leptin. Supershift, chromatin immunoprecipitation and co-immunoprecipitation revealed formation of a single complex between IRF-1 and CREB in response to leptin; importantly, recruitment of this complex to the THBS1 promoter mediated leptin-induced TSP-1 transcription. Finally, binding sequence decoy oligomer and site-directed mutagenesis revealed that regulatory elements for both IRF-1 (-1019 to -1016) and CREB (-1198 to -1195), specific to the distal THBS1 promoter, were required for leptin-induced TSP-1 transcription. Taken together, these findings demonstrate that leptin promotes a co-operative association between IRF-1 and CREB on the THBS1 promoter driving TSP-1 transcription in VSMC.
Polyunsaturated fatty acids (PUFAs) modulate voltage-gated K+ channel inactivation by an unknown site and mechanism. Effects of -6 and -3 PUFAs were investigated on the heterologously expressed Kv1.4 channel. PUFAs inhibited wild-type Kv1.4 during repetitive pulsing as a result of slowing of recovery from inactivation. In a mutant Kv1.4 channel lacking N-type inactivation, PUFAs reversibly enhanced C-type inactivation (KD, 15-43 μM). C-type inactivation was affected by extracellular H+ and K+ as well as PUFAs, and there was an interaction among the three: the effect of PUFAs was reversed during acidosis and abolished on raising K+. Replacement of two positively-charged residues in the extracellular pore (H508 and K532) abolished the effects of the PUFAs (and extracellular H+ and K+) on C-type inactivation, but had no effect on the lipoelectric modulation of voltage sensor activation, suggesting two separable interaction sites/mechanisms of action of PUFAs. Charge calculations suggest that the acidic head group of the PUFAs raises the pKa of H508 and this reduces the K+ occupancy of the selectivity filter, stabilising the C-type inactivated state.
The proton-coupled folate transporter (PCFT) mediates folate absorption across the brush-border membrane of the proximal small intestine and is required for folate transport across the choroid plexus into the cerebrospinal fluid. In this study, the functional role and accessibility of the seven PCFT Trp residues was assessed by the substituted-cysteine accessibility method. Six Trp residues at a lipid-aqueous interface tolerated Cys substitution in terms of protein stability and function. W85C, W202C, and W213C were accessible to N-biotinyl aminoethylmethanethiosulfonate; W48C and W299C were accessible only after treatment with dithiotreitol (DTT), consistent with modification of these residues by an endogenous thiol-reacting molecule and their extracellular location. Neither W107C nor W333C was accessible (even after DTT) consistent with their cytoplasmic orientation. Biotinylation was blocked by pemetrexed only for the W48C (after DTT), W85C, W202C residues. Function was impaired only for the W299C PCFT mutant located in the 4th external loop between the 7th and 8th transmembrane helices. Despite its aqueous location, function could only be fully preserved with Phe, and to a lesser extent, Ala substitutions. There was a 6.5-fold decrease in the pemetrexed influx Vmax and a 3.5- and 6-fold decrease in the influx Kt and Ki ,respectively, for the W299S PCFT. The data indicate that the hydrophobicity of the W299 residue is important for function suggesting that during the transport cycle this residue interacts with the lipid membrane thereby impacting on the oscillation of the carrier and, indirectly, on the folate binding pocket.
Background Human subcutaneous fat tissue consists of two layers: superficial adipose tissue (SAT) and deep adipose tissue (DAT). Some recent reports suggest that a disproportionate accumulation of DAT is related to obesity-associated metabolic complications. However, the differences in adipocyte function between SAT and DAT are unclear. Materials and Methods To clarify the differences in human adipocyte characteristics between SAT and DAT, human ceiling culture-derived proliferative adipocytes (ccdPAs) were primary cultured from SAT and DAT of three lean females. Differences in adipogenic differentiation potential and sensitivity to exogenous adipogenic factors were examined. Epigenetic modification of the CpG island DNA methylation levels of genes related to adipogenesis was measured. Results In histological analyses, the mean adipocyte size in SAT was significantly larger than that in DAT (8741 ± 416 vs. 7732 ± 213 μm2, P < 0.05). Primary cultured adipocytes from SAT showed significantly greater adipogenesis than did those of DAT. Sensitivity to partial adipogenic stimulation was significantly different between ccdPAs of SAT and DAT. PPAR- protein expression and leptin protein secretion from ccdPAs were significantly higher in SAT than DAT. DNA methylation levels of PPAR- were significantly lower in ccdPAs of SAT than DAT. Conclusion Adipocyte size was larger in SAT than DAT in vivo. This is consistent with the findings of an in vitro study that, compared with ccdPAs in DAT, ccdPAs in SAT have higher adipogenic potential and lower DNA methylation levels of PPAR-.
We tested the hypotheses that (i) a decrease in activation of skeletal muscles at short sarcomere lengths (SLs) is caused by an inhibition of Ca2+ release from the sarcoplasmic reticulum (SR), and (ii) the decrease in Ca2+ would be caused by an inhibition of action potential conduction from the periphery to the core of the fibres. Intact, single fibres dissected from the flexor digitorum brevis from mice were activated at different SLs, and intracellular Ca2+ was imaged with confocal microscopy. Force decreased at SLs shorter than 2.1µm, while Ca2+ concentration decreased at SLs below 1.9µm. The concentration of Ca2+ at short SL was lower at the core than at the peripheries of the fibre. When the external concentration of Na+ was decreased in the experimental media, impairing action potential conduction, Ca2+ gradients were observed in all SLs. When caffeine was used in the experimental media, the gradients of Ca2+ were abolished. We concluded that there is an inhibition of Ca2+ release from the sarcoplasmic reticulum (SR) at short SLs, which results from a decreased conduction of action potential from the periphery to the core of the fibres
Maintenance of corneal hydration is dependent on the active transport properties of the corneal endothelium. We tested the hypothesis that lactate efflux, facilitated by buffering, is a component of endothelial transport. Rabbit corneas were perfused with Bicarbonate-Rich (BR) or Bicarbonate-Free (BF) ringer of varying buffering power, while corneal thickness was measured. Perfusate was collected and analyzed for lactate efflux. In BF with no added HEPES, the maximal corneal swelling rate was 30.0±4.1µm/hr, compared to 5.2±0.9 µm/hr in BR. Corneal swelling decreased directly with [HEPES], such that with 60 mM HEPES corneas swelled at 7.5±1.6µm/hr. Perfusate [lactate] increased directly with [HEPES]. Similarly, reducing the [HCO3-] increased corneal swelling and decreased lactate efflux. Corneal swelling was inversely related to ringer buffering power (β), while lactate efflux was directly related to β. Ouabain (100 μM) produced maximal swelling and reduction in lactate efflux, whereas carbonic anhydrase inhibition and an MCT1 inhibitor produced intermediate swelling and decreases in lactate efflux. Conversely, 10μM adenosine reduced the swelling rate to 4.2±0.8 μm/hr and increased lactate efflux by 25%. We found a strong inverse relation between corneal swelling and lactate efflux (r=0.98, p<0.0001). Introducing lactate in the ringer transiently increased corneal thickness, reaching a steady-state (0±0.6 μm/hr) within 90 minutes. We conclude that corneal endothelial function does not have an absolute requirement for bicarbonate; rather it requires a perfusate with high buffering power. This facilitates lactic acid efflux, which is directly linked to water efflux, indicating that lactate flux is a component of the corneal endothelial pump.
We previously showed that sodium tanshinone IIA sulfonate (STS) inhibited store-operated Ca2+ entry (SOCE) through store-operated Ca2+ channels (SOCC) via down regulating the expression of transient receptor potential canonical proteins (TRPC), which contribute to the formation of SOCC. The detailed molecular mechanisms by which STS inhibits SOCE and downregulates TRPC, however, remain largely unknown. We have previously shown that under hypoxic conditions, inhibition of protein kinase G (PKG) and peroxisome proliferator-activated receptor (PPAR) signaling axis results in the up-regulation of TRPC. This suggests that strategies targeting the restoration of this signaling pathway may be an effective treatment strategy for pulmonary hypertension. In this study, our results demonstrated that STS treatment can effectively prevent the hypoxia mediated inhibition of the PKG-PPAR signaling axis in rat distal pulmonary artery smooth muscle cells (PASMCs) and distal pulmonary arteries (PA). These effects of STS treatment were blocked by pharmacological inhibition or specific small interfering RNA knockdown of either PKG or PPAR. Moreover, targeted PPAR agonist markedly enhanced the beneficial effects of STS. These results comprehensively suggest that STS treatment can prevent hypoxia mediated increases in intracellular calcium homeostasis and cell proliferation, by targeting and restoring the hypoxia-inhibited PKG-PPAR signaling pathway in PASMCs.
Sphingomyelin synthase (SMS) catalyzes the conversion of phosphatidylcholine and ceramide to sphingomyelin and diacylglycerol. We previously showed that SMS1 deficiency leads to a reduction in expression of the K+ channel KCNQ1 in the inner ear, causing hearing loss. However, it remains unknown whether this change in expression is attributable to a cellular process or a systemic effect in the knockout animal. Here, we examined whether manipulation of SMS1 activity affects KCNQ1/KCNE1 currents in individual cells. To this end, we expressed the KCNQ1/KCNE1 channel in human embryonic kidney 293T cells, and evaluated the effect of SMS1 manipulations on the channel using whole-cell recording. Application of tricyclodecan-9-yl-xanthogenate, a non-specific inhibitor of SMSs, significantly reduced current density and altered channel voltage dependence. Knockdown of SMS1 by an shRNA, however, reduced current density alone. Consistent with this, overexpression of SMS1 increased the current density without changing channel properties. Furthermore, application of a protein kinase D (PKD) inhibitors also suppressed current density without changing channel properties; this effect was non-additive with that of SMS1 shRNA. These results suggest that SMS1 positively regulates KCNQ1/KCNE1 channel density in a PKD-dependent manner.
The K+:Cl- cotransporters (KCC1-KCC4) encompass a branch of the SLC12 family of electroneutral cation-coupled chloride cotransporters that translocate ions out of the cell to regulate various factors, including cell volume and intracellular chloride concentration, among others. L-WNK1 is an ubiquitously expressed kinase that is activated in response to osmotic stress and intracellular chloride depletion, and it is implicated in two distinct hereditary syndromes: the renal disease pseudohypoaldosteronism type II (PHAII) and the neurological disease hereditary sensory neuropathy 2 (HSN2). The effect of L-WNK1 on the K+:Cl- cotransporter (KCCs) activity is unknown. Using Xenopus laevis oocytes and HEK-293 cells, we show that the activation of KCCs by cell swelling was prevented by L-WNK1 coexpression. In contrast, the activity of the Na+:K+:2Cl- cotransporter NKCC1 was remarkably increased by L-WNK1 coexpression. The negative effect of L-WNK1 on the KCCs is kinase dependent. Elimination of the SPAK binding site or the HQ motif required for the WNK-WNK interaction prevented the effect of L-WNK1 on KCCs, suggesting a required interaction between L-WNK1 molecules and SPAK. Together, our data support that NKCC1 and KCCs are coordinately regulated by L-WNK1 isoforms.
The Iroquois homeobox gene (Irx5) is essential in the embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 is involved in the regulation of cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies to determine relative expression of Irx5 expression in the peripheral vasculature using primary human endothelial cells (HUVECs), VSMCs, and intact carotid arteries demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization and the presence of IRX5 protein was examined by immunohistochemistry after two weeks. Results indicate markedly elevated IRX5 signal at 14 days compared to uninjured controls Total RNA was isolated from injured and uninjured arteries and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression from 3-14days post-injury when compared to controls. Irx5 genetic gain- and loss-of-function studies resulted in modulation of DNA synthesis, using thymidine and BrdU incorporation assays in primary rat aortic VSMCs (RASMCs). Quantitative RT-PCR results revealed modulation of p27kip1, E2f1, and Pcna expression in Irx5 transduced VSMCs when compared to control conditions. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation when compared to control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S cell cycle checkpoint control and apoptosis.
NLRP3 is a cytosolic protein that nucleates assembly of inflammasome signaling platforms which facilitate caspase-1 mediated IL-1β release and other inflammatory responses in myeloid leukocytes. NLRP3 inflammasomes are assembled in response to multiple pathogen- or environmental stress-induced changes in basic cell physiology, including the destabilization of lysosome integrity and activation of K+-permeable channels/transporters in the plasma membrane (PM). However, the quantitative relationships between lysosome membrane permeabilization (LMP), induction of increased PM K+ permeability, and activation of NLRP3 signaling are incompletely characterized. We used Leu-Leu-O-methyl ester (LLME), a soluble lysosomotropic agent, to quantitatively track the kinetics and extent of LMP in relation to NLRP3 inflammasome signaling responses (ASC oligomerization, caspase-1 activation, IL-1β release) and PM cation fluxes in murine bone marrow-derived dendritic cells (BMDC). Treatment of BMDC with submillimolar (<1 mM) LLME induced slower and partial increases in LMP that correlated with robust NLRP3 inflammasome activation and K+ efflux. In contrast, supramillimolar (>2 mM) LLME elicited extremely rapid and complete collapse of lysosome integrity that was correlated with suppression of inflammasome signaling. Supramillimolar LLME also induced dominant negative effects on inflammasome activation by the canonical NLRP3 agonist nigericin; this inhibition correlated with an increase in NLRP3 ubiquitination. LMP elicited rapid BMDC death by both inflammasome-dependent pyroptosis and inflammasome-independent necrosis. LMP also triggered Ca2+ influx which attenuated LLME-stimulated NLRP3 inflammasome signaling but potentiated LLME-induced necrosis. Taken together, these studies reveal a previously unappreciated signaling network that defines the coupling between LMP, changes in PM cation fluxes, cell death, and NLRP3 inflammasome activation.
Green tea catechins, especially (-)-epigallocatechin gallate (EGCG), have been reported to circulate in the placenta of animals and blood of humans after consumption. Whether EGCG regulates activity of human villous trophoblasts (HVT) is unknown. This study investigated the pathways involved in EGCG modulation of trophoblast mitogenesis. EGCG inhibited trophoblast proliferation in a dose-dependent and time-dependent manner, as indicated by the number of cells and incorporation of bromodeoxyuridine (BrdU). EGCG was more effective than other green tea catechins in inhibiting cell growth. EGCG also increased the phosphorylation of the MAPK pathway proteins, ERK1/2, and p38, but not JNK. Furthermore, EGCG had no effects on the total amounts of ERK1/2, p38 MAPK, and JNK proteins. This suggests that EGCG selectively affects particular MAPK subfamilies. Pretreatment with specific inhibitors of ERK1/2, p38 MAPK, and AMP-activated protein kinase (AMPK) antagonized EGCG-induced decreases in both cell number and BrdU incorporation. These inhibitors also blocked EGCG-induced increases in the levels of pERK1/2, pp38, and pAMPK proteins, respectively. Moreover, EGCG was similar to the PI3K inhibitors wortmannin and LY294002 to decrease protein kinase B (AKT) phosphorylation, cell number and BrdU incorporation. These data imply that EGCG inhibits the growth of HVT through the ERK, p38, AMPK and AKT pathways.
Anoctamin-1 (ANO1; also termed TMEM16A) is a Ca2+-activated Cl- (ClCa) channel expressed in arterial myocytes that regulates membrane potential and contractility. Signaling mechanisms that control ANO1 activity in arterial myocytes are poorly understood. In cerebral artery myocytes, ANO1 channels are activated by local Ca2+ signals generated by plasma membrane non-selective cation channels, but the molecular identity of these proteins is unclear. Arterial myocytes express several different non-selective cation channels, including multiple members of the transient receptor potential receptor (TRP) family. The goal of this study was to identify localized ion channels that control ANO1 currents in cerebral artery myocytes. Co-immunoprecipitation and immunofluorescence resonance energy transfer microscopy experiments indicate that ANO1 and TRP canonical 6 (TRPC6) channels are present in the same macromolecular complex and locate in close spatial proximity in the myocyte plasma membrane. In contrast, ANO1 is not nearby TRPC3, TRPM4, or inositol trisphosphate receptor 1 (IP3R1) channels. Hyp9, a selective TRPC6 channel activator, stimulated Cl- currents in myocytes that were blocked by T16Ainh-A01, an ANO1 inhibitor, ANO1 knockdown using siRNA, and equimolar replacement of intracellular EGTA with BAPTA, a fast Ca2+ chelator that abolishes local Ca2+ signaling. Hyp9 constricted pressurized cerebral arteries and this response was attenuated by T16Ainh-A01. In contrast T16Ainh-A01 did not alter depolarization-induced (60 mM K+) vasoconstriction. These data indicate that TRPC6 channels generate a local intracellular Ca2+ signal that activates nearby ANO1 channels in myocytes to stimulate vasoconstriction.
The mitochondrial (UPRmt) and the endoplasmic reticulum (UPRER) unfolded protein responses are important for cellular homeostasis during stimulus-induced increases in protein synthesis. Exercise triggers the synthesis of mitochondrial proteins, regulated in part by PGC-1 . To investigate the role of the UPR in exercise-induced adaptations, we subjected rats to 3-hrs of chronic contractile activity (CCA) for 1, 2, 3, 5 or 7 days, followed by 3-hrs recovery. Mitochondrial biogenesis signaling, through PGC-1 mRNA, increased 14-fold after 1 day of CCA. This resulted in 10-32% increases in COX activity, indicative of mitochondrial content, between days 3-7, as well as increases in the autophagic degradation of p62 and LC3-II protein. Preceding these adaptations, UPRER transcripts ATF4, XBP1s, and BiP were elevated (1.3-3.8-fold) from 1-3 days, while CHOP and chaperones BiP and HSP70 were elevated at both mRNA and protein levels (1.5-3.9-fold) from 1-7 days of CCA. Mitochondrial chaperones CPN10, HSP60, and mtHSP70, protease ClpP, and regulatory protein SirT3 of the UPRmt were concurrently induced 10-80% between 1-7 days. To test the role of the UPR in CCA-induced remodeling, we treated animals with the ER stress suppressor TUDCA and subjected them to 2 or 7 days of CCA. TUDCA attenuated CHOP and HSP70 protein induction, however this failed to impact mitochondrial remodeling. Our data indicate that signaling to the UPR is rapidly activated following acute contractile activity, that this is attenuated with repeated bouts, and that the UPR is involved in chronic adaptations to CCA, however this appears to be independent of CHOP signaling.
The expression of caveolin-1 (Cav1) in corneal epithelium is associated with regeneration potency. We used Cav1-/- mice to study the role of Cav1 in modulating corneal wound healing. Western blot and whole cell patch-clamp were employed to study the effect of Cav1 deletion on Kir4.1 current density in corneas. We found that Ba2+-sensitive K+ currents in primary cultured murine corneal epithelial cells (pMCE) from Cav1-/- were dramatically reduced (602pA) in comparison to those from WT (1300pA). As a consequence, membrane potential was elevated in pMCE from Cav1-/- comparing to that from WT (-43±7.5 vs -58±4.0 mV, respectively). Western blot showed that either inhibition of Cav1 expression or Ba2+ incubation stimulated phosphorylation of EGFR. The transwell migration assay showed that Cav1 genetic inactivation accelerated cell migration. The regrowth efficiency of human corneal epithelial cells (HCE) transfected with siRNA-Cav1 or negative control was evaluated by scrape injury assay. With the presence of mitomycin C (10 µg/ml) to avoid the influence of cell proliferation, Cav1 inhibition with siRNA significantly increased migration as compared to control siRNA in HCE. This promoting effect by siRNA-Cav1 could not be further enhanced by cotransfection with siRNA-Kcnj10. By using corneal debridement, we found that wound healing was significantly accelerated in Cav1-/- compared to WT mice (70±10% vs 36±3%, p<0.01). Our findings imply that the mechanism by which Cav-1 knockout promotes corneal regrowth is, at least partially, due to the inhibition of Kir4.1 which stimulates EGFR signaling.
Vitamin C (ascorbic acid, AA) is indispensable for normal metabolism of all mammalian cells including pancreatic acinar cells (PAC). PAC obtain AA from their surrounding via transport across the cell membrane. Chronic alcohol exposure negatively affects body AA homeostasis; it also inhibits uptake of other micronutrients into PAC but its effect on AA uptake is not clear. We examined this issue using both in vitro (266-6 cells) and in vivo (mice) models of chronic alcohol exposure. First, we determined the relative expression of the AA transporters-1 & 2 (i. e, SVCT-1 and SVCT-2) in mouse and human PAC and found SVCT-2 to be the predominent transporter. Chronic exposure of 266-6 cells to alcohol significantly inhibited AA uptake, and caused a marked reduction in SVCT-2 expression at the protein, mRNA, and hnRNA levels. Similarly, chronic alcohol feeding of mice significantly inhibited AA uptake and caused a marked reduction in level of expression of the SVCT-2 protein, mRNA and hnRNA. These findings suggest possible involvement of transcriptional mechanism(s) in mediating chronic aclohol effect on AA uptake by PAC. We also observed significant epigentic changes (histone modifications) in the Slc23a2 gene (reduction in H3K4me3 level and an increase in H3K27me3 level) in the alcohol exposed 266-6 cells. These findings show that chronic alcohol exposure inhibits PAC AA uptake and that the effect is mediated, in part, at the level of transcription of the Slc23a2 gene and may involve epigenetic mechanism(s).
Proteomics studies have identified SPAK and OSR1 in exosomes isolated from body fluids such as blood, saliva, urine. Because proteomics studies likely overestimate the number of exosome proteins, we sought to confirm and extend this observation using traditional biochemical and cell biology methods. We utilized HEK293 cells in culture to verify the packaging of these Ste20 kinases in exosomes. Using a series of centrifugation and filtration steps of conditioned culture medium isolated from HEK293 cells, we isolated nanovesicles in the range of 40-100 nm. We show that these small vesicles express the tetraspanin protein CD63 and lack endoplasmic reticulum and Golgi markers, consistent with these being exosomes. We show by Western blot and immuno-gold analyses that these exosomes express SPAK, OSR1, and NKCC1. We show that exosomes are not only secreted by cells, but also accumulated by adjacent cells. Indeed, exposing cultured cells to exosomes produced by other cells expressing a fluorescently labeled kinase resulted in the kinase finding its way into the cytoplasm of these cells, consistent with the idea of exosomes serving as cell to cell communication vessels. Similarly, co-culturing cells expressing different fluorescently-tagged proteins resulted into the exchange of proteins between cells. In addition, we show that both SPAK and OSR1 kinases entering cells through exosomes are preferentially expressed at the plasma membrane and that the kinases in exosomes are functional and maintain NKCC1 in a phosphorylated state.
Mitochondrial Ca2+ homeostasis-composed of the balance of Ca2+ influx and efflux-is responsible for the control of numerous cellular functions, including energy metabolism, the generation of reactive oxygen species, the spatiotemporal dynamics of Ca2+ signaling, as well as cell growth and death. Recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) provides new possibilities for applying genetic approaches to study the mitochondrial Ca2+ influx mechanism in various cell types and tissues. In addition, the successive discovery of various auxiliary subunits associated with the MCU suggests that mitochondrial Ca2+ uptake is not solely regulated by a single protein (MCU), but likely by a macromolecular protein complex referred to as MCU-protein complex (mtCUC). Moreover, recent reports have shown the potential role of MCU post-translational modifications in the regulation of mitochondrial Ca2+ uptake through mtCUC. These observations indicate that mtCUCs form a local signaling complex at the inner mitochondrial membrane, which could significantly regulate mitochondrial Ca2+ handling as well as numerous mitochondrial and cellular functions. In this review, we discuss the current literature on mitochondrial Ca2+ uptake mechanisms, with a particular focus on the structure and function of mtCUC as well as its regulation by signal transduction pathways, highlighting current controversies and discrepancies.
Muscle wasting is the hallmark of cancer cachexia and is associated with poor quality of life and increased mortality. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has important biological effects in the treatment of muscular dystrophy. To verify whether VPA could ameliorate muscle wasting induced by cancer cachexia, we explored the role of VPA in two cancer cachectic mouse models [induced by colon-26 (C26) adenocarcinoma or Lewis lung carcinoma (LLC)] and atrophied C2C12 myotubes [induced by C26 cell conditioned medium (CCM) or LLC cell conditioned medium (LCM)]. Our data demonstrated that treatment with VPA increased the mass and cross-sectional area (CSA) of skeletal muscles in tumor-bearing (TB) mice. Furthermore, treatment with VPA also increased the diameter of myotubes cultured in conditioned medium. The skeletal muscles in cachectic mice or atrophied myotubes treated with VPA exhibited reduced levels of CCAAT/enhancer binding protein beta (C/EBPβ), resulting in atrogin1 downregulation and the eventual alleviation of muscle wasting and myotube atrophy. Moreover, atrogin1 promoter activity in myotubes was stimulated by CCM via activating the C/EBPβ-responsive cis-element and subsequently inhibited by VPA. In contrast to the effect of VPA on the levels of C/EBPβ, the levels of inactivating forkhead box O3 (FoxO3a) were unaffected. In summary, VPA attenuated muscle wasting and myotube atrophy and reduced C/EBPβ binding to atrogin1 promoter locus in the myotubes. Our discoveries indicate that HDAC inhibition by VPA might be a promising new approach for the preservation of skeletal muscle in cancer cachexia.
The 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, and AMPK is activated in skeletal muscle in response to exercise and energy storage utilization. The diffusibility properties of AMPK α and β isoforms were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus (EDL) and oxidative soleus muscles that had their surface membrane removed by mechanical dissection. After 1 and 10 min washes, ~60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. Following in vitro stimulation that resulted in ~80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, determined by adding a wash step containing amylase. Stimulation of EDL muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target, acetyl carboxylase-CoA. This, however, had no effect on the proportion of total β2-AMPK that was phosphorylated in whole muscle homogenates, measured using immunoprecipitation. These findings suggest that in rat skeletal muscle, β2-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate β2-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of β2-AMPK in skeletal muscle.
Pulmonary arterial hypertension (PAH) is a progressive, life-threatening disease for which there is currently no curative treatment available. Pathologic changes in this disease involve remodeling of the pulmonary vasculature, including marked proliferation of pulmonary artery smooth muscle cells (PASMCs). Recently, the bioactive lipid sphingosine-1-phosphate (S1P) and its activating kinase, sphingosine kinase 1 (SphK1), have been shown to be up-regulated in PAH and promote PASMC proliferation. The mechanisms regulating the transcriptional upregulation of SphK1 in PASMCs are unknown. In this study, we investigated the role of platelet derived growth factor (PDGF), a PAH-relevant stimuli associated with enhanced PASMC proliferation, on SphK1 expression regulation. In human PASMCs (hPASMCs), PDGF significantly increased SphK1 mRNA and protein expression and induced cell proliferation. Selective inhibition of SphK1 attenuated PDGF-induced hPASMC proliferation. In silico promoter analysis for SphK1 identified several binding sites for early growth response protein 1 (Egr-1), a PDGF-associated transcription factor. Luciferase assays demonstrated that PDGF activates the SphK1 promoter in hPASMCs, and truncation of the 5' promoter reduced PDGF-induced SphK1 expression. Stimulation of hPASMCs with PDGF induced Egr-1 protein expression, and direct binding of Egr-1 to the SphK1 promoter was confirmed by chromatin immunoprecipitation analysis. Inhibition of ERK signaling prevented induction of Egr-1 by PDGF. Silencing of Egr-1 attenuated PDGF-induced SphK1 expression and hPASMC proliferation. These studies demonstrate that SphK1 is regulated by PDGF in hPASMCs via the transcription factor Egr-1, promoting cell proliferation. This novel mechanism of SphK1 regulation may be a therapeutic target in pulmonary vascular remodeling in PAH.
Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are the downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibitions of calpain-2 by using calpain inhibitor MDL28170 and calpain-2 siRNA attenuated Akt phosphorylations at serine-473 (S473) and threonine-308 (T308) as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylations at S473 and T308. Moreover, knockout of calpain attenuated Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable specific TGFβ receptor inhibitor SB431542 attenuated Akt phosphorylations at both S473 and T308 induced by PDGF and by overexpressed calpain-2 in PASMCs. Furthermore, SB-431452 and knocking down ALK5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGFβ1 using a cell-impermeable TGFβ1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308. Further, inhibition of mTORC2 by knocking down its component protein Rictor prevented Akt phosphorylations at S473 and T308 induced by PDGF and by overexpressed calpain-2. These data provide first evidence supporting that calpain-2 up-regulates PDGF-induced Akt phosphorylation in pulmonary vascular remodeling via an intracrine TGFβ1/mTORC2 mechanism.
Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA, lithocholic acid (LCA), stimulate Cl- secretion in human colonic T84 cells (AJP 305:C447-56, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50µM, 15') drastically (>90%) inhibited forskolin-stimulated short-circuit current (Isc), it did not alter carbachol -stimulated Isc. LCA did not alter basal Isc, transepithelial resistance, cell viability or cytotoxicity. LCA's inhibitory effect was dose-dependent, acted faster from the apical membrane, rapid and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues, 8Br-cAMP, lubiprostone or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA-specific, since CDCA, cholic acid or taurodeoxycholic acid did not alter forskolin or carbachol action. While LCA alone had no effect on [cAMP]i, it decreased forskolin-stimulated [cAMP]i by 90%. Although LCA caused a small increase in [Ca2+]i, chelation by BAPTA-AM did not reverse LCA's effect on Isc. LCA action does not appear to involve known BA receptors, FXR, VDR, M3 or TGR5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD98059. Surprisingly PD98059 did not reverse LCA's effect on Isc. Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical CFTR Cl- current, and inhibits a basolateral K+ current. In summary, 50µM LCA greatly inhibits cAMP-stimulated Cl- secretion, making low doses of LCA of potential therapeutic interest for diarrheal diseases.
Peripheral arterial disease (PAD) is a common circulatory disorder of the lower limb arteries that reduces functional capacity and quality of life of patients. Despite relatively effective available treatments, PAD is a serious public health issue associated with a significant morbidity and mortality. Ischemia-reperfusion cycles occurring during PAD are responsible for insufficient oxygen supply, mitochondriopathy, free radical production, inflammation and lead events that contribute to myocyte death and remote organ failures. However, the mitochondrial and cellular chronological events that occur during the ischemic period and at the moment of reperfusion have been poorly reviewed in skeletal muscle fibers. Thus, after a review of the basal myocyte state and the normal mitochondrial biology, we will discuss the physiopathology of ischemia and reperfusion from a mitochondrial and cellular point of view. We will first describe the chronology of the deleterious biochemical and mitochondrial mechanisms activated by ischemia-reperfusion. We will then develop skeletal muscle ischemia-reperfusion injury to the muscle environment, mitochondrial dynamics and inflammation. A better chronological understanding of the events underlying ischemia-reperfusion will allow us to identify key actors in the development of this pathology and point to new suitable therapies. Emerging data on mitochondrial dynamics should identify new molecular and therapeutic targets and develop protective strategies against PAD.
Nitration of both protein-bound and free tyrosine by reactive nitrogen species results in the formation of nitrotyrosine (NT). We previously reported that free NT impairs microtubule polymerization and uncouples endothelial nitric oxide synthase (eNOS) function in pulmonary artery endothelial cells (PAEC). Since microtubules modulate mitochondrial function, we hypothesized that increased NT levels during inflammation and oxidative stress will induce mitochondrial dysfunction in PAEC. PAEC isolated from fetal lambs were exposed to varying concentrations of NT. At low concentrations (1-10 µM), free NT increased nitration of mitochondrial electron transport chain (ETC) protein subunits complexes I-V and state III oxygen consumption. Higher concentrations of NT (50 µM) caused decreased microtubule acetylation, impaired eNOS interactions with mitochondria, and decreased ETC protein levels. We also observed increases in heat-shock-protein-90 (hsp90) nitration, mitochondrial superoxide formation, and fragmentation of mitochondria in PAEC. Our data suggest that free NT accumulation may impair microtubule polymerization and exacerbate reactive oxygen species-induced cell damage by causing mitochondrial dysfunction.
Periaxin, a PDZ domain protein expressed preferentially in myelinating Schwann cells and lens fibers, plays a key role in membrane scaffolding and cytoarchitecture. Little is known, however, about how periaxin (Prx) is anchored to the plasma membrane. Here we report that Ankyrin-B (AnkB), a well characterized adaptor protein involved in linking the spectrin-actin cytoskeleton to integral membrane proteins, is required for membrane association of Prx in lens fibers and colocalizes with Prx in hexagonal fiber cells. Under AnkB haploinsufficiency, Prx accumulates in the soluble fraction with a concomitant loss from the membrane-enriched fraction of mouse lenses. Moreover, AnkB haploinsufficiency-induced age-dependent disruptions in fiber cell hexagonal geometry and radial alignment, and decreased compressive stiffness in mouse lenses parallel the changes observed in Prx null mouse lens. Both AnkB and Prx deficient mice exhibit disruptions in membrane organization of the spectrin-actin network and the dystrophin-glycoprotein complex in lens fiber cells. Taken together, these observations reveal that AnkB is required for Prx membrane anchoring and for maintenance of lens fiber cell hexagonal geometry, membrane skeleton organization and biomechanics.
The extracellular calcium-sensing receptor, CaSR, is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative (wild-type, WT) and Cre-positive SM22αCaSRflox/flox (knock-out, KO) mice showed and endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared to WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca2+) concentrations (1-5 mM) evoked contraction in WT, but only relaxation in KO aortae. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared to WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either NO-synthase inhibition with L-NAME or by a high salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca2+ concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.
Arginylation is a posttranslational modification that plays a global role in mammals. Mice lacking the enzyme arginyltransferase in skeletal muscles exhibit reduced contractile forces that have been linked to a reduction in myosin cross-bridge formation. The role of arginylation in passive skeletal myofibril forces has never been investigated. In this study, we used single sarcomere and myofibril measurements and observed that lack of arginylation leads to a pronounced reduction in passive forces in skeletal muscles. Mass spectrometry indicated that skeletal muscle titin, the protein primarily linked to passive force generation, is arginylated on five sites located within the A-band, an important area for protein-protein interactions. We propose a model of passive force regulation by arginylation through modulation of protein-protein binding between the titin molecule and the thick filament.
Mechanical stretch can activate muscle and myotube protein synthesis through mTORC1 signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated if media containing cachectic factors derived from Lewis Lung Carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes pre-incubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMPK phosphorylation in myotubes independent of stretch. Both stretch and LLC independently increased ERK1/2, p38 and NF-B phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either LIF or gp130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMPK inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that gp130 signaling is associated with the basal stretch response in myotubes.
Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca2+ signaling and its effects on anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca2+] ([Ca2+]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca2+ eliminated the plateau phase increase of [Ca2+]cyt in lung cancer cells, indicating that the plateau phase of [Ca2+]cyt increase is due to Ca2+ influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Pro-apoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the anti-apoptotic protein Bcl-2 demonstrated more sensitivity to ATP than pro-apoptotic protein Bax. Decreasing extracellular Ca2+ or chelating intracellular Ca2+ with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca2+]cyt through Ca2+ influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression.
The effectiveness and stability of epithelial barrier depend on apical junctional complexes (AJCs), which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the post-transcriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HuR are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3'-untranslated region (3'-UTR) of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs, therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions.
Human airway epithelial cells express β-adrenergic receptors (β-AR) that regulate mucociliary clearance by stimulating transepithelial anion transport and ciliary beat frequency. Previous studies using airway epithelial cells showed that stimulation with isoproterenol increased cell migration and wound repair by a cAMP-dependent mechanism. In the present study, impedance sensing arrays were used to measure cell migration and epithelial restitution following wounding of confluent normal human bronchial epithelial (NHBE) cells and Calu-3 cells by electroporation. Stimulation with epinephrine or the β2-selective agonist salbutamol significantly delayed wound closure and reduced the mean surface area of lamellipodia protruding into the wound. Treatment with the β-AR bias agonist carvedilol or isoetharine also produced a delay in epithelial restitution similar in magnitude to epinephrine and salbutamol. Measurements of extracellular-signal-regulated kinase (ERK) phosphorylation following salbutamol or carvedilol stimulation showed no significant change in the level of phosphorylation compared to untreated control cells. However, inhibition of PP2A phosphatase activity completely blocked the delay in wound closure produced by β-agonists. In CFTR silenced Calu-3 cells, salbutamol did not inhibit wound repair, suggesting that β-agonist stimulation and loss of CFTR expression inhibit cell migration through a common pathway. Confocal images of the basal membrane of Calu-3 cells labeled with anti-β1 integrin antibody showed that treatment with epinephrine or carvedilol reduced the level of activated integrin in the membrane. These findings suggest that β-AR agonists delay airway epithelial repair by a G-protein and cAMP-independent mechanism involving the PP2A phosphatase and a reduction in β1 integrin activation in the basal membrane.
Background: Inflammation-dominated sympathetic sprouting adjacent to the necrotic region following myocardial infarction (MI) has been implicated in the etiology of arrhythmias resulting in sudden cardiac death; however, the mechanisms responsible remain to be elucidated. Although being a key immune mediator, the role of the Notch has yet to be explored. Objective: We investigated whether Notch regulates macrophage responses to inflammation and affects cardiac sympathetic reinnervation in rats undergoing MI. Methods and Results: MI was induced by coronary artery ligation. A high level of NICD was observed in the macrophages that infiltrated the infarct area at 3 days post-MI. The administration of the Notch inhibitor N-N-(3,5-difluorophenacetyl-l-alanyl)-S-phenylglycine-t-butyl ester (DAPT) (i.v. 30 minutes before MI and then daily until sacrifice) decreased the number of macrophages and significantly increased the M2 macrophage activation profile in the early stages as well as attenuated the expression of nerve growth factor (NGF). Eventually, NGF-induced sympathetic hyperinnervation was blunted, as assessed by the immunofluorescence of tyrosine hydroxylase (TH). At 7 days post-MI, the arrhythmia score of programmed electric stimulation in the vehicle-treated infarcted rats was higher than that in rats treated with DAPT. Further deterioration in cardiac function and decreases in the plasma levels of TNF-α and IL-1β were also detected. In vitro studies revealed that LPS/IFN- upregulated the surface expression of NGF in M1 macrophages in a Notch-dependent manner. Conclusions: Notch inhibition during the acute inflammatory response phase is associated with the downregulation of NGF, probably through a macrophage-dependent pathway, thus preventing the process of sympathetic hyperinnervation.
Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis type-I/III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of 6 pathological FLNB mutants that have been linked to skeletal abnormalities. The subfractionation assay showed a greater accumulation of the FLNB ABD mutants, W148R and E227K, to the cytoskeleton, compared with the wild type protein. Ectopic expression of FLNB-W148R, and the E227K protein with a lesser extent, induced formation of prominent F-actin accumulations, the consequent rearrangements of focal adhesions, myosin-II and septin filaments, and results in a delayed directional migration of the cells. The W148R protein-induced cytoskeletal re-arrangement was partially attenuated by the inhibition of myosin-II, PAK or ROCK. The expression of a single-head ABD fragment with the mutations partially mimicked the rearrangement induced by the dimer. The F-actin clustering through the interaction with the mutant FLNB ABD may limit the cytoskeletal reorganization preventing normal skeletal development.
The Na,K-ATPase α2 isoform in skeletal muscle is rapidly stimulated during muscle use and plays a critical role in fatigue resistance. The acute mechanisms which stimulate α2 activity are not completely known. This study examines whether phosphorylation of phospholemman (PLM/FXYD1), a regulatory subunit of Na,K-ATPase, plays a role in the acute stimulation of α2 in working muscles. Mice lacking PLM (PLM KO) have a normal content of α2 subunit and show normal exercise capacity, in contrast to the greatly reduced exercise capacity of mice which lack α2 in the skeletal muscles. In PLM KO mice, neither nerve-evoked contractions in vivo or fatiguing contractions of isolated muscles induces a change in total PLM or PLM phosphorylated at Ser63 or Ser68. Isolated muscles of PLM KO mice maintain contraction and resist fatigue as well as WT. Rb+ transport by the α2 Na,K-ATPase is stimulated to the same extent in contracting WT and contracting PLM KO muscles. Phosphorylation of sarcolemmal membranes prepared from WT but not PLM KO skeletal muscles stimulates the activity of both α1 and α2 in a PLM-dependent manner. The stimulation occurs by an increase in Na+ affinity without significant change in Vmax, and is more effective for α1 than α2. These results demonstrate that phosphorylation of PLM is capable of stimulating the activity of both isozymes in skeletal muscle; however, contractile activity alone is not sufficient to induce PLM phosphorylation. Importantly, acute stimulation of α2, sufficient to support exercise and oppose fatigue, does not require PLM or its phosphorylation.
Adenosine modulates different functional activities in many cells of the gastrointestinal tract; some of them are believed to be mediated by interaction with its four G-protein-coupled receptors. The renewed interest in the adenosine A2B receptor (A2BR) subtype can be traced by studies in which the introduction of new genetic and chemical tools has widened the pharmacological and structural knowledge of this receptor as well as its potential therapeutic use in cancer and inflammation- or hypoxia-related pathologies. In the acid-secreting parietal cells of the gastric mucosa, the use of various radioligands for adenosine receptors suggested the presence of the A2 adenosine receptor subtype(s) on the cell surface. Recently, we confirmed A2BR expression in native, non-transformed parietal cells at rest by using flow cytometry and confocal microscopy. In this study, we show that A2BR is functional in primary rabbit gastric parietal cells, as indicated by the fact that agonist binding to A2BR increased adenylate cyclase activity and acid production. In addition, both acid production and radioligand binding of adenosine analogues to isolated cell membranes were potently blocked by selective A2BR antagonists, whereas ligands for A1, A2A and A3 adenosine receptors failed to abolish activation. We conclude that rabbit gastric parietal cells possess functional A2BR proteins that are coupled to Gs and stimulate HCL production upon activation. Whether adenosine- and A2BR-mediated functional responses play a role in human gastric pathophysiology is yet to be elucidated.
Metastasis contributes to over 90% of cancer-related deaths, and is initiated when cancer cells detach from the primary tumor, invade the basement membrane and enter the circulation as circulating tumor cells (CTCs). While metastasis is viewed as an inefficient process with most CTCs dying within the bloodstream, it is evident that some CTCs are capable of resisting hemodynamic shear forces to form secondary tumors in distant tissues. We hypothesized that nuclear lamins A and C act as key structural components within tumor cells necessary to resist destruction from elevated shear forces of the bloodstream. Herein, we show that, compared to non-malignant epithelial cells, tumor cells are resistant to elevated fluid shear forces in vitro that mimic those within bloodstream, as evidenced by significant decreases in cellular apoptosis and necrosis. Upon knockdown of lamin A/C, tumor cell resistance to fluid shear stress was significantly reduced, with significantly increased cell death compared to parental tumor cell and non-targeting controls. Interestingly, lamin A/C knockdown increased shear stress-induced tumor cell apoptosis, but did not significantly affect cellular necrosis. These data demonstrate that lamin A/C is an important structural component that enables tumor cell resistance to fluid shear stress-mediated death in the bloodstream, and may thus facilitate survival and hematogenous metastasis of CTCs.
Obesity is associated with increased fibrinogen production and fibrin formation which produces fibrin degradation products (FDP-E and FDP-D). Fibrin and FDP's both contribute to inflammation which would be expected to suppress glucose uptake and insulin signaling in adipose tissue, yet the effect of FDP-E and FDP-D on adipocyte function and glucose disposal is completely unknown. We tested the effects of FDP's on inflammation in 3T3-L1 adipocytes and primary macrophages and adipocyte glucose uptake in vitro. High-fat fed mice increased hepatic fibrinogen mRNA expression 9-fold over chow-fed mice with concomitant increases in plasma fibrinogen protein levels. Obese mice also displayed increased fibrinogen content of epididymal fat pads. We treated cultured 3T3-L1 adipocytes and primary macrophages with FDP-E, FDP-D, or fibrinogen degradation products (FgnDP-E). FDP-D and FgnDP-E had no effect on inflammation or glucose uptake. Cytokine mRNA expression in RAW264.7 macrophage-like cells and 3T3-L1 adipocytes treated with FDP-E induced inflammation with maximal effects at 100nM and 6 hours. Insulin stimulated 3H-2-deoxy-D-glucose uptake was reduced by 71% in adipocytes treated with FDP-E. FDP-E, but not FDP-D or FgnDP-E induces inflammation in macrophages and adipocytes and decreases glucose uptake in in vitro. FDP-E may contribute toward obesity associated acute inflammation and glucose intolerance although its chronic role in obesity remains to be elucidated.
Enteropathogenic E. coli is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl-/HCO3- exchanger, SLC26A3 (DRA) contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. The current study investigated DRA trafficking under basal conditions and in response to EPEC utilizing Caco-2 cells. Apical Cl-/HCO3- exchange activity was measured as DIDS-sensitive 125I uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 μM) increased apical Cl-/HCO3- exchange activity. Dynasore, a dynamin inhibitor also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice administered colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent upon EPEC virulence genes espG1/G2. Intriguingly, EPEC induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis.
Unloading or disuse rapidly results in skeletal muscle atrophy, switching to fast type fibers, and decreased resistance to fatigue. The recovery process is of major importance in rehabilitation for varies clinical conditions. Here we studied mouse soleus muscle during 60 days of reloading after 4 weeks of hindlimb suspension. Unloading produced significant atrophy of soleus muscle with decreased contractile force and fatigue resistance, accompanied by switches of myosin isoforms from IIa to IIx and IIb, and fast troponin T to more low molecular weight splice forms. The total mass, fiber size and contractile force of soleus muscle recovered to control levels after 15 days of reloading. However, the fatigue resistance showed a trend of worsening during this period with significant infiltration of inflammatory cells at days 3 and 7, indicating reloading injuries that were accompanied by active regeneration with up-regulations of filamin-C, alpha B-crystallin and desmin. The fatigue resistance partially recovered after 30 to 60 days of reloading. The expression of peroxisome proliferator-activated receptor coactivator 1 α (PGC-1α) and mitofusin-2 showed changes parallel to that of fatigue resistance after unloading and during reloading, suggesting a causal role of decreased mitochondrial function. Slow fiber contents in the soleus muscle were increased after 30-60 days of reloading to become significantly higher than the normal level, indicating a secondary adaption to compensate for the slow recovery of fatigue resistance.
Oxidative stress or reduced expression of naturally occurring antioxidants during aging has been identified as a major culprit in the neuronal cells/tissue degeneration. Peroxiredoxin (Prdx) 6, a protective protein with GSH peroxidase and acidic calcium-independent phospholipase A2 activities, acts as a rheostat in regulating cellular physiology by clearing reactive oxygen species (ROS) and thereby optimizing gene regulation. We found that under stress, the neuronal cells displayed reduced expression of Prdx6 protein and mRNA with increased levels of ROS, and the cells subsequently underwent apoptosis. Using Prdx6 fused to TAT transduction domain, we showed evidence that Prdx6 was internalized in human brain cortical neuronal cells, HCN-2, and mouse hippocampal cells, HT22. The cells transduced with Prdx6 conferred resistance against oxidative stress inducers paraquat, H2O2 and glutamate. Furthermore, Prdx6 delivery ameliorated damage to neuronal cells by optimizing ROS levels and overstimulation of NF-B. Intriguingly, transduction of Prdx6 increased the expression of endogenous Prdx6, suggesting that protection against oxidative stress was mediated by both extrinsic and intrinsic Prdx6. The results demonstrate that Prdx6 expression is critical to protecting oxidative stress-evoked neuronal cell death. We propose that local or systemic application of Prdx6 can be effective means of delaying/postponing neuronal degeneration.
PO2 cycling, often referred to as intermittent hypoxia, involves exposing tissues to brief cycles of low oxygen environments immediately followed by hyperoxic conditions. After experiencing long-term hypoxia, muscle can be damaged during the subsequent reintroduction of oxygen, which leads to muscle dysfunction via reperfusion injury. The protective effect and mechanism behind PO2 cycling in skeletal muscle during reoxygenation have yet to be fully elucidated. We hypothesize that PO2 cycling effectively increases muscle fatigue resistance through reactive oxygen species (ROS), protein kinase B (Akt), extracellular signal-regulated kinase (ERK), and certain mitochondrial channels during reoxygenation. Using a dihydrofluorescein fluorescent probe, we detected the production of ROS in mouse diaphragmatic skeletal muscle in real time under confocal microscopy. Muscles treated with PO2 cycling displayed significantly attenuated ROS levels (n = 5; p < 0.001) as well as enhanced force generation when compared to controls during reperfusion (n = 7; p < 0.05). We also used inhibitors for signaling molecules or membrane channels such as ROS, protein kinase B (Akt), extracellular signal-regulated kinase (ERK), as well as chemical stimulators to close mitochondrial ATP-sensitive potassium channel (KATP) or open mitochondrial permeability transition pore (mPTP). All these blockers or stimulators abolished improved muscle function with PO2 cycling treatment. This current investigation has discovered a correlation between KATP and mPTP and the PO2 cycling pathway in diaphragmatic skeletal muscle. Thus, we have identified a unique signaling pathway which may involve ROS, Akt, ERK and mitochondrial channels responsible for PO2 cycling protection during reoxygenation conditions in the diaphragm.
Skeletal muscles present a non-crossbridge increase in sarcomere stiffness and tension upon Ca2+ activation, referred to as static stiffness and static tension, respectively. It has been hypothesized that this increase in tension is caused by Ca2+-dependent changes in the properties of titin molecules. To verify this hypothesis, we investigated the static tension in muscles containing different titin isoforms. Permeabilized myofibrils were isolated from the psoas, soleus and heart ventricle from the rabbit, and tested in pCa 9.0 and pCa 4.5, before and after extraction of troponin C, thin filaments and treatment with the acto-myosin inhibitor blebbistatin. The myofibrils were tested with stretches of different amplitudes in sarcomere lengths (SLs) varying between 1.93-3.37µm for the psoas, 2.68-4.21µm for the soleus, and 1.51µm-2.86µm for the ventricle. Using gel electrophoresis, we confirmed that the three muscles tested have different titin isoforms. The static tension was present in psoas and soleus myofibrils, but not in ventricle myofibrils, and higher in psoas myofibrils than in soleus myofibrils. These results suggest that the increase in the static tension is directly associated with Ca2+-dependent change in titin properties, and not associated with changes in titin-actin interactions.
The potassium channel Kv7.1 plays critical physiological roles in both heart and epithelial tissues. In heart, Kv7.1 and the accessory subunit KCNE1 forms the IKs current, which is enhanced by PKA mediated phosphorylation. The observed current increase requires both phosphorylation of Kv7.1 and the presence of KCNE1. However, PKA also stimulates Kv7.1 currents in epithelial tissues, such as colon, where the channel does not co-assemble with KCNE1. Here, we demonstrate that PKA activity significantly impacts the subcellular localization of Kv7.1 in Madin Darby Canine Kidney cells. While PKA inhibition reduced the fraction of channels at the cell surface, PKA activation increased it. We show that PKA inhibition lead to intracellular accumulation of Kv7.1 in late endosomes/lysosomes. By mass spectroscopy we identified eight phosphorylated residues on Kv7.1, however, none appeared to play a role in the observed response. Instead, we found that PKA acted by regulating endocytic trafficking involving the ubiquitin ligase Nedd4-2. We show that a Nedd4-2 resistant Kv7.1-mutant displayed significantly reduced intracellular accumulation upon PKA inhibition. Similar effects were observed upon siRNA knockdown of Nedd4-2. However, although Nedd4-2 is known to regulate Kv7.1 by ubiquitylation, biochemical analyses demonstrated that PKA did not influence the amount of Nedd4-2 bound to Kv7.1 or the ubiquitylation level of the channel. This suggests that PKA influences Nedd4-2-dependent Kv7.1 transport though a different molecular mechanism. In summary, we identify a novel mechanism whereby PKA can increase Kv7.1 current levels, namely by regulating Nedd4-2 dependent Kv7.1 transport.
Highly organized thick filaments in skeletal muscle cells are formed from ~300 myosin molecules. Each thick filament-associated myosin molecule is thought to be constantly exchanged. However, the mechanism of myosin replacement remains unclear, as does the source of myosin for substitution. Here, we investigated the dynamics of myosin exchange in the myofibrils of cultured myotubes by fluorescent recovery after photobleaching (FRAP), and found that myofibrillar myosin is actively replaced with an exchange half-life (t1/2) of ~3 h. Myosin replacement was not disrupted by the absence of the microtubule system or by actomyosin interactions, suggesting that known cytoskeletal systems are dispensable for myosin substitution. Intriguingly, myosin replacement was independent of myosin binding protein C (Mybpc), which links myosin molecules together to form thick filaments. This implies that an individual myosin molecule rather than a thick filament functions as an exchange unit. Furthermore, the myosin substitution rate was decreased by the inhibition of protein synthesis, suggesting that newly synthesized myosin as well as pre-existing cytosolic myosin contributes to myosin replacement in myofibrils. Notably, incorporation and release of myosin occurred simultaneously in myofibrils, but rapid myosin release from myofibrils was observed without protein synthesis. Collectively, our results indicate that myosin shuttles between myofibrils and the non-myofibrillar cytosol to maintain a dynamic equilibrium in skeletal muscle cells.
In this study, we characterized ammonia and ammonium (NH3/NH4+) transport by the Rhesus Associated Glycoproteins RhAG, Rhbg and Rhcg expressed in Xenopus oocytes. To do so we used ion-selective microelectrodes & two-electrode voltage clamp to measure changes in intracellular pH (pHi), surface pH (pHs) and whole cell currents (I) induced by NH3/NH4+ and methyl amine/ammonium (MA/MA+). These measurements allowed us to define signal-specific signatures to distinguish NH3 from NH4+ transport and to determine how transport of NH3 and NH4+ differ among RhAG, Rhbg and Rhcg. Our data indicated that expressing Rh glycoproteins in oocytes generally enhanced NH3/NH4+ transport and that transport of MA/MA+ by Rh proteins induced different cellular changes than transport of NH3/NH4+. Our results support the following conclusions: 1) RhAG and Rhbg transport both the ionic NH4+ and neutral NH3 species. 2) Transport of NH4+ is electrogenic. 3) Like Rhbg, RhAG transport of NH4+ masks NH3 transport. 4) Rhcg is likely to be a predominantly NH3 transporter with no evidence of enhanced NH4+ transport by this transporter. The dual role of Rh proteins as NH3 and NH4+ transporters is a unique property and may be critical in understanding how transepithelial secretion of NH3/NH4+ occurs in the renal collecting duct.
The lymphatics have emerged as critical players both in the progression and resolution of inflammation. The goal of this study was to identify specific microRNAs (miRNAs) that regulate lymphatic inflammatory processes. Rat mesenteric lymphatic endothelial cells (LECs) were exposed to the proinflammatory cytokine tumor necrosis factor-α (TNF-α) for different time points (2hr, 24hr and 96hr) and miRNA profiling was carried out by Real time PCR arrays. Our data demonstrate a specific set of miRNAs that are differentially expressed (>1.8 fold and/or p<0.05) in LECs in response to TNF-α and are involved in inflammation, angiogenesis, endothelial to mesenchymal transition (EndMT), cellular proliferation and senescence. We further characterized the expression of miR-9 that was induced in LECs and in inflamed rat mesenteric lymphatics. Our results showed that miR-9 overexpression, significantly repressed NF-B expression, thereby suppressing inflammation but promoted LEC tube formation as well as the expression of the pro-lymphangiogenic molecules eNOS and VEGFR3. LEC viability, proliferation and EndMT were also significantly induced by miR-9. This study provides the first evidence of a distinct profile of miRNAs associated with LECs during inflammation. It also identifies the critical dual role of miR-9 in fine-tuning the balance between lymphatic inflammatory and lymphangiogenic pathways.
Myostatin (MSTN) is a key negative regulator of muscle growth and development, and an increase of muscle mass is achieved by inhibiting MSTN signaling. In the current study, five alternative splicing isoforms of MSTN mRNAs in avian species were identified in various tissues. Among these five, three truncated forms of myostatin, MSTN-B, -C, and -E created premature stop codons and produced partial MSTN prodomains encoded from exon 1. MSTN-B is the second dominant isoform following full-length MSTN-A, and their expression was dynamically regulated during muscle development of chicken, turkey, and quail in vivo and in vitro. To clarify the function of MSTN-B, two stable cell lines of quail myoblasts (QM7) were generated to overexpress MSTN-A or MSTN-B. Interestingly, MSTN-B promoted both cell proliferation and differentiation similar to the function of the MSTN prodomain to counteract the negative role of MSTN on myogenesis. The co-immunoprecipitation assay revealed that MSTN-B binds to MSTN-A and reduces the generation of mature MSTN. Furthermore, the current study demonstrated that the partial prodomain encoded from exon 1 is critical for binding of MSTN-B to MSTN-A. Altogether, these data imply that alternative splicing isoforms of MSTN could negatively regulate pro-myostatin processing in muscle cells and prevent MSTN-mediated inhibition of myogenesis in avian species.
The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out 'deep' proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 GB of spectral data. As a result, we identified 6766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over 8 orders of magnitude of protein abundance. The data are provided to users as a public database (https://helixweb.nih.gov/ESBL/Database/mpkFractions/. The MS data were mapped back to their gel slices to generate 'virtual western blots' for each protein. For most of the 6766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, either with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, post-translational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular weight <20 kD.
Vasopressin controls osmotic water transport in the renal collecting duct through regulation of aquaporin-2. We carried out bioinformatic analysis of quantitative proteomic data from the accompanying paper to investigate mechanisms involved. The experiments used SILAC (Stable Isotope Labeling by Amino acids in Cell culture) in cultured mpkCCD cells to quantify each protein species in each of 5 differential-centrifugation (DC) fractions with or without the vasopressin analog (dDAVP). The mass spectrometry data and parallel western blotting experiments confirmed that dDAVP addition is associated with an increase of aquaporin-2 abundance in the 17,000 Xg pellet and a corresponding decrease in the 200,000 Xg pellet. Remarkably, all subunits of the cytoplasmic ribosome also increased in the 17,000 Xg pellet in response to dDAVP (P<10-34) with a concomitant decrease in the 200,000 Xg pellet. Eukaryotic translation initiation complex 3 (eIF-3) subunits underwent parallel changes (P<10-6). These findings are consistent with translocation of assembled ribosomes and eIF-3 complexes into the rough endoplasmic reticulum in response to dDAVP. Conversely, there was a systematic decrease in small GTPase abundances in the 17,000 Xg fraction. In contrast, most proteins showed no systematic redistribution among DC fractions including protein kinases. 246 of the 521 protein kinases coded by the mouse genome were identified, but many fewer were found to colocalize with aquaporin-2 among DC fractions. Bayes' Rule was used to integrate the new colocalization data with prior data in order to identify protein kinases most likely to phosphorylate aquaporin-2 at Ser256 (Camk2b>Camk2d>Prkaca) and Ser261 (Mapk1=Mapk3>Mapk14).
Obesity, a known risk factor for pancreatic cancer, is associated with inflammation and insulin resistance. Pro-inflammatory PGE2, and elevated IGF-1 related to insulin resistance, are both shown to play critical roles in pancreatic cancer progression. We aimed at exploring a potential crosstalk between the PGE2 signaling and IGF-1/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway in pancreatic cancer, which may be a key to unraveling the obesity-cancer link. In PANC-1 human pancreatic cancer cells, we showed that PGE2 stimulated mTORC1 activity independently of Akt, as evaluated by downstream signaling events. Subsequently using pharmacological and genetic approaches, we demonstrated that PGE2-induced mTORC1 activation is mediated by EP4/cAMP/PKA, as well as an EP1/Ca2+-dependent pathway. The cooperative roles of the two pathways were supported by the maximal inhibition achieved with the combined pharmacological blockade, and the co-existence of highly expressed EP1 (mediating Ca2+ response) and EP2 or 4 (mediating cAMP/PKA pathway) in PANC-1 and a prostate cancer line PC-3, which also robustly exhibited PGE2-induced mTORC1 activation, as identified from a screen in various cancer cell lines. Importantly, we showed a reinforcing interaction between PGE2 and IGF-1 on mTORC1 signaling, with an increased IL-23 production as a cellular outcome. Together, our data reveal a previously unrecognized mechanism of PGE2-stimulated mTORC1 activation mediated by EP4/cAMP/PKA and EP1/Ca2+ signaling, which may be of great importance in elucidating the promoting effects of obesity in pancreatic cancer. Ultimately, a precise understanding of these molecular links may provide novel targets for efficacious interventions devoid of adverse effects.
Menkes disease is a fatal neurodegenerative disorder arising from a systemic copper deficiency caused by loss-of-function mutations in a ubiquitously expressed copper transporter, ATP7A. Although this disorder reveals an essential role for copper in the developing human nervous system, the role of ATP7A in the pathogenesis of signs and symptoms in affected patients, including severe mental retardation, ataxia and excitotoxic seizures, remains unknown. To directly examine the role of ATP7A within the central nervous system, we generated Atp7aNes mice in which the Atp7a gene was specifically deleted within neural- and glial cell precursors without impairing systemic copper homeostasis, and compared these mice to the Brindled mutant (mo-br), a murine model of Menkes disease in which Atp7a is defective in all cells. Whereas mo-br mice displayed neurodegeneration, demyelination and 100% mortality prior to weaning, the Atp7aNes mice showed none of these phenotypes, exhibiting only mild sensorimotor deficits, increased anxiety and susceptibility to NMDA-induced seizure. Our results indicate that the pathophysiology of severe neurological signs and symptoms in Menkes disease are the result of copper deficiency within the central nervous system secondary to impaired systemic copper homeostasis, and do not arise from an intrinsic lack of ATP7A within the developing brain. Furthermore the sensorimotor deficits, hypophagia, anxiety, and sensitivity to NMDA-induced seizure in the Atp7aNes mice reveal unique autonomous requirements for ATP7A in the nervous system. Taken together these data reveal essential roles for copper acquisition into the central nervous system in early development and suggest novel therapeutic approaches in affected patients.
Activation of β-PDGFR (platelet-derived growth factor receptor) is associated with prostate cancer (PCa) progression and recurrence after prostatectomy. Analysis of the β-PDGFR ligands in PCa revealed association between PDGF-D expression and Gleason score as well as tumor stage. During the course of studying the functional consequences of PDGF ligand-specific β-PDGFR signaling in PCa, we discovered a novel function of PDGF-D for activation/shedding of the serine protease matriptase leading to cell invasion, migration, and tumorigenesis. The present study showed that PDGF-D, not PDGF-B, induces extracellular acidification which correlates with increased matriptase activation. A cDNA microarray analysis revealed that PDGF-D/β-PDGFR signaling upregulates expression of the acidosis regulator carbonic anhydrase IX (CAIX), a classic target of the transcriptional factor HIF-1α. Cellular fractionation displayed a strong HIF-1α nuclear localization in PDGF-D expressing cells. Treatment of vector control or PDGF-B expressing cells with the HIF-1α activator, CoCl2 led to increased CAIX expression accompanied by extracellular acidosis and matriptase activation. Furthermore, the analysis of the CAFTD cell lines, variants of the BPH-1 transformation model, showed that increased PDGF-D expression is associated with enhanced HIF-1α activity, CAIX induction, cellular acidosis and matriptase shedding. Importantly, shRNA-mediated knockdown of CAIX expression effectively reversed extracellular acidosis and matriptase activation in PDGF-D transfected BPH-1 cells and in CAFTD variants that express endogenous PDGF-D at a high level. Taken together, these novel findings reveal a new paradigm in matriptase activation involving PDGF-D-specific signal transduction leading to extracellular acidosis.
Understanding differences in gene expression that increase risk for pulmonary arterial hypertension (PAH) is essential to understanding the molecular basis for disease. Previous studies on patient samples were limited by either end-stage disease effects or by use of non-adherent cells which are not ideal to model vascular cells in vivo. These studies addressed the hypothesis that pathological processes associated with PAH may be identified via a genetic signature common across multiple cell types. Expression array experiments were initially conducted analyzing cell types at different stages of vascular differentiation (mesenchymal stromal and endothelial) derived from PAH patient specific induced pluripotent stem cells (iPS cells). Molecular pathways which were altered in the PAH cell lines were then compared to fibroblasts from 21 patients, including both idiopathic and heritable PAH. Wnt was identified as a target pathway and validated in vitro using primary patient mesenchymal and endothelial cells. Taken together, our data suggest that the molecular lesions that cause PAH are present in all cell types evaluated regardless of origin and that stimulation of the Wnt signaling pathway was a common molecular defect in both heritable and idiopathic PAH.
The calcium sensing receptor (CaSR) has played an important role as a target in the treatment of a variety of disease states over the last 20 plus years. In this review, we give an overview of the receptor at the cellular level and then provide details as to how this receptor has been targeted to modulate cellular ion transport mechanisms. As a member of the G protein-coupled receptors family, it has a high degree of homology with a variety of other members in this class, which could explain why this receptor has been identified in so many different tissues throughout the body. This diversity of locations throughout the body sets it apart from other members of the family, and may explain how the receptor interacts with so many different organ systems in the body to modulate the physiology and pathophysiology. The receptor is unique in that it has two large exofacial lobes that sit in the extracellular environment and sense changes in a wide variety of environmental cues including: salinity, pH, amino acids concentration, polyamines to name just a few. It is for this reason that there has been a great deal of research associated with normal receptor physiology over the last 20 years. With the ongoing research in more recent years a focus on the pathophysiology has emerged and the effects of receptor mutations on cellular and organ physiology. We hope that this review will enhance and update the knowledge about the importance of this receptor and stimulate future potential investigations focused around this receptor in cellular, organ, and systemic physiology and pathophysiology.
Skeletal muscle atrophy occurs in response to a variety of conditions including chronic kidney disease, diabetes, cancer, and elevated glucocorticoids. MicroRNAs (miR) may play a role in the wasting process. Activation of the Forkhead box O3 (FoxO3) transcription factor causes skeletal muscle atrophy in patients, animals, and cultured cells by increasing the expression of components of the ubiquitin-proteasome and autophagy-lysosome proteolytic systems. To identify microRNAs that potentially modulate the atrophy process, an in silico target analysis was performed and miR-182 was predicted to target FoxO3 mRNA. Using a combination of immunoblot analysis, qPCR, and FoxO3 3'-UTR luciferase reporter genes, miR-182 was confirmed to regulate FoxO3 expression in C2C12 myotubes. Transfection of miR-182 into muscle cells decreased FoxO3 mRNA 30% and FoxO3 protein 67% (P<0.05) and also prevented a glucocorticoid-induced upregulation of multiple FoxO3 gene targets including MAFbx/Atrogin-1, ATG12, Cathepsin L, and LC3. Treatment of C2C12 myotubes with dexamethasone (Dex) (1 µM, 6 h) to induce muscle atrophy decreased miR-182 expression by 63% (P<0.05). Similarly, miR-182 was decreased 44% (P<0.05) in the gastrocnemius muscle of rats injected with streptozotocin to induce diabetes compared to controls. Finally, miR-182 was present in exosomes isolated from the media of C2C12 myotubes and Dex increased its abundance. These data identify miR-182 as an important regulator of FoxO3 expression that participates in the control of atrophy-inducing genes during catabolic diseases.
Enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium are attaching-and-effacing (A/E) pathogens that cause intestinal inflammation and diarrhea. The bacteria adhere to the intestinal epithelium, destroy microvilli and induce actin-filled membranous pedestals, but do not invade into the mucosa. Adherence leads to activation of several host cell kinases, including FYN, n-SRC, YES, ABL and ARG, phosphorylation of the bacterial translocated intimin receptor, and actin polymerization and pedestal formation in cultured cells. However, marked functional redundancy appears to exist between kinases, and their physiological importance in A/E pathogen infections has remained unclear. To address this question, we employed a novel dynamic in vitro infection model that mimics transient and short-term interactions in the intestinal tract. Screening of a kinase inhibitor library and RNA interference experiments in vitro revealed that ABL and PDGF receptor kinases, as well as p38 MAP kinase have unique, indispensable roles in early attachment of EPEC to epithelial cells under dynamic infection conditions. Studies with mutant EPEC showed that the attachment functions of ABL and PDGFR were independent of the intimin receptor, but required bacterial bundle-forming pili. Furthermore, inhibition of ABL and PDGFR with imatinib protected against infection of mice with modest loads of C. rodentium, whereas the kinases were dispensable for high inocula or late after infection. These results indicate that ABL and PDGFR have indispensable roles in early A/E pathogen attachment to intestinal epithelial cells and for in vivo infection with limiting inocula, but are not required for late intimate bacterial attachment or high inoculum infections.
N-Acyl-homoserine lactones (AHL) are quorum-sensing molecules in bacteria that play important roles in regulating virulence gene expression in pathogens such as Pseudomonas aeruginosa. The present study compared responses between undifferentiated and differentiated Caco-2 cells to N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL). A low concentration of 3-oxo-C12-HSL (30 μM) is sufficient to reduce viability accompanied by apoptosis via the suppression of phosphorylation by Akt in undifferentiated Caco-2 cells. The suppression of Akt phosphorylation appears specific in 3-oxo-C12-HSL, because other AHLs did not influence the phosphorylation status of Akt. The reduced viability induced by 3-oxo-C12-HSL was partially recovered by constitutively active Akt overexpression in undifferentiated Caco-2 cells. Since mucin is considered a vital component of the gut barrier, we investigated whether mucin protects cellular functions induced by 3-oxo-C12-HSL in undifferentiated Caco-2 cells. The results showed that mucin protected undifferentiated Caco-2 cells from apoptosis induced by 3-oxo-C12-HSL. 3-Oxo-C12-HSL did not induce cell death in differentiated Caco-2 cells that expressed higher levels of mucin 3 (MUC3) than undifferentiated Caco-2 cells. In addition, 3-oxo-C12-HSL promoted cell death in undifferentiated Caco-2 cells transfected with MUC3 siRNA and reduced MUC3 expression in undifferentiated Caco-2 cells. Therefore, MUC3 might be responsible for the survival of undifferentiated intestinal epithelial cells in the presence of 3-oxo-C12-HSL through regulating Akt phosphorylation. In conclusion, 3-oxo-C12-HSL might influence the survival of undifferentiated intestinal epithelial cells as well as interactions between these cells and pathogens.
We tested the hypothesis that non-weightbearing-induced muscle weakness (i.e. specific force) results from decreases in myosin protein quantity (i.e., myosin content per half-sarcomere and the ratio of myosin to actin) and quality (i.e., force per half-sarcomere and population of myosin heads in the strong-binding state during muscle contraction) in single myosin heavy chain (MHC) type II fibers. Fisher-344 rats were assigned to weightbearing control (CON) or non-weightbearing (NWB). The NWB rats were hindlimb unloaded for two weeks. Diameter, force, and MHC content were determined in permeabilized single fibers from the semimembranosus (SM) muscle. MHC isoform and the ratio of MHC to actin in each fiber were determined by gel electrophoresis and silver staining techniques. The structural distribution of myosin from spin-labeled fiber bundles during maximal isometric contraction was evaluated using electron paramagnetic resonance (EPR) spectroscopy. Specific force (peak force per cross sectional area) in MHC type IIB and IIXB fibers from NWB was significantly reduced by 38% and 18%, respectively. MHC content per half-sarcomere was significantly reduced by 21%. Two weeks of hindlimb unloading resulted in a reduced force per half-sarcomere by 52% and fraction of myosin strong-binding during contraction by 34%. The results suggest that reduced myosin and actin content (quantity) and myosin quality concomitantly contribute to non-weightbearing-related muscle weakness.
Inflammation-induced vascular endothelial dysfunction can allow plasma proteins to cross the vascular wall causing edema. Proteins may traverse the vascular wall through two main paracellular and transcellular transport pathways. Paracellular transport involves changes in endothelial cell (EC) junction proteins, while transcellular transport involves caveolar transcytosis. Since both processes are associated with filamentous actin formation the two pathways are interconnected. Therefore, it is difficult to differentiate the prevailing role of one or the other pathways during various pathologies causing an increase in vascular permeability. Using a newly developed dual-tracer probing method, we differentiated transcellular from paracellular transport during conditions of increased content of fibrinogen (Fg), called hyperfibrinogenemia (HFg). Roles of cholesterol and sphingolipids in formation of functional caveolae were assessed using a cholesterol chelator, methyl-beta-cyclodextrin (MβCD) and the de novo sphingolipid synthesis inhibitor myriocin. Fg-induced formation of functional caveolae was defined by association and co-localization of sodium-potassium ATPase (Na+/K+-ATPase) and plasmalemmal vesicle-associated protein-1 (PV-1) using Försters' Resonance Energy Transfer (FRET) and Total Internal Reflection Fluorescence (TIRF) microscopies, respectively. HFg increased permeability of the EC layer mainly through the transcellular pathway. While MβCD blocked Fg-increased transcellular and paracellular transports, myriocin affected only the transcellular transport. Pial venular leakage of albumin was lesser in myriocin-treated HFg mice. HFg induced greater formation of functional caveolae as indicated by co-localization of Na+/K+-ATPase with PV-1 by FRET and TIRF. Our results suggest that elevated blood levels of Fg alter cerebrovascular permeability mainly by affecting caveolae-mediated transcytosis through modulation of de novo sphingolipid synthesis.
Thyroid hormones L-thyroxine (T4) and 3,5,3'-triiodo-L-thyronine (T3) have been shown to initiate short- and long-term effects via a plasma membrane receptor site located on integrin αvβ3. Also insulin-like growth factor-1 (IGF-1) activity is known to be subject to regulation by this integrin. To investigate the possible cross-talk between T4 and IGF-1 in rat L6 myoblasts, we have examined integrin αvβ3-mediated modulatory actions of T4 on glucose uptake, measured through carrier-mediated 2-deoxy-[3H]-D-glucose uptake, and on cell proliferation stimulated by IGF-1, assessed by cell counting, [3H]-thymidine incorporation and FACS analysis. IGF-1 stimulated glucose transport and cell proliferation via the cell surface IGF-1 receptor (IGF1R) and, downstream of the receptor, by the phosphatidylinositol 3-kinase signal transduction pathway. Addition of 0.1 nM free T4 caused little or no cell proliferation, but prevented both glucose uptake and proliferative actions of IGF1. These actions of T4 were mediated by an Arg-Gly-Asp (RGD)-sensitive pathway, suggesting the existence of crosstalk between IGF1R and the T4 receptor located near the RGD recognition site on the integrin. An RGD-sequence-containing integrin inhibitor, a monoclonal antibody to αvβ3, and the T4 metabolite tetraiodothyroacetic acid all blocked the inhibition by T4 of IGF-1-stimulated glucose uptake and cell proliferation. Western blotting confirmed roles for activated phosphatidylinositol 3-kinase and extracellular regulated kinase 1/2 (ERK1/2) in the effects of IGF-1, and also showed a role for ERK1/2 in the actions of T4 that modified the effects of IGF-1. We conclude that thyroid hormone inhibits IGF-1-stimulated glucose uptake and cell proliferation in L6 myoblasts.
Tissue growth and function depend on vascularization, and vascular insufficiency or excess exacerbates many human diseases. Identifying the biological processes involved in angiogenesis will dictate strategies to modulate reduced or excessive vessel formation. Here, we examine the essential role of pericytes. Their heterogeneous morphology, distribution, origins, and physiology have been described. Using double transgenic Nestin-GFP/NG2-DsRed mice, we had identified two pericyte subsets. In this work, we found that Nestin-GFP-/NG2-DsRed+ (type-1) and Nestin-GFP+/NG2-DsRed+ (type-2) attach to the walls of small and large blood vessels in vivo, and in vitro, type-2, but not type-1, spark endothelial cells to form new vessels. Matrigel assay showed that only type-2 pericytes participate in normal angiogenesis. Moreover, when cancer cells were transplanted into Nestin-GFP/NG2-DsRed mice, type-1 pericytes did not penetrate the tumor, while type-2 were recruited during its angiogenesis. As inhibiting angiogenesis is a promising strategy in cancer therapy, type-2 pericytes may provide a cellular target susceptible to signaling and pharmacological manipulation in treating malignancy. This work also reports the potential of type-2 pericytes to improve blood perfusion in ischemic hindlimbs, indicating their potential for treating ischemic illnesses.
Vasoactive Intestinal Peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating CFTR-dependent chloride secretion that contributes to the airways innate defense mechanism. We had previously reported that prolonged VIP stimulation of VPAC1 receptor in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we have identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in-situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its co-localization and interaction with the scaffolding protein NHERF1, a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by PKC, of the actin-binding protein complex ERM (Ezrin-Radiaxin-Moesin) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR co-localization and interaction with CAL (CFTR Associated ligand), another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.
NHE3 is a brush border (BB) Na+/H+ antiporter which accounts for the majority of physiologic small intestinal and renal Na+ absorption. It is regulated physiologically and in disease via changes in endocytosis/exocytosis. Paradoxically, NHE3 is fixed to the microvillar (MV) actin cytoskeleton and has little basal mobility.. This fixation requires NHE3 binding to the multi-PDZ domain scaffold proteins NHERF1 and NHERF2 and to ezrin. Coordinated release of NHE3 from the MV cytoskeleton has been demonstrated during both stimulation and inhibition of NHE3. However, the signaling molecules involved in coordinating NHE3 trafficking and cytoskeletal association have not been identified. This question was addressed by studying LPA stimulation of NHE3 in polarized renal proximal tubule OK cells which occurs via apical LPA5 receptors and is NHERF2 dependent and mediated by EGFR, Rho/ROCK and ERK. NHE3 activity was determined by BCECF/fluorometry and NHE3 microvillar mobility by FRAP/confocal microscopy using NHE3-EGFP. Apical LPA (3µM)/LPA5R stimulated NHE3 activity, increased NHE3 mobility, and decreased the NHE3/NHERF2 association. The LPA stimulation of NHE3 was also PKC dependent. PKC was necessary for LPA stimulation of NHE3 mobility and NHE3/NHERF2 association. Moreover, the LPA induced translocation to the membrane of PKC was both ERK and phospholipase C dependent with ERK acting upstream of PLC. We conclude that LPA stimulation of NHE3 exocytosis includes a signaling pathway that regulates fixation of NHE3 to the MV cytoskeleton. This involves a signaling module consisting of ERK-PLC-PKCwhich dynamically and reversibly releases NHE3 from NHERF2 to contribute to the changes in NHE3 MV mobility
We have previously shown that ischemic preconditioning (IPC) protection against necrosis in whole hearts and both fresh and cultured cardiomyocytes, as well as improved regulatory volume decrease (RVD) to hypo-osmotic swelling in cardiomyocytes, are abrogated through Cl- channel blockade, pointing to a role for enhanced cell volume regulation in IPC. To further define this cardioprotective mechanism, cultured rabbit ventricular cardiomyocytes were preconditioned either by 10-minute simulated ischemia (SI) followed by 10-minute simulated reperfusion (SR), by 10-minute exposure/10-minute washout of remote ischemic preconditioning (rIPC) plasma dialysate (from rabbits subjected to repetitive limb ischemia), or by adenoviral transfection with the constitutively active PKC gene. These interventions were done prior to subjecting the cardiomyocytes to either 60-minute or 75-minute SI/60-minute SR to assess cell necrosis (by trypan blue staining), 30-minute SI to assess ischemic cell swelling, or 30-minute hypo-osmotic (200 mOsm) stress (HS) to assess cell volume regulation. Necrosis after SI/SR and both the SI- and HS-induced swelling was reduced in preconditioned cardiomyocytes compared to control cardiomyocytes (neither preconditioned nor transfected). These effects on necrosis and cell swelling were blocked by either Cl- channel blockade or dominant-negative knockdown of IK1 channels with adenoviruses, suggesting that Cl- and K+ movement across the sarcolemma are critical for cell volume regulation and thereby cell survival under hypoxic/ischemic conditions. Our results define enhanced cell volume regulation as a key common mechanism of cardioprotection by preconditioning in cardiomyocytes.
In the central nervous system, L-type voltage gated calcium channels (LTCCs) come in two isoforms, namely Cav1.2 and Cav1.3 channels. It has been shown previously that these channels differ in biophysical properties, subcellular localization and in the coupling to the gene transcription machinery. In previous work on rat hippocampal neurons we have identified an excitatory cation conductance and an inhibitory potassium conductance as important LTCC coupling partners. Notably, a stimulus-dependent interplay of LTCC-mediated Ca2+ influx and activation of these Ca2+-dependent conductances was found to give rise to characteristic voltage responses. However, the contribution of Cav1.2 and Cav1.3 to these voltage responses remained unknown. Hence, the relative contribution of the LTCC isoforms therein was the focus of the current study on hippocampal neurons derived from genetically modified mice, which either lack a LTCC isoform (Cav1.3 knock-out mice) or express a dihydropyridine-insensitive LTCC isoform (Cav1.2DHP-- knock-in mice). We identified common and alternate ion channel couplings of Cav1.2 and Cav1.3 channels. Whereas hyperpolarizing Ca2+-dependent conductances were coupled to both Cav1.2 and Cav1.3 channels, an afterdepolarizing potential was only induced by the activity of Cav1.2 channels. Unexpectedly, the activity of Cav1.2 channels was found at relatively hyperpolarized membrane voltages. Our data add important information about the differences between Cav1.2 and Cav1.3 channels, that furthers our understanding of the physiological and pathophysiological neuronal roles of these calcium channels. Moreover, our findings suggest that Cav1.3 knock-out mice together with Cav1.2DHP-- knock-in mice provide valuable models for future investigation of hippocampal LTCC-dependent afterdepolarizations.
Vascular Gqα signaling has been shown to contribute to cardiac hypertrophy. In addition, angiotensin II (Ang II) was shown to induce VSMC hypertrophy through Gqα signaling, however, the studies on the role of Gqα and PLCβ1 proteins in VSMC hypertrophy in animal model are lacking. The present study was therefore undertaken to examine the role of Gqα/PLCβ1 proteins and the signaling pathways in VSMC hypertrophy using spontaneously hypertensive rats (SHR). VSMC from 16 week-old SHR and not from 12 week-old SHR exhibited enhanced levels of Gqα/PLCβ1 proteins as compared to age-matched Wistar-Kyoto (WKY) rats as determined by Western blotting. However, protein synthesis as determined by 3[H]leucine incorporation was significantly enhanced in VSMC from both 12 and 16 week-old SHR as compared to VSMC from age-matched WKY rats. Furthermore, the knockdown of Gqα/PLCβ1 in VSMC from 16 week-old SHR by antisense and siRNA resulted in attenuation of protein synthesis. In addition, the enhanced expression of Gqα/PLCβ1 proteins, enhanced phosphorylation of ERK1/2 and enhanced protein synthesis in VSMC from SHR were attenuated by Ang II AT1 and endothelin-1 (ET-1) ETA receptor antagonists, losartan and BQ123 respectively but not by ETB receptor antagonist, BQ788. In addition, PD98059 decreased the enhanced expression of Gqα/PLCβ1 and protein synthesis in VSMC from SHR. These results suggest that the enhanced levels of endogenous Ang II and ET-1 through the activation of AT1 and ETA receptors respectively and MAP kinase signaling, enhanced the expression of Gqα/PLCβ1 proteins in VSMC from 16 week-old SHR and result in VSMC hypertrophy.
Slower, more oxidative muscle fibers are more resistant to the dystrophic pathology in Duchenne muscular dystrophy (DMD) patients as well as in the pre-clinical mdx mouse model of DMD. Therefore, one therapeutic strategy for DMD focuses on promoting expression of the slow, oxidative myogenic program. In the current study, we explored the therapeutic potential of stimulating the slow, oxidative phenotype in mdx mice by feeding 6 week-old animals with the natural phenol resveratrol (RSV; ~100 mg/kg/day) for 6 weeks. SIRT1 activity and protein levels increased significantly, as well as PGC-1α activity, in the absence of alterations in AMPK signaling. These adaptations occurred concomitant with evidence of a fast, glycolytic, to slower, more oxidative fiber type conversion, including mitochondrial biogenesis and increased expression of slower myosin heavy chain isoforms. These positive findings raised the question of whether increased exposure to RSV would result in greater therapeutic benefits. We discovered that an elevated RSV dose of ~500 mg/kg/day across a duration of 12 weeks was clearly less effective at muscle remodeling in mdx mice. This treatment protocol failed to influence SIRT1 or AMPK signaling and did not result in a shift towards a slower, more oxidative phenotype. Taken together, this study demonstrates that RSV can stimulate SIRT1 and PGC-1α activation, which in turn may promote expression of the slow, oxidative myogenic program in mdx mouse muscle. The data also highlight the importance of selecting an appropriate dosage regimen of RSV to maximize its potential therapeutic effectiveness for future application in DMD patients.
Hyposalivation resulting from salivary gland dysfunction leads to poor oral health and greatly reduces the quality of life of patients. Current treatments for hyposalivation are limited. However, regenerative medicine to replace dysfunctional salivary glands represents a revolutionary approach. The ability of dispersed salivary epithelial cells or salivary gland-derived progenitor cells to self-organize into acinar-like spheres or branching structures that mimic the native tissue holds promise for cell-based reconstitution of a functional salivary gland. However, the mechanisms involved in salivary epithelial cell aggregation and tissue reconstitution are not fully understood. This study investigated the role of the P2Y2 nucleotide receptor (P2Y2R), a G protein-coupled receptor that is upregulated following salivary gland damage and disease, in salivary gland reconstitution. In vitro results with the rat parotid acinar Par-C10 cell line indicate that P2Y2R activation with the selective agonist UTP enhances the self-organization of dispersed salivary epithelial cells into acinar-like spheres. Other results indicate that the P2Y2R-mediated response is dependent on EGF receptor activation via the metalloproteases ADAM10/ADAM17 or the α5β1 integrin/Cdc42 signaling pathway, which leads to activation of the MAPKs JNK and ERK1/2. Ex vivo data using primary submandibular gland (SMG) cells from wild-type and P2Y2R-/- mice confirmed that UTP-induced migratory responses required for acinar cell self-organization are mediated by the P2Y2R. Overall, this study suggests that the P2Y2R is a promising target for salivary gland reconstitution and identifies the involvement of two novel components of the P2Y2R signaling cascade in salivary epithelial cells, the α5β1 integrin and the Rho GTPase Cdc42.
Our previous experiment confirmed that high mobility group box chromosomal protein 1 ( HMGB1 ) was involved in the pathogenesis of Lupus nephritis ( LN ) by up-regulating the proliferation of mouse mesangial cells ( MMC ) through the cyclin D1/CDK4/p16 system, but the precise mechanism is still unknown. Therefore，in the present study, we demonstrated that HMGB1 induced the proliferation of MMC in time- and concentration-dependent manner, down-regulated phosphatase and tensin homolog deleted on chromosome ten ( PTEN ) expression, increased the level of Akt serine 473 phosphorylation, and induced p65 subunit nuclear translocation. The over-expression of PTEN prevented the up-regulation of HMGB1-induced proliferation by blocking the activation of Akt. The knock-down of Akt by siRNA technology and blocking the nuclear factor-kappa B (NF-B) pathway using pyrrolidine dithiocarbamate (PDTC) and SN50, inhibitors of NF-B, both attenuated the HMGB1-induced proliferation by counteracting the activation of the cyclin D1. In addition, while sh-Akt partly blocked the nuclear translocation of the p65 subunit, PDTC did not affect the activation of the Akt induced by HMGB1 in MMC cells. These findings indicate that high mobility group box chromosomal protein 1 induced the proliferation of mouse mesangial cells by activating the PTEN/PI3K/Akt/NF-B signalling pathway.
Mitochondria are dynamic organelles, capable of altering their morphology and function. However the mechanisms governing these changes are not fully elucidated, particularly in muscle cells. We demonstrated that oxidative stress with H2O2 resulted in a 41% increase in fragmentation of the mitochondrial reticulum in myoblasts within 3 hours of exposure, an effect that was preceded by a reduction in membrane potential. Using live-cell imaging, we monitored mitochondrial motility and found that oxidative stress resulted in a 30% reduction in the average velocity of mitochondria. This was accompanied by parallel reductions in both organelle fission and fusion. The attenuation in mitochondrial movement was abolished by the addition of N-acetylcysteine. To investigate whether H2O2-induced fragmentation was mediated by Drp1, we incubated cells with mDivi1, an inhibitor of Drp1 translocation to mitochondria. mDivi1 attenuated oxidative stress-induced mitochondrial fragmentation by 27%. Moreover, we demonstrated that exposure to H2O2 upregulated ER-unfolded protein response markers prior to the initiation of mitophagy signaling and the mitochondrial-unfolded protein response. These findings indicate that oxidative stress is a vital signaling mechanism in the regulation of mitochondrial morphology and motility.
The signaling pathways mediating sustained contraction of mouse colonic longitudinal smooth muscle and the mechanisms involved in hypercontractility of this muscle layer in response to cytokines and TNBS-induced colitis have not been fully explored. In control longitudinal smooth muscle cells, ACh acting via m3 receptors activated sequentially Gα12, RhoGEF (LARG), and the RhoA/Rho kinase pathway. There was abundant expression of MYPT1, minimal expression of CPI-17, and a notable absence of a PKC/CPI-17 pathway. LARG expression was increased in longitudinal muscle cells isolated from muscle strips cultured for 24 h with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice. The increase in LARG expression was accompanied by a significant increase in ACh-stimulated Rho kinase and ZIP kinase activities, and sustained muscle contraction. The increase in LARG expression, Rho kinase and ZIP kinase activities, and sustained muscle contraction was abolished in cells pre-treated with the Jun kinase inhibitor, SP600125. Expression of the MLCP activator, telokin, and MLCP activity were also decreased in longitudinal muscle cells from TNBS-treated mice or from strips treated with IL-1β or TNF-α. In contrast, previous studies had shown that sustained contraction in circular smooth muscle is mediated by sequential activation of Gα13, p115RhoGEF, and a dual RhoA-dependent pathways involving phosphorylation of MYPT1 and CPI-17. In colonic circular smooth muscle cells isolated from TNBS-treated mice or from strips treated with IL-1β or TNF-α, CPI-17 expression and sustained muscle contraction were decreased. The disparate changes in the two muscle layers contribute to intestinal dysmotility during inflammation.
Understanding vascular growth and maturation in developing tumors has important implications for tumor progression, spread, and ultimately host survival. Modulating the signaling of endothelial G-protein coupled receptors in blood and lymphatic vessels can enhance or limit tumor progression. S1PR1 is a GPCR for circulating lysophospholipid S1P that is highly expressed in blood and lymphatic vessels. Using the S1PR1-eGFP mouse model in combination with intravital imaging and pharmacologic modulation of S1PR1 signaling, we show that boundary conditions of high and low S1PR1 signaling retard tumor progression by enhancing or destabilizing neovasculature integrity, respectively. In contrast, mid-range S1PR1 signaling, achieved by receptor antagonist titration, promotes abundant growth of small, organized vessels and thereby enhances tumor progression. Furthermore, in vivo S1PR1 antagonism supports lung colonization by circulating tumor cells. Regulation of endothelial S1PR1 dynamically controls vascular integrity and maturation and thus modulates angiogenesis, tumor growth and hematogenous metastasis.
Tissue fibrosis occurs with excessive extracellular matrix (ECM) deposition from myofibroblasts resulting in tissue scarring and inflammation. It is driven by multiple mediators, such as the G-protein coupled receptor ligands lysophosphatidic acid (LPA) and endothelin (ET-1) as well as signaling by transforming growth factor β (TGFβ), connective tissue growth factor (CTGF), and integrins. Fibrosis contributes to 45% of deaths in the developed world. As current therapeutic options for tissue fibrosis are limited and organ transplantation is the only effective treatment for end-stage disease, there is an imminent need for efficacious antifibrotic therapies. This review discusses the various molecular pathways involved in fibrosis. It highlights the Rho GTPase signaling pathway and its downstream gene transcription output through myocardin-related transcription factor (MRTF) and serum response factor (SRF) as a convergence point for targeting this complex set of diseases.
Homeostasis and maturation of the mammalian intestinal epithelium are preserved through strict regulation of cell proliferation, apoptosis and differentiation, but the exact mechanism underlying this process remains largely unknown. c-Jun N-terminal kinase 2 (JNK2) is highly expressed in the intestinal mucosa and its activation plays an important role in proliferation and also mediates apoptosis in cultured intestinal epithelial cells (IECs). Here, we investigated the in vivo function of JNK2 in the regulation of intestinal epithelial homeostasis and maturation by using a targeted gene deletion approach. Targeted deletion of the jnk2 gene increased cell proliferation within the crypts in the small intestine and disrupted mucosal maturation as indicated by decreases in the height of villi and villus/crypt ratio. JNK2 deletion also decreased susceptibility of the intestinal epithelium to apoptosis. JNK2-deficient intestinal epithelium was associated with an increase in the level of the RNA-binding protein HuR and with a decrease in the abundance of CUG-binding protein 1 (CUGBP1). In studies in vitro, JNK2 silencing protected IEC-6 cells against apoptosis and this protection was prevented by inhibiting HuR. Ectopic overexpression of CUGBP1 repressed IEC-6 cell proliferation, whereas CUGBP1 silencing enhanced cell growth. These results indicate that JNK2 is essential for maintenance of normal intestinal epithelial homeostasis and maturation under biological conditions by differentially modulating HuR and CUGBP1.
Heparanase (HPSE1) is known to be involved in mechanisms of metastatic tumour cell migration. This enzyme selectively cleaves heparan sulfate proteoglycans (HSPG), which are ubiquitously expressed in mammals and are known to be involved in regulating the activity of an array of inflammatory mediators. In the present study, we have investigated the effects of human recombinant heparanase, the inactive precursor of this enzyme (pro-heparanase) and enzymatically inactivated heparanase, on inflammatory cell recruitment in the rat and on human leukocyte-endothelial adhesion in vitro. Intraperitoneal injection of heparanase (500 μg) induced a significant inflammatory cell infiltrate in the rat, assessed by peritoneal lavage four hours later. Intravital microscopy of the mesenteric microcirculation of anaesthetized rats showed an increase in rolling and adherent cells in post-capillary venules that was sensitive to heparin, a non-selective inhibitor of heparanase activity. In vitro, heparanase augmented the adhesion of human neutrophils and mononuclear cells to human umbilical vein endothelial cells in a concentration-dependent manner. Pro-heparanase had similar effects to the active enzyme with respect to leukocyte accumulation in the peritoneal cavity and adhesion in vitro. However, heat-inactivated heparanase induced cell adhesion in vitro but was without effect in vivo. Together, these data indicate a role for heparanase in inflammatory cell trafficking in vivo that appears to require enzymatic activity.
The metabolic activity of articular chondrocytes is influenced by osmotic alterations that occur in articular cartilage secondary to mechanical load. The mechanisms that sense and transduce mechanical signals from cell swelling and initiate volume regulatory are poorly understood. The purpose of this study was to investigate how the expression of two putative osmolyte channels (TRPV4 and BKCa) in chondrocytes is modulated in different osmotic conditions and to examine a potential role for MAPKs in this process. Isolated equine articular chondrocytes were subjected to anisosmotic conditions and the expression of the TRPV4 andBKCa channels and also ERK1/2 and p38 MAPKs protein phosphorylation were investigated using western blotting. Results indicated that TRPV4 contributes to the early stages of hypo-osmotic stress, while BKCa is involved in responses to elevated of intracellular Ca2+ and mediating regulatory volume decrease (RVD). ERK1/2 is phosphorylated by hypo-osmotic stress (P<0.001), and p38 MAPK is phosphorylated by hyper-osmotic stress (P<0.001). In addition this study demonstrated the importance of endogenous ERK1/2 phosphorylation in the expression of TRPV4 channel where blocking ERK1/2 by specific inhibitor (PD89059) significantly decreased endogenous level of the TRPV4 channel in cells exposed to hypo-osmotic stress and below endogenous levels in iso-osmotic conditions (P<0.001).
Platelet-derived growth factor BB and its receptor (PDGFRβ) play pivotal role in development of renal glomerular mesangial cells. Their roles in increased mesangial cell proliferation during mesangioproliferative glomerulonephritis have long been noted; but the signaling mechanism regulating these changes remains poorly understood. We examined the role of a recently identified MAPK, Erk5, in this process. PDGF increased the activating phosphorylation of Erk5 and tyrosine phosphorylation of proteins in a time-dependent manner. A pharmacologic inhibitor of Erk5, XMD8-92, abrogated PDGF-induced DNA synthesis and mesangial cell proliferation. Similarly, expression of dominant negative Erk5 or siRNAs against Erk5 blocked PDGF-stimulated DNA synthesis and proliferation. Inhibition of Erk5 attenuated expression of cyclin D1 mRNA and protein, resulting in suppression of CDK4-mediated phosphorylation of the tumor suppressor protein pRb. Expression of cyclin D1 or CDK4 prevented the dominant negative Erk5- or siErk5-mediated inhibition of DNA synthesis and mesangial cell proliferation induced by PDGF. We have previously shown that PI 3 kinase contributes to PDGF-induced proliferation of mesangial cells. Inhibition of PI 3 kinase blocked PDGF-induced phosphorylation of Erk5. Since PI 3 kinase acts through Akt, we determined the role of Erk5 on Akt phosphorylation. XMD8-92, dominant negative Erk5 and siErk5 inhibited phosphorylation of Akt by PDGF. Interestingly, we found inhibition of PDGF-induced Erk5 phosphorylation by a pharmacological inhibitor of Akt kinase and kinase dead Akt in mesangial cells. Thus our data unfold the presence of a positive feedback microcircuit between Erk5 and Akt downstream of PI 3 kinase nodal point for PDGF-induced mesangial cell proliferation.
FXYD5 (dysadherin or RIC) is a transmembrane auxiliary subunit of the Na+/K+ ATPase shown to increase its Vmax. FXYD5 has also been identified as a cancer associated protein whose expression in tumor derived cell lines impairs cytoskeletal organization and increases cell motility. Previously, we have demonstrated that the expression of FXYD5 in M1 cells derived from mouse kidney collecting duct, impairs the formation of tight and adherence junctions. The current study aimed to further explore effects of FXYD5 at a single cell level. It was found that in M1 as well as three other cell lines, FXYD5 inhibits transformation of adhered single cells from the initial radial shape to a flattened, elongated shape, in the first stage of monolayer formation. This is also correlated to less ordered actin cables and fewer focal points. Structure-function analysis has demonstrated that the transmembrane domain of FXYD5, and not its unique extracellular segment, mediates the inhibition of change in cell shape. This domain has been shown before to be involved in the association of FXYD5 with the Na+/K+ ATPase which leads to the increase in Vmax. Furthermore, specific transmembrane point mutations in FXYD5 that either increase or decrease its effect on cell elongation had a corresponding effect on the co-immunoprecipitation of FXYD5 with αNa+/K+ ATPase . These findings lend support to the possibility that FXYD5 affects cell polarization through its transmembrane domain interaction with the Na+/K+ ATPase. Yet interaction of FXYD5 with other proteins can not be excluded.
We have previously shown that in addition to the well-known secreted isoform of prostatic acid phosphatase (sPAP), a transmembrane isoform exists (TMPAP) which interacts with snapin (a SNARE associated protein) and regulates the endo-/exocytic pathways. We have also shown that PAP has 5'-ectonucleotidase and thiamine monophosphatase activity, and elicits anti-nociceptive effects in mouse models of chronic inflammatory and neuropathic pain. Therefore, in order to determine the physiological role of PAP in a typical exocrine organ, we studied the submandibular salivary gland (SMG) of PAP-/- and wild-type C57BL/6J mice by microarray analyses, microRNA sequencing, activity tests, immunohistochemistry and biochemical and physiological analyses of saliva. We show that PAP is the main acid phosphatase in the wild-type male mouse saliva, accounting for 50% of the total acid phosphatase activity, and it is only expressed in the granular convoluted tubules of the SMGs, where it is the only 5'-ectonucleotidase. The lack of PAP in the male PAP-/- mice was associated with a significant increase in the salivation volume under secretagogue stimulation, overexpression of genes related to cell proliferation (Mki67, Aurkb, Birc5) and immune response (Irf7, Cxcl9, Ccl3, Fpr2), and upregulation of miR-146a in SMG. An increased and sustained acinar cell proliferation was detected without signs of glandular hyperplasia. Our results indicate that in PAP-/- mice SMG homeostasis is maintained by an innate immune response. Additionally, we suggest that in male mice PAP via its 5'-ectonucleotidase activity and production of adenosine can elicit analgesic effects when animals lick their wounds.
A significant amount of research has been conducted to examine the pathologic processes and epigenetic mechanisms contributing to peripheral hypertension. However, few studies have been carried out to understand the vascular remodeling behind pulmonary hypertension; they include peripheral artery (PA) muscularization, medial hypertrophy and neointima formation in proximal arteries, and plexiform lesion formation. Similarly, there is minimal research examining the epigenetic principles such as DNA methylation, histone modification, and miRNA regulation that contribute to vascular remodeling. Understanding these principles may be a key in developing new and more effective treatments for pulmonary hypertension. The purpose of this review is to summarize epigenetic research conducted in the field of hypertension that could possibly be used to understand the epigenetics of pulmonary hypertension. Possible future therapies that could be pursued using information from these studies include selective HDAC inhibitors (HDIs), exogenous administration of miRNAs, inhibitors for miRNA-21, miRNA-145, miRNA-138, and targeted DNA methyltransferases (DNMTs). All of these could potentially be used to silence pro-proliferative or anti-apoptotic genes that can lead to decreased SMC proliferation. Epigenetics may provide a glimmer of hope for the eventual improved treatment of this highly morbid and debilitating disease.
Erythropoietin-Producing human Hepatocellular carcinoma (Eph) receptors are largest family of receptor tyrosine kinases (RTKs) that mediate various cellular and developmental processes. The degrees of expression of these key molecules control the cell-cell interactions. Although Eph receptors and their ligand Ephrins role are well studied in developmental processes, their function in tobacco smoke (TS) induced epithelial barrier dysfunction is unknown. We hypothesized that TS may induce permeability in bronchial airway epithelial cell (BAEpC) monolayer by modulating receptor EphA2 expression, actin cytoskeleton, adherens junction and focal adhesion proteins. Here we report that in BAEpCs, acute TS exposure significantly up regulated EphA2, EphrinA1 expression, disrupted the actin filaments, decreased E-Cadherin expression, and increased protein permeability whereas the focal adhesion protein Paxillin was un-affected. Silencing the receptor EphA2 expression with silencing interference RNA (siRNA) significantly attenuated TS induced hyper-permeability in BAEpCs. In addition, when BAEpCs monolayer transfected with EphA2 expressing plasmid and treated with recombinant EphrinA1, the trans-epithelial electrical resistance decreased significantly. Furthermore, TS down regulated E-Cadherin expression and induced hyper-permeability across BAEpC monolayer in Erk1/Erk2, p38 and JNK MAPK dependent manner. TS induced hyper-permeability in BAEpCs monolayer by targeting cell-cell adhesions, and interestingly cell-matrix adhesions were un-affected. The present data suggests that TS causes a significant damage to the BAEpCs via induction of EphA2 and down regulation of E-Cadherin. Induction of EphA2 in the BAEpCs exposed to TS may be an important signaling event in the pathogenesis of TS-induced epithelial injury.
The PACAP-selective PAC1 receptor (Adcyap1r1) is a G protein coupled receptor (GPCR) that activates both adenylyl cyclase and phospholipase C. Similar to many other GPCRs, our previous studies have shown that the PAC1 receptor is internalized after ligand binding to form signaling endosomes, which recruit additional second messenger pathways. Using a HEK293 PAC1Hop1-EGFP receptor cell line, we have examined how different PAC1 receptor signaling mechanisms contribute to MEK/ERK activation. Unlike PAC1 receptor-stimulated adenylyl cyclase/cAMP production in the plasma membrane, PACAP-mediated ERK phosphorylation was partly dependent on receptor internalization as determined by treatment with pharmacological inhibitors of endocytosis or reducing temperature, which also suppressed receptor internalization. Stimulation of cAMP generation by forskolin or exposure to the cell permeable cAMP analogues bromo-cAMP and dibutyryl-cAMP had minimal effects on ERK phosphorylation in this system. The ability of reduced temperature (24°C) to consistently suppress ERK activation to a greater extent than by the endocytosis inhibitors Pitstop 2 and dynasore indicated that other mechanisms in addition to PAC1 internalization/endosome activation were involved. Inhibition of PAC1 receptor-stimulated PLC/DAG/PKC signaling with BimI also attenuated ERK phosphorylation and direct PKC activation with phorbol ester increased ERK phosphorylation in a temperature-dependent manner. The inhibition of both PAC1 receptor endocytosis and PKC activation completely blocked PACAP-stimulated ERK activation. PACAP augmented phosphorylated ERK staining uniformly over the cytoplasm and nucleus, and PKC signaling facilitated nuclear phosphorylated ERK translocation. In sum, our results show that PACAP/PAC1 receptor endocytosis and PLC/DAG/PKC activation represent two complementary mechanisms contributing to PACAP-induced ERK activation.
Mammalian target of rapamycin, mTOR is a serine/threonine kinase that modulates translation in response to growth factors, alterations in nutrient availability and following hypoxia and DNA damage. Here we demonstrate that mTOR activity in Ehrlich Lettré ascites (ELA) cells is transiently increased within minutes following osmotic cell swelling and that inhibition of phosphatidylinositol-3-phosphatase (PTEN), which counteracts the upstream phosphatidylinositol kinase PI3K, potentiates mTOR activity. PTEN inhibition concomitantly potentiates swelling induced taurine release via the volume sensitive transporter for organic osmolytes (VSOAC) and enhances swelling induced inhibition of taurine uptake via the taurine specific transporter TauT. Chronic osmotic stress, i.e., exposure to hypotonic or hypertonic media for 24 hours, reduces and increases mTOR activity in ELA cells, respectively. Using rapamycin we demonstrate that mTOR inhibition is accompanied by reduction in TauT activity and increase in VSOAC activity in cells expressing high (NIH3T3 fibroblasts) or low (ELA) amounts of mTOR protein. The effect of mTOR inhibition on TauT activity reflects reduced TauT mRNA, TauT protein abundance and an overall reduction in protein synthesis, whereas the effect on VSOAC is mimicked by catalase inhibition and correlates with reduced catalase mRNA abundance. Hence, mTOR activity favors loss of taurine following hypoosmotic cell swelling, i.e., release via VSOAC and uptake via TauT during acute hypotonic exposure is potentiated and reduced, respectively by phosphorylation involving mTOR and/or upstream to mTOR kinases. Decrease in TauT activity during chronic hypotonic exposure, on the other hand, involves reduction in expression/activity of TauT and enzymes in antioxidative defense.
The pineal gland regulates circadian rhythm through the synthesis and secretion of melatonin. The rise of intracellular Ca2+ concentration ([Ca2+]i) following nicotinic acetylcholine receptor (nAChR) stimulation due to parasympathetic nerve activity downregulates melatonin production. Important characteristics and roles of Ca2+ mobilization due to nAChR stimulation remain to be clarified. We report here that spontaneous Ca2+ oscillations can be observed in approximately 15% of the pinealocytes in slice preparations from rat pineal glands when this dissociation procedure is done within 6 h from a dark-to-light change. The frequency and half-life of [Ca2+]i rise were 0.86 min-1 and 19 s, respectively. Similar spontaneous Ca2+ oscillations were recorded in 17% of rat pinealocytes that was primary-cultured for several days. Simultaneous measurement of [Ca2+]i and membrane potential revealed that spontaneous Ca2+ oscillations were triggered by periodic membrane depolarizations. Spontaneous Ca2+ oscillations in cultured pinealocytes were abolished by extracellular Ca2+ removal or application of nifedipine, a blocker of voltage-dependent Ca2+ channel (VDCC). In contrast, blockers of intracellular Ca2+-release channels, 2-aminoethoxydiphenylborate and ryanodine have no effect. Our results also reveal that, in 23% quiescent pinealocytes, Ca2+ oscillations were observed following the withdrawal of nicotine. Norepinephrine-induced melatonin secretion from whole pineal glands was significantly decreased by the co-application of acetylcholine (ACh). This inhibitory effect of ACh was attenuated by nifedipine. In conclusion, both spontaneous and evoked Ca2+ oscillations, are due to membrane depolarization following activation of VDCCs. This consists of VDCC 1F subunit, and the associated Ca2+ influx can strongly regulate melatonin secretion in pineal glands.
Resolution of acute inflammation is an active process locally controlled by a novel genus of specialized pro-resolving mediators (SPM) that orchestrate key resolution responses. Hence, it is of general interest to identify individual bioactive mediators, profile their biosynthetic pathways with related isomers as well as their relation(s) to classic eicosanoids in mammalian tissues. Lipid mediator (LM)-SPM levels and signature profiles of their biosynthetic pathways were investigated using liquid chromatography-tandem mass spectrometry (LC-MS-MS)-based LM-metabololipidomics. LM and SPM were identified using ≥6 diagnostic ions and chromatographic behavior matching with both authentic and synthetic materials. This approach was validated using the composite reference plasma (SRM1950) of 100 healthy individuals. Using targeted LM-metabololipidomics, we profiled LM and SPM pathways in human peripheral blood (plasma and serum) and lymphoid organs. In these, we identified endogenous SPM-metabolomes, namely the potent lipoxins (LX), resolvins (Rv), protectins (PD) and maresins (MaR). These included RvD1, RvD2, RvD3, MaR1 and NPD1/PD1, which were identified in amounts within their bioactive ranges. In plasma and serum, principal component analysis (PCA) identified signature profiles of eicosanoids and SPM clusters. Plasma-SPM increased with omega-3 and acetylsalicylic acid intake that correlated with increased phagocytosis of E. coli in whole blood. These findings demonstrate an approach for identification of SPM pathways (e.g. resolvins, protectins and maresins) in human blood and lymphoid tissues that were in amounts commensurate with their pro-resolving, organ protective and tissue regeneration functions. LM-metabololipidomics coupled with calibration tissues and physiologic changes documented herein provide a tool for functional phenotypic profiling.
Hyperoxic acute lung injury (HALI) is characterized by inflammation and epithelial cell death. CLOCK genes are master regulators of circadian rhythm also implicated in inflammation and lung diseases. However, the relationship of CLOCK genes in hyperoxia-induced lung injury has not been studied. This study will determine if HALI alters CLOCK gene expression. To test this, wild-type and NALP3–/– mice were exposed to room air or hyperoxia for 24, 48, or 72 hr. In addition, mice were exposed to different concentrations of hyperoxia (50, 75, 100% O2) or room air for 72 hr. The mRNA and protein levels of lung CLOCK genes, based on qPCR and Western blot analysis respectively, and their target genes are significantly elevated in mice exposed to hyperoxia compared to controls. Alterations in CLOCK genes are associated with increased inflammatory markers in bronchoalveolar lavage fluid of hyperoxic mice compared to controls. Histological examination of mice lungs exposed to hyperoxia show increased inflammation and alveolar congestion compared to controls. Our results indicate sequential increase in CLOCK gene expression in lungs of mice exposed to hyperoxia compared to controls. Additionally, data suggests dose-dependent increase in CLOCK gene expression with increased oxygen concentrations. To validate if the expression changes related to CLOCK genes are indeed associated with inflammation, NALP3-/- was introduced to analyze loss of function in inflammation. Western blot analysis showed significant CLOCK gene downregulation in NALP3-/- mice compared to wild-type controls. Together, our results demonstrate that hyperoxia-mediated lung inflammation is associated with alterations in CLOCK gene expression.
The iberiotoxin-sensitive large conductance voltage- and Ca2+-activated potassium (BK) channels (maxi-K+-channels) hyperpolarize the cell membrane thus supporting Ca2+-entry through Ca2+-release activated Ca2+-channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK-channels, cRNA encoding Ca2+-insensitive BK-channels (BKM513I+899-903) was injected into Xenopus oocytes with or without cRNA encoding wild type JAK2, gain-of-function V617FJAK2 or inactive K882EJAK2. K+-conductance was determined by dual electrode voltage clamp, and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells iberiotoxin-sensitive K+-current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result the K+-current in BKM513I+899-903-expressing oocytes was significantly increased following coexpression of JAK2 or V617FJAK2, but not K882EJAK2. Coexpression of BK-channel with V617FJAK2 but not K882EJAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel and V617FJAK2 expressing oocytes to JAK2 inhibitor AG490 (40 µM) significantly decreased K+-current. Inhibition of channel insertion by brefeldin A (5 µM) decreased the K+-current to a similar extent in oocytes expressing BK-channel alone and in in oocytes expressing BK-channel and V617FJAK2. Iberiotoxin (50 nM)-sensitive K+-current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 µM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK-channel mRNA and protein abundance. In conclusion, JAK2 up-regulates BK-channels by increasing channel protein abundance in the cell membrane.
The reversible inhibition of mitochondrial respiration by complex I inhibition at the onset of reperfusion decreases injury in buffer perfused hearts. Administration of acidic reperfusate for a brief period at reperfusion decreases cardiac injury. We asked if acidification treatment decreased cardiac injury during reperfusion by inhibiting complex I. Exposure of isolated mouse heart mitochondria to acidic buffer decreased the complex I substrate stimulated respiration, whereas respiration with complex II substrates was unaltered. Evidence of the rapid and reversible inhibition of complex I by an acidic environment was obtained at the level of isolated complex, intact mitochondria and in situ mitochondria in digitonin permeabilized cardiac myocytes. Moreover, ischemia-damaged complex I was also reversibly inhibited by an acidic environment. In the buffer-perfused mouse heart, reperfusion with pH 6.6 buffer for the initial 5 min decreased infarction. Compared to untreated hearts, acidification treatment markedly decreased the mitochondrial generation of reactive oxygen species and improved mitochondrial calcium retention capacity and inner mitochondrial membrane integrity. The decrease in infarct size achieved by acidic reperfusion approximates the reduction obtained by a reversible, partial blockade of complex I at reperfusion. Extracellular acidification decreases cardiac injury during reperfusion in part via the transient and reversible inhibition of complex I leading to a reduction of oxyradical generation accompanied by a decreased susceptibility to mitochondrial permeability transition during early reperfusion.
Mechanical stimulation of osteoblasts activates many cellular mechanisms including the release of ATP. Binding of ATP to purinergic receptors is key to load-induced osteogenesis. Osteoblasts also respond to fluid shear stress (FSS) with increased actin stress fiber formation (ASFF) that we postulate is in response to activation of the P2Y2 receptor (P2Y2R). Further, we predict that ASFF increases cell stiffness and reduces the sensitivity to further mechanical stimulation. We found that siRNA suppression of P2Y2R attenuated ASFF in response to FSS and ATP treatment. In addition, RhoA GTPase was activated within 15 minutes after the onset of FSS or ATP treatment and mediated ASFF following P2Y2R activation via the ROCK1/LIM Kinase 2/cofilin pathway. We also observed that ASFF in response to FSS or ATP treatment increased the cell stiffness and was prevented by knocking down P2Y2R. Finally, we confirmed that the enhanced cell stiffness and ASFF in response to RhoA GTPase activation during FSS drastically reduced the mechanosensitivity of the osteoblasts based on the [Ca2+]i response to consecutive bouts of FSS. These data suggest that osteoblasts can regulate their mechanosensitivity to continued load through P2Y2R activation of the RhoA GTPase signaling cascade, leading to ASFF and increased cell stiffness.
The process of wound healing must be tightly regulated to achieve successful restoration of injured tissue. Previously, we demonstrated that when injury to corneal epithelium occurs, nucleotides and neuronal factors are released to the extracellular milieu, generating a Ca2+ wave from the origin of the wound to neighboring cells. In the present study we sought to determine how the communication between epithelial cells in the presence or absence of neuronal wound media is affected by hypoxia. A signal-sorting algorithm was developed to determine dynamics of Ca2+ signaling between neuronal and epithelial cells. The crosstalk between activated corneal epithelial cells in response to neuronal wound media demonstrated that injury-induced Ca2+ dynamic patterns were altered in response to decreased oxygen levels. These alterations were associated with an overall decrease in ATP, and changes in purinergic receptors-mediated Ca2+ mobilization and localization of N-methyl-D-aspartate (NMDA) receptors. In addition we used the cornea in an organ culture wound model to examine how hypoxia impedes re-epithelialization after injury. There was a change in the recruitment of paxillin to the cell membrane and deposition of fibronectin along the basal lamina, both factors in cell migration. Our results provide evidence that complex Ca2+-mediated signaling occurs between sensory neurons and epithelial cells after injury and is critical to wound healing. Information revealed by these studies will contribute to an enhanced understanding of wound repair under compromised conditions and provide insight into ways to effectively stimulate proper epithelial repair.
Brain edema forms rapidly in the early hours of ischemic stroke by increased secretion of Na, Cl and water into the brain across an intact blood-brain barrier (BBB), together with astrocyte swelling as they take up the ions and water crossing the BBB. Our previous studies provided evidence that luminal BBB Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) participate in ischemia-induced edema formation. NKCC1 and two NHE isoforms, NHE1 and NHE2, reside predominantly at the luminal BBB membrane. Both NKCC and NHE activities of cerebral microvascular endothelial cells (CMEC) are rapidly stimulated by the ischemic factors hypoxia, aglycemia and arginine vasopressin (AVP) and inhibition of these transporters by bumetanide and HOE642, respectively, reduces brain Na uptake and edema in the rat middle cerebral artery occlusion model of stroke. The present study was conducted to further explore BBB NHE responses to ischemia. Here, we examined whether ischemic factor-stimulated NHE activity is sustained over several hours, when the majority of edema forms during stroke. We also examined whether ischemic factors alter NHE1 and/or NHE2 protein abundances. Finally, we conducted initial studies of ERK1/2 MAP kinase involvement in BBB NHE and NKCC responses to ischemia. We found that hypoxia, aglycemia and AVP all increase CMEC NHE activity through 5 hr and that NHE1 but not NHE2 abundance is increased by 1-5 hr exposures to these factors. Further, we found that the factors rapidly increase BBB ERK1/2 activity and that ERK1/2 inhibition reduces or abolishes ischemic factor stimulation of NKCC and NHE activities.
The human solute carrier (SLC26) family of anion transporters consists of 10 members (SLCA1-11, SLC10 being a pseudogene) that encode membrane proteins containing ~12 transmembrane (TM) segments with putative N-glycosylation sites (-NXS/T-) in extracellular loops and a C-terminal cytosolic STAS domain. All 10 members of the human SLC26 family, FLAG-tagged at the N-terminus, were transiently expressed in HEK-293 cells. While most proteins were observed to contain both high mannose and complex oligosaccharides, SLC26A2 was mainly in the complex form, SLC26A4 in the high mannose form, and SLC26A8 was not N-glycosylated. Mutation of the putative N-glycosylation sites showed that most members contain multiple N-glycosylation sites in the second extra-cytosolic (EC) loop, except SLC26A11 which was N-glycosylated in EC loop 4. Immunofluorescence staining of permeabilized cells localized the proteins to the plasma membrane and the endoplasmic reticulum (ER), with SLC26A2 highly localized to the plasma membrane. N-glycosylation was not a necessary requirement for cell surface expression as the localization of non-glycosylated proteins was similar to their wild-type counterparts, although a lower level of cell-surface biotinylation was observed. No immunostaining of intact cells was observed for any SLC26 members, demonstrating that the N-terminal FLAG tag was located in the cytosol. Topological models of the SLC26 proteins that contain an even number of transmembrane segments with both the N- and C-termini located in the cytosol and utilized N-glycosylation sites defining the positions of two EC loops are presented.
We examined whether class IIa histone deacetylases (HDACs) play a role in mitogenic signaling mediated by protein kinase D1 (PKD1) in IEC-18 intestinal epithelial cells. Our results show that class IIa HDAC 4, 5 and 7 are prominently expressed in these cells. Simulation with angiotensin II (ANG II), a potent mitogen for IEC-18 cells, induced a striking increase in the phosphorylation of HDAC4 at Ser246 and Ser632, HDAC5 at Ser259 and Ser498 and HDAC7 at Ser155. Treatment with the PKD family inhibitors kb NB 142-70 and CRT0066101 or siRNA-mediated knockdown of PKD1 prevented ANG II-induced phosphorylation of HDAC 4, 5 and 7. A variety of PKD1 activators in IEC-18 cells, including vasopressin, lysophosphatidic acid or phorbol esters, also induced HDAC4, 5 and 7 phosphorylation. Using endogenously and ectopically expressed HDAC5, we show that PKD1-mediated phosphorylation of HDAC5 induces its nuclear extrusion into the cytoplasm. In contrast, HDAC5 with Ser259 and Ser498 mutated to Ala was localized to the nucleus in both unstimulated and stimulated cells. Treatment of IEC-18 cells with specific inhibitors of class IIa HDACs, including MC1568 and TMP269, prevented cell cycle progression, DNA synthesis and proliferation induced in response to GPCR/PKD1 activation. The PKD1/class IIa HDAC axis also functions in intestinal epithelial cell in vivo, since an increase in the phosphorylation of HDAC4/5 and HDAC7 was demonstrated in lysates of cryptal cells from PKD1 transgenic mice as compared with matched non-transgenic littermates. Collectively, our results reveal a PKD1/classIIa HDAC axis in intestinal epithelial cells leading to mitogenic signaling.
Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. Oct4 is a critical regulator of pluripotency whose level is precisely controlled in mouse embryonic stem cells (mESC). However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. We specifically examine the functional role of the OCT4A isoform in hESC by using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiologic levels. (We also designed and generated a comparable series of vectors for the overexpression and knockdown of OCT4B, which were not functional.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.
Pattern recognition receptors (PRR), toll-like receptors (TLR), and nucleotide-oligomerization domain containing proteins (NOD) play critical roles in mediating inflammation and modulating functions in white adipocytes in obesity. However, the role of PRR activation in brown adipocytes, which are recently found to be present in adult humans, has not been studied. Here we report that mRNA of TLR4, TLR2, NOD1 and NOD2, are up-regulated, paralleled with up-regulated mRNA of inflammatory cytokine and chemokine in BAT of the obese mice. During brown adipocyte differentiation, mRNA and protein expression of NOD1and TLR4, but not TLR2 and NOD2, are also increased. Activation of TLR4, TLR2, or NOD1 in brown adipocytes induces activation of NF-B and MAPK signaling pathways, leading to inflammatory cytokines/chemokine mRNA expression and/or protein secretion. Moreover, activation of TLR4, TLR2, or NOD1, attenuates both basal and isoproterenol-induced uncoupling protein 1 expression without affecting mitochondrial biogenesis and lipid accumulation in brown adipocytes. Cellular bioenergetics measurements confirm that attenuation of UCP-1 expression by PRR activation is accompanied by suppression of both basal and isoproterenol-stimulated oxygen consumption rates, isoproterenol-induced uncoupled respiration from proton leak and coupling efficiency; however, maximal respiration and ATP-coupled respiration are not changed. Further, the attenuation of UCP-1 by PRR activation appears to be mediated through downregulation of the UCP-1 promoter activities. Taken together, our results demonstrate the role of selected PRR activation in inducing inflammation and down-regulation of UCP-1 expression and mitochondrial respiration in brown adipocytes. Our results uncover novel targets in BAT for obesity treatment and prevention.
Pseudomonas aeruginosa secrete N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression, and micromolar concentrations are found in the airway surface liquid of infected lungs. Exposure of the airway surface to C12 caused a sudden loss of transepithelial resistance (RT) within one hour, and was characterized by a degradation and relocation of the tight junction protein ZO-1 from the apical to the basolateral pole of polarized human airway cultures (Calu-3 and primary tracheal epithelia). These effects were blocked by ZVAD-fmk, a pancaspase blocker, indicating that tight junction degradation was an early event in C12-triggered apoptosis. Short-time (10 min) pretreatment with thapsigargin (1 uM), an inhibitor of Ca2+ uptake into the ER, was found to be protective against the C12-induced airway epithelial barrier breakdown, and also against other apoptosis-related effects including activation of caspase 3/7 (executioner caspases in apoptosis), release of endoplasmic reticulum-targeted roGFP into the cytosol and depolarization of mitochondrial membrane potential. Pretreatment of Calu-3 airway cell monolayers with BAPTA/AM (to buffer cytosolic [Ca2+] (Cacyto) or Ca2+-free solutions + BAPTA/AM reduced C12-activation of apoptotic events, suggesting that C12-triggered apoptosis may involve Ca2+. Because C12 and thapsigargin both reduced [Ca2+] in the endoplasmic reticulum (Caer) and increased Cacyto, while thapsigarin increased mitochondrial [Ca2+] (Camito) and C12 reduced Camito, it is proposed that thapsigargin, may reduce C12-induced apoptosis in host cells not by raising Cacyto but by preventing C12-induced decreases in Camito.
GLUT1 is the primary glucose transport protein in human red blood cells (RBCs) but also transports oxidized vitamin C (dehydroascorbic acid; DHA). A recent study suggests that RBC GLUT1 transports DHA as its primary substrate and that only a subpopulation of GLUT1 transports sugars. This conclusion is based on measurements of cellular glucose and DHA equilibrium spaces rather than steady-state transport rates. We have characterized RBC transport of DHA and 3-O-methylglucose (3-OMG; a transported, non metabolizable sugar). Steady-state 3-OMG and DHA uptake in the absence of intracellular substrate are characterized by similar Vmax (0.16 ± 0.01 and 0.13 ± 0.02 mmol/L/min respectively) and Km(app) (1.4 ± 0.2 and 1.6 ± 0.7 mM respectively). 3-OMG and DHA compete for uptake with Ki(app) of 0.7 ± 0.4 mM and 1.1 ± 0.1 mM respectively. Uptake measurements using RBC inside-out-membrane vesicles (IOVs) demonstrate that 3-OMG and DHA compete at the cytoplasmic surface of the membrane with Ki(app) of 0.7 ± 0.1 and 0.6 ± 0.1 mM respectively. Intracellular 3-OMG stimulates unidirectional uptake of 3-OMG and DHA. These findings indicate that DHA and 3-OMG bind at mutually exclusive sites at both exo- and endofacial surfaces of GLUT1 and are transported via the same GLUT1 complex.
Skeletal muscle is the major tissue disposing of dietary glucose, a function regulated by insulin-elicited signals that impart mobilization of GLUT4 glucose transporters to the plasma membrane. This phenomenon, also central to adipocyte biology, has been the subject of intense and productive research for decades. We focus on muscle cell studies scrutinizing insulin signals and vesicle traffic in a spatiotemporal manner. Using the analogy of an integrated circuit to approach the intersection between signal transduction and vesicle mobilization, we identify signalling relays ('software') that engage structural/mechanical elements ('hardware') to enact the rapid mobilization and incorporation of GLUT4 into the cell surface. We emphasize how insulin signal transduction switches from tyrosine through lipid and serine phosphorylation down to activation of small G proteins of the Rab and Rho families; describe key negative regulation step of Rab GTPases through the GAP activity of the Akt substrate AS160; and focus on the mechanical effectors engaged by Rabs 8A and 10 (the molecular motor Myosin Va), and the Rho GTPase Rac1 (actin filament branching and severing through Arp2/3 and cofilin). Finally, we illustrate how actin filaments interact with Myosin 1c and α-Actinin4 to promote vesicle tethering as preamble to fusion with the membrane.
Proline-rich protein tyrosine kinase 2 (Pyk2) is a member of focal adhesion kinase family. We studied Pyk2 role in cutaneous wound repair using Pyk2-null mice. We report that the rate of wound closure was delayed in Pyk2-null compared with control mice. To examine whether impaired wound healing of Pyk2-null mice was caused by a keratinocyte cell-autonomous defect, the capacities of primary keratinocytes from Pyk2-null and wild-type (WT) littermates to heal scratch wounds in vitro were compared. The rate of scratch wound repair by Pyk2-null keratinocytes was decreased compared with that by WT cells. Moreover, cultured human epidermal keratinocytes overexpressing dominant-negative mutant of Pyk2 failed to heal scratch wounds. Conversely, stimulation of Pyk2-dependent signaling via WT Pyk2 overexpression induced accelerated scratch wound closure, and was associated with increased expression of matrix metalloproteinases (MMPs) -1, -9, and -10. The Pyk2-stimulated increase in the rate of scratch wound repair was abolished by co-expression of dominant-negative mutant of Protein Kinase C (PKC) and by GM-6001, a broad spectrum inhibitor of MMP activity. These results suggest that Pyk2 is essential for skin wound re-epithelialization both in vivo and in vitro, and that it regulates epidermal keratinocyte migration via a pathway that requires PKC and MMP functions.
In the renal collecting duct, binding of arginine vasopressin (AVP) to the V2 receptor triggers signaling changes that regulate osmotic water transport. Short-term regulation of water transport is dependent on vasopressin-induced phosphorylation of aquaporin-2 (AQP2) at Ser256. The protein kinase that phosphorylates this site is not known. Here we use Bayes' theorem to rank all 521 rat protein kinases with regard to the likelihood of a role in Ser256 phosphorylation based on prior data and new experimental data. First, prior probabilities were estimated from previous transcriptomic and proteomic profiling data, kinase substrate specificity data, and evidence for kinase regulation by vasopressin. This ranking was updated using new experimental data describing the effects of several small-molecule kinase inhibitors with known inhibitory spectra (H89, KN62, KN93, and GSK-650394) on Ser256-AQP2 phosphorylation in inner medullary collecting duct suspensions. The top-ranked kinase was calcium/calmodulin-dependent protein kinase II (CAMK2) followed by protein kinase A (PKA) and protein kinase B (AKT). LC-MS/MS-based in vitro phosphorylation studies compared the ability of three highly ranked kinases to phosphorylate AQP2 and other IMCD proteins, viz. PKA, CAMK2 and serum/glucocorticoid-regulated kinase (SGK). All three proved capable of phosphorylating Ser256, although both CAMK2 and PKA were more potent than SGK. The in vitro phosphorylation experiments also identified candidate protein kinases for several additional phosphoproteins with likely roles in collecting duct regulation including Nedd 4-2, Map4k4 and 3-phosphoinositide-dependent protein kinase 1. We conclude that the use of Bayes' theorem is an effective means of integrating data from multiple data sets in physiology.
Many skeletal muscle diseases are associated with progressive fibrosis leading to impaired muscle function. Collagen within the extracellular matrix is the primary structural protein providing a mechanical scaffold for cells within tissues. During fibrosis collagen not only increases in amount, but also undergoes post-translational changes that alter its organization that is thought to contribute to tissue stiffness. Little, however, is known about collagen organization in fibrotic muscle and its consequences for function. In order to investigate the relationship between collagen content and organization with muscle mechanical properties, we studied mdx mice, a model for Duchenne muscular dystrophy (DMD) that undergoes skeletal muscle fibrosis, and age-matched control mice. We determined collagen content both histologically, with picosirius red staining, and biochemically, with hydroxyproline quantification. Collagen content increased in the mdx soleus and diaphragm muscles, which was exacerbated by age in the diaphragm. Collagen packing density, a parameter of collagen organization, was determined using circularly polarized light microscopy of picosirius red stained sections. EDL and soleus muscle had proportionally less dense collagen in mdx muscle, while the diaphragm did not change packing density. The mdx muscles had compromised strength as expected, yet only the EDL had a significantly increased elastic stiffness. The EDL and diaphragm had increased dynamic stiffness and a change in relative viscosity. Unexpectedly, passive stiffness did not correlate with collagen content and only weakly correlated with collagen organization. We conclude that muscle fibrosis does not lead to increased passive stiffness, and that collagen content is not predictive of muscle stiffness.
Sodium glucose cotransporters (SGLTs) mediate the translocation of carbohydrates across the brush border membrane of different organs such as intestine, kidney and brain. The human SGLT5 (hSGLT5) in particular, is localized in the kidney were it is responsible for mannose and fructose reabsorption from the glomerular filtrate as confirmed by more recent studies on hSGLT5 knockout mice. Here we characterize the functional properties of hSGLT5 expressed in a stable T-Rex- HEK-293 cell line using biochemical and electrophysiological assays. We confirmed that hSGLT5 is a sodium/mannose transporter that is blocked by phlorizin. Li+ and H+ ions were also able to drive mannose transport. Transport was electrogenic with a 1:1 Na+:mannose coupling. Our results moreover indicate that substrates require a pyranose ring with an axial hydroxyl group (-OH) on carbon 2 (C-2). Compared to Na+ /glucose cotransport, the level of function of Na+/mannose cotransport in rat kidney slices was low.
Notch signaling plays a critical role in controlling proliferation and differentiation of pulmonary arterial smooth muscle cells (PASMC). Upregulated Notch ligands and Notch3 receptors in PASMC have been reported to promote the development of pulmonary vascular remodeling in patients with pulmonary arterial hypertension (PAH) and in animals with experimental pulmonary hypertension. Activation of Notch receptors by their ligands leads to the cleavage of the Notch intracellular domain (NICD) to the cytosol by -secretase; NICD then translocates into the nucleus to regulate gene transcription. In this study, we examined whether short-term activation of Notch functionally regulates store-operated Ca2+ entry (SOCE) in human PASMC. Treatment of PASMC with the active fragment of human Jagged-1 protein (Jag-1) for 15-60 min significantly increased the amplitude of SOCE induced by passive deletion of Ca2+ from the intracellular stores, the sarcoplasmic reticulum (SR). The Jag-1-induced enhancement of SOCE was time-dependent: the amplitude was maximized at 30 min of treatment with Jag-1, which was closely correlated with the time course of Jag-1-mediated increase in NICD protein level. The scrambled peptide of Jag-1 active fragment had no effect on SOCE. Inhibition of -secretase by N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) significantly attenuated the Jag-1-induced augmentation of SOCE. In addition to the short-term effect, prolonged treatment of PASMC with Jag-1 for 48 hrs also markedly enhanced the amplitude of SOCE. These data demonstrate that short-term activation of Notch signaling enhances SOCE in PASMC; the NICD-mediated functional interaction with store-operated Ca2+ channels (SOC) may be involved in the Jag-1-mediated enhancement of SOCE in human PASMC.
The function of an individual protein is typically defined by protein-protein interactions orchestrating the formation of large complexes critical for a wide variety of biological processes. Over the last decade the analysis of purified protein complexes by mass spectrometry became a key technique to identify protein-protein interactions. However, mass spectrometry based interactomics is time-consuming and is becoming more prone to identification of false-positive interactors with constantly increasing sensitivity of mass spectrometers. In addition, overexpression of proteins may often yield false-positive interactors while suitable antibodies to pull-down endogenous proteins are often unavailable or might interfere with the binding of interaction partners. To overcome these technical issues, we present a fast and straight-forward approach for analyses of interacting proteins combining a Flp-in single-copy cellular integration system and single-step affinity purification with single-shot mass spectrometry analysis. We applied this protocol to the analysis of the YAP and TAZ interactome. YAP and TAZ are the downstream effectors of the mammalian Hippo tumor suppressor pathway. Our study provides comprehensive interactomes for both, YAP and TAZ and does not only confirm the majority of previously described interactors but, strikingly, revealed uncharacterized interaction partners that affect YAP/TAZ TEAD dependent transcription. Among these newly identified candidates are Rassf8, Thymopoetin and the transcription factors CEBPβ/ and CBFB. In addition, our data allowed insights into complex stoichiometry and uncovered discrepancies between the YAP and TAZ interactomes. Taken together, the stringent approach presented here could help to significantly sharpen the understanding of protein-protein networks.
Excess enzyme-mediated protein O-GlcNAcylation is known to occur with diabetes mellitus. A characteristic of diabetic cardiomyopathy is the development of myocardial fibrosis. The role that enhanced protein O-GlcNAcylation plays in modulating the phenotype of cardiac fibroblasts is unknown. To address this issue, rat cardiac fibroblasts (CF) were cultured in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for 48 hours. Results demonstrate that CF cultured in HG have higher levels (~50%) of overall protein O-GlcNAcylation vs. NG cells. Key regulators of collagen synthesis such as TGF-β1, SMADs 2/3, and SMAD 7 protein levels, including those of arginase I and II, were altered, leading to increases in collagen levels. The nuclear transcription factor Sp1 and arginase II evidence excess O-GlcNAcylation in HG cells. Expression in CF of an adenovirus coding for the enzyme N-acetylglucosaminidase which removes O-GlcNAc moieties from proteins, decreased Sp1 and arginase II O-GlcNAcylation and restored HG induced perturbations in CF back to NG levels. These findings may have important pathophysiological implications for the development of diabetes-induced cardiac fibrosis.
Aims: The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel activity. Methods: Ventricular tissue was obtained from adult guinea pig hearts using collagenase. Ca2+ channel activity was monitored using the patch clamp technique. Proteins were purified using wheat germ agglutinin-Sepharose and the concentration was determined using the Coomassie Brilliant Blue technique. ATP binding to Cav1.2 channel was examined using the photoaffinity method. Results: EDA-ATP-Biotin maintains Ca2+ channel activity in inside-out membrane patches. ATP directly bound to Cav1.2 channel in a dose-dependent manner, and at least two molecules of ATP bound to one molecule of Cav1.2 channel. Low levels of CaM increased ATP binding to Cav1.2 channel, but higher levels of CaM decreased ATP binding to Cav1.2 channel. In addition, Ca2+ was another regulator for ATP binding to Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of N-terminal region (amino acids 6-140) and proximal C-terminal region (amino acids 1509-1789) of the main subunit (α1C) of Cav1.2 channel. Conclusions: Our data suggest that ATP might regulate Cav1.2 channel activity by directly binding to Cav1.2 channel in a dose-dependent manner. In addition, ATP binding effect to Cav1.2 channel was both CaM- and Ca2+-dependent.
Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function reduces chloride secretion and increased sodium uptake, but it is not clear why CFTR mutation also results in progressive lung inflammation and infection. We previously demonstrated that CFTR-silenced airway cells migrate more slowly during wound repair than CFTR-expressing controls. In addition, CFTR-deficient cells and mouse models have been reported to have altered sphingolipid levels. Here, we investigated the hypothesis that reduced migration in CFTR-deficient airway epithelial cells results from altered sphingolipid composition. We used cell lines derived from a human airway epithelial cell line (Calu-3) stably transfected with CFTR short hairpin RNA (CFTR-silenced) or non-targeting short hairpin RNA (controls). Cell migration was measured by electric cell substrate impedance sensing (ECIS). Lipid analyses, addition of exogenous glycosphingolipids, and immunoblotting were performed. We found that levels of the glycosphingolipid, GM1 ganglioside, were ~60% lower in CFTR-silenced cells than in controls. CFTR-silenced cells exhibited reduced levels of activated ß1-integrin, phosphorylated tyrosine 576 of focal adhesion kinase (pFAK) and phosphorylation of Crk-associated substrate (pCAS). Addition of GM1 (but not GM3) ganglioside to CFTR-silenced cells restored activated ß1-integrin, pFAK and pCAS to near control levels and partially restored (~40%) cell migration. Our results suggest that decreased GM1 in CFTR-silenced cells depresses ß1-integrin signaling, which contributes to the delayed wound repair observed in these cells. These findings have implications for the pathology of cystic fibrosis, where altered sphingolipid levels in airway epithelial cells could result in a diminished capacity for wound repair after injury.
Adrenal neuroendocrine chromaffin cells receive excitatory synaptic input from the sympathetic nervous system and secrete hormones into the peripheral circulation. Under basal sympathetic tone, modest amounts of freely-soluble catecholamine are selectively released through a restricted fusion pore formed between the secretory granule and plasma membrane. Upon activation of the sympatho-adrenal stress reflex, elevated stimulation drives fusion pore expansion, resulting in increased catecholamine secretion and facilitating release of co-packaged peptide hormones. Thus, regulated expansion of the secretory fusion pore is a control point for differential-hormone release of the sympatho-adrenal stress response. Previous work has shown syndapin 1 deletion to alter transmitter release and dynamin 1/syndapin 1 interaction as necessary for coupled endocytosis in neurons. Dynamin has also been shown to be involved in regulation of fusion pore expansion in neuroendocrine chromaffin cells through an activity-dependent association with syndapin. However, it is not known which syndapin isoform(s) contribute to pore dynamics in neuroendocrine cells. Nor is it known at what stage of the secretion process dynamin/syndapin associate to modulate pore expansion. Here we investigate the expression and localization of syndapin isoforms and determine which are involved in mediating fusion pore expansion. We show all syndapin isoforms are expressed in the adrenal medulla. Mutation of the SH3 dynamin-binding domain of all syndapin isoforms shows that fusion pore expansion and catecholamine release are limited specifically by the mutation of syndapin 3. The mutation also disrupts targeting of syndapin 3 to the cell periphery. Syndapin 3 exists in a persistent colocalized state with dynamin 1.
Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC) activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK) in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via PKC-dependent pathway and hypothesized that PKC-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKC. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKC from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by siRNA targeting PKC, revealed that PKC is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, αSMA and vimentin. We conclude that PKC/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKC and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases.
We recently demonstrated the abundant presence of cardiomyocytes in the wall of thoracic veins of adult mouse and rat. The highly differentiated morphology and myofilament protein contents of the venous cardiomyocytes suggested contractile functions. Here we further investigated the contractility of mouse and rat azygos venous rings in comparison with that of atrial strips and ventricular papillary muscle. X-gal staining of transgenic mouse vessels expressing lacZ under a cloned cardiac troponin T promoter demonstrated that the venous cardiomyocytes are discontinuous from atrial myocardium and aligned in the wall of thoracic veins perpendicular to the vessel axis. Histological sections displayed sarcomeric striations in the venous cardiomyocytes, which indicate an encirclement orientation of myofibrils in the vessel wall. Mechanical studies found that the rings of mouse and rat azygos vein produce strong cardiac type twitch contractions when stimulated with electrical pacing in contrast to the weak and slow smooth muscle contractions induced using 90 mM KCl. The twitch contraction and relaxation of mouse azygos veins further exhibited cardiac type of β-adrenergic responses. Quantitative comparison showed that the contractions of venous cardiomyocytes are slightly slower than that of atrium muscle but significantly faster than that of ventricular papillary muscle. These novel findings indicate that the cardiomyocytes abundant in the wall of rodent thoracic veins possess fully differentiated cardiac muscle phenotype despite their anatomical and functional segregations from the heart.
SGLT1-based oral rehydration solution (ORS) used in the management of acute diarrhoea does not substantially reduce stool output, despite the fact that glucose stimulates the absorption of sodium and water. To explain this phenomenon, we investigated the possibility that glucose might also stimulate anion secretion. Transepithelial electrical measurements, isotope flux measurements in Ussing chambers, fluid movement in isolated ileal sacs and electrical measurements in patch clamp studies were used to study the effect of glucose on active chloride and fluid secretion in mouse small intestinal cells and human Caco-2 cells. Confocal fluorescence laser microscopy and immunohistochemistry measured intracellular changes in calcium, SGLT1 and calcium-activated chloride channel (anoctamin 1) expression. In addition to enhancing active sodium absorption, glucose increased intracellular calcium and stimulated electrogenic chloride secretion. Calcium imaging studies showed increased intracellular calcium when intestinal cells were exposed to glucose. Niflumic acid, but not glibenclamide, inhibited glucose-stimulated chloride secretion in mouse small intestine and decreased whole-cell conductance in Caco-2 cells. In ileal intestinal sacs incubated with cholera toxin and glucose, decreased fluid secretion was seen only when the glucose-stimulated Cl secretion was blocked using niflumic acid. These observations establish that glucose not only stimulates active Na absorption, a well-established phenomenon, but also induces a Ca-activated chloride secretion. Glucose-induced chloride secretion may explain the failure of glucose-based ORS to markedly reduce stool output in acute diarrhoea. The present results have immediate potential to improve the outcome in the treatment of acute and/or chronic diarrhoeal diseases.
Cardiac fibrosis, a known risk factor for heart disease, is typically caused by uncontrolled proliferation of fibroblasts and excessive deposition of extracellular matrix proteins in the myocardium. Cyclin dependent kinase 1 (CDK1) is involved in the control of G2/M transit phase of the cell cycle. Here, we showed that Isoproterenol (ISO) induced cardiac fibrosis is associated with increased levels of CDK1 exclusively in fibroblasts in the adult mouse heart. Treatment of primary embryonic ventricular cell cultures with ISO (a non-selective β-adrenergic receptor agonist) increased CDK1 protein expression in fibroblasts and promoted their cell cycle activity. QPCR analysis confirmed that ISO increases CDK1 transcription in a transient manner. Further, the ISO responsive element was mapped to the proximal -100bp sequence of the CDK1 promoter region using various 5' flanking sequence deletion constructs. Sequence analysis of the -100bp CDK1 minimal promoter region revealed two putative NF-Y binding elements. Overexpression of the NF-YA subunit in primary ventricular cultures significantly increased the basal activation of -100bp CDK1 promoter construct but not the ISO induced transcription of the minimal promoter construct. In contrast, dominant negative NF-YA expression decreased the basal activity of the minimal promoter construct and ISO treatment fully rescued the dominant negative effects. Furthermore, site directed mutagenesis of the distal NF-Y binding site in the -100bp CDK1 promoter region completely abolished both basal and ISO induced promoter activation of the CDK1 gene. Collectively, our results raise an exciting possibility that targeting CDK1 or NF-Y in the diseased heart may inhibit fibrosis and subsequently confer cardioprotection.
Statins and aspirin deliver well-established cardiovascular benefits resulting in their increased use as combined polypills to decrease risk of stroke and heart disease. However, the direct endothelial effect of combined statin/aspirin cotreatment remains unclear. Histamine is an inflammatory mediator that increases vascular permeability and so we examined the effect of treating human umbilical vein endothelial cells (HUVECs) for 24-hours with 1μM simvastatin and 100μM aspirin on histamine responsiveness. Subsequent histamine (1μM) challenge increased Ca2+i concentration, an effect that was significantly inhibited by combined simvastatin/aspirin pretreatment but not when then the compounds were given separately, even at 10-fold higher concentrations. In contrast, the Ca2+i mobilization response to ATP challenge (10μM) was not inhibited by combined simvastatin/aspirin pretreatment. The H1 receptor antagonist pyrilamine significantly inhibited both histamine-induced Ca2+i mobilization and extracellular signal-regulated kinase (ERK) activation, whereas ranitidine (H2 receptor antagonist) was without effect. However, combined simvastatin / aspirin pretreatment failed to decrease H1 receptor protein expression ruling out receptor downregulation as the mechanism of action. Histamine-induced ERK activation was also inhibited by atorvastatin pretreatment, while simvastatin further inhibited histamine-induced vascular endothelial cadherin phosphorylation as well as altering HUVEC morphology and inhibiting actin polymerization. Therefore, in addition to the known therapeutic benefits of statins and aspirin, here we provide initial cellular evidence that combined statin/aspirin treatment inhibits histamine responsiveness in HUVECs.
The objective of the present study was to determine the impact of simulated apnea with intermittent hypoxia (IH) on endothelial barrier function and assess the underlying mechanism(s). Experiments were performed on human lung micro-vascular endothelial cells exposed to IH consisting alternating cycles of 1.5% O2 for 30s followed by 20% O2 for 5 min. IH decreased trans-endothelial electrical resistance (TEER) suggesting attenuated endothelial barrier function. The effect of IH on TEER was stimulus-dependent and reversible after re-oxygenation. IH exposed cells exhibited stress fiber formation and redistribution of cortactin, vascular endothelial-cadherins and zona occludens-1, junction proteins along with increased intercellular gaps at cell-cell boundaries. Extracellular signal regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) were phosphorylated in IH exposed cells. Inhibiting either ERK or JNK prevented IH-induced decrease in TEER and the reorganization of the cytoskeleton and junction proteins. IH increased reactive oxygen species (ROS) levels and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride, a membrane permeable anti-oxidant prevented ERK and JNK phosphorylation as well as IH-induced changes in endothelial barrier function. These results demonstrate that IH via ROS-dependent activation of MAP kinases leads to re-organization of cytoskeleton and junction proteins resulting in endothelial barrier dysfunction.
c-Abl is a nonreceptor protein tyrosine kinase that has a role in regulating smooth muscle cell proliferation and contraction. The role of c-Abl in smooth muscle cell migration has not been investigated. Here, c-Abl was found in the leading edge of smooth muscle cells. Knockdown of c-Abl by RNAi attenuated smooth muscle cell motility as evidenced by time-lapse microscopy. Furthermore, the actin-associated proteins cortactin and profilin-1 (Pfn-1) have been implicated in cell migration. In this report, cell adhesion induced cortactin phosphorylation at Tyr-421, an indication of cortactin activation. Phospho-cortactin and Pfn-1 were also found in the cell edge. Pfn-1 directly interacted with cortactin in vitro. Silencing of c-Abl attenuated adhesion-induced cortactin phosphorylation and Pfn-1 localization in the cell edge. To assess the role of cortactin/Pfn-1 coupling, we developed a cell permeable peptide. Treatment with the peptide inhibited the interaction of cortactin with Pfn-1 without affecting cortactin phosphorylation. Moreover, treatment with the peptide impaired the recruitment of Pfn-1 to the leading edge and cell migration. Finally, β1-integrin was required for the recruitment of c-Abl to the cell edge. Inhibition of actin dynamics impaired the spatial distribution of c-Abl. These results suggest that β1 integrin may recruit c-Abl to the leading cell edge, which may regulate cortactin phosphorylation in response to cell adhesion. Phosphorylated cortactin may facilitate the recruitment of Pfn-1 to the cell edge, which promotes localized actin polymerization, leading edge formation and cell movement. Conversely, actin dynamics may strengthen the recruitment of c-Abl to the leading edge.
Interstitial cells of Cajal (ICC) generate slow waves in gastrointestinal (GI) muscles. Previous studies have suggested that slow wave generation and propagation depends upon a voltage-dependent Ca2+ entry mechanism with the signature of a T-type Ca2+ conductance. We studied voltage-dependent inward currents in isolated ICC. ICC displayed two phases of inward current upon depolarization: a low voltage-activated inward current and a high voltage-activated current. The latter was of smaller current density and blocked by nicardipine. Ni2+ (30μM) or mibefradil (1μM) blocked the low voltage-activated current. Replacement of extracellular Ca2+ with Ba2+ did not affect the current, suggesting that either charge carrier was equally permeable. Half-activation and half-inactivation occurred at -36 mV and -59 mV, respectively. Temperature sensitivity of the Ca2+ current was also characterized. Increasing temperature (20° to 30°C) augmented peak current from -7 to -19 pA and decreased the activation time from 20.6 to 7.5 ms (Q10=3.0). Molecular studies showed expression of Cacna1g (Cav3.1) and Cacna1h (Cav3.2) in ICC. The temperature dependence of slow waves in intact jejunal muscles of wildtype and Cacna1h-/- mice was tested. Reducing temperature decreased the upstroke velocity significantly. Upstroke velocity was also reduced in muscles of Cacna1h-/- mice and Ni2+ or reduced temperature had little effect on these muscles. Our data show that a T-type conductance is expressed and functional in ICC. With previous studies our data suggest that T-type current is required for entrainment of pacemaker activity within ICC and for active propagation of slow waves in ICC networks.
Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell protruding regions the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.
Regulated urine concentration takes place in the renal collecting duct upon arginine vasopressin (AVP) stimulation, where subapical vesicles containing aquaporin-2 (AQP2) are inserted into the apical membrane instantly increasing water reabsorption and urine concentration. The reabsorped water exits via basolateral AQP3 and AQP4. Upon long-term stimulation with AVP or during thirst, expression levels of both AQP2 and AQP3 are increased, however, there is so far no evidence for short-term AVP regulation of AQP3 or AQP4. To facilitate the increase in trans-epithelial water transport, AQP3 may be short-term regulated via changes in protein-protein interactions, incorporation into lipid rafts and/or changes in steady state turn-over, which could result in changes in the diffusion behavior of AQP3. Thus, we measured AQP3 diffusion coefficients upon stimulation with the AVP mimic forskolin, to reveal if AQP3 could be short-term regulated by AVP. k-Space Image Correlation Spectroscopy (kICS) analysis of time-lapse image sequences of basolateral EGFP-tagged AQP3 (AQP3-EGFP) revealed that forskolin mediated elevation of cAMP increased the diffusion coefficient by 58% from 0.0147 ± 0.0082 µm2/sec (control) to 0.0232 ± 0.0085 µm2/sec (forskolin, p<0.05). Quantum dot conjugated antibody labeling also revealed a significant increase in AQP3 diffusion upon forskolin treatment by 44% (0.0104 ± 0.0040 µm2/sec (control) vs. 0.0150 ± 0.0016 µm2/sec (forskolin, p<0.05)). Immunoelectron microscopy showed no obvious difference in AQP3-EGFP expression levels or localization in the plasma membrane upon forskolin stimulation. Thus, AQP3-EGFP diffusion is altered upon increased cAMP, which may correspond to basolateral adaptations in response to the increased apical water readsorption.
Airway epithelium, which forms the first barrier towards environmental insults, is disturbed by cigarette smoking, a major risk factor for developing COPD. A-kinase anchoring proteins (AKAP) maintain endothelial barrier function and coordinate subcellular localization of protein kinase A. However, the role of AKAPs in epithelial barrier function is unknown. We studied the role of AKAPs in regulating human bronchial epithelial (HBE) barrier. Cigarette smoke extract (CSE) reduced barrier function in 16HBE cells and the expression of the adhesion molecule E-cadherin specifically at the cell membrane. In addition, CSE reduced the protein expression of the AKAP family member AKAP9 at the cell membrane. The expression of AKAP5 and AKAP12 was unaffected by CSE. AKAP9 interacted and co-localized with E-cadherin at the cell membrane, suggesting that the reduction of both proteins may be related. Interestingly, disruption of AKAP-PKA interactions by st-Ht31 prevented CSE-induced reduction of E-cadherin and AKAP9 protein expression and subsequent loss of barrier function. Silencing of AKAP9 reduced the functional epithelial barrier and prevented the ability of st-Ht31 to restore membrane localization of E-cadherin. Our data suggest the possibility of a specific role for AKAP9 in the maintenance of the epithelial barrier. E-cadherin, but not AKAP9, protein expression was reduced in lung tissue from COPD patients compared to controls. However, AKAP9 mRNA expression was decreased in primary bronchial epithelial cells from current smokers compared to non-/ex-smokers. In conclusion, our results indicate that AKAP proteins, most likely AKAP9 maintain bronchial epithelial barrier by regulating the E-cadherin expression at the cell membrane.
Microvesicles may represent a new tool of intercellular communication. Two different microvesicle types have been identified: membrane-derived vesicles (abbreviated EVs) and exosomes. The former is originated by direct budding from the plasma membrane, while the latter origins from ectocytosis of multivesicular bodies. Recently, a particular attention has been focused on the capacity of EVs to alter the phenotype of neighboring cells towards the phenotype of EV producing cells. It has been observed that stem cells are an abundant source of EVs. The interaction of stem cells with the microenvironment (i.e. stem cell niche) has a critical role in determining stem cell phenotype. The stem cell niche hypothesis predicts that stem cell number can be limited by the availability of niches that release the necessary signals for self-renewal and survival. Consequently, the niche provides a mechanism to control and limit stem cell number. In this context, EVs may have a fundamental role by transfer genetic information between cells. In fact, they are able to transfer to target cells both mRNA and miRNA, which may be involved in phenotypic changes of target cells toward the phenotype of EV producing cells. The exchange of genetic information may be bidirectional. In fact, after tissue damage, EV-mediated transfer of genetic information may reprogram the phenotype of stem cells to acquire features of the injured tissue cells. In addition, stem cell derived EVs may induce de-differentiation of cells which have surveyed injury with a re-entry in cell cycle and the possibility of tissue regeneration.
Foxo family transcription factors contribute to muscle atrophy by promoting transcription of the ubiquitin ligases MuRF1 and MAFbx/atrogin1. Foxo transcriptional effectiveness is largely determined by its nuclear cytoplasmic distribution, with unphosphorylated Foxo1 transported into nuclei and phosphorylated Foxo1 transported out. We expressed the fluorescent fusion protein Foxo1-GFP in cultured adult mouse flexor digitorum brevis muscle fibers, and tracked the time course of nuclear (N) to cytoplasmic (C) Foxo1-GFP mean pixel fluorescence in living fibers by confocal imaging. We previously showed that IGF1, which activates the Foxo kinase Akt/PKB, caused a rapid marked decline in N/C, whereas inhibition of Akt caused a modest increase in N/C [1]. Here we develop a 2 state mathematical model for Foxo1 nuclear cytoplasmic redistribution, where Foxo phosphorylation- dephosphorylation is assumed to be fast compared to nuclear influx and efflux. C is constant due to the much larger cytoplasmic than nuclear volume. Analysis of N/C time courses reveals that IGF1 strongly increased unidirectional nuclear efflux, indicating similarly increased fractional phosphorylation of Foxo1 within nuclei. Unidirectional nuclear influx was decreased in IGF1, indicating increased cytoplasmic fractional phosphorylation of Foxo1. Inhibition of Akt increased Foxo1 unidirectional nuclear influx, consistent with block of Foxo1 cytoplasmic phosphorylation, but did not decrease Foxo1 unidirectional nuclear efflux, indicating that Akt may not be involved in Foxo1 nuclear efflux under control conditions. New solution change experiments show that cultured fibers release factors into the medium that maintain low nuclear Foxo1. This study demonstrates the power of quantitative modelling of observed nuclear fluxes.
Intense muscle contraction induces high rates of ATP hydrolysis with resulting increases in inorganic phosphate (Pi), H+ and ADP, factors thought to induce fatigue by interfering with steps in the cross-bridge cycle. Force inhibition is less at physiological temperatures; thus the role of low pH in fatigue has been questioned. Therefore, effects of pH 6.2 and collective effects with 30 mM Pi on the pCa-force relationship were assessed in skinned fast and slow rat skeletal muscle fibers at 15 and 30°C. At 30°C, the pH 6.2 and 30 mM Pi condition significantly depressed peak force in all fiber types with the greatest effect in type IIx fibers. Across fiber types, Ca2+ sensitivity was depressed by low pH and low pH plus high Pi, with the greater effect at 30°C. For type IIx fibers at 30°C, the pCa50 was 5.36 at pH 6.2 (no added Pi) and 4.98 at pH 6.2, 30 mM Pi, compared to 6.58 for control (pH 7, no added Pi). At 30°C, n2, reflective of thick filament cooperativity, was unchanged by low cell pH but was depressed from 5.02 to 2.46 in type IIx fibers with pH 6.2, 30 mM Pi. With acidosis, activation thresholds of all fiber types required higher free Ca2+ at both 15 and 30°C. With the exception of type IIx fibers, the Ca2+ required to reach activation threshold increased further with added Pi. In conclusion, it is clear that fatigue-inducing effects of low cell pH and elevated Pi at near-physiological temperatures are substantial.
The existence of a local renin-angiotensin system (RAS) in neurons was first postulated forty years ago. Further studies indicated intraneuronal generation of angiotensin II (ANG II). However, the function and signaling mechanisms of intraneuronal ANG II remained elusive. Since angiotensin II type 1 receptor, AT1, is the major type of receptor mediating the effects of ANG II, we used intracellular microinjection and concurrent calcium and voltage imaging to examine the functionality of intracellular AT1 receptor in neurons. We show here that intracellular administration of ANG II produces a dose-dependent elevation in cytosolic Ca2+ concentration, [Ca2+]i, in hypothalamic neurons, that is sensitive to AT1 receptor antagonism. Endo-lysosomal, but not Golgi apparatus disruption, prevents the effect of microinjected ANG II on [Ca2+]i. Additionally, the ANG II-induced Ca2+ response is dependent on microautophagy and sensitive to inhibition of phospholipase C or antagonism of inositol 1,4,5-trisphosphate receptors. Furthermore, intracellular application of ANG II produces AT1-mediated depolarization of hypothalamic neurons, which was dependent on [Ca2+]i increase and on cation influx via transient receptor potential canonical channels. In summary, in the present study we provide evidence that intracellular ANG II activates endo-lysosomal AT1 receptors in hypothalamic neurons. Our results point to the functionality of a novel intraneuronal angiotensinergic pathway extending the current understanding of intracrine angiotensin II signaling.
Cardiac injury induces myocyte apoptosis and necrosis, resulting in the secretion and/or release of intracellular proteins. Currently, myocardial injury can be detected by analysis of a limited number of biomarkers in blood or coronary artery perfusate. However, the complete proteomic signature of protein release from necrotic cardiac myocytes is unknown. Therefore, we undertook a proteomic-based study of proteins released from cultured neonatal rat cardiac myocytes in response to H2O2 (necrosis) or staurosporine (apoptosis) to identify novel specific markers of cardiac myocyte cell death. Necrosis and apoptosis resulted in the identification of 147 and 79 proteins, respectively. Necrosis resulted in a relative increase in the amount of many proteins including the classical necrotic markers LDH, HMGB1, myoglobin, enolase and 14-3-3 proteins. Additionally, we identified several novel markers of necrosis including HSP90, α-actinin, and Trim72, many of which were elevated over control levels earlier than classical markers of necrotic injury. In contrast, the majority of identified proteins remained at low levels during apoptotic cell death, resulting in no candidate markers for apoptosis being identified. Blotting for a selection of these proteins confirmed their release during necrosis but not apoptosis. We were able to confirm the presence of classical necrotic markers in the extracellular milieu of necrotic myocytes. We also were able to identify novel markers of necrotic cell death with relatively early release profiles compared to classical protein markers of necrosis. These results have implications for the discovery of novel biomarkers of necrotic myocyte injury, especially in the context of ischemia/reperfusion injury.
The lens is proposed to have an internal microcirculation system consisting of continuously circulating ionic fluxes which play an essential role in maintaining lens transparency. One of the key components of this system is the sodium leak conductance. Here we investigate the contribution of Cx46 hemichannels to the basal membrane permeability of peripheral fiber cells isolated from transgenic mouse lenses lacking Cx50 or both Cx50 and Cx46 (dKO) using the whole cell patch clamp technique. Our results show that Cx46 hemichannels were largely closed at a resting voltage of -60 mV in the presence of millimolar divalent cation concentrations. However, even though the vast majority of these channels were closed at -60 mV, a small, persistent, inward current could still be detected. This current could be mostly blocked by exposure to 1 mM La3+ and was not observed in fiber cells isolated from dKO mouse lenses suggesting that it was due to Cx46 hemichannels. In addition, Cx50-/- fiber cells showed increased open channel noise and a depolarized resting potential as compared with dKO fiber cells. Exposure of Cx50-/- fiber cells to La3+ hyperpolarized the resting potential to -58 mV which is similar to the value of resting potential measured in dKO fiber and significantly reduced the open channel noise. In conclusion, these results suggest that Cx46 hemichannels may contribute to the sodium leak conductance in lens fiber cells.
Oxidant injury contributes to acute lung injury (ALI). We previously reported that activation of protein kinase GI (PKGI) post-transcriptionally increased the key antioxidant enzymes catalase and glutathione peroxidase 1 (Gpx-1) and attenuated oxidant-induced cytotoxicity in mouse lung microvascular endothelial cells (MLMVEC). The present studies tested the hypothesis that the antioxidant effect of PKGI is mediated via inhibition of the c-Abl tyrosine kinase. We found that activation of PKGI with the cGMP analogue 8pCPT-cGMP inhibited c-Abl activity and decreased c-Abl expression in wild type but not PKGI-/- MLMVEC. Treatment of wild-type MLMVEC with atrial natriuretic peptide also inhibited c-Abl activation. Moreover, treatment of MLMVEC with the c-Abl inhibitor imatinib increased catalase and GPx-1 protein in a post-transcriptional fashion. In imatinib-treated MLMVEC, there was no additional effect of 8pCPT-cGMP on catalase or GPx-1. The imatinib-induced increase in antioxidant proteins was associated with an increase in extracellular H2O2 scavenging by MLMVEC, attenuation of oxidant-induced endothelial barrier dysfunction, and prevention of oxidant-induced endothelial cell death. Finally, in the isolated perfused lung, imatinib prevented oxidant-induced endothelial toxicity. We conclude that cGMP, through activation of PKGI, inhibits c-Abl, leading to increased key antioxidant enzymes and resistance to lung endothelial oxidant injury. Inhibition of c-Abl by active PKGI may be the downstream mechanism underlying PKGI-mediated antioxidant signaling. Tyrosine kinase inhibitors may represent a novel therapeutic approach in oxidant-induced ALI.
Since the seminal studies of Otto Warburg in the 1920s it has been widely recognized that cancers grow glycolytically even in the presence of oxygen. This generates an abundance of protons in a gradient across most solid tumors with an acidic core and an alkaline rim. Whether and how this proton gradient may also serve in an autocrine fashion on these tumors is unclear. Here we demonstrate that human glioma cells form spheroids which act as a viable three-dimensional tumor model, forming physiologically relevant extracellular pH (pHe) and cell proliferation gradients. Using fluorescent cell cycle trackers we determined that the rate of cell proliferation is directly dependent on pHe, and that cells adjust their growth rate according to their position within the pH gradient. We further show that glioma cells sense pH via H+-sensitive K+ channels that translate changes in pH into changes in membrane voltage. These channels are tonically active and blocked by acidic pHe, quinine, and ruthenium red. Blockade of this K+ conductance either by acidic pHe or drug inhibition depolarized both glioma cells and tumor spheroids and prevented them from passing through the hyperpolarization-dependent G1-to-S phase cell cycle checkpoint, thereby inhibiting cell division. In this way, pHe directly determines the proliferative state of glioma cells.
Cellular mechanisms to account for the low Na+ concentration in human milk are poorly defined. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short circuit current (Isc; a sensitive indicator of net ion transport), suggesting activity of the epithelial Na+ channel, ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride sensitive Isc at all time points tested (2h to 7d), an effect that is not reduced with Ctx washout for 12 hours. Amiloride sensitive Isc remains elevated by Ctx in the presence of inhibitors for PKA (H-89, Rp-cAMP), PI3K (LY294002) and protein trafficking (brefeldin A). Additionally, the Ctx B subunit, alone, does not replicate these effects. RT-PCR and western blot analyses indicate no significant increase in either the mRNA or protein expression for α, β, or, -ENaC subunits. Ctx increases the abundance of both β- and -ENaC in the apical membrane. Additionally, Ctx increases both phosphorylated and nonphosphorylated Nedd4-2 expression. These results demonstrate that human mammary epithelia express ENaC, which can account for the low Na+ concentration in milk. Importantly, the results suggest that Ctx increases the expression, but reduces the activity of the E3 ubiquitin ligase, Nedd4-2, which would tend to reduce the ENaC retrieval and increase steady-state membrane residency. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na+ transport, which may have inferences for epithelial ion transport regulation in other tissues throughout the body.
Grape-seed procyanidins (GSPE) modulate glucose homeostasis and it was suggested that GSPE may achieve this by enhancing the secretion of incretin hormones such as glucagon-like peptide-1 (GLP-1). Therefore, the aim of the present study is to examine in detail the effects of GSPE on intestinal endocrine cells (STC-1). GSPE was found to modulate plasma membrane potential in enteroendocrine cells, inducing depolarization at low concentrations (0.05 mg/L) and hyperpolarization at high concentrations (50 mg/L), and surprisingly this was also accompanied by suppressed GLP-1 secretion. Furthermore, how GSPE affects STC-1 cells under nutrient-stimulated conditions (i.e. glucose, linoleic acid and L-proline) was also explored, and we found that the higher GSPE concentration was effective in limiting membrane depolarization and reducing GLP-1 secretion. Next, it was also examined whether GSPE affected mitochondrial membrane potential, finding that this too is altered by GSPE, however this does not appear to explain the observed effects on plasma membrane potential and GLP-1 secretion. In conclusion, our results show that grape-seed procyanidins modulate cellular membrane potential and nutrient-induced enteroendocrine hormone secretion in STC-1 cells.
Large conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. Protein kinase C (PKC) modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the interactions between PKC and BK channels in DSM remains unknown. Here, we provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies on DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with phorbol 12-myristate 13-acetate (PMA) inhibited the spontaneous transient BK currents (TBKCs) in native freshly-isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode), upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated the intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. The results supported the concept that PKC activation leads to a reduction in BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility.
Intracellular calcium (Ca2+) plays pivotal roles in distinct cellular functions through global and local signaling in various subcellular compartments; and subcellular Ca2+ signal is the key factor for independent regulation of different cellular functions. In vascular smooth muscle cells, sub-sarcolemmal Ca2+ is an important regulator of excitation-contraction coupling, and nucleoplasmic Ca2+ is crucial for excitation-transcription coupling. However, information on Ca2+ signals in these subcellular compartments is limited. To study the regulation of the subcellular Ca2+ signals, genetically encoded Ca2+ indicators (cameleon), D3cpv, targeting the plasma membrane (PM), cytoplasm and nucleoplasm were transfected into rat pulmonary arterial smooth muscle cells (PASMCs) and Ca2+ signals were monitored using laser scanning confocal microscopy. In situ calibration showed that the Kd for Ca2+ of D3cpv was comparable in the cytoplasm and nucleoplasm, but it was slightly higher in the PM. Stimulation of digitonin permeabilized cells with IP3 elicited a transient elevation of [Ca2+] with similar amplitude and kinetics in the nucleoplasm and cytoplasm. Activation of G-protein coupled receptors by endothelin-1 and angiotensin-II preferentially elevated the subsarcolemmal Ca2+ signal with higher amplitude in the PM region than the nucleoplasm and cytoplasm. In contrast, the receptor tyrosine kinase activator, platelet derived growth factor, elicited Ca2+ signals with similar amplitudes in all three regions, except the rise-time and decay-time were slightly slower in the PM region. These data clearly revealed compartmentalization of Ca2+ signals in the subsacrolemmal regions and provide the basis for further investigations of differential regulation of subcellular Ca2+ signals in PASMCs.
Progressive fibrosis is a pathological hallmark of many chronic diseases responsible for organ failure. The dynamic fibrogenic process is known to be regulated by multiple soluble mediators that may be therapeutically intervened. The failing hamster heart exhibits marked fibrosis and increased expression of secreted Frizzled-related protein 2 (sFRP2) amenable to reversal by mesenchymal stem cell (MSC) therapy. Given the previous demonstration that sFRP2-null mice subjected to myocardial infarction exhibited reduced fibrosis and improved function, we tested whether antibody-based sFRP2 blockade might counteract the fibrogenic pathway and repair cardiac injury. Cardiomyopathic hamsters were injected intraperitoneally twice a week each with 20 μg of sFRP2 antibody. Echocardiography, histology and biochemical analyses were performed after one month. sFRP2 antibody increased left ventricular ejection fraction from 40 ± 1.2% to 49 ± 6.5%, whereas saline and IgG control exhibited a further decline to 37 ± 0.9% and 31 ± 3.2%, respectively. Functional improvement is associated with a ~50% reduction in myocardial fibrosis, ~65% decrease in apoptosis and ~75% increase in wall thickness. Consistent with attenuated fibrosis, both MSC therapy and sFRP2 antibody administration significantly increased the activity of myocardial matrix metalloproteinase-2. Gene expression analysis of the hamster heart and cultured fibroblasts identified Axin2 as a downstream target, the expression of which was activated by sFRP2 but inhibited by therapeutic intervention. sFRP2 blockade also increased myocardial levels of VEGF and HGF along with increased angiogenesis. These findings highlight the pathogenic effect of dysregulated sFRP2, which may be specifically targeted for anti-fibrotic therapy.
Skeletal muscle atrophy is prevalent in chronic diseases and microRNAs (miRs) may play a key role in the wasting process. miR-23a was previously shown to inhibit the expression of atrogin-1 and MuRF1 in muscle. It also was reported to be regulated by NFATc3 in cardiomyocytes. The objective of this study was to determine if miR-23a is regulated during muscle atrophy and to evaluate the relationship between calcineurin (Cn)/nuclear factor of activated T-cells (NFAT) signaling and miR-23a expression in skeletal muscle cells during atrophy. miR-23a was decreased in the gastrocnemius of rats with acute streptozotocin (STZ)-induced diabetes, a condition known to increase atrogin-1 and MuRF1 expression and cause atrophy. Treatment of C2C12 myotubes with Dexamethasone (Dex) for 48 hours also reduced miR-23a as well as RCAN1.4 mRNA, which is transcriptionally regulated by NFAT. Both NFATc3 nuclear localization and the amount of miR-23a decreased rapidly within 1 hour of Dex administration suggesting a link between Cn signaling and miR-23a. Compared to primary myotubes from wild type mice, myotubes from CnAα-/- or CnAβ-/- mice had a lower level of miR-23a. Dex did not further suppress miR-23a in the Cn-deficient myotubes. Overexpression of CnAβ in C2C12 myotubes prevented Dex-induced suppression of miR-23a. Finally, miR-23a was present in exosomes isolated from the media of C2C12 myotubes and Dex increased its exosomal abundance. Dex did not alter the number of exosomes released into the media. We conclude that atrophy-inducing conditions down regulate miR-23a in muscle by mechanisms involving attenuated Cn/NFAT signaling and selective packaging into exosomes.
Iron homeostasis is achieved by regulating the intestinal absorption of the metal and its recycling by macrophages. Iron export from enterocytes or macrophages to blood plasma is thought to be mediated by ferroportin under the control of hepcidin. Although ferroportin was identified over a decade ago, little is understood about how it works. We expressed in Xenopus oocytes a human ferroportin-EGFP fusion protein and observed using confocal microscopy its exclusive plasma-membrane localization. As a first step in its characterization, we established an assay to detect functional expression of ferroportin by microinjecting oocytes with 55Fe and measuring efflux. Ferroportin expression increased the first-order rate constants describing 55Fe efflux up to 300-fold over control. Ferroportin-mediated 55Fe efflux was saturable, temperature-dependent (activation energy, Ea 17 kcal.mol–1), maximal at extracellular pH 7.5, and inactivated at extracellular pH < 6.0. We estimated that ferroportin reacts with iron at its intracellular aspect with apparent affinity < 10–7 M. Ferroportin expression also stimulated efflux of 65Zn and 57Co but not of 64Cu, 109Cd, or 54Mn. Hepcidin treatment of oocytes inhibited efflux of 55Fe, 65Zn, and 57Co. Whereas hepcidin administration in mice resulted in a marked hypoferremia within 4 h, we observed no effect on serum zinc levels in those same animals. We conclude that ferroportin is an iron-preferring cellular metal-efflux transporter with a narrow substrate profile that includes cobalt and zinc. Whereas hepcidin strongly regulated serum iron levels in the mouse, we found no evidence that ferroportin plays an important role in zinc homeostasis.
While white adipose tissue (AT) is an energy storage depot, brown AT is specialized in energy dissipation. Uncoupling protein 1 (UCP1)-expressing adipocytes with a different origin than classical brown adipocytes have been found in white AT. These "brite" (brown-in-white) adipocytes may represent a therapeutic target to counteract obesity. Bone morphogenetic proteins (BMPs) play a role in the regulation of adipogenesis. Based on studies with murine cells, BMP4 is assumed to induce stem cell commitment to the white adipocyte lineage, whereas BMP7 promotes brown adipogenesis. There is evidence for discrepancies between mouse and human AT. Therefore we compared the effect of BMP4 and BMP7 on white-to-brown transition in primary human adipose stem cells (hASCs) from subcutaneous AT. Long-term exposure of hASCs to recombinant BMP4 or BMP7 during differentiation increased adipogenesis, as determined by lipid accumulation and PPAR expression. Not only BMP7, but also BMP4 increased UCP1 expression in hASCs and decreased expression of the white-specific marker TCF21. The ability of hASCs to induce UCP1 in response to BMP4 and BMP7 markedly differed between donors and could be related to the expression of the brite marker CD137. However, mitochondrial content and oxygen consumption was not increased in hASCs challenged with BMP4 and BMP7. In conclusion, we showed for the first time that BMP4 has similar effects on white-to-brown transition as BMP7 in our human cell model. Thus, the roles of BMP4 and BMP7 in adipogenesis cannot always be extrapolated from murine to human cell models.
Breast cancer is the second leading cause of cancer mortality in women, estimated at nearly 40,000 deaths and more than 230,000 new cases diagnosed in the U.S. this year alone. One of the defining characteristics of breast cancer is the radiographic presence of microcalcifications. These palpable mineral precipitates are commonly found in the breast after formation of a tumor. Since free Ca2+ plays a crucial role as a second messenger inside cells, we hypothesize that these chelated precipitates may be a result of dysregulated Ca2+ secretion associated with tumorigenesis. Transient and sustained elevations of intracellular Ca2+ regulate cell proliferation, apoptosis and cell migration, and offer numerous therapeutic possibilities in controlling tumor growth and metastasis. During lactation, a developmentally determined program of gene expression controls the massive transcellular mobilization of Ca2+ from the blood into milk by the coordinated action of Calcium Transporters, including pumps, channels, sensors and buffers, in a functional module that we term CALTRANS. Here we assess the evidence implicating genes that regulate free and buffered Ca2+ in normal breast epithelium and cancer cells and discuss mechanisms that are likely to contribute to the pathological characteristics of breast cancer.
Large-conductance Ca2+-activated K+ channels (BK) regulate action potential (AP) properties and excitability in many central neurons. However, the properties and functional role of BK channels of parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus (NA) have not yet been well characterized. In this study, the tracer X-rhodamine-5 (and 6)-isothiocyanate (XRITC) was injected into the pericardial sac to retrogradely label PCMNs in FVB mice at 7-9 days postnatal. Two days later, XRITC-labeled PCMNs in brain stem slices were identified. Using excised patch single-channel recordings, we identified voltage-gated and Ca2+-dependent BK channels in PCMNs. The majority of BK channels exhibited persistent channel opening during voltage holding. These BK channels had a conductance of 237 pS. The channel dwell time increased exponentially as the membrane potential depolarized. Occasionally, some BK channels showed a transient channel opening or fast inactivation. Using whole-cell voltage clamp, we found that BK channel mediated outward currents had both transient and persistent components. Using whole-cell current clamp, we found that application of IBTX increased spike half-width in single APs and trains, and reduced the spike frequency-dependent AP broadening in trains. In addition, BK channel blockade suppressed fast afterhyperpolarization (fAHP) amplitude following APs. Furthermore, BK channel blockade significantly decreased spike frequency and spike frequency adaption (SFA). Collectively, these results demonstrate that PCMNs have BK channels which significantly regulate AP repolarization, fAHP, SFA and spike frequency. We conclude that activation of BK channels underlies one of the mechanisms for facilitation of PCMN excitability.
Using microarray analysis, we found that aging sarcopenia is associated with a sharp increase in the mRNA of the matricellular protein CCN1 (Cyr61/CTGF/Nov). CCN1 mRNA was up-regulated 113-fold in muscle of aged versus young rats. CCN1 protein was increased in aging muscle in both rats (2.8-fold) and mice (3.8-fold). When muscle progenitor cells (MPCs) were treated with recombinant CCN1, cell proliferation was decreased but there was no change in the myogenic marker myoD. However, the CCN1 treated MPCs did express a senescence marker (SA-βgal). Interestingly, we found CCN1 increased p53, p16Ink4A and pRP (hypophosphorylated retinoblastoma protein) protein levels, all of which can arrest cell growth in MPCs. When MPCs were treated with aged rodent serum CCN1 mRNA increased by 7-fold and protein increased by 3-fold suggesting the presence of a circulating regulator. Therefore, we looked for a circulating regulator. Wnt-3a, a stimulator of CCN1 expression, was increased in serum from elderly humans (2.6-fold) and aged rodents (2.0-fold) compared with young controls. We transduced C2C12 myoblasts with wnt-3a and found that CCN1 protein was increased in a time and dose dependent manner. We conclude that in aging muscle, the circulating factor, wnt-3a, acts to increase CCN1 expression, prompting muscle senescence by activating cell arrest proteins.
Protein phosphatase 1 (PP1) and Ca2+/calmodulin-dependent protein kinase (CaMKII) are up-regulated in heart disorders. Alterative splicing factor (ASF), a major splice factor for CaMKII splicing, can be regulated by both protein kinase and phosphatase. Here we determine the role of PP1 isoforms in ASF-mediated splicing of CaMKII in cells. We found that (1) PP1, but not α or β isoform, enhanced the splicing of CaMKII in HEK293T cells; (2) PP1 promoted the function of ASF, evidenced by the existence of ASF-PP1 association as well as the PP1 overexpression- or silencing-mediated change in CaMKII splicing in ASF-transfected HEK293T cells; (3) CaMKII splicing was promoted by overexpression of PP1 and impaired by application of PP1 inhibitor 1 (I1PP1) or pharmacological inhibitor tautomycetin in primary cardiomyocytes; (4) CaMKII splicing and enhancement of ASF-PP1 association induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) were potentiated by overexpression of PP1 and suppressed by inhibition of PP1 with I1PP1 or tautomycetin in primary cardiomyocytes; (5) Functionally, overexpression and inhibition of PP1 respectively potentiated or suppressed the apoptosis and Bax/Bcl-2 ratio, which were associated with the enhanced activity of CaMKII in OGD/R-stimulated cardiomyocytes; (6) CaMKII was required for the OGD/R induced- and PP1 exacerbated-apoptosis of cardiomyocytes, evidenced by a specific inhibitor of CaMKII KN93, but not its structural analog KN92, attenuating the apoptosis and Bax/Bcl-2 ratio in OGD/R and PP1-treated cells. In conclusion, our results show that PP1 promotes the alternative splicing of CaMKII through its interacting with ASF, exacerbating OGD/R-triggered apoptosis in primary cardiomyocytes.
A constant provision of ATP is of necessity for cardiac contraction. As the heart progresses towards failure following a myocardial infarction (MI) it undergoes metabolic alterations that have the potential to compromise the ability to meet energetic demands. This study evaluated the efficacy of MSC transplantation into the infarcted heart to minimize impairments in the metabolic processes that contribute to energy provision. Seven and 28 days following the MI and MSC transplantation, MSC administration minimized cardiac systolic dysfunction. Hyperinsulinemic-euglycemic clamps coupled with 2-[14C]deoxyglucose administration were employed to assess systemic insulin sensitivity and tissue-specific, insulin-mediated glucose uptake 36 days following the MI in the conscious, unrestrained, C57BL6 mouse. The improved systolic performance in MSC-treated mice was associated with a preservation of in vivo insulin-stimulated cardiac glucose uptake. Conserved glucose uptake in the heart was linked to the ability of the MSC treatment to diminish the decline in insulin signalling as assessed by Akt phosphorylation. The MSC treatment also sustained mitochondrial content, ADP-stimulated oxygen flux and mitochondrial oxidative phosphorylation efficiency in the heart. Maintenance of mitochondrial function and density was accompanied by preserved peroxisome proliferator-activated receptor coactivator 1α (PGC-1α); a master regulator of mitochondrial biogenesis. These studies provide insight into mechanisms of action that lead to an enhanced energetic state in the infarcted heart following MSC transplantation that may assist in energy provision and dampen cardiac dysfunction.
Efficient repair of the epithelial tissue, which is frequently exposed to insults, is necessary to maintain its functional integrity. It is therefore necessary to better understand the biological and molecular determinants of tissue regeneration and to develop new strategies to promote epithelial repair. Interestingly, a growing body of evidence indicates that many members of the large and widely-expressed family of K+ channels are involved in the regulation of cell migration and proliferation, key processes of epithelial repair. The first goal of this review is to briefly summarize the complex mechanisms, including cell migration, proliferation and differentiation, engaged after epithelial injury. We then present evidence implicating K+ channels in the regulation of these key repair processes. The mechanisms whereby K+ channels may control epithelial repair processes are also described. In particular, changes in membrane potential, K+ concentration, cell volume, intracellular calcium and signaling pathways following modulation of K+ channel activity, as well as physical interaction of K+ channels with the cytoskeleton or integrins are presented. Finally, the challenges to efficient, specific and safe targeting of K+ channels for therapeutic applications to improve epithelial repair in vivo are discussed.
Elevated levels of serotonin (5-HT) and endothelin-1 (ET-1) may be involved in cardiovascular complications of diabetes mellitus. Data suggests supra-physiological concentrations of 5-HT (10-6 M) potentiate ET-1's ability to stimulate DNA synthesis and vascular smooth muscle cell (VSMC) proliferation in vitro via activation of mitogen activated protein kinase (p42/44 MAPK) and janus kinase 2 (JAK2) pathways. Additionally, 5-HT enhances agonist-induced contractions via p42/44 MAPK and an unknown tyrosine kinase. However, the exact mechanisms of the 5-HT/ET-1 interactions and whether these effects occur at physiological levels (10-9 M) are unknown. Therefore, we hypothesized that interactions between 5-HT and ET-1 at physiological concentrations in VSMC enhanced activation of both p42/44 MAPK and JAK2 pathways contributing to vascular growth and contractile responses. Using rat VSMC and Western Blot analysis our data suggest no effect of acute (30 minutes) pre-incubation with 5-HT (10-9 M) and/or ET-1 (10-9 M) on the activation of either pathway in normal or high glucose conditions. To determine if there was altered vascular reactivity in intact vessels we tested the effects of 5-HT and ET-1 interaction using myographs to measure isometric contractions of rat thoracic aortic rings. 5-HT (10-9 M) and ET-1 (10-12 M) stimulate enhanced contractile responses to each other that were inhibited by JAK2 and p42/44 MAPK antagonists. Our findings demonstrate that both 5-HT and ET-1 at physiological concentrations could interact with each other and activate p42/44 MAPK and JAK2 signaling pathways to cause an increase in smooth muscle contraction which could lead to altered vascular function.
Do you know the sex of your cells? Not a question that is frequently heard around the lab bench, yet thanks to recent research is probably one that should be asked. It is self-evident that cervical epithelial cells would be derived from female tissue and prostate cells from a male subject (exemplified by HeLa and LnCaP respectively), yet beyond these obvious examples, it would be true to say that the gender of cell lines derived from non-reproductive tissue, such as lung, intestine, kidney, for example, is given minimal if any thought. After all, what possible impact could the presence of a Y chromosome have on the biochemistry and cell biology of tissues such as the exocrine pancreatic acini? Intriguingly, recent evidence has suggested that far from being irrelevant, genes expressed on the sex chromosomes can have a marked impact on the biology of such diverse tissues as neurons and renal cells. It is also policy of the American Journal of Physiology, that the "........source of all cells utilized (species, sex etc) should be clearly indicated" when submitting an article for publication, an instruction that is rarely followed. In this review we discuss recent data arguing that the gender of cells being used in experiments can impact the cell's biology, and provide a table outlining the gender of cell lines that have appeared in AJP Cell over the last decade.
Cell shrinkage and dehydration are essential characteristics of apoptosis, and the loss of as much as half of the initial cell volume is not uncommon. This phenomenon is usually explained by the efflux of potassium and chloride ions. We reexamine this hypothesis based on the available data for ion concentrations and taking into account the requirements for osmotic equilibrium and electroneutrality. In addition to ion loss, the possible impacts of several other processes are discussed: the efflux of low-molecular osmolytes, acidification of the cytosol, the effects of water channels and pumps, heterogeneity of intracellular water and dissociation of apoptotic bodies. We conclude that most mammalian cells are theoretically capable of reducing their volume by 15-20% through ion loss or a decrease in cytosolic pH, although in reality the contribution of these mechanisms to apoptotic shrinkage may be smaller. Transitions between osmotically active and inactive water pools might influence cell volume as well; these mechanisms are poorly understood but are amenable to experimental study. Dissociation of apoptotic bodies is a separate mechanism of volume reduction; it should be monitored closely, which can be best achieved by measuring intracellular water rather than cell volume.
Sodium/Potassium/Chloride Cotransporter (NKCC1) proteins play important roles in Na+ and K+ concentrations in key physiological systems, including cardiac, vascular, renal, nervous and sensory systems. NKCC1 levels and functionality are altered in certain disease states, and tend to decline with age. A sensitive, effective way of regulating NKCC1 protein expression has significant bio-therapeutic possibilities. The purpose of the present investigation was to determine if the naturally occurring hormone aldosterone (ALD) could regulate NKCC1 protein expression. Application of ALD to a human cell line (HT-29) revealed that ALD can regulate NKCC1 protein expression, quite sensitively and rapidly, independent of mRNA expression changes. Utilization of a specific inhibitor of mineralocorticoid receptors, eplerenone, implicated these receptors as part of the ALD mechanism of action. Further experiments with cycloheximide (protein synthesis inhibitor) and MG132 (proteasome inhibitor) revealed that ALD can upregulate NKCC1 by increasing protein stability, i.e., reducing ubiquitination of NKCC1. Having a procedure for controlling NKCC1 protein expression opens the doors for therapeutic interventions for diseases involving the mis-regulation or depletion of NKCC1 proteins, for example during aging.
Mechanisms underlying ethanol (EtOH)-induced detrusor smooth muscle (DSM) relaxation and increased urinary bladder capacity remain unknown. We investigated whether the large conductance Ca2+-activated K+ (BK) channels or L-type voltage-dependent Ca2+ channels (VDCCs), major regulators of DSM excitability and contractility, are targets for EtOH by patch-clamp electrophysiology (conventional and perforated whole cell, and excised patch single-channel) and isometric tension recordings using guinea pig DSM cells and isolated tissue strips, respectively. EtOH at 0.3% v/v (~50 mM) enhanced whole cell BK currents at +30 mV and above, determined by the selective BK channel blocker paxilline. In excised patches recorded at +40 mV and ~300 nM intracellular [Ca2+], EtOH (0.1-0.3%) affected single BK channels (mean conductance ~205 pS and blocked by paxilline) by increasing the open channel probability, number of open channel events and open dwell-time constants. The amplitude of single BK channel currents and unitary conductance were not altered by EtOH. Conversely, at ~10 μM but not ~2 μM intracellular [Ca2+], EtOH (0.3%) decreased the single BK channel activity. EtOH (0.3%) affected transient BK currents (TBKCs) by either increasing frequency or decreasing amplitude, depending on the basal level of TBKC frequency. In isolated DSM strips, EtOH (0.1-1%) reduced the amplitude and muscle force of spontaneous phasic contractions. The EtOH-induced DSM relaxation, except at 1%, was attenuated by paxilline. EtOH (1%) inhibited L-type VDCC currents in DSM cells. In summary, we reveal the involvement of BK channels and L-type VDCCs in mediating EtOH-induced urinary bladder relaxation accommodating alcohol-induced diuresis.
The anti-inflammatory function of Tanshionone IIA (TIIA), an active natural compound from Chinese herbal medicine Danshen, has been well-recognized and therefore TIIA was widely used to treat various inflammatory conditions associated with cardiac and lung diseases. Mucin 1 (Muc1) plays important anti-inflammatory roles in resolution of acute lung inflammation. In this study, we investigated the effects of TIIA on lipopolysaccharide (LPS)-induced acute lung inflammation, as well as its relationship to Muc1 expression in mouse lung and MUC1 in human alveolar epithelial cells. TIIA pretreatment significantly inhibited LPS-induced pulmonary inflammation in both Muc1 wild type (Muc1+/+) and knockout (Muc1-/-) mice, as manifested by reduced neutrophils infiltration, and reduced TNF-α and KC levels in bronchoalveolar lavage (BAL) fluid. The inhibitory effects of TIIA on airway inflammation were associated with reduced expression of Muc1 in Muc1+/+ mouse lung. Moreover, pretreatment with TIIA significantly inhibited LPS-induced MUC1 expression and TNF-α release in A549 alveolar epithelial cells. TNF-α upregulated MUC1 mRNA and protein expression in A549 cells, which was inhibited by pretreatment with TIIA. The LPS induced-MUC1 expression was blocked when A549 cells were transfected with siRNA targeting for TNF-α receptor 1 (TNFR1). Further, TIIA inhibited LPS-induced nuclear translocation of NF-B and upregulation of TLR4 in A549 cells. Taken together, these results demonstrated that TIIA suppressed LPS-induced acute lung inflammation regardless the presence of Muc1, and TIIA inhibited LPS- and TNF-α-induced MUC1/Muc1 expression in airway epithelial cells, suggesting MUC1/Muc1 does not account for the mechanisms of the anti-inflammatory effects of TIIA in the airway.
Proteostasis is the maintenance of the proper function of cellular proteins. Hypertonic stress disrupts proteostasis and causes rapid and widespread protein aggregation and misfolding in the nematode C. elegans. Optimal survival in hypertonic environments requires degradation of damaged proteins. Inhibition of protein synthesis occurs in response to diverse environmental stressors and may function in part to minimize stress induced protein damage. We recently tested this idea directly and demonstrated that translation inhibition by acute exposure to cycloheximide suppresses hypertonicity induced aggregation of polyglutamine::YFP (Q35::YFP) in body wall muscle cells. In this paper, we further characterized the relationship between protein synthesis and hypertonic stress induced protein damage. We demonstrate that inhibition of translation reduces hypertonic stress induced formation and growth of Q35::YFP, Q44::YFP and -synuclein aggregates, misfolding of paramyosin and ras GTPase and aggregation of multiple endogenous proteins expressed in diverse cell types. Activation of GCN-2 kinase signaling during hypertonic stress inhibits protein synthesis via phosphorylation of eIF-2 . Inhibition of GCN-2 activation prevents the reduction in translation rate and greatly exacerbates the formation and growth of Q35::YFP aggregates and the aggregation of endogenous proteins. The current studies together with our previous work provide the first direct demonstration that hypertonic stress induced reduction in protein synthesis minimizes protein aggregation and misfolding. Reduction in translation rate also serves as a signal that activates osmoprotective gene expression. The cellular proteostasis network thus plays a critical role in minimizing hypertonic stress induced protein damage, degrading stress damaged proteins and in cellular osmosensing and signaling.
The proximal tubule (PT) reabsorbs the majority of sodium, bicarbonate, chloride ions, phosphate, glucose, water, and plasma proteins from the glomerular filtrate. Despite the critical importance of endocytosis for PT cell function, the organization of the endocytic pathway in these cells remains poorly understood. We have used immunofluorescence and live-cell imaging to dissect the itinerary of apically internalized fluid and membrane cargo in polarized primary cultures of PT cells isolated from mouse kidney cortex. Cells from the S1 segment could be distinguished from more distal PT segments by their robust uptake of albumin and comparatively low expression of -glutamyltranspeptidase. Rab11a in these cells is localized to variously sized spherical compartments that resemble the apical vacuoles observed by electron microscopy analysis of PT cells in vivo. These Rab11a-positive structures are highly dynamic and receive both membrane and fluid phase cargo. Moreover, we observed segregation of membrane proteins and fluid phase cargoes into separate carriers emanating from Rab11a-positive compartments. In contrast, fluid phase cargoes are largely excluded from Rab11a-positive compartments in immortalized kidney cell lines. The unusual morphology and sorting capacity of Rab11a compartments in primary PT cells may reflect a unique specialization of these cells to accommodate the functional demands of handling a high endocytic load.
Connexin 37 (Cx37) suppresses cell proliferation when expressed in rat insulinoma (Rin) cells, an effect also manifest in vivo during vascular development and in response to tissue injury. Mutant forms of Cx37 with non-functional channels but normally localized, wild-type carboxyl termini are not growth suppressive. Here we determined whether the carboxyl terminal (CT) domain is required for Cx37-mediated growth suppression and whether the Cx37 pore-forming domain can be replaced with the Cx43 pore-forming domain and still retain growth suppressive properties. We show that despite forming functional gap junction channels and hemichannels, Cx37 with residues subsequent to 273 replaced with a V5-epitope tag (Cx37-273tr*V5) had no effect on the proliferation of Rin cells, did not facilitate G1 cell cycle arrest with serum deprivation, and did not prolong cell cycle time comparably to the wild-type protein. The chimera Cx43*CT37, comprising the pore forming domain of Cx43 and CT of Cx37, also did not suppress proliferation, despite forming functional gap junctions with a permselective profile similar to wild-type Cx37. Differences in channel behavior of both Cx37-273tr*V5 and Cx43*CT37 relative to their wild-type counterparts and failure of the Cx37-CT to interact as the Cx43-CT does with the Cx43 cytoplasmic loop suggest that the Cx37-CT and pore-forming domains are both essential to growth suppression by Cx37.
Infections with enteric pathogens like enterotoxigenic Escherichia coli (ETEC) is a major health issue worldwide and while diarrhea is the major problem, prolonged, severe and dual infections with multiple pathogens may also compromise the nutritional status of the infected individuals. There is almost nothing currently known about the effect of ETEC infection on intestinal absorptions of water-soluble vitamins including thiamin. We examined the effect of ETEC infection on intestinal uptake of the thiamin using as a model the human-derived intestinal epithelial Caco-2 cells. The results showed that infecting confluent Caco-2 monolayers with live ETEC (but not with boiled/killed ETEC or nonpathogenic E. coli) or treatment with bacterial culture supernatant led to a significant inhibition in thiamin uptake. This inhibition appears to be caused by a heat-labile and secreted ETEC component, and is mediated via activation of the epithelial adenylate cyclase system. The inhibition in thiamin uptake by ETEC was associated with a significant reduction in expression of hTHTR-1 & -2 at the protein and mRNA levels as well as in the activity of the SLC19A2 and SLC19A3 promoters. Dual infection of Caco-2 cells with ETEC and EPEC (enteropathogenic E.coli) led to compounded inhibition in intestinal thiamin uptake. These results show for the first time that infection of human intestinal epithelial cells with ETEC causes a significant inhibition in intestinal thiamin uptake. This inhibition is mediated by a secreted heat-labile toxin, and is associated with a decrease in the expression of intestinal thiamin transporters.
Due to evolutionary pressure there are many complex interactions at the interface between pathogens and eukaryotic host cells wherein host cells attempt to clear invading microorganisms and pathogens counter these mechanisms to colonize replication niches. One striking observation from studies focused on this interface is that pathogens have multiple mechanisms to modulate and disrupt normal cellular physiology to establish replication niches and avoid clearance. The precision by which pathogens exert their effects on host cells makes them excellent tools to answer questions about cell physiology of eukaryotic cells. Furthermore, an understanding of these mechanisms at the host-pathogen interface will benefit our understanding of how pathogens cause disease. In this review, we describe a few examples of how pathogens disrupt normal cellular physiology and protein trafficking at epithelial cell barriers to underscore how pathogens modulate cellular processes to cause disease and how this knowledge has been utilized to learn about cellular physiology.
In a recent mass spectrometry screen we have identified 108 new proteins that were modified endogenously by covalent binding of biotin; members of the heat shock superfamily of proteins, including heat shock protein 72 (HSP72), were overrepresented among the biotinylated proteins. Mammals respond to infections by secreting extracellular HSP72 (eHSP72) which elicits an immune response. Here, we identified five biotinylation sites in HSP72 using mass spectrometry and site-directed mutagenesis. We used co-immunoprecipitation, mass spectrometry, and limited proteolysis assays to demonstrate that HSP72 interacts physically with the protein biotin ligase, holocarboxylase synthetase (HLCS) leading to the biotinylation of residues K112, K128 K348, K361, K415, and probably additional lysines. Finally, we demonstrated that HLCS-dependent biotinylation of eHSP72 increases the expression of the chemokine RANTES by the HEK293 human embryonic kidney cells. In conclusion, we report a novel endogenous modification of HSP72 and demonstrated that the binding of biotin to eHSP72 poises cells for a strong immune response.
As tumor cells metastasize from the primary tumor location to a distant secondary site, they encounter an array of biologically and physically heterogeneous microenvironments. While it is well established that biochemical signals guide all stages of the metastatic cascade, mounting evidence indicates that physical cues also direct tumor cell behavior, including adhesion and migration phenotypes. Physical cues acting on tumor cells in vivo include extracellular matrix mechanical properties, dimensionality, and topography, as well as interstitial flow, hydrodynamic shear stresses, and local forces due to neighboring cells. State-of-the-art technologies have recently enabled us and other researchers to engineer cell microenvironments that mimic specific physical properties of the cellular milieu. Through integration of these engineering strategies, along with physics, molecular biology, and imaging techniques, we have discovered new insights into tumor cell adhesion and migration mechanisms. In this review, we focus on the extravasation and invasion sections of the metastatic cascade. We first discuss the physical role of the endothelium during tumor cell extravasation and invasion, and how contractility of both endothelial and tumor cells contributes to the ability of tumor cells to exit the vasculature. Next, we examine how matrix dimensionality and stiffness co-regulate tumor cell adhesion and migration beyond the vasculature. Finally, we summarize how tumor cells translate and respond to physical cues through mechanotransduction. Due to the critical role of tumor cell mechanotransduction during the metastatic cascade, targeting signaling pathways involved in tumor cell mechano-sensing of physical stimuli may prove to be an effective therapeutic strategy for cancer.
Increase in matrix protein content in the kidney is a cardinal feature of diabetic kidney disease. While renal matrix protein content is increased by chronic hyperglycemia, whether it is regulated by acute elevation of glucose and insulin has not been addressed. In this study, we aimed to evaluate if short duration of combined hyperglycemia and hyperinsulinemia, mimicking metabolic environment of pre-diabetes and early type 2 diabetes, induces kidney injury. Normal rats were subjected to either saline infusion (control, n=4) or seven hours of combined hyperglycemic-hyperinsulinemic clamp (HG+HI clamp; n=6). During the clamp, plasma glucose and plasma insulin were maintained at about 350 mg/dl and 16 ng/ml, respectively. HG+HI clamp increased the expression of renal cortical TGFβ and renal matrix proteins, laminin and fibronectin. This was associated with the activation of SMAD3, Akt, mTOR complexes and Erk signaling pathways and their downstream target events in the initiation and elongation phases of mRNA translation, an important step in protein synthesis. Additionally, HG+HI clamp provoked renal inflammation as shown by the activation of toll like receptor 4 (TLR4) and infiltration of CD68 positive monocytes. Urinary F2t isoprostane excretion, an index of renal oxidant stress, was increased in the HG+HI clamp rats. We conclude that even a short duration of hyperglycemia and hyperinsulinemia contributes to activation of pathways that regulate matrix protein synthesis, inflammation and oxidative stress in the kidney. This finding could have implications for the control of short term rises in blood glucose in diabetic individuals at risk of developing kidney disease.
Chronic myeloid leukemia (CML) is a hematopoietic stem/progenitor cell disorder in which Bcr-Abl oncoprotein inhibits cell differentiation. Differentiation induction is currently considered an alternative strategy for treating CML. Activin A, a member of the transforming growth factor-β superfamily, induces erythroid differentiation of CML cells through the p38 mitogen-activated protein kinase (MAPK) pathway. In this study, pretreatment of K562 CML stem/progenitor cell line with activin A followed by a subtoxic concentration of Bcr-Abl inhibitor imatinib strongly induced growth inhibition and apoptosis compared to the simultaneous co-treatment scheme. Imatinib induced growth inhibition and apoptosis following activin A pretreatment were dose- and time-dependent manners. Imatinib-induced growth inhibition and apoptosis was also dependent on dosing of activin A pretreatment. More than 90% of the activin A-induced increases of glycophorin A-positive cells were sensitive to imatinib. However, only the part of original glycophorin A-positive cells after activin A treatment were sensitive to imatinib. Activin A/imatinib sequential treatment decreased Bcr-Abl, procaspase-3, Mcl-1 and Bcl-xL. This treatment also induced cleavage of procaspase-3/PARP. The reduction of erythroid differentiation in p38 MAPK dominant negative mutants or by shRNA knockdown of p38 MAPK decreased the sequential treatment-mediated growth inhibition and apoptosis. Furthermore, the same inhibition level of MDR1 expression was observed under activin A alone, activin A/imatinib sequential treatment or co-treatment. The p38 MAPK inhibitor SB203580 can restore activin A-inhibited MDR1 expression. Taken together, our results suggest that a subtoxic concentration of imatinib could exhibit strong cytotoxicity against erythroid differentiated K562 CML cells.
Nuclear factor-kappaB (NF-B) is a master regulator of genes, which control a large number of cellular processes including angiogenesis and inflammation. We recently demonstrated that Cytochrome P450 1B1 (Cyp1B1)-deficiency in endothelial cells (EC) and pericytes (PC) results in increased oxidative stress, alterations in migration, attenuation of capillary morphogenesis, sustained activation of NF-B, and increased thrombospondin-2 (TSP2) expression, an endogenous inhibitor of angiogenesis. Based on a growing body of evidence that phenylethyl isothiocyanate (PEITC) and pyrrolidine diothiocarbamate (PDTC) function as antioxidants and suppressors of NF-B activation, we investigated their potential ability to restore a normal phenotype in Cyp1B1-deficient (cyp1b1-/-) vascular cells. PEITC and PDTC inhibited NF-B activity and expression in cyp1b1-/- EC and PC. We also observed restoration of migration and capillary morphogenesis of cyp1b1-/- EC, and decreased cellular oxidative stress in cyp1b1-/- EC and PC without restoring normal TSP2 levels. In addition, expression of a dominant negative IBα, a suppressor of NF-B activation, decreased NF-B activity without affecting TSP2 expression in these cells. In contrast, knockdown of TSP2 expression resulted in attenuation of NF-B activity in cyp1b1-/- vascular cells. Furthermore, expression of TSP2 in wild type (cyp1b1+/+) cells resulted in increased NF-B activity. Together our results demonstrate an important role for TSP2 in modulation of NF-B activity, and attenuation of angiogenesis. Thus, Cyp1B1 expression in vascular cells plays an important role in the regulation of vascular homeostasis through modulation of the cellular reductive state, TSP2 expression, and NF-B activation.
Physical exercise has potent therapeutic and preventive effects against metabolic disorders. A number of studies have suggested that 5'AMP-activated protein kinase (AMPK) plays a pivotal role in regulating carbohydrate and lipid metabolism in contracting skeletal muscles, while several genetically manipulated animal models revealed significance of AMPK-independent pathways. In order to elucidate significance of AMPK and AMPK-independent signals in contracting skeletal muscles, we conducted a metabolomic analysis which compared the metabolic effects of 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) stimulation with the electrical contraction ex vivo in isolated rat epitrochlearis muscles, in which both α1 and α2 isoforms of AMPK and glucose uptake were equally activated. The metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry detected 184 peaks and successfully annotated 132 small molecules. AICAR stimulation exhibited high similarity to the electrical contraction in overall metabolites. Principal component analysis (PCA) demonstrated that the major principal component characterized common effects whereas the minor principal component distinguished the difference. PCA and a factor analysis suggested a substantial change in redox status as a result of AMPK activation. We also found decrease in reduced glutathione levels in both AICAR-stimulated and contracting muscles. The muscle contraction-evoked influences related to the metabolism of amino acids, in particular: aspartate, alanine or lysine, are supposed to be independent of AMPK activation. Our results substantiate significance of AMPK activation in contracting skeletal muscles and provide novel evidence that AICAR stimulation closely mimics the metabolomic changes in the contracting skeletal muscles.
The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a single proton in exchange for one extracellular sodium. The human protein contains a membrane domain of approximately 500 amino acids, and a regulatory, cytosolic domain of approximately 315 amino acids. NHE1 is activated by a number of hormones including endothelin (ET), and by calcium. The regulatory tail possesses an inhibitory calmodulin binding domain and inhibition of NHE1 is relieved by binding of a calcium-calmodulin (CaM) complex. We examined the dynamics of ET-1 and calcium regulation of binding to NHE1 in vivo. Cerulean was linked to the NHE1 protein cytoplasmic C-terminus. This was stably transfected into AP-1 cells that are devoid of their own NHE1 protein. The protein was expressed and targeted properly and retained NHE1 activity comparable to the wild type. We examined the in vivo coupling of NHE1 to CaM by FRET using CaM linked to the fluorescent protein Venus. CaM interaction with NHE1 was dynamic. Removal of serum, reduced CaM interaction with NHE1. Addition of the calcium ionophore ionomycin, increased the interaction between CaM and NHE1. We expressed an ET receptor in AP-1 cells and also found that there was a time dependent association of NHE1 with CaM in vivo that was dependent on ET treatment. The results are the first demonstration of the in vivo association of NHE1 and CaM through ET- dependent signaling pathways.
In older adults, changes in skeletal muscle composition are associated with increased fibrosis, loss of mass, and decreased force, which can lead to dependency, morbidity, and mortality. Understanding the biological mechanisms responsible is essential to sustaining and improving their quality of life. Compared to young mice, aged mice take longer to recover from muscle injury; their tissue fibrosis is more extensive, and regenerated myofibers are smaller. Strong evidence indicates that cells called pericytes, embedded in the basement membrane of capillaries, contribute to the satellite-cell pool and muscle growth. In addition to their role in skeletal muscle repair, after tissue damage, they detach from capillaries and migrate to the interstitial space to participate in fibrosis formation. Here we distinguish two bona fide pericyte subtypes in the skeletal muscle interstitium, type-1 (Nestin-GFP-/NG2-DsRed+) and type-2 (Nestin-GFP+/NG2-DsRed+), and characterize their heretofore unknown specific roles in the aging environment. Our in vitro results show that type-1 and type-2 pericytes are either fibrogenic or myogenic, respectively. Transplantation studies in young animals indicate that type-2 pericytes are myogenic, while type-1 pericytes remain in the interstitial space. In older mice, however, the muscular regenerative capacity of type-2 pericytes is limited, and type-1 pericytes produce collagen, contributing to fibrous tissue deposition. We conclude that in injured muscles from aging mice, the pericytes involved in skeletal muscle repair differ from those associated with scar formation.
Rett syndrome (RTT) is a neurodevelopmental disorder with symptoms starting 6-18 months after birth, while what underlies the delayed onset is unclear. Allopregnanolone (Allop) is a metabolite of progesterone, and a potent modulator of GABAA-ergic currents whose defects are seen in RTT. Allop changes its concentrations during the perinatal period, which may affect central neurons via the GABAA-ergic synaptic transmission, contributing to the onset of the disease. To determine whether the Mecp2 disruption affects the Allop modulation, we performed the studies in brain slices obtained from wild-type (WT) and Mecp2–/Y mice. Allop dose-dependently suppressed locus coeruleus (LC) neuronal excitability in WT mice, while the Mecp2-null neurons showed significant defects. Using optogenetic approaches, channelrhodopsin was specifically expressed in GABA-ergic neurons in which optical stimulation evoked action potentials. In LC neurons of WT mice, Allop exposure increased the amplitude of GABAA-ergic inhibitory postsynaptic currents (IPSCs) evoked by optical stimulation, and prolonged the IPSC decay time. Consistently, Allop augmented both frequency and amplitude of GABAA-ergic spontaneous IPSCs (sIPSCs), and expended the sIPSC decay time. The Allop-induced potentiation of sIPSCs was deficient in Mecp2–/Y mice. Surprisingly, the impairment occurred at 3 weeks of the postnatal age, while no significant difference in the Allop modulation was observed in 1-2 weeks between the WT and Mecp2–/Y mice. These results indicate that the modulation of GABAA-ergic synaptic transmission by Allop is impaired in LC neurons of Mecp2-null mice at a time when RTT-like symptoms manifest, suggesting a potential mechanism for the delayed onset of the disease.
To address pivotal roles of cell surface F1/FO ATP synthase in the development of acidic microenvironment in tumor tissues, we investigated effects of shear stress stimulation on the cultured human breast cancer cells, MDA-MB-231 and MDA-MB-157 or human melanoma cells, SK-Mel-1. Shear stress stimulation (0.5-5.0 dyn/cm2), the levels of which are similar to those produced by the interstitial flow, induced strength-dependent co-release of ATP and H+ from the cells, which triggered CO2 gas excretion. In contrast, the same level of shear stress stimulation did not induce significant ATP release and CO2 gas excretion from the control human mammary epithelial cells (HMEC). Marked immunocytochemical and mRNA expression of cell surface F1/FO ATP synthase, vacuolar-ATPase (V-ATPase), carbonic anhydrase type IX, and ectonucleoside triphosphate diphosphohydrolase (ENTPDase) 3 were detected in MDA-MB-231 cells, but little or no expression on the HMEC. Pretreatment with cell surface F1/FO ATP synthase inhibitors, but not cell surface V-ATPase inhibitors caused a significant reduction of the shear stress stimulation-mediated ATP release and CO2 gas excretion from MDA-MB-231 cells. The ENTPDase activity in the shear stress-loaded MDA-MB-231 cell culture medium supernatant increased significantly in a time-dependent manner. In addition, MDA-MB-231 cells displayed strong staining for purinergic 2Y1 (P2Y1) receptors on their surfaces, and the receptors partially co-localized with ENTPDase 3. These findings suggest that cell surface F1/FO ATP synthase, but not V-ATPase may play key roles in the development of interstitial flow-mediated acidic microenvironment in tumor tissues through the shear stress stimulation-induced ATP and H+ co-release and CO2 gas production.
To withstand physiological loading during lifetime, human synovial joints are covered and protected by a layer of low-friction, load-bearing tissue called articular cartilage. The unique mechanical function of articular cartilage largely depends on the composition and structural integrity of cartilage matrix. The matrix is produced by the highly specialized resident cells called chondrocytes. Under physiological loading, chondrocytes maintain the balance between degrading and synthesizing matrix macromolecules. By contrast, under excessive loading or injury, the degradation exceeds the synthesis causing joint degeneration and eventually osteoarthritis. Hence, the mechanoresponses of chondrocytes play an important role in the development of osteoarthritis. Despite its clear importance, the mechanobiology of articular chondrocytes is yet not well understood. To summarize our current understanding, here we review the studies on effect of mechanical forces on both mechanical and biological properties of articular chondrocytes. First, we present viscoelastic properties of structures like cell nucleus, chondrocyte, pericellular matrix, and chondron. Then, we present how these properties change in osteoarthritis. Finally, we discuss the responses of both normal and osteoarthritic chondrocytes to variety of mechanical stimuli. Studies reviewed here may provide novel insights in pathogenesis of osteoarthritis, and may help in developing effective biophysical treatment.
The presence of N-methyl D-aspartate receptor (NMDAR) was previously shown in rat red blood cells (RBCs) and in UT-7/Epo human myeloid cell line differentiating into erythroid lineage. Here we have characterized the subunit composition of the NMDAR and monitored its function during human erythropoiesis and in circulating RBCs. Expression of the NMDARs subunits was assessed in erythroid progenitors during ex vivo erythropoiesis and in circulating human RBCs using qPCR, and flow cytometry. Receptor activity was monitored using a radiolabeled antagonist binding assay, live imaging of Ca2+ uptake, patch clamp and monitoring of cell volume changes. The receptor tetramers in erythroid precursor cells are composed of the NR1, NR2A, 2C, 2D, NR3A and 3B subunits of which the glycine-binding NR3A and 3B and glutamate-binding NR2C and 2D subunits prevailed. Functional receptor is required for survival of erythroid precursors. Circulating RBCs retain a low number of the receptor copies that is higher in young cells compared to mature and senescent RBC populations. In circulating RBCs the receptor activity is controlled by plasma glutamate and glycine. Modulation of the NMDAR activity in RBCs by agonists or antagonists is associated with the alterations in whole-cell ion currents. Activation of the receptor results in the transient Ca2+ accumulation, cell shrinkage, alteration in the intracellular pH which is associated with the change in hemoglobin oxygen affinity. Thus, functional NMDARs are present in erythroid precursor cells and in circulating RBCs. These receptors contribute to intracellular Ca2+ homeostasis and modulate oxygen delivery to peripheral tissues.
Calcium flux in the podocytes is critical for normal and pathophysiological regulation of this type of cells, and excessive calcium signaling results in podocytes damage and improper glomeruli function. Purinergic activation of P2 receptors is a powerful and rapid signaling process; however, the exact physiological identity of P2 receptors subtypes in podocytes remains essentially unknown. The goal of this study was to determine P2 receptors profile in podocytes of the intact Sprague Dawley rat glomeruli using available pharmacological tools. Glomeruli were isolated by differential sieving, loaded with Fluo-4/Fura Red cell permeable calcium indicators and purinergic response in the podocytes was analyzed with ratiometric confocal fluorescence measurements. Various P2 receptors activators were tested and compared to the effect of ATP; specifically, UDP, MRS 2768, MRS 2365, bzATP, αβMethylene, 2-meSADP, MRS 4062, and MRS 2768 were analyzed. Antagonists (MRS 2500, 5-BDBD, A438079 and NF449) were tested when 10 μM ATP was applied as the EC50 for ATP activation of the calcium influx in the podocytes was determined to be 10.7 ± 1.5 μM. Several agonists including MRS 2365 and 2-meSADP caused calcium flux. Importantly, only P2Y1 specific antagonist MRS 2500 (1 nM) precluded the effects of ATP concentrations of the physiological range. Immunohistochemical analysis confirmed that P2Y1 receptors are highly expressed in the podocytes. We conclude that P2Y1 receptor signaling is the predominant P2Y purinergic pathway in the glomeruli podocytes and P2Y1 might be involved in the pathogenesis of glomerular injury and could be a target for treatment of kidney diseases.
Inflammatory angiogenesis involves the induction of a novel gene ZC3H12A encoding MCP-1-induced protein-1 (MCPIP1) that has deubiquitinase and antidicer RNAse activities. If and how these enzymatic activities of MCPIP1 mediate the biological functions of MCPIP1 is unknown. Present studies with human umbilical vein endothelial cells suggest that MCPIP-induced angiogenesis is mediated via hypoxia-inducible factor (HIF-1α), vascular endothelial growth factor (VEGF) and silent information regulator (SIRT -1) induction that results in the inhibition of angiogenesis inhibitor, thrombospondin-1. MCPIP1 expression inhibited the production of the anti-angiogenic microRNAs -20b and -34a that repress the translation of HIF-1α and SIRT-1, respectively. RNase dead MCPIP mutant, D141N, not only did not induce angiogenesis but also inhibited the production of miR-20b and -34a suggesting that the anti-dicer RNase activity of MCPIP1 is involved in MCPIP mediated angiogenesis. Mimetics of miR-20b and -34a inhibited MCPIP1-induced angiogenesis confirming that MCPIP1suppresses the biogenesis of miRs-20b and -34a. Furthermore, our results indicate that MCPIP expression induces nuclear translocation of HIF-1α. We show that under hypoxia angiogenesis is mediated via induction of MCPIP1 and under normoxia, in vitro, MCPIP deubiquitinates ubiquitinated HIF-1α and the stabilized HIF-1α enters the nucleus to promote the transcription of its target genes, cyclooxygenase-2 and VEGF, suggesti ng that the deubiquitinase activity of MCPIP may also promoteangiogenesis. The present results show for the first time that the anti-dicer RNase activity of MCPIP1 is critical in mediating a biological function of MCPIP, namely angiogenesis.
To elucidate how tumor cells produce energy in oxygen-depleted microenvironments, we sought the possibility of mitochondrial electron transport without oxygen. We produced well-controlled oxygen gradients (O2) in monolayer-cultured cells. We then visualized oxygen levels and mitochondrial membrane potential (m) in individual cells by using the red shift of green fluorescent protein (GFP) fluorescence and a cationic fluorescent dye, respectively. In this 2-dimensional tissue model, m was abolished in cells located approximately >500 µm from the oxygen source (anoxic front, AF), indicating limitations in diffusional oxygen delivery. This result perfectly matched O2 determined by GFP. In cells pretreated with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase domain containing protein (PHD) inhibitor, AF was expanded to 1500-2000 µm from the source. In these cells, tissue O2 were substantially decreased, indicating that the PHD pathway activation suppressed mitochondrial respiration. The expansion of AF and the reduction of O2 were much more prominent in a cancer cell line (Hep3B) than in the equivalent fibroblast-like cell line (COS-7). Hence, the results indicate that PHD pathway-activated cells can sustain m despite significantly decreased electron flux to complex IV. Complex II inhibition abolished the effect of DMOG in expanding AF, although the tissue O2 remained shallow. Separate experiments demonstrated that complex II plays a substantial role in sustaining m in DMOG-pretreated Hep3B cells with complex III inhibition. From these results, we conclude that PHD pathway activation can sustain m in an otherwise anoxic microenvironment by decreasing tissue O2 while activating oxygen-independent electron transport in mitochondria.
The tubulin cytoskeleton plays a key role in maintaining the characteristic quiescent discoid shape of resting platelets. Upon activation, platelets undergo a dramatic change in shape; however, little is known how the microtubule system contributes to regulating platelet shape and function. Here we investigated the role of the covalent modification of α-tubulin by acetylation in the regulation of platelet physiology during activation. Superresolution microscopy analysis of the platelet tubulin cytoskeleton showed that the marginal band together with an interconnected web of finer tubulin structures collapsed upon platelet activation with the GPVI-agonist, collagen related peptide (CRP). Western blot analysis revealed that α-tubulin was acetylated in resting platelets and deacetylated during platelet activation. Tubacin, a specific inhibitor of the tubulin deacetylase HDAC6, prevented tubulin deacetylation upon platelet activation with CRP. Inhibition of HDAC6 upregulated tubulin acetylation and disrupted the organization of the platelet microtubule marginal band without significantly affecting platelet volume changes in response to CRP stimulation. HDAC6 inhibitors also inhibited platelet aggregation in response to CRP and blocked platelet signaling events upstream of platelet Rho GTPase activation. Together these findings support a role for acetylation signaling in controlling the resting structure of the platelet tubulin marginal band as well as in the coordination of signaling systems that drive platelet cytoskeletal changes and aggregation.
Inactivation of tumor suppressor genes via promoter hypermethylation may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). We have previously shown that acid-induced p16 gene promoter hypermethylation may depend on activation of NADPH oxidase NOX5-S in BAR-T cells and OE33 EA cells. DNA methyltransferase 1 (DNMT1) is known to participate in maintaining established patterns of DNA methylation in dividing cells and may play an important role in the development of cancer. Therefore, we examined whether DNMT1 is involved in acid induced p16 gene promoter hypermethylation in BAR-T cells. We found that the acid significantly increased p16 gene promoter methylation, decreased p16 mRNA and increased cell proliferation, effects which may depend on activation of DNMT1 in BAR-T cells. DNMT1 is overexpressed in EA cells FLO and EA tissues. Acid treatment upregulated DNMT1 mRNA expression and increased DNMT1 promoter activity. Acid-induced increase in DNMT1 mRNA expression and promoter activity were significantly decreased by knockdown of NOX5-S and NF-B1 p50. Conversely, overexpression of NOX5-S, p50 or p65 significantly increased DNMT1 promoter activity. Knockdown of NOX5-S significantly decreased the acid-induced increase in luciferase activity in cells transfected with pNFB-Luc. An NF-B binding element GGGGTATCCC was identified in the DNMT1 gene promoter. We conclude that the acid-induced increase in p16 gene promoter methylation, downregulation of p16 mRNA and increase in cell proliferation may depend on activation of DNMT1 in BAR-T cells. Acid-induced DNMT1 expression may depend on sequential activation of NOX5-S and NF-B1 p50.
Chronic heart failure (CHF) is characterized by decreased cardiac parasympathetic and increased cardiac sympathetic nerve activity. This autonomic imbalance increases the risk of arrhythmias and sudden death in the patients with CHF. We hypothesized that the molecular and cellular alterations of cardiac postganglionic parasympathetic (CPP) neurons located in the intracardiac ganglia (ICG) and sympathetic (CPS) neurons existed in the stellate ganglia (SG) possibly link to the cardiac autonomic imbalance in CHF. Rat CHF was induced by left coronary artery ligation. Single cell real-time PCR and immunofluorescent data showed that L (Cav1.2 and Cav1.3), P/Q (Cav 2.1), N (Cav 2.2), and R (Cav 2.3) types of Ca++ channels were expressed in the CPP and CPS neurons, but CHF only decreased the mRNA and protein expression of N-type Ca++ channels in the CPP neurons and did not affect the mRNA and protein expression of all Ca++ channel subtypes in the CPS neurons. Patch clamp recording confirmed that CHF reduced N-type Ca++ currents and cell excitability in the CPP neurons and enhanced N-type Ca++ currents and cell excitability in the CPS neurons. N-type Ca++ channel blocker (1 µM -conotoxin GVIA) lowered Ca++ currents and cell excitability in the CPP and CPS neurons from sham and CHF rats. These results suggest that CHF reduces the N-type Ca++ channel currents and cell excitability in the CPP neurons and enhances the N-type Ca++ currents and cell excitability in the CPS neurons, which may contribute to the cardiac autonomic imbalance in CHF.
Important functions of the vascular endothelium, including permeability, production of antithrombotic factors, and control of vascular tone, are regulated by changes in intracellular Ca2+. The molecular identities and regulation of Ca2+ influx channels in the endothelium are incompletely understood, in part because of experimental difficulties associated with application of patch-clamp electrophysiology to native endothelial cells. However, advances in confocal and total internal reflection fluorescence microscopy and the development of fast, high affinity Ca2+-binding fluorophores have recently allowed for direct visualization and characterization of single channel transient receptor potential (TRP) channel Ca2+ influx events in endothelial cells. These events, called "TRP channel Ca2+ sparklets," have been optically recorded from primary endothelial cells and the intact endothelium, and the biophysical properties and fundamental significance of these Ca2+ signals in vasomotor regulation have been characterized. This review will first briefly discuss the role of endothelial cell TRP channel Ca2+ influx in endothelium-dependent vasodilation, will describe improved methods for recording unitary TRP channel activity using optical methods, and will highlight discoveries regarding the regulation and physiological significance of TRPV4 Ca2+ sparklets in the vascular endothelium enabled by this new technology. Perspectives on the potential use of these techniques to evaluate changes in TRP channel Ca2+ influx activity associated with endothelial dysfunction are offered.
Zebrafish lateral-line hair cells are an in vivo model for studying hair cell development, function, and ototoxicity. However, molecular identification and properties of the mechanotransducer (MET) channel in hair cells are still controversial. In this study, a noninvasive electrophysiological technique, the scanning ion-electrode technique (SIET), was applied for the first time to investigate properties of MET channels in intact zebrafish embryos. Using a Ca2+-selective microelectrode to deflect hair bundles and simultaneously record the Ca2+ flux, the inward Ca2+ flux was detected at stereocilia of hair cells in 2~4-days post-fertilization embryos. The Ca2+ influx was blocked by MET channel blockers (BAPTA, La3+, Gd3+ and curare). In addition, 10 µM aminoglycoside antibiotics (neomycin and gentamicin) were found to effectively block the Ca2+ influx within 10 min. Elevating the external Ca2+ level (0.2 to 2 mM) neutralized the effects of neomycin and gentamicin. However, elevating the Mg2+ level up to 5 mM neutralized the blockade of gentamicin but not neomycin. This study demonstrated MET channel-mediated Ca2+ entry at hair cells and showed the SIET to be a sensitive approach for functionally assaying the MET channel in zebrafish.
Mutations in Cx50 cause dominant cataracts in both humans and mice. The exact mechanisms by which mutations cause these variable phenotypes are poorly understood. We examined the functional properties of gap junctions made by three Cx50 mutations, V44E, D47N and V79L, expressed in mammalian cell lines. V44E trafficked to the plasma membrane properly and formed gap junctional plaques. However, the mutant did not form functional gap junctions when expressed alone, or with wildtype (WT) Cx46 and Cx50, indicating that V44E is a dominant negative inhibitor of WT connexin function. In contrast, D47N subunits did not localize to junctional plaques or form functional homotypic gap junctions; however, mixed expression of D47N and WT subunits of either Cx50 or Cx46 resulted in functional intercellular channels, with high levels of coupling. Single channel studies indicated that D47N formed heteromeric channels with WT Cx46 with unique properties. Unlike either V44E or D47N, V79L formed functional homotypic intercellular channels. However, the mutation caused an alteration in voltage gating and a reduction in the open probability, resulting in much lower levels of conductance in cells expressing V79L alone, or together with WT connexin subunits. Thus, each mutation produced distinct changes in the properties of junctional coupling. V44E failed to form intercellular channels in any configuration, D47N formed only heteromeric channels with WT connexins and V79L formed homotypic and heteromeric channels with altered properties. These results suggest that unique interactions between mutant and wild-type lens connexins might underlie the development of various cataract phenotypes in humans.
Vacuolar ATPases (V-ATPases) are highly conserved proton pumps that regulate organelle pH. Epithelial luminal pH is also regulated by cAMP-dependent traffic of specific subunits of the V-ATPase complex from endosomes into the apical membrane. In the intestine, cAMP-dependent traffic of cystic fibrosis transmembrane conductance regulator (CFTR) channels and the sodium hydrogen exchanger (NHE3) in the brush border regulate luminal pH. V-ATPase was found to co-localize with CFTR in intestinal CFTR High Expresser (CHE) cells recently. Moreover, apical traffic of V-ATPase and CFTR in rat Brunner's glands was shown to be dependent on cAMP/PKA. These observations support a functional relationship between V-ATPase and CFTR in the intestine. The current study examined V-ATPase and CFTR distribution in intestines from wild-type, CFTR-/- mice and polarized intestinal CaCo-2 BBe cells following cAMP stimulation and inhibition of CFTR/V-ATPase function. Co- immunoprecipitation studies examined V-ATPase interaction with CFTR. The pH sensitive dye BCECF determined proton efflux and its dependence on V-ATPase/CFTR in intestinal cells. cAMP increased V-ATPase/CFTR co-localization in the apical domain of intestinal cells, and redistributed the V-ATPase Voa1 and Voa2 trafficking subunits from the basolateral membrane to the BBM. Voa1 and Voa2 subunits were localized to endosomes beneath the terminal web in untreated CFTR -/- intestine, but redistributed to the subapical cytoplasm following cAMP treatment. Inhibition of CFTR or V-ATPase significantly decreased pHi in cells, confirming their functional interdependence. These data establish that V-ATPase traffics into the brush border membrane to regulate proton efflux and this activity is dependent on CFTR in the intestine.
Hypoglossal motoneurons (HNs) control tongue movement and play a role in maintenance of upper airway patency. Defects in these neurons may contribute to the development of sleep apnea and other cranial motor disorders including Rett syndrome (RTT). HNs are modulated by norepinephrine (NE) through α-adrenoceptors. Although postsynaptic mechanisms are known to play a role in this effect, how NE modulates the synaptic transmissions of HNs remains poorly understood. More importantly, the NE system is defective in RTT, while how the defect affects HNs is unknown. Believing that information of NE modulation of HNs may help the understanding of RTT and the design of new therapeutical interventions to motor defects in the disease, we performed these studies in which glycinergic IPSCs and intrinsic membrane properties were examined in wild-type (WT) and Mecp2–/Y mice, a mouse of model of RTT. We found that activation of α1-adrenoceptor facilitated glycinergic synaptic transmission and excited HNs. These effects were mediated by both pre- and postsynaptic mechanisms. The latter effect involved an inhibition of barium-sensitive G-protein dependent K+ currents. The pre- and postsynaptic modulations of the HNs by α1-adrenoceptors were not only retained in Mecep2-null mice but also markedly enhanced, which appears to be a compensatory mechanism for the deficiencies in NE and GABA-ergic synaptic transmission. The existence of the endogenous compensatory mechanism is an encouraging finding, as it may allow therapeutical modalities to alleviate motoneuronal defects in RTT.
Defective colonic Na+ and Cl- absorption is a feature of active ulcerative colitis (UC), but little is known about changes in colonic K+ transport. We therefore investigated colonic K+ transport in a rat model of dextran sulfate-induced colitis. Colitis was induced in rat distal colon using 5% dextran sulfate sodium (DSS). Short-circuit current (Isc, indicating electrogenic ion transport) and 86Rb (K+ surrogate) fluxes were measured in colonic mucosa mounted in Ussing chambers under voltage clamp conditions in the presence of mucosal orthovanadate (a P-type ATPase inhibitor). Serum aldosterone was measured by immunoassay. Control animals exhibited zero net K+ flux. By contrast, DSS-treated animals exhibited active K+ secretion, which was inhibited by 98%, 76% and 22% by Ba2+ (nonspecific K+ channel blocker), iberiotoxin (IbTX; BK channel blocker) and TRAM-34 (IK channel blocker), respectively. Apical BK channel α-subunit mRNA abundance and protein expression, and serum aldosterone levels in DSS-treated animals, were enhanced 6-, 3- and 6-fold respectively, compared with controls. Increasing intracellular Ca2+ with carbachol (CCH), or intracellular cAMP with forskolin (FSK), stimulated both active Cl- secretion and active K+ secretion in controls, but had no or little effect in DSS-treated animals. In DSS-induced colitis, active K+ secretion involves up-regulation of apical BK channel expression which may be aldosterone-dependent, whereas Cl- secretion is diminished. Since similar ion transport abnormalities occur in patients with UC, diarrhoea in this disease may reflect increased colonic K+ secretion (rather than increased Cl- secretion), as well as defective Na+ and Cl- absorption.
Hypertonic saline (HS) inhalation therapy benefits Cystic Fibrosis (CF) patients (8; 11). Surprisingly, these benefits are long-lasting and are diminished by the epithelial Na+ channel blocker amiloride (8). Our aim was to explain these effects. Human bronchial epithelial (hBE) cells from CF lungs were grown in inserts and were used in three experimental approaches: i) Ussing chambers to measure amiloride-sensitive short circuit currents (INa); ii) Continuous perfusion Ussing chambers; and iii) The airway surface of the inserts was exposed to a small volume (30 µl) of isosmotic or HS solution as the inserts were kept in their incubation tray and were subsequently used to measure INa under isosmotic conditions (near "thin-film" experiments) (35). HS solutions (660 mOsm/Kg H2O) were prepared by adding additional NaCl to the isosmotic buffer. The transepithelial short-circuit current (ISC), conductance (GT) and capacitance (CT) were measured by transepithelial impedance analysis (6; 33). Exposure to apical HS inhibited INa, GT and CT. The INa inhibition required 60 minutes of re-exposure to the isosmotic solution to recover 75%. The time of exposure to HS required to inhibit INa was < 2.5 minutes. Under near "thin-film" conditions apical exposure to HS inhibited INa but as osmotically-driven water moved to the apical surface, the aqueous apical volume increased leading to an amiloride-insensitive decrease in its osmolality and to recovery of INa that lagged behind the osmotic recovery. Amiloride significantly accelerated the recovery of INa following exposure to HS. Conclusions: Exposure to HS inhibits hBE INa and Amiloride diminishes this effect.
Glucagon-like peptide-1 (GLP-1), secreted from gut L-cells upon nutrient intake, forms the basis for novel drugs against type 2 diabetes (T2D). Secretion of GLP-1 has been suggested to be impaired in T2D and in conditions associated with hyperlipidemia and insulin resistance. Further, recent studies support lipotoxicity of GLP-1-producing cells in vitro. However, little is known about the regulation of L-cell viability/function, the effects of insulin signaling or potential effects of stable GLP-1 analogues and DPP-4 inhibitors. We determined effects of insulin as well as possible autocrine action of GLP-1 on viability/apoptosis of GLP-1-secreting cells, in the presence/absence of palmitate, while also assessing direct effects on function. The studies were performed using the GLP-1-secreting cell line GLUTag, and palmitate was used to simulate hyperlipidemia. Our results show that palmitate-induced production of reactive oxygen species, caspase-3 activity and reduced cell viability are significantly attenuated by pre-incubation with insulin/exendin-4. The indicated lipoprotective effect of insulin/exendin-4 was not detectable in the presence of the GLP-1R antagonist exendin (9-39), and attenuated in response to pharmacological inhibition of Epac signaling, while protein kinase A inhibition had no significant effect. Insulin/exendin-4 also significantly stimulate acute and long term GLP-1 secretion in the presence of glucose, suggesting novel beneficial effects of insulin signaling and GLP-1R activation on glycemia through enhanced mass of GLP-1 producing cells and enhanced GLP-1 secretion. In addition, the effects of insulin indicate that not only is GLP-1 important for insulin secretion but altered insulin signaling may contribute to an altered GLP-1 secretion.
Hydroxyurea is currently the only FDA-approved drug that ameliorates the pathophysiology of sickle cell anemia. Unfortunately, substantial inter-patient variability in the pharmacokinetics (PK) of hydroxyurea may result in variation of the drug's efficacy. However, little is known about mechanisms that modulate hydroxyurea PK. Recent in vitro studies identifying hydroxyurea as a substrate for organic anion transporting polypeptide (OATP1B) transporters prompted the current investigation assessing the role of OATP1B transporters in modulating hydroxyurea PK. Using wild-type and Oatp1b knockout (Oatp1b-/-) mice, hydroxyurea PK was analyzed in vivo by measuring 14C-hydroxyurea distribution in plasma, kidney, liver, urine, or the exhaled 14CO2 metabolite. Plasma levels were significantly reduced by 20% in Oatp1b-/- mice compared to wild-type (AUC of 38.64 μg-hr/ml or 48.45 μg-hr/ml, respectively) after oral administration; whereas no difference was observed between groups following intravenous administration. Accumulation in the kidney was significantly decreased by 2-fold in Oatp1b-/- mice (356.9 pmol/g vs. 748.1 pmol/g), which correlated with a significant decrease in urinary excretion. , Hydroxyurea accumulation in the liver was also decreased (136.6 pmol/g vs. 107.3 pmol/g in wild-type or Oatp1b-/- mice, respectively) correlating with a decrease in exhaled 14CO2. These findings illustrate that deficiency of Oatp1b transporters alters the absorption, distribution, and elimination of hydroxyurea thus providing the first in vivo evidence that cell membrane transporters may play a significant role in modulating hydroxyurea PK. Future studies to investigate other transporters and their role in hydroxyurea disposition are warranted for understanding the sources of variation in hydroxyurea's PK.
Endothelial cell dysfunction is implicated in cardiovascular diseases including diabetes. The decrease in nitric oxide (NO) bioavailability is the hallmark of endothelial dysfunction and it leads to attenuated vascular relaxation and atherosclerosis followed by the decrease in blood flow. In the heart, decreased coronary blood flow is responsible for insufficient oxygen supply to cardiac myocytes, and subsequently increases the incidence of cardiac ischemia. In this study, we investigate whether and how reactive oxygen species (ROS) in mitochondria contributes to coronary endothelial dysfunction in type 2 diabetic mice. Type 2 diabetic mice were induced by a high-fat diet combined with a single injection of low dose streptozotocin. Acetylcholine (ACh)-induced vascular relaxation was significantly attenuated in diabetic coronary arteries (CAs) compared to control CAs. The pharmacological approach reveals that NO-dependent relaxation, but not hyperpolarization- or prostacyclin- dependent relaxation, was decreased in CAs of diabetic mice. Attenuated ACh-induced relaxation in diabetic CAs was restored by treatment of mitoTempol (a mitochondria-specific superoxide anion scavenger) toward control level. Mouse coronary endothelial cells (MCECs) isolated from type 2 diabetic mice exhibited a significant increase in mitochondrial ROS concentration and decreased in superoxide dismutase 2 (SOD2) protein expression compared with control MCECs. Furthermore, protein ubiquitination of SOD2 was significantly increased in MCECs isolated from diabetic mice. These results suggest that augmented SOD2 ubiquitination leads to the increase in mitochondrial ROS concentration in diabetic MCECs, and attenuates coronary vascular relaxation in Type 2 diabetic mice.
When hypertonicity is imposed with sufficient intensity and acuteness, cells die. Here we investigated cellular pathways involved in the death using cell lines derived from renal epithelia. We found that hypertoncity rapidly induced activation of intrinsic cell death pathway - release of cytochrome c and activation of caspase-3 and caspase-9 - and extrinsic pathway - activation of caspase-8. Likewise, a lysosomal pathway of cell death characterized by partial lysosomal rupture and release of cathepsin B from lysosomes to the cytosol was also activated. Relationship among the pathways was examined using specific inhibitors. Caspase inhibitors did not affect cathepsin B release into the cytosol by hypertonicity. In addition, cathepsin B inhibitors and caspase inhibitors did not affect hypertoncity-induced cytochrome c release suggesting that the three pathways were independently activated. Combined inhibition of caspases and cathepsin B conferred significantly more protection from hypertonicity-induced cell death than inhibition of caspase or cathepsin B alone indicating that all the three pathways contributed to the hypertonicity-induced cell death. Similar pattern of sensitivity to the inhibitors was observed in two other cell lines derived from renal epithelia. We conclude that multiple cell death pathways are independently activated early in response to lethal hypertonic stress in renal epithelial cells.
Canonical transient receptor potential-6 channels (TRPC6) have been implicated in the pathophysiology of glomerular diseases. TRPC6 channels are typically activated by diacylglycerol (DAG) during phospholipase C (PLC)-dependent transduction cascades. TRPC6 can also be activated by reactive oxygen species (ROS). We have previously shown that podocin is required for DAG analogs to produce robust activation of TRPC6 channels in podocytes. Here we show that endogenous TRPC6 channels in immortalized podocytes reciprocally co-immunoprecipitate with the catalytic subunit of the NADPH oxidase NOX2 (gp91phox). The NOX2-TRPC6 interaction was not detected in cells stably expressing an shRNA targeting podocin, although NOX2 and TRPC6 were present at normal levels. Application of a membrane permeable DAG analog (OAG) increased generation of ROS in podocytes, but this effect was not detected in podocin knockdown cells. OAG also increased steady-state surface expression of the NOX2 regulatory subunit p47phox. In whole-cell recordings, TRPC6 activation by OAG was reduced in podocytes pretreated with the NOX2 inhibitor apocynin, by the pan-NOX inhibitor diphenylene iodonium (DPI) and by tempol, a ROS quencher. Cholesterol depletion and disruption of lipid rafts using methyl-β-cyclodextrin (MBCD) reduced activation of podocyte TRPC6 channels by OAG, and also eliminated the NOX2-TRPC6 interaction as assessed by co-immunoprecipitation. These data suggest that active NOX2 complex assembles with TRPC6 at podocin-organized sterol-rich raft domains, and becomes catalytically active in response to DAG. The localized production of ROS contributes to TRPC6 activation by chemical stimuli such as DAG. Podocin appears to be necessary for assembly of the NOX2-TRPC6 complex in lipid rafts.
Molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor at term are incompletely known. More than 10% of babies born worldwide are premature and one-million die annually. Preterm labor results in preterm delivery in 50% of cases in the US explaining 75% of fetal morbidity and mortality. There is no FDA-approved treatment to prevent preterm delivery. Nitric oxide mediated relaxation of human uterine smooth muscle is independent of global elevation of cGMP following activation of soluble guanylyl cyclase. S-nitrosation is a likely mechanism to explain cGMP-independent relaxation to nitric oxide and may reveal S-nitrosated proteins as new therapeutic targets for the treatment of preterm labor. Employing S-nitrosoglutathione as an NO donor, we identified 110 proteins that are S-nitrosated in one or more states of human pregnancy. Using area under the curve of extracted ion chromatograms as well as normalized spectral counts to quantify relative expression levels for 62 of these proteins, we show that 26 proteins demonstrate statistically significant S-nitrosation differences in myometrium from spontaneously laboring preterm patients compared to non-laboring patients. We identified proteins that were up-S-nitrosated as well as proteins that were down-S-nitrosated in preterm laboring tissues. Identification and relative quantification of the S-nitrosoproteome provides a fingerprint of proteins that can form the basis of hypothesis-directed efforts to understand the regulation of uterine contraction-relaxation and the development of new treatment for preterm labor.
It is becoming increasingly apparent that the dynamics of glycans reflect the physiological state of cells involved in several cell functions including growth, response to signal molecules, migration, as well as adhesion to, interaction with and recognition of other cells. Presence of glycoconjugates in human placenta suggests their major role in maternal-fetal exchanges, intercellular adhesion, cellular metabolism and villous vessels branching. Although several studies have described glycoconjugate distribution in the human placenta descriptions of their physiological function and control mechanisms during placental development are lacking. In this study we investigated the developmental distribution and regulation of placental core 1 O- and N-glycans focusing on early and late first trimester human pregnancy. In order to define the control mechanisms of the oligosaccharide chains during early placentation process, chorionic villous explants and human trophoblast cell lines were exposed to various oxygen levels. We found that oxygen tension regulates changes in core-1 O-glycan (the disaccharide Galβ1-3 GalNAc) epitope expression levels. Moreover, by double affinity chromatography and subsequent analysis with mass spectrometry we identified in the Heat Shock Protein 90 alpha (HSP90α) a good candidate as carrier of the Galβ1-3 GalNAc epitope at low oxygen tension. Our results support a fundamental role of oxygen tension in modulating glycosylation of proteins during placental development.
Muscle contraction during exercise is a major stimulus for the release of peptides and proteins (myokines) that are supposed to take part in the beneficial adaptation to exercise. We hypothesize that application of an in vitro exercise stimulus as electric pulse stimulation (EPS) to human myotubes enables the investigation of the molecular response to exercise in a clearly defined model. We applied EPS for 24 h to primary human myotubes and studied the whole genome-wide transcriptional response as well as the release of candidate myokines. We observed 183 differentially regulated transcripts with fold-changes > 1.3. The transcriptional response resembles several properties of the in vivo situation in the skeletal muscle after endurance exercise, namely significant enrichment of pathways associated with interleukin and chemokine signaling, lipid metabolism, and anti-oxidant defense. Multiplex immunoassays verified the translation of the transcriptional response of several cytokines into high secretion levels (IL6, IL8, CXCL1, LIF, CSF3, IL1B, TNF) and the increased secretion of further myokines such as angiopoietin-like 4. Notably, EPS did not induce the release of creatine kinase. Inhibitor studies and immunoblotting revealed the participation of ERK1/2, JNK and NFB-dependent pathways in the upregulation of myokines. To conclude, our data highlight the importance of skeletal muscle cells as endocrine cells. This in vitro exercise model is not only suitable to identify exercise-regulated myokines but it might be applied to primary human myotubes obtained from different muscle biopsy donors to study the molecular mechanisms of the individual response to exercise.
There is a global epidemic of obesity, and obesity is known to inhibit AMP-activated protein kinase (AMPK) activity and impairs myogenesis. Myogenin mediates the fusion of myoblasts into myotubes, a critical step in myogenesis. We observed that inhibition of AMPKα1 down-regulates myogenin expression and myogenesis, but the underlying mechanisms are unclear. We postulated that AMPK regulates myogenin expression through phosphorlytion of histone deacetylase 5 (HDAC5). In C2C12 cells, HDAC5 knockdown increased while HDAC5 stablization by MC1568 reduced myogenin expression. Consistently, we observed that myogenin promoter activity was negatively regulated by HDAC5. Using C2C12 cells with AMPK 1 Knockdown, and primary myoblasts prepared from wild-type and AMPKα1 KO mice, we further demonstrate that AMPK 1 regulates HDAC5 phosphorylation at Ser 259 and 498. Mutation of these two sites to Ala in HDAC5 abolished the regulatory role of AMPK 1 on myogenin expression, clearly showing the necessity of these phosphorylation sites in mediating myogenin expression. In aggregate, these data show that AMPK inhibition down-regulates myogenin transcription and myogenesis through phosphorylation of HDAC5, mediated mainly by AMPK 1. These data demonstrate that AMPK is a key molecular target for promoting myogenesis and muscular regeneration. Because drugs activating AMPK activity, such as metformin, is widely available, our finding has critical clinical implications to ensure proper muscle development and regeneration in obese subjects and under other pathophysiological conditions where AMPK activity is attenuated.
Alternative splicing of the CaV α1 subunit adds to the functional diversity of Ca2+ channels. A variant with a 73nt deletion in exon 15 of Cav1.2 α1 (Cav1.273) produced by alternative splicing that predicts a truncated protein has been described but its function if any is unknown. We sought to determine if by analogy to other truncated CaV α1 subunits, Cav1.273 acts as an inhibitor of wild type Cav1.2 currents. HEK293 cells were transfected with Cav1.273 in either a pIRES vector with CD8 or in pcDNA3.1 with a V5/his C-terminal tag plus β2 and α21 accessory subunits, and pEGFP. Production of Cav1.273 protein was confirmed by Western blot and immunofluorescence. Voltage clamp studies revealed the absence of functional channels in transfected cells. In contrast, cells transfected with full length Cav1.2 plus accessory subunits and pEGFP exhibited robust Ca2+ currents. A7r5 cells exhibit endogenous Cav1.2-based currents that were greatly reduced (>80%) without a change in voltage dependent activation when transfected with Cav1.273-IRES-CD8 compared to empty vector or to pIRES-CD8 controls. Transfection of A7r5 cells with an analogous Cav2.373-IRES-CD8 had no effect on Ca2+ currents. Immunofluorescence showed intracellular but not plasma membrane localization of Cav1.273-V5/his as well as colocalization with an endoplasmic reticulum marker, ER Lights. Expression of Cav1.273 α1 subunits in A7r5 cells inhibits endogenous Cav1.2 currents. The fact that this variant arises naturally by alternative splicing raises the possibility that it may represent a physiological mechanism to modulate Cav1.2 functional activity.
The goal of this study was to determine whether transforming growth factor β1 (TGF-β1) affects epithelial cells lining the vas deferens, an organ that is universally affected in cystic fibrosis male patients. In PVD9902 cells, which are derived from porcine vas deferens epithelium, TGF-β1 exposure significantly reduced short circuit current (Isc) stimulated by forskolin or a cell membrane-permeant cAMP analog, 8-pCPT-cAMP, suggesting that TGF-β1 affects targets of the cAMP signaling pathway. Electrophysiological results indicated that TGF-β1 reduces the magnitude of current inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) channel blockers. Real-time RT-PCR revealed that TGF-β1 down-regulates the abundance of mRNA coding for CFTR, while biotinylation and western blot showed that TGF-β1 reduces both total CFTR and apical cell surface CFTR abundance. These results suggest that TGF-β1 causes a reduction in CFTR expression, which limits CFTR-mediated anion secretion. TGF-β1 associated attenuation of anion secretion was abrogated by SB431542, a TGF-β1 receptor I inhibitor. Signaling pathway studies showed that the effect of TGF-β1 on Isc was reduced by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). TGF-β1 exposure also increased the amount of phospho-p38 MAPK substantially. In addition, anisomycin, a p38 MAPK activator, mimicked the effect of TGF-β1, which further suggests that TGF-β1 affects PVD9902 cells through a p38 MAPK pathway. These observations suggest that TGF-β1, via TGF-β1 receptor I and p38 MAPK signaling, reduces CFTR expression to impair CFTR-mediated anion secretion, which would likely compound the effects associated with mild CFTR mutations, and ultimately would compromise male fertility.
Colorectal cancer metastases can appear on the peritoneum, in lymph nodes, liver, and lungs, suggesting both hematogenous and lymphatic spreading of the primary tumor. While antithrombotic agents have been shown to reduce both long-term incidence and metastasis, the role of coagulation in facilitating metastasis is ill-defined. We investigated the kinetics and molecular mechanisms of metastatic colon adenocarcinoma cell recruitment to thrombi under shear flow, ex vivo. Platelet aggregates were formed by perfusing citrated anticoagulated whole blood over immobilized fibrinogen or fibrillar collagen. Thrombi were formed by perfusing recalcified whole blood over fibrinogen or fibrillar collagen in the presence of coagulation. Cultured colon adenocarcinoma cells (SW620) were perfused either during or following platelet aggregate or thrombus formation. The degree of transient tumor cell interactions (recruitment, rolling, and release) and the number of firmly adhered tumor cells were quantified using fluorescence microscopy. Platelet aggregates and thrombi formed on either fibrinogen or fibrillar collagen supported SW620 cell interactions and adhesion under shear. Thrombi or fibrin supported a greater degree of SW620 cell interactions and adhesion as compared to platelet aggregates or fibrinogen, respectively, demonstrating that coagulation promoted SW620 cell recruitment under shear. Interestingly, in the absence of anticoagulation, we observed SW620 preferentially binding to thrombus-bound polymorphonuclear leukocytes (PMNs). The addition of purified PMNs to thrombi resulted in a doubling of the number of interacting and bound SW620 cells. Since thrombi often accumulate and activate leukocytes, our findings suggest that leukocytes may play a role in localizing metastases to sites of thrombogenesis.
Most G-protein-coupled receptors (GPCRs) do not generate membrane currents in response to ligand-receptor binding (LRB). Here, we describe a novel technique using endocytosis as a bioassay that can detect activation of a GPCR in a way analogous to patch-clamp recording of an ion channel in a living cell. The confocal imaging technique, termed FM endocytosis imaging (FEI), can record ligand-GPCR binding with high temporal (sec) and spatial (μm) resolution. LRB leads to internalization of an endocytic vesicle, which can be labeled by a styryl FM dye and visualized as a fluorescent spot. Distinct from the GFP-labeling method, FEI can detect LRB endocytosis mediated by essentially any receptors (GPCRs or receptors of tyrosine kinase) in a native cell/cell line. Three modified versions of FEI permit promising applications in functional GPCR studies and drug-screening in living cells: (1) LRB can be recorded in "real time" (time scale of seconds); (2) internalized vesicles mediated by different GPCRs can be discriminated by different colors; and (3) a high throughput method can screen ligands of a specific GPCR.
The epithelial sodium channel (ENaC) plays an important role in homeostasis of blood pressure and of the airway surface liquid, and excess function of ENaC results in refractory hypertension (in Liddle's Syndrome) and impaired mucociliary clearance (in Cystic Fibrosis). The regulation of ENaC by molecular chaperones, such as the 70 kDa heat shock protein Hsc70, is not completely understood. Our previously published data suggested that Hsc70 negatively affects ENaC activity and surface expression in Xenopus oocytes, we investigated the mechanism by which Hsc70 acts upon ENaC in epithelial cells. In MDCK cells stably expressing epitope-tagged αβ-ENaC and with tetracycline-inducible overexpression of Hsc70, treatment with 5 μg/ml Doxycycline increased total Hsc70 expression 20%. This increase in Hsc70 expression led to a decrease in ENaC activity and surface expression that corresponded to an increased rate of functional ENaC retrieval from the cell surface. In addition, Hsc70 overexpression decreased the association of newly synthesized ENaC subunits. These data support the hypothesis that Hsc70 inhibits ENaC functional expression at the apical surface of epithelia by regulating both ENaC biogenesis and ENaC trafficking at the cell surface.
Large conductance, Ca2+-activated K+ channels, commonly referred to as BK channels, have a major role in flow-induced K+ secretion in the distal nephron. With-no-lysine kinase 4 (WNK4) is a serine-threonine kinase expressed in the distal nephron that inhibits ROMK activity and renal K+ secretion. WNK4 mutations have been described in individuals with familial hyperkalemic hypertension (FHHt), a Mendelian disorder characterized by low-renin hypertension and hyperkalemia. As BK channels also have an important role in renal K+ secretion, we examined whether they are regulated by WNK4 in a manner similar to ROMK. BK channel activity was inhibited in a rabbit intercalated cell line transfected with WNK4 or a WNK4 mutant found in individuals with FHHt. Co-expression of an epitope-tagged BK α subunit with WNK4 or the WNK4 mutant in HEK293 cells reduced BK α subunit plasma membrane and whole cell expression. A region within WNK4 encompassing the autoinhibitory domain and a coiled coil domain was required for WNK4 to inhibit BK α subunit expression. The relative fraction of BK α subunit that was ubiquitinated was significantly increased in cells expressing WNK4, compared with controls. Our results suggest that WNK4 inhibits BK channel activity, in part, by increasing channel degradation through an ubiquitin-dependent pathway. Based on these results, we propose that WNK4 provides a cellular mechanism for the coordinated regulation of two key secretory K+ channels in the distal nephron, ROMK and BK.
Chronic hypoxia (Chox) augments chemoafferent activity in sensory fibers innervating carotid body (CB) chemoreceptor type I cells, however, the underlying mechanisms are poorly understood. Here, we tested the hypothesis that enhanced paracrine signaling via adenosine (Ado) A2b receptors is involved. Dissociated rat CB cultures were exposed for 24 hr to normoxia (Nox; 21% O2), chronic hypoxia (Chox; 2% O2), or the hypoxia mimetic deferoxamine (DFX), and catecholamine (CAT) secretion from type I cells was monitored by amperometry. Compared to Nox controls, Chox and DFX-treated type I cells showed a more robust CAT secretion after acute exposure to acid hypercapnia (10% CO2; pH 7.1) and high K+ (75 mM). Exogenous Ado increased CAT secretion in a dose-dependent manner, and the EC50 was right shifted from ~21 µM Ado in Nox cells to ~78 µM in Chox cells. Ado-evoked secretion in Nox and Chox cells was markedly inhibited by MRS 1754, an A2b receptor blocker, but was unaffected by SCH58261, an A2a receptor blocker. Similarly MRS 1754, but not SCH58261, partially inhibited high K+-evoked CAT secretion, suggesting a contribution from paracrine activation of A2b receptors by endogenous Ado. CB chemostimuli, acid hypercapnia and hypoxia, elicited a MRS 1754-sensitive rise in intracellular Ca2+ that was more robust in Chox and DFX-treated cells compared to Nox cells. Taken together, these data suggest that paracrine Ado A2b receptor signalling contributes to stimulus-evoked CAT secretion in normoxic and chronically hypoxic CB chemoreceptors; however, the effects of Ado are more robust after chronic hypoxia.
Volumetric muscle loss (VML) results in a large void deficient in the requisite materials for regeneration for which there is no definitive clinical standard of care. Autologous minced muscle grafts (MG), which contain the essential components for muscle regeneration, may embody an ideal tissue engineering therapy for VML. The purpose of this study was to determine if orthotopic transplantation of MG acutely after VML in the tibialis anterior muscle of male Lewis rats promotes functional tissue regeneration. Herein we report that over the first 16 weeks post-injury, MG transplantation 1) promotes remarkable regeneration of innervated muscle fibers within the defect area (i.e., de novo muscle fiber regeneration), 2) reduced evidence of chronic injury in the remaining muscle mass as compared to non-repaired muscles following VML (i.e., transplantation attenuated chronically up-regulated TGFß1 gene expression and the presence of centrally located nuclei in 30% of fibers observed in non-repaired muscles), and 3) significantly improves net torque production (i.e., ~55% of the functional deficit in non-repaired muscles was restored). Additionally, voluntary wheel running was shown to reduce the heightened accumulation of extracellular matrix deposition observed within the regenerated tissue of MG-repaired sedentary rats 8 weeks post-injury (Collagen 1% Area: Sedentary vs. Runner, ~41 vs. 30%), which may have been the result of an augmented inflammatory response (i.e., M1 (CCR7) and M2 (CD163) macrophage expression was significantly greater in runner than sedentary MG-repaired muscles two weeks post-injury). These findings support further exploration of autologous minced muscle grafts for the treatment of VML.
Flow impingement at arterial bifurcations causes high frictional force (or wall shear stress, WSS), and flow acceleration and deceleration in the branches create positive and negative streamwise gradients in WSS (WSSG), respectively. Intracranial aneurysms tend to form in regions with high WSS and positive WSSG. However, little is known about the responses of endothelial cells (ECs) to either positive or negative WSSG under high WSS conditions. We used cDNA microarrays to profile gene expression in cultured ECs exposed to positive or negative WSSG for 24 hours in a flow chamber where WSS varied between 3.5 and 28.4 Pa. Gene ontology and biological pathway analysis indicated that positive WSSG favored proliferation, apoptosis, and extracellular matrix processing while decreasing expression of pro-inflammatory genes. To determine if similar responses occur in vivo, we examined EC proliferation and expression of the matrix metalloproteinase, ADAMTS1, under high WSS and WSSG created at the basilar terminus of rabbits after bilateral carotid ligation. Precise hemodynamic conditions were determined by computational fluid dynamic simulations from 3D angiography and mapped on immunofluorescence staining for the proliferation marker, Ki-67, and ADAMTS1. Both proliferation and ADAMTS1 were significantly higher in ECs under positive WSSG than in adjacent regions of negative WSSG. Our results indicate that WSSG elicits distinct EC gene expression profiles and particular biological pathways including increased cell proliferation and matrix processing. Such ECs responses may be important in understanding the mechanisms of intracranial aneurysm initiation at regions of high WSS and positive WSSG.
Hematopoietic stem cells (HSC) are maintained and their division/proliferation and quiescence are regulated in the microenvironments, niches, in the bone marrow. Although diabetes is known to induce abnormalities in HSC mobilization and proliferation through chemokine and chemokine receptors, little is known about the interaction between long-term HSCs (LT-HSCs) and osteopontin-positive (OPN) cells in endosteal niche. To examine this interaction, LT-HSCs and OPN cells were isolated from streptozotocin-induced diabetic and non-diabetic mice. In diabetic mice, we observed a reduction in the number of LT-HSCs and OPN cells and impaired expression of Tie2, β-catenin and N-cadherin on LT-HSCs, and β1-integrin, β-catenin, angiopoietin-1 and CXCL12 on OPN cells. In an in vitro co-culture system, LT-HSCs isolated from non-diabetic mice exposed to diabetic OPN cells showed abnormal mRNA expression levels of Tie2 and N-cadherin. Conversely, in LT-HSCs derived from diabetic mice exposed to non-diabetic OPN cells, the decreased mRNA expressions of Tie2, β-catenin and N-cadherin were restored to normal levels. The effects of diabetic or non-diabetic OPN cells on LT-HSCs shown in this co-culture system were confirmed by the co-injection of LT-HSCs and OPN cells into bone marrow of irradiated non-diabetic mice. Our results provide new insight into the treatment of diabetes-induced LT-HSC abnormalities, and suggest that the replacement of OPN cells may represent a novel treatment strategy.
A better understanding of the control of body fat distribution and muscle development is of the upmost importance for both human and animal physiology. This requires a better knowledge of the features and physiology of adult stem cells in adipose tissue and skeletal muscle. Thus, the objective of the current study was to determine the type and proportion of these cells in growing and adult pigs. The different cell subsets of stromal vascular cells isolated from these tissues were characterized by flow cytometry using cell surface markers (CD11b, CD14, CD31, CD34, CD45, CD56 and CD90). Adipose and muscle cells were predominantly positive for the CD34, CD56 and CD90 markers. The proportion of positive cells changed with age especially in intermuscular adipose tissue and skeletal muscle where the percentage of CD90+ cells markedly increased in adult animals. Further analysis using co-immunostaining indicates that eight populations with proportions ranging from 12 to 30% were identified in at least one tissue at 7 days of age, i.e. CD90+/CD34+, CD90+/CD34-, CD90+/CD56+, CD90+/CD56-, CD90-/CD56+, CD56+/CD34+, CD56+/CD34- and CD56-/CD34+. Adipose tissues appeared to be a less heterogeneous tissue than skeletal muscle with two main populations (CD90+/CD34-, CD90+/CD56-) compared to five or more in muscle during the studied period. In culture, cells from adipose tissue and muscle differentiated into mature adipocytes in adipogenic medium. In myogenic conditions, only cells from muscle could form mature myofibers. Further studies are now needed to better understand the plasticity of those cell populations throughout life.
The rates of H2S and HS- transport across the human erythrocyte membrane were estimated by measuring rates of dissipation of pH gradients in media containing 250 µM H2S/HS-. Net acid efflux is caused by H2S/HS- acting analogously to CO2/HCO3- in the Jacobs-Stewart cycle. The steps are: 1) H2S efflux through the lipid bilayer and/or a gas channel; 2) extracellular H2S deprotonation; 3) HS- influx in exchange for Cl-, catalyzed by the anion exchange protein AE1; 4) intracellular HS- protonation. Net acid transport by the Cl-/HS-/H2S cycle is more efficient than by the Cl-/HCO3-/CO2 cycle be-cause of the rapid H2S/HS- interconversion in both cells and medium. The rates of acid transport were analyzed by solving the mass flow equations for the cycle to produce estimates of the HS- and H2S transport rates. The data indicate that HS- is a very good substrate for AE1; the Cl-/HS- exchange rate is about 1/3 as rapid as Cl-/HCO3- exchange. The H2S permeability coefficient must also be high (>10-2 cm s-1; half-time <0.003s) to account for the pH equilibration data. The results imply that 1) H2S and HS- enter erythrocytes very rapidly in the microcirculation of H2S-producing tissues, thereby acting as a sink for H2S and lowering the local extracellular concentration; and 2) the fact that HS- is a substrate for a Cl-/HCO3- exchanger indicates that some effects of exogenous H2S/HS- may not result from a regulatory role of H2S but rather from net acid flux by H2S and HS- transport in a Jacobs-Stewart cycle.
The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-ROCK-F-actin signaling in arteriolar derived smooth muscle cells to increase expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microVSM cDNA library and the α2C-AR C-terminus to identify a novel interaction with the actin cross-linker, filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and co-immunoprecipitation experiments in heterologous HEK 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSMs, α2C-ARs co-localized with filamin on intracellular filaments and at the plasma membrane. siRNA mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser-2113. Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.
Resolution agonists are endogenous mediators that drive inflammation to homeostasis. We earlier demonstrated in vivo activity of resolvins and lipoxins on regenerative periodontal wound healing. The goal of this study was to determine the impact of resolvin D1 (RvD1) on the function of human periodontal ligament fibroblasts (PDL), which are critical for wound healing during regeneration of the soft and hard tissues around teeth. Primary cells were cultured from biopsies obtained from three individuals free of periodontal diseases. Peripheral blood mononuclear cells were isolated by density gradient centrifugation from whole blood of healthy volunteers. PGE2, LTB4, LXA4 in culture supernatants were measured by ELISA. The direct impact of RvD1 on PDL fibroblast proliferation was measured and wound closure was analyzed in vitro using a fibroblast culture "scratch assay". PDL fibroblast function in response to RvD1 was further characterized by basic FGF production by ELISA. IL-1β and TNF-α enhanced the production of PGE2. Treatment of PDL cells and monocytes with 0.1-10 ng/ml RvD1 (0.27-27nM) reduced cytokine induced production of PGE2, and up-regulated LXA4 production by both PDL cells and monocytes. RvD1 significantly enhanced PDL fibroblast proliferation and wound closure as well as basic FGF release. The results demonstrate that anti-inflammatory and pro-resolution actions of RvD1 with up-regulation of arachidonic acid derived endogenous resolution pathways (LXA4) and suggest resolution pathway synergy establishing a novel mechanism for the pro-resolution activity of -3 DHA-derived resolution agonist RvD1.
KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K+ current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g. E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubule-dependent compartment within the Endoplasmic Reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 minutes was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4-5 hours after drug wash out. Coexpression studies with a dominant-negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential.
SLC4A11, a member of the Solute Linked Cotransporter 4 family that is comprised predominantly of bicarbonate transporters, was described as an electrogenic 2Na+/ B(OH)4- (Borate) cotransporter and a Na+/ 2OH- cotransporter. The goal of the current study was to confirm and/or clarify the function of SLC4A11. In HEK293 cells transfected with SLC4A11 we tested if SLC4A11 is a: 1) Na+:HCO3- cotransporter, 2) Na+:OH- (H+) transporter, and/or 3) Na+:B(OH)4- cotransporter. CO2/HCO3-perfusion yielded no significant differences in rate or extent of pHi changes or Na+ flux in SLC4A11-transfected compared to control cells. Similarly, in CO2/HCO3-, acidification on removal of Na+ and alkalinization on Na+ add-back were not significantly different between control and transfected indicating that SLC4A11 does not have Na+:HCO3- cotransport activity. In the absence of CO2/HCO3-, SLC4A11-transfected cells showed higher resting [Na+i] (25 vs 17 mM), increased NH4+ induced acidification and increased acid recovery rate (160%) after an NH4 pulse. Na+ efflux and influx were faster (80%) following Na+ removal and add-back, respectively indicative of Na+:OH- (H+) transport by SLC4A11. The increased alkalinization recovery was confirmed in NHE-deficient PS120 cells demonstrating that SLC4A11 is a bonafide Na+:OH- (H+) transporter and not an activator of NHEs. SLC4A11 mediated H+ efflux is inhibited by EIPA (EC50: 0.1 µM). The presence of 10 mM borate did not alter dpHi/dt or pH during a Na+-free pulse in SLC4A11-transfected cells. In summary our results show that SLC4A11 is not a bicarbonate or borate-linked transporter, but has significant EIPA-sensitive Na+: OH- (H+) and NH4+ permeability.
Aquaporin 6 (AQP6) is unique among mammalian AQPs in being an anion channel with negligible water permeability. However, the point mutation Asn60Gly converts AQP6 from an anion channel into a water channel. In the present study of human AQP5, we mutated Leu51 (corresponding to residue 61 in AQP6), the side chain of which faces the central pore. We evaluated function in Xenopus oocytes by two-electrode voltage clamp, video measurements of osmotic H2O permeability (Pf), microelectrode measurements of surface pH (pHS) to assess CO2 permeability, and surface biotinylation. We found that AQP5-L51R does not exhibit the H2O or CO2 permeability of the wild-type protein but instead has a novel pCMBS-sensitive current. The double mutant AQP5-L51R/C182S renders the conductance insensitive to pCMBS, demonstrating that the current is intrinsic to AQP5. AQP5-L51R has the anion permeability sequence I– > NO3- NO2- > Br– > Cl– > HCO3- > Gluconate. Of other L51 mutants, L51T (polar uncharged) and L51V (nonpolar) retain H2O and CO2 permeability and do not exhibit anion conductance. L51D and L51E (negatively charged) have no H2O or CO2 permeability. L51K (positively charged) has an intermediate H2O and CO2 permeabilities and anion conductance. L51H is unusual in having a relatively low CO2 permeability and anion conductance, but a moderate Pf. Thus, positively charged mutations of L51 can convert AQP5 from a H2O/CO2 channel into an anion channel. However, the paradoxical effect of L51H is consistent with the hypothesis that CO2, in part, takes a pathway different from H2O through AQP5.
Anion Exchanger 1 or Band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis associated mutations (R730C, E758K and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared to WT AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared to WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1, and conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.
Connective Tissue Growth Factor (CCN2/CTGF) mediates TGFβ induced fibrosis. Drug-induced gingival overgrowth is tissue specific. Here the role of the phosphoinositol 3 kinase (PI3K) pathway in mediating TGFβ1 stimulated CCN2/CTGF expression in primary human adult gingival fibroblasts and human adult lung fibroblasts was compared. Data indicate that PI3K inhibitors attenuate up-regulation of TGFβ1-induced CCN2/CTGF expression in human gingival fibroblasts independent of reducing JNK MAP kinase activation. Pharmacologic inhibitors and siRNA mediated knockdown studies indicate that calcium-dependent isoforms and an atypical isoform of protein kinase C (PKC) do not mediate TGFβ1 stimulated CCN2/CTGF expression in gingival fibroblasts. As glycogen synthase kinase-3-beta (GSK3β) can undergo phosphorylation by the PI3K/pathway, effects of GSK3β inhibitor kenpaullone and siRNA knockdown were investigated. Data in gingival fibroblasts indicate that kenpaullone attenuates TGFβ1 mediated CCN2/CTGF expression. Activation of the Wnt canonical pathways with Wnt3a, which inhibits GSK3β, similarly inhibits TGFβ1 stimulated CCN2/CTGF expression. In contrast, inhibition of GSK3β by Wnt3a does not inhibit, but modestly stimulates CCN2/CTGF levels in primary human adult lung fibroblasts and is β-catenin-dependent, consistent with previous studies performed in other cell models. These data identify a novel pathway in gingival fibroblasts in which inhibition of GSK3β attenuates CCN2/CTGF expression. In adult lung fibroblasts inhibition of GSK3β modestly stimulates TGFβ1 regulated CCN2/CTGF expression. These studies have potential clinical relevance to the tissue-specificity of drug-induced gingival overgrowth.
O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of a single β-N-GlcNAc unit to target proteins, has been shown to act as a transcriptional regulator. In the current study, we discovered that OGT exerted inhibitory effects on the LPS-driven activation of NF-B and iNOS. In response to LPS, OGT exhibited an increased interaction with the transcriptional corepressor, mSin3A. Furthermore, mSin3A, HDAC1, and HDAC2 displayed increased binding to the iNOS promoter in response to LPS. Treatment with GlcN, in contrast, inhibits LPS-induced inflammation and decreased LPS-mediated recruitment of OGT, mSin3A and HDACs. LPS treatment also resulted in the hypo-O-GlcNAcylation of mSin3A, which was reversed by GlcN. When the effect of the HDAC inhibitor, trichostatin A (TSA), on LPS- and/or GlcN-mediated iNOS protein/mRNA induction was investigated, the results revealed that TSA dose-dependently enhanced iNOS expression in response to LPS and/or GlcN. In addition, histone acetyltransferases (HATs), p300 and CBP enhanced LPS- and/or GlcN-induced iNOS protein expression. These results collectively suggest that OGT inhibits LPS-driven NF-B activation and subsequent iNOS transcription by modulating histone acetylation either directly or indirectly.
Foxo1 controls the expression of proteins which carry out processes leading to skeletal muscle atrophy, making Foxo1 of therapeutic interest in conditions of muscle wasting. The transcription of Foxo1-regulated proteins is dependent on the translocation of Foxo1 to the nucleus, which can be repressed by insulin-like growth factor-1 (IGF-1) treatment. The role of Foxo1 in muscle atrophy has been explored at length, but whether Foxo1 nuclear activity affects skeletal muscle excitation-contraction (EC) coupling has not yet been examined. Here, we use cultured adult mouse skeletal muscle fibers to investigate the effects of Foxo1 overexpression on EC coupling. Fibers expressing Foxo1-GFP exhibit an inability to contract, impaired propagation of action potentials, and ablation of calcium transients in response to electrical stimulation compared to fibers expressing GFP alone. Evaluation of the T-tubule system morphology, the membranous system involved in the radial propagation of the action potential, revealed an intact T-tubule network in fibers overexpressing Foxo1-GFP. Interestingly, long-term IGF-1 treatment of Foxo1-GFP fibers, which maintains Foxo1-GFP outside the nucleus, prevented the loss of normal calcium transients, indicating that Foxo1 translocation and the atrogenes it regulates affect the expression of proteins involved in the generation and/or propagation of action potentials. A reduction in the sodium channel Nav1.4 expression in fibers overexpressing Foxo1-GFP was also observed in the absence of IGF-1. We conclude that increased nuclear activity of Foxo1 prevents the normal muscle responses to electrical stimulation, and that this indicates a novel capability of Foxo1 to disable the functional activity of skeletal muscle.
We investigated the mechanisms underlying the activation of a transient BK current (tBK) in rabbit urethral smooth muscle cells (RUSMCs), using the perforated patch clamp technique at 37°C. The tBK current required an elevation in intracellular Ca2+ resulting from RyR activation via Ca2+-induced Ca2+-release, triggered by Ca2+ influx through L-type Ca2+ (CaV) channels. Carbachol inhibited tBK current by reducing both Ca2+ influx and Ca2+ release and altered the shape of spike complexes recorded under current clamp. The tBK currents were blocked by iberiotoxin and penitrem A (300 nM and 100 nM, respectively) and were also inhibited when external Ca2+ ([Ca2+]o) was removed or the CaV channel inhibitors, nifedipine (10 μM) and cadmium (Cd2+, 100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM) and tetracaine (100 μM), suggesting that RyR-mediated Ca2+ release contributed to the activation of the tBK current. When IP3Rs were blocked with 2APB (100 μM) the tBK current was not reduced in amplitude. However, when Ca2+ release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM) the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-DAMP (1 μM¬). Under current clamp, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst and these effects were reduced in the presence of 2APB.
Vitamin B2 (riboflavin, RF) is essential for normal human health. Mammals obtain RF from exogenous sources via intestinal absorption, and prevent its urinary loss by re-absorption in the kidneys. Both these absorptive events are carrier-mediated and involve specific RF transporters (RFVTs). Chronic alcohol consumption in humans is associated with a high prevalence of RF deficiency and sub-optimal levels, but little is known about the effect of chronic alcohol exposure on physiological and molecular parameters of the intestinal and renal RF transport events. We addressed these issues using rats chronically fed an alcohol liquid diet and pair-fed controls as a model. The results showed that chronic alcohol feeding significantly inhibits carrier-mediated RF transport across the intestinal brush border and basolateral membrane domains of the polarized enterocytes. This inhibition was associated with a parallel reduction in the expression of the rat RFVT-1 and -3 at the protein, mRNA, and heterogenous nuclear RNA (hnRNA) levels. Chronic alcohol feeding also caused a significant inhibition in RF uptake in the colon. Similarly, a significant inhibition in carrier-mediated RF transport across the renal brush border and basolateral membrane domains was observed, which again was associated with a significant reduction in level of expression of RFVT-1 and 3 at the protein, mRNA and hnRNA levels. These findings demonstrate that chronic alcohol exposure impairs both intestinal absorption and renal re-absorption processes of RF and that these effects are, at least in part, mediated via transcriptional mechanism(s) involving the slc52a1 and slc52a3 genes.
Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (iCell Cardiomyocytes) with ion channel activities that are remarkably similar to adult cardiomyocytes. Here, we extend this characterization to cardiac ion transporters . Additionally, we document facile molecular biological manipulation of iCell Cardiomyocytes to overexpress and knockdown transporters and regulatory proteins. Na/Ca exchange (NCX1) and Na/K pump currents were recorded via patch clamp, and Na/H and Cl/OH exchange were recorded via oscillating proton-selective microelectrodes during patch clamp. Flux densities of all transport systems are similar to those of non-rodent adult cardiomyocytes. NCX1 protein and NCX1 currents decline after NCX1 siRNA transfection with similar time courses (2 d), and an NCX1-Halo® fusion protein is internalized after its extracellular labeling by AlexaFluor488 Ligand with a similar time course. Loss of the cardiac regulatory protein, phospholemman (PLM), occurs over a longer time course (60 h) after PLM siRNA transfection. Similar to multiple previous reports for adult cardiomyocytes, Na/K pump currents in iCell Cardiomyocytes are not enhanced by activating cAMP production with either maximal or submaximal cytoplasmic Na and using either forskolin or isoproterenol to activate adenylate cyclases. Finally, we describe Ca influx-dependent changes of iCell cardiomyocyte capacitance (Cm). Large increases of Cm occur during Ca influx via NCX1, thereby documenting large internal membrane reserves that can fuse to the sarcolemma, and subsequent declines of Cm document active endocytic processes. Together, these results document a great potential of iCell Cardiomyocytes for both short- and long-term studies of cardiac ion transporters and their regulation.
Resveratrol (REV) is a naturally occurring phytoalexin that inhibits neuronal K+ channels; however, the molecular mechanisms behind the effects of REV and the relevant α-subunit are not well defined. Using a patch clamp technique, cultured cerebellar granule cell and HEK-293 cells transfected with the Kv2.1 and Kv2.2 α-subunits, we investigated the effect of REV on Kv2.1 and Kv2.2 α-subunits. Our data have demonstrated that REV significantly suppressed Kv2.2 but not Kv2.1 currents with a fast, reversible and mildly concentration-dependent manner, and shifted the activation or inactivation curve of Kv2.2 channels. Activating or inhibiting cAMP/PKA pathway did not abolish the inhibition of Kv2.2 current by REV. In contrast, activation of PKC with PMA mimicked the inhibitory effect of REV on Kv2.2 with modifying the activation or inactivation properties of Kv2.2 channels and eliminated any further inhibition by REV. PKC and PKC- inhibitor completely eliminated the REV-induced inhibition of Kv2.2. Moreover, the effect of REV on Kv2.2 was reduced by pre-incubation with antagonists of GPR30 receptor and shRNA for GPR30 receptor. Western blotting results indicated that the levels of PKC-α and PKC-β were significantly increased in response to REV application. Our data reveal, for the first time, that REV inhibited Kv2.2 currents through PKC-dependent pathways and a nongenomic action of the oestrogen receptor GPR30.
The concentration of free Glutamate (Glu) in rat's milk is approximately 10 times higher than that in plasma. Previous work has shown that mammary tissue actively transports circulatory leucine (Leu), which is transaminated to synthesize other amino acids such as Glu and aspartate (Asp). To investigate the molecular basis of Leu transport and its conversion into Glu in the mammary gland, we characterized the expression of Leu transporters and [3H] Leu uptake in rat mammary cells. Gene expression analysis indicated that mammary cells express two Leu transporters, LAT1 and LAT2, with LAT1 being more abundant than LAT2. This transport system is sodium-independent and transports large neutral amino acids. The Leu transport system in isolated rat mammary cells could be specifically blocked by LAT1 inhibitors BCH and triiodothyronine (T3). In organ cultures, Glu secretion was markedly inhibited by these LAT1 inhibitors. Furthermore, the profiles of Leu uptake inhibition by amino acids in mammary cells were similar to those reported for LAT1. In vivo, concentrations of free Glu and Asp increased in milk by oral gavage with Leu at 6, 12 and 18 days of lactation. These results indicate that the main Leu transporter in mammary tissue is LAT1, and the Leu transporter is a limiting factor for the synthesis and release of Glu and Asp into milk. Our studies provide the bases for the molecular mechanism of Leu transport in mammary tissue by LAT1 and its active role on free Glu secretion in milk, which confer umami taste in suckling pups.
The mechanisms governing maintenance of quiescence during pregnancy are unknown. This study characterizes a stretch-activated, TEA-insensitive K+ current found in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that the K+ currents described can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until term. The results of this study demonstrate that in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF using arachidonic acid or application of sodium bicarbonate causing intracellular acidification, respectively. Similarly, outward currents were inhibited following the application of 10 μM fluphenazine by 51.2 ± 9.8% after activation by arachidonic acid, and 73.9 ± 4.2% after activation using sodium bicarbonate. In HEK293 cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF using arachidonic acid or sodium bicarbonate, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 μM fluphenazine following activation by arachidonic acid and 91.6 ± 3.4% after sodium bicarbonate activation. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with interfering RNA (siRNA) or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.
The activity of persistent Ca2+ sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady state Ca2+ entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca2+ sparklets by PKCα and c-Src, both of which increase whole-cell Cav1.2 current: 1) Does c-Src activation enhance persistent Ca2+ sparklet activity? 2) Does PKCα activate c-Src to produce persistent Ca2+ sparklets? Using TIRF microscopy, Ca2+ sparklets were recorded from voltage-clamped tsA-201 cells co-expressing wild type (WT) or mutant Cav1.2c (the neuronal isoform of Cav1.2) constructs ± active or inactive PKCα/c-Src. Cells expressing Cav1.2c exhibited both low-activity and persistent Ca2+ sparklets. Persistent Ca2+ sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or co-expression of kinase-dead c-Src. Cav1.2c constructs mutated at one of the two C-terminal residues (Y2122F, Y2139F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y2122F but not Y2139F Cav1.2c abrogated the potentiating effect of c-Src on Ca2+ sparklet activity. We could not detect a significant change in persistent Ca2+ sparklet activity or density in cells co-expressing Cav1.2c + PKCα, regardless of whether WT or Y2122F Cav1.2c was used, or after PP2 application, suggesting that PKCα does not act upstream of c-Src to produce persistent Ca2+sparklets. However, our results indicate that persistent Ca2+ sparklet activity is promoted by the action of c-Src on residue Y2122 of the Cav1.2c C-terminus.
The blood-brain barrier (BBB) physiologically isolates the brain from the blood and thus plays a vital role in brain homeostasis. Ion transporters play a critical role in this process by effectively regulating access of chemicals to the brain. Organic anion transporting polypeptides (Oatp's) transport a wide range of amphipathic substrates and are involved in efflux of chemicals across the vertebrate BBB. The anatomical complexity of the vascularized vertebrate BBB, however, creates challenges for experimental analysis of these processes. The Drosophila BBB is structurally less complex, facilitating measurement of solute transport. Here we investigate a physiological function for organic anion transporting polypeptide 58Dc in transporting small organic anions across the BBB. We made use of genetic manipulation, immunocytochemistry and molecular techniques to supplement a whole animal approach used to study the BBB. This whole animal approach makes use of microinjecting the traceable small organic anion fluorescein into the haemolymph. This research shows that the organic anion transporting polypeptide 58Dc is involved in maintaining a chemical barrier against fluorescein permeation into the brain. Oatp58Dc was found to be expressed in the perineurial and subperineurial glia as well as postmitotic neurons. We specifically targeted knockdown of Oatp58Dc expression in the perineurial and subperineurial glia. This revealed that expression of Oatp58Dc in the perineurial glia is sufficient to maintain the barrier against fluorescein influx into the brain. Our results show that Oatp58Dc contributes to maintaining a functional barrier against fluorescein influx past the blood-brain barrier into the brain.
The chemical structures of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) resemble those of small-molecules that inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We therefore tested the acute effects of T3, T4 and reverse T3 (rT3) on recombinant wild-type human CFTR using the patch-clamp technique. When added directly to the intracellular solution bathing excised membrane patches, T3, T4 and rT3 (all tested at 50 μM) inhibited CFTR in several ways: they reduced strongly CFTR open probability by impeding channel opening; they decreased moderately single-channel current amplitude and they promoted transitions to sub-conductance states. To investigate the mechanism of CFTR inhibition, we studied T3. T3 (50 μM) had multiple effects on CFTR gating kinetics, suggestive of both allosteric inhibition and open-channel blockade. Channel inhibition by T3 was weakly voltage-dependent and stronger than the allosteric inhibitor genistein, but weaker than the open-channel blocker glibenclamide. Raising the intracellular ATP concentration abrogated T3 inhibition of CFTR gating, but not the reduction in single-channel current amplitude nor the transitions to sub-conductance states. The decrease in single-channel current amplitude was relieved by membrane depolarization, but not the transitions to sub-conductance states. We conclude that T3 has complex effects on CFTR consistent with both allosteric inhibition and open-channel blockade. Our results suggest that there are multiple allosteric mechanisms of CFTR inhibition, including interference with ATP-dependent channel gating and obstruction of conformational changes that gate the CFTR pore. CFTR inhibition by thyroid hormones has implications for the development of innovative small-molecule CFTR inhibitors.
The function Bax and/or Bak in constituting a gateway for mitochondrial apoptosis in response to apoptotic stimuli has been unequivocally demonstrated. However, recent work has suggested that Bax/Bak may have unrecognized non-apoptotic functions related to mitochondrial function in non-stressful environments. Wild type (WT) and Bax/Bak double knockout (DKO) mice were used to determine alternative roles for Bax and Bak in mitochondrial morphology and protein import in skeletal muscle. The absence of Bax and/or Bak altered mitochondrial dynamics by regulating protein components of the organelle fission and fusion machinery. Moreover, DKO mice exhibited defective mitochondrial protein import, both into the matrix and outer membrane compartments, which was consistent with our observations of impaired membrane potential and attenuated expression of protein import machinery (PIM) components in IMF mitochondria. Furthermore, the cytosolic chaperones Hsp90 and BiP were markedly increased with the deletion of Bax/Bak, indicating that the cytosolic environment related to protein folding may be changed in DKO mice. Interestingly, endurance training fully restored the deficiency of protein import in DKO mice, likely via the upregulation of PIM components, and through improved cytosolic chaperone protein expression. Thus, our results emphasize novel roles for Bax and/or Bak in mitochondrial function, and provide evidence, for the first time, of a curative function of exercise training in ameliorating a condition of defective mitochondrial protein import.
Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42 and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21 activated kinases (PAKs), including GIT1, βPIX and GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling and PS exposure in platelets.
Efficient skeletal muscle repair and regeneration require coordinated remodeling of the extracellular matrix (ECM). Previous reports have indicated that Matrix Metalloproteases (MMPs) play the pivotal role in ECM remodeling during muscle regeneration. The goal of the current study was to determine if the interstitial collagenase, MMP-13, was involved in the muscle repair process. Using intramuscular cardiotoxin injections to induce acute muscle injury, we found that MMP-13 expression and activity transiently increased during the regeneration process. In addition, muscles from mdx mice, which exhibit chronic injury, also had elevated MMP-13 expression and protein. In differentiating C2C12 cells, a murine myoblast cell line, Mmp13 expression was most pronounced after myoblast fusion and during myotube formation. We tested the necessity of MMP-13 activity for the proliferation, differentiation, migration and fusion of C2C12 cells by pharmacologic inhibition of MMP-13, and found a dramatic blockade of myoblast migration, as well as a delay in differentiation. In contrast, C2C12 cells with stable overexpression of MMP-13 showed enhanced migration, without affecting myoblast maturation. Taken together, these results support a primary role for MMP-13 in myoblast migration, which lead to secondary effects on differentiation.
Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive channel in pulmonary arterial smooth muscle cells (PASMCs). Its upregulation by chronic hypoxia is associated with enhanced myogenic tone and genetic deletion of trpv4 suppresses the development of chronic hypoxic pulmonary hypertension (CHPH). Here we further examine the roles of TRPV4 in agonist-induced pulmonary vasoconstriction and in the enhanced vasoreactivity in CHPH. Initial evaluation of TRPV4-selective antagonists HC-067047 and RN-1734 in KCl-contracted PAs of trpv4-/- mice found that submicromolar HC-067047 was devoid of off-target effect on pulmonary vasoconstriction. Inhibition of TRPV4 with 0.5 µM HC-067047 significant reduced the sensitivity of serotonin (5-HT) induced contraction in WT PAs, but had no effect on endothelin-1 or phenylephrine-activated response. Similar shift in the concentration-response curve of 5-HT was observed in trpv4-/- PAs, confirming specific TRPV4 contribution to 5-HT-induced vasoconstriction. 5-HT-induced Ca2+ response was attenuated by HC-067047 in WT PASMCs but not in trpv4-/- PASMCs, suggesting TRPV4 is a major Ca2+ pathway for 5-HT-induced Ca2+ mobilization. Chronic exposure (3-4 weeks) of WT mice to 10% O2 caused significant increase in 5-HT-induced maximal contraction, which was partially reversed by HC-067047. In concordance, the enhancement of 5-HT-induced contraction was significantly reduced in PAs of CH trpv4-/- mice. These results suggest unequivocally that TRPV4 contributes to 5-HT-dependent pharmco-mechanical coupling, and plays a major role in the enhanced pulmonary vasoreactivity to 5-HT in CHPH. Since TRPV4 participates in multiple pathological changes in CHPH, it can be considered as a potential target for the treatment of pulmonary hypertension.
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely recommended for several chronic conditions, including osteoarthritis, rheumatoid arthritis, and cardiovascular disease (CVD). For patients with CVD, low-dose aspirin in particular is recommended as a primary prevention strategy against myocardial infarction. However, long-term intake of NSAIDs can cause damage to the mucosal barrier surrounding the GI tract, leading to the formation of gastric ulcers. While microencapsulation of NSAIDs has been shown to reduce these upper GI effects, sustained release in the lower GI tract and colon may cause epithelial erosion due to increased acidification. In this study, we investigated the role of the NSAIDs aspirin and indomethaci on Na+/H+ exchanger (NHE) activity in rat colonic crypts. Through comparing rates of pH recovery between control and NSAID perfusion runs, we were able to determine that both aspirin and indomethacin increase hydrogen extrusion into the colonic lumen. Treatment with NHE3 inhibitor 5-ethylisopropyl amiloride (EIPA) and NHE1 inhibitor amiloride further demonstrated that aspirin and indomethacin may preferentially upregulate activity of the apical Na+/H+ exchanger NHE3. Our results therefore suggest that prolonged clinical exposure to NSAIDs may affect colonic tissue at the site of selected NHE isoforms resulting in modulation of transport and barrier function.
The anion transport inhibitor DIDS is known to reduce aqueous humor (AH) secretion but questions remain about anion-dependence of the effect. In some tissues, DIDS is reported to cause Na,K-ATPase inhibition. Here, we report on the ability of DIDS to inhibit Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE) and investigate the underlying mechanism. Porcine NPE cells were cultured to confluence on permeable supports, treated with drugs added to both sides of the membrane, and then used for 86Rb uptake measurements or homogenized to measure Na,K-ATPase activity or to detect protein phosphorylation. DIDS inhibited ouabain-sensitive 86Rb uptake, activated Src family kinase (SFK) and caused a reduction of Na,K-ATPase activity. PP2, an SFK inhibitor, prevented the DIDS responses. In BCECF-loaded NPE, DIDS was found to reduce cytoplasmic pH (pHi). PP2-sensitive Na,K-ATPase activity inhibition, 86Rb uptake suppression and SFK activation were observed when a similar reduction of pHi was imposed by low pH medium or an ammonium chloride withdrawal maneuver. PP2 and the ERK inhibitor U0126 prevented robust ERK1/2 activation in cells exposed to DIDS or subjected to pHi reduction but U0126 did not prevent SFK activation or the Na,K-ATPase activity response. The evidence points to an inhibitory influence of DIDS on NPE Na,K-ATPase activity by a mechanism that hinges upon SFK activation associated with a reduction of cytoplasmic pH.
Platelet-derived growth factor (PDGF) stimulates vascular smooth muscle cell (VSMC) migration and neointimal formation in response to injury. We previously identified IQ-domain GTPase-activating protein 1 (IQGAP1) as a novel VEGF receptor2 binding scaffold protein involved in endothelial migration. However, its role in VSMC migration and neointimal formation in vivo is unknown. Here we show that PDGF stimulation rapidly promotes IQGAP1 association with PDGF receptor β (PDGFR) as well as IQGAP1 tyrosine phosphorylation in cultured VSMC. Overexpression or knockdown of IQGAP1 enhances or inhibits PDGFR autophosphorylation (p-PDGFR), respectively. Immunofluorescence and cell fractionation analysis reveals that PDGF-induced p-PDGFR localized in focal adhesions (FAs), but not caveolae/lipid rafts, is inhibited by IQGAP1 knockdown with siRNA. PDGF stimulation promotes IQGAP1 association with PDGFR/FAs signaling proteins complex. Functionally, IQGAP1 siRNA inhibits PDGF-induced FAs formation as well as VSMC migration induced by PDGF. In vivo, IQGAP1 expression is markedly increased at neointimal VSMC in wire-injured femoral arteries. Mice lacking IQGAP1 exhibit impaired neointimal formation in response to vascular injury. In summary, IQGAP1, through interaction with PDGFR and FAs signaling proteins, promotes activation of PDGFR in FAs as well as FA formation, which may contribute to VSMC migration and neointimal formation after injury. Our findings provide insight into IQGAP1 as a potential therapeutic target for vascular migration-related diseases.