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ABSTRACT: Many signaling molecules and pathways that regulate gap junctions (GJs) protein expression and function are, in fact, also controlled by GJs. We, therefore, speculated an existence of the GJ channel-mediated self-regulation of GJs. Using a cell culture model in which nonjunctional connexin43 (Cx43) hemichannels were activated by cadmium (Cd), we tested this hypothesis.
Incubation of Cx43-transfected LLC-PK1 cells with Cd led to an increased expression of Cx43. This effect of Cd was tightly associated with JNK activation. Inhibition of JNK abolished the elevation of Cx43. Further analysis revealed that the changes of JNK and Cx43 were controlled by GSH. Supplement of a membrane-permeable GSH analogue GSH ethyl ester or GSH precursor N-acetyl-cystein abrogated the effects of Cd on JNK activation and Cx43 expression. Indeed, Cd induced extracellular release of GSH. Blockade of Cx43 hemichannels with heptanol or Cx43 mimetic peptide Gap26 to prevent the efflux of GSH significantly attenuated the Cx43-elevating effects of Cd.
Collectively, our results thus indicate that Cd-induced upregulation of Cx43 is through activation of nonjunctional Cx43 hemichannels. Our findings thus support the existence of a hemichannel-mediated self-regulation of Cx43 and provide novel insights into the molecular mechanisms of Cx43 expression and function.
PLoS ONE 01/2013; 8(2):e58057. · 4.09 Impact Factor
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ABSTRACT: The gap junction protein connexin43 (Cx43) was markedly increased in podocytes in a rat model of nephrosis induced by puromycin. However, the mechanisms and roles of the altered Cx43 in podocytes are still unclear. Given that oxidative stress mediates podocyte injury under a variety of pathological situations, we examined the possible involvement of an oxidative stress-related mechanism in the regulation of Cx43. Incubation of podocytes with puromycin led to a time- and concentration-dependent loss of cell viability, which was preceded by an elevation in Cx43 levels. Concomitantly, puromycin also induced NOX4 expression and promoted superoxide (O(2)(-)) generation. Inhibition of NADPH oxidase with apocynin and diphenyleneiodonium chloride or addition of the superoxide dismutase mimetic tempol completely abrogated, whereas the O(2)(-) donors menadione and 2,3-dimethoxy-1,4-naphthoquinone reproduced, the effects of puromycin on Cx43 expression and cell injury. Further analysis demonstrated that treatment of podocytes with several structurally different gap-junction inhibitors significantly attenuated the cytotoxicity of puromycin. Our results thus indicate that NADPH oxidase-mediated upregulation of Cx43 contributes to podocyte injury.
Free radical biology & medicine 07/2012; 53(6):1286-97. · 5.42 Impact Factor
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ABSTRACT: Gap junctions (GJs) play an important role in the control of bladder contractile response and in the regulation of various immune inflammatory processes. Here, we investigated the possible interaction between inflammation and GJs in bladder smooth muscle cells (BSMCs). Stimulation of BSMCs with IL1β and TNFα increased connexin43 (Cx43) expression and function, which was associated with increased phosphorylation of vasodilator-stimulated phosphoprotein. Inhibition of PKA with H89 or down-regulation of CREB with specific siRNAs largely abolished the Cx43-elevating effect. Further analysis revealed that IL1β/TNFα induced NFκB-dependent inducible NO synthase (iNOS) expression. Inhibition of iNOS with G-nitro-l-arginine methyl ester abrogated and an exogenous NO donor mimicked the effect of the cytokines on Cx43. Intraperitoneal injection of LPS into mice also induced bladder Cx43 expression, which was largely blocked by an iNOS inhibitor. Finally, the elevated Cx43 was found to negatively regulate iNOS expression. Dysfunction of GJs with various blockers or down-regulation of Cx43 with siRNA significantly potentiated the expression of iNOS. Fibroblasts from Cx43 knock-out (Cx43(-/-)) mice also displayed a significantly higher response to the cytokine-induced iNOS expression than cells from Cx43 wild-type (Cx43(+/+)) littermates. Collectively, our study revealed a previously unrecognized reciprocal regulation loop between cytokine-induced NO and GJs. Our findings may provide an important molecular mechanism for the symptoms of bladder infection. In addition, it may further our understanding of the roles of GJs in inflammatory diseases.
Journal of Biological Chemistry 09/2011; 286(48):41552-62. · 4.77 Impact Factor
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ABSTRACT: Gap junctions (GJs) play an important role in the control of bladder contractile response and in the regulation of various
immune-inflammatory processes. Here, we investigated the possible interaction between inflammation and GJs in bladder smooth
muscle cells (BSMCs). Stimulation of BSMCs with IL1β and TNFα increased connexin43 (Cx43) expression and function, which was
associated with increased phosphorylation of VASP. Inhibition of PKA with H89 or downregulation of CREB with specific siRNAs
largely abolished the Cx43-elevating effect. Further analysis revealed that IL1β/TNFα induced NFκB-dependent iNOS expression.
Inhibition of iNOS with G-nitro-L-argininemethylester abrogated, whereas an exogenous NO donor mimicked the effect of the
cytokines on Cx43. Intraperitoneal injection of LPS into mice also induced bladder Cx43 expression, which was largely blocked
by an iNOS inhibitor. Finally, the elevated Cx43 was found to negatively regulate iNOS expression. Dysfunction of GJs with
various blockers or downregulation of Cx43 with siRNA significantly potentiated the expression of iNOS. Fibroblasts from Cx43
knockout (Cx43−/−) mice also displayed a significantly higher response to the cytokine-induced iNOS expression than cells
from Cx43 wild-type (Cx43+/+) littermates. Collectively, our study revealed a previously unrecognized reciprocal regulation
loop between cytokine-induced NO and GJs. Our findings may provide an important molecular mechanism for the symptoms of bladder
infection. In addition, it may further our understanding of the roles of GJs in inflammatory diseases.
Journal of Biological Chemistry 09/2011; · 4.77 Impact Factor
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Yuan Chi,
Kai Li, Qiaojing Yan,
Schuichi Koizumi,
Liye Shi,
Shuhei Takahashi,
Ying Zhu,
Hiroyuki Matsue,
Masayuki Takeda,
Masanori Kitamura,
Jian Yao
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ABSTRACT: Flufenamic acid (FFA) is a nonsteroidal anti-inflammatory drug (NSAID). It has anti-inflammatory and antipyretic properties. In addition, it modulates multiple channel activities. The mechanisms underlying the pharmacological actions of FFA are presently unclear. Given that AMP-activated protein kinase (AMPK) has both anti-inflammatory and channel-regulating functions, we examined whether FFA induces AMPK activation. 1) Exposure of several different types of cells to FFA resulted in an elevation of AMPKα phosphorylation at Thr172. This effect of FFA was reproduced by functionally and structurally similar mefenamic acid, tolfenamic acid, niflumic acid, and meclofenamic acid. 2) FFA-induced activation of AMPK was largely abolished by the treatment of cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (an intracellular Ca(2+) chelator) or depletion of extracellular Ca(2+), whereas it was mimicked by stimulation of cells with the Ca(2+) ionophore 5-(methylamino)-2-({(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl}methyl)-1,3-benzoxazole-4-carboxylic acid (A23187) or ionomycin. 3) FFA triggered a rise in intracellular Ca(2+), which was abolished by cyclosporine, a blocker of mitochondrial permeability transition pore. Cyclosporine also abolished FFA-induced activation of AMPK. 4) Inhibition of Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ) with 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609) or down-regulation of CaMKKβ with short interfering RNA largely abrogated FFA-induced activation of AMPK. 5) FFA significantly suppressed nuclear factor-κB activity and inducible nitric-oxide synthase expression triggered by interleukin-1β and tumor necrosis factor α. This suppression was also largely abrogated by STO-609. Taken together, we conclude that FFA induces AMPK activation through the Ca(2+)-CaMKKβ pathway. Activation of AMPK is a presently unrecognized important mechanism underlying the pharmacological effects of FFA.
Journal of Pharmacology and Experimental Therapeutics 07/2011; 339(1):257-66. · 3.83 Impact Factor
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ABSTRACT: In kidney, the role of cell proliferation, differentiation, apoptosis, inflammatory mediators and cytokines expression is closely related with cell signaling pathways, including tyrosine kinase pathway, transforming growth factor-beta/Smad pathway, Rho/Rho-associated coiled-coil forming protein kinase pathway, phosphoinositol pathway, cyclic nucleotide pathway, nuclear factor kappaB pathway and so on. Some Chinese herbs and their extracts, such as rhubarb and triptolide, as well as some Chinese herbal prescriptions, such as astragalus-angelica mixture and Chailing decoction, not only could ameliorate proliferation, differentiation and apoptosis of renal cell by regulating cell signaling pathways, but also could control target gene transcription, expression and its biological effects through inhibiting the phosphorylation of key signaling molecules.
Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica 01/2011; 36(1):85-91.
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ABSTRACT: We investigated the potential involvement of connexin hemichannels in cadmium ions (Cd(2+))-elicited cell injury. Transfection of LLC-PK1 cells with a wild-type connexin43 (Cx43) sensitized them to Cd(2+)-elicited cell injury. The cell susceptibility to Cd(2+) was increased by depletion of glutathione (GSH) with DL-buthionine-[S,R]-sulfoximine, and decreased by N-acetyl-cysteine or glutathione reduced ethyl ester. Fibroblasts derived from Cx43 wild-type (Cx43+/+) and knockout (Cx43-/-) fetal littermates displayed different susceptibility to Cd(2+). Cd(2+) induced a higher concentration of reactive oxygen species, a stronger activation c-Jun N-terminal kinase, and significantly more severe cell injury in Cx43+/+ fibroblasts, as compared with Cx43-/- fibroblasts. Cd(2+) caused a reduction in intracellular GSH, whereas it elevated extracellular GSH. This effect of Cd(2+) was more dramatic in Cx43+/+ than Cx43-/- fibroblasts. Treatment of Cx43+/+ fibroblasts with Cd(2+) caused a Cx43 hemichannel-dependent influx of Lucifer Yellow and efflux of ATP. Collectively, our study demonstrates that Cx43 sensitizes cells to Cd(2+)-initiated cytotoxicity, possibly through hemichannel-mediated effects on intracellular oxidative status.
Antioxidants & Redox Signaling 01/2011; 14(12):2427-39. · 8.20 Impact Factor
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ABSTRACT: Multi-glycoside from Tripterygium wilfordii Hook f. (GTW) is used for treatment of progressive glomerulonephritis (GN) in China. We have previously reported the beneficial effects of GTW on acute GN induced by an anti-Thy-1.1 monoclonal antibody (mAb). In the present study, the effect and potential mechanisms of GTW on the chronic irreversible model of GN were investigated.
Progressive GN was induced in rats by two intravenous injections of anti-Thy-1.1 mAb 1-22-3. Daily oral administration of GTW was started before the second injection of mAb until the day of sacrifice. Ten rats were randomly divided into a control (vehicle-treated) and a GTW-treated group, and sacrificed on day 45 after the first injection of mAb 1-22-3. Proteinuria was determined on days 0, 1, 3, 5, 7, 10, 14, 20, 25, 30, 35, 40, and 45. Blood biochemical parameters, morphological changes of mesangium, glomerular infiltration of macrophage and T lymphocyte, and glomerular mRNA expression of cytokines (TGF-beta, IL-2, and IFN-gamma) were examined from the samples taken at terminal sacrifice.
GTW treatment significantly ameliorated proteinuria, renal function, prolonged mesangial lesions and inflammatory cell accumulation in glomerulus. In addition, it significantly reduced the glomerular mRNA expression for TGF-beta, IL-2, and IFN-gamma.
GTW ameliorates prolonged glomerular lesions presumably through suppression of cytokine production (TGF-beta, IL-2, and IFN-gamma). GTW could be an effective therapeutic agent for treatment of chronic renal diseases.
Nephron Experimental Nephrology 10/2009; 114(1):e7-e14. · 1.86 Impact Factor
