The Research On Function Of AR In TGF-β1 Induced Expression Of Fibronectin In Human Mesangial Cells

Glomerulosclerosis is a final common outcome of glomerular injury in various types of human glomerular diseases,characterized by extracellular matrix(ECM) proteins accumulation,represents a common pathway of different renal diseases.There is increasing evidence that the imbalance of ECM synthesis and degradation may be an important mechanism of glomerulosclerosis.Glomerular MsC plays an important role in the regulation of ECM metabolism.Recent research points are focused on the MsC and regulation ECM metabolism,as well as its correlation with the development of glomerulosclerosis.As resident cells in glomerulus,the mesangial cells play an important role not only in maintaining the normal structure and functions of glomerulus,but also in acute glomerulonephritis and progressive glomerulosclerosis.Several reports have implicated that MsC excretes various cellular factors,PDGF,TGF-β1 and IL6.Recent researches have demonstrated that transforming growth factor-β1(TGF-β1) in kidney diseases,including glomerulosclerosis,since the MsC high expression of TGF-β1 can result in ECM deposition by inducing ECM proteins expression.Aldose reductase,a member of the aldo-keto reductase superfamily,can catalyse the reduction of a wide range of aldehydes including glucose.This enzyme represents the first and the rate-limiting step of the polyol pathway.Increased polyol pathway activity has been linked to the aetiology of hyperglycaemic injury and inhibition of AR in preventing or delaying diabetes-related changes in the lens,retina,peripheral nerve and kidney.Our group has found that AR gene was one of TGF-β1-responsive genes by SSH-PCR.As one of TGF-β1-responsive genes,whether AR has any relations to glomerulosclerosis just like TGF-β1 under normal glucose concentration remains unclear.Fibronectin(FN) is a heterodimeric extracellular matrix glycoprotein implicated in a number of physiological events during embryogenesis,angiogenesis,thrombosis,as well as inflammation.It is a major component of the ECM.FN plays an important role during development and wound healing by promoting cell adhesion,migration and cytoskeletal organization.However,FN is often observed following oncogenic transformation,leading to decreased adhesion and increased metastatic potential.Overproduction of matrix components including FN is the main pathological finding in tissue fibrosis.In this study we used molecular biologic technologies and cell culture to examine the expression of TGF-β1 and AR in HMC and the relations between them,and to observe the effect of AR in TGF-β1-induced expression of ECM components,fibronectin,and to analyze the effect related signaling pathways.The purpose of our research was to elucidate the function and significance of AR in glomerulosclerosis,and provide new experiments data for enriching the pathologic mechanism of glomerulosclerosis.PartⅠTGF-β1 induced expression of AR,FN and MAPKs signaling pathway phosphorylation in cultured human mesangial cellsObjective To explore the effect of TGF-β1 induced expression of AR,FN and MAPKs phosphorylation in cultured human mesangial cells.Methods Cultured HMCs was treated with recombinant human TGF-β1 in different concentrations or for different time.The expression of AR,FN and MAPKs were examined by Western Blot analysis,respectively.Results The cultured HMC treated with TGF-β1 showed increased expression of AR,FN and phosphorylation MAPKs.When using the single concentration,1ng/ml TGF-β1,the high expression were observed when incubated for 15min,60min is the most effective in AR and 120min in FN.When using different concentrations for 24h incubation,the high expression was observed when used from1ng/ml to 8ng/ml TGF-β1,and reached a peak at 1ng/ml,TGF-β1 activated the three MAPK cascades p38,ERK and JNK pathway.The maximum activation of p38 was observed after 60 min of stimulation,ERK and JNK was observed after 30 min of stimulation.The activation of JNK and p38 by TGF-β1 was sustained up to 120 min,ERK activation was more sustained a longer time.Conclusions The recombinant human cytokine TGF-β1 can up-regulate the expression of AR, FN and MAPKs in dose and time-dependent manners in cultured HMC,which provide new proof for our former conclusion,namely AR gene was one of TGF-β1-responsive genes. PartⅡThe effect of inhibitors of AR,MAPKs and PKC on Expression of FN by TGF-β1 inducedObjective To elucidate the role of AR and signal pathway in TGF-β1-induced expression of FN and explore the expression of FN and AR by inhibit the activation of AR,MAPKs and PKC.Methods Cultured HMCs were treated with inhibitors of AR(sorbinil and zopolrestat), inhibitors of p38,JNK and ERK(SB203580,SP600125 and U0126) in HMC,then the different groups of cells were treated with TGF-β1(1ng/ml) for 1h.The expression of AR and FN were examined by Western Blot analysis,respectively.Results The normal HMC showed not reduced expression of FN after incubation with single inhibitors of AR and MAPKs.Pre-incubation of cells with sorbinil or zopolrestat, then the different groups of cells were treated with TGF-β1(1ng/ml).More than 2.9 folds decrease in the protein level of FN was observed in cells treated with zopolrestat.Similar results were observed in cells treated with sorbinil,which showed that ARI could reduce the expression of FN.The results showed that TGF-β1-induced expression of FN protein levels was suppressed by G_O丨¨6983,SB203580,SP600125 and U0126,indicating that the PKC, ERK,JNK and p38-mediated signaling pathways are involved in the TGF-β1-induced up-regulation of FN protein expression.TGF-β1-induced up-regulation of FN was significantly suppressed in together with inhibitors of AR,ERK,JNK and PKC.In contrast, co-incubated inhibitor of AR,p38 did not suppressed FN expression.Conclusions These results indicate that TGF-β1-induced FN expressing could be attributed to AR activation,which is regulated,at least in part,by PKC,ERK,JNK and p38 signaling pathways.PartⅢThe effect of AR on TGF-β1-induced ECM components and the related signaling pathwayObjective To further clarify the role of AR in TGF-β1 induced FN expression,explore the effect of AR and AR on TGF-β1-induced expression of FN and the related signaling pathway.Methods Restriction endonucleases digestion and ligation was used to reconstructing eukaryotic expression plasmid pcDNA3-AR and siRNA.Lipofectin2000 was used to transfect AR vectors into HMC.RT-PCR,Real-Time PCR,Western Blot and immunofluorescence analysis were performed to verify the transfection and RNAi.Western Blot was used to analyse the expression of fibronectin and MAPKs proteins with or without the stimulation of TGF-β1.Results The HMC transfected with AR showed increased expression of AR and FN (P＜0.05).After stimulation of TGF-β1,the HMC transfected with AR showed increased expression of FN.The HMC knock-down of AR gene with RNAi showed decreased expression of AR and FN(P＜0.05).After stimulation of TGF-β1,the results showed decreased expression of FN.The HMC transfected with AR and knocked down AR gene showed obviously increased or reduced expression of FN(P＜0.05).There was no change in expression of total MAPKs in different groups of HMC with or without stimulation of TGF-β1.The normal HMC showed increased expression of phospho-ERK,phospho-JNK, and phospho-p38 after stimulation of TGF-β1,and the HMC knocked down AR gene with RNAi showed reduced expression of phospho-JNK,phospho-p38 and phospho-ERK (p＜0.05).Conclusions AR can regulates the expression of FN with the stimulation of TGF-β1 as one of the responsive genes of TGF-β1,which may have relations with the activation of JNK-MAPK,ERK-MAPK and p38-MAPK signaling pathways.These data indicate that AR may probably play a role in the pathogenesis of glomerulosclersis. 1.TGF-β1 can induce the expression of AR and FN in time and dose-dependent manners, which show the controllability of AR gene as one of TGF-β1-responsive genes.2.These results showed that ARI could reduce the expression of FN,indicating that the PKC,ERK,JNK and p38-mediated signaling pathways are involved in the TGF-β1-induced up-regulation of FN protein expression.3.AR may regulate the TGF-β1-induced expression of FN,one of ECM components, probably via MAPKs and PKC signaling pathway,which can be considered one of new function of AR under normal glucose concentration in glomerulosclerosis.