The Effect Of Modulating Smad3 Expression On Extracellular Matrix Metabolism In Cultured Rat Mesangial Cells

IntroductionIt is established that Smad3 as a mediator plays a critical role in the development of organ fibrosis by in vitro and in vivo studies.Smad3 mostly participate in TGF-β1 induced organ fibrosis.The deletion of Smad3 gene may decrease epithelial-to-mesenchymal transformation(EMT)and the expression levels of collagens,plasminogen activator inhibitor-1 and tissue inhibitor of metalloprotease-1 genes induced by TGF-β1 in vitro.Null mice knocked-out Smad3 gene are resistant to radiation-induced cutaneous fibrosis,bleomycin-induced pulmonary fibrosis,carbon tetrachloride-induced hepatic fibrosis as well as streptozotocin-induced diabetic renal fibrosis,and showed that abrogated EMT presented in proliferative vitreoretinopathy,ocular capsule injury and renal interstitial fibrosis resulting from unilateral ureteral obstruction.The inhibition of Smad3 by means of inhibitory Smad7 upregulation or treatment with the small molecule,halofuginone,dramatically reduces fibrotic degree of various organs in animal models.Recently it is reported that IFN-γexerts antifibrotic effects/in vitro and in vivo. IFN-γcan inhibit extracellular matrix(ECM)synthesis in hepatic and renal fibrosis animal models.The antifibrotic effects were proved to be correlated with the downstream signal pathway of IFN-γ,including Jak1/Stat1 pathway and CK-Ⅱ/Yb-1 pathway,and their crosstalk with TGF-β/smad signal pathway.The main purpose of the present research is to elucidate the role of smad3 gene on the expression of ECM components,such as fibronectin(FN),typeⅣcollagen (ColⅣ),and matrix metalloproteinase-2(MMP-2),tissue inhibitor of metalloproteinase-2(TIMP-2)in cultured rat MsC through the methods of gene stably expression and RNA interference,to explore the effect of IFN-γon the expression of TGF-β1/Smad signal pathway,MMP-2 and TIMP-2,and try to elucidate the effect of TGFβ1/Smad signal pathway on the pathogenesis of glomerulosclerosis,providing the therotical and experimental basis for IFN-γ therapeutic intervention in animal models with chronic progressive glomerulonephritis.PartⅠThe effect of Smad3 gene transfection on ECM metabolism in cultured rat MsCObjectives To elucidate the effect of Smad3 overexpression on the synthesis of ColⅣ,FN,the expression of MMP-2/TIMP-2,and the effect of Smad3 on FN promoter activity in MsCs transfected with Smad3 gene.Methods MsCs stably expressing Smad3 protein,constructed by transfection of the recombinant plasmid pIRES-EGFP-Smad3 and screening positive clones,were treated with TGF-β1.The expression of ColⅣ,FN,MMP-2/TIMP-2 were evaluated by real-time RT-PCR and western blot analysis respectively.MsCs stably expressing Smad3 protein were further transfected with pGL2F1900 plasmid to detect the effect of Smad3 on the activity of FN promoter with a reporter gene firefly luciferase(Fluc).Results Increased expression of ColⅣand FN(fold induction:mRNA 2.5 and 1.3, protein 3.1 and 1.5 respectively)(P＜0.05),and increased expression and secretion of MMP-2 and TIMP-2(fold induction:mRNA 1.8 and 2.5,protein 2.1 and 3.5, respectively)(P＜0.05)were detected when MsCs stably expressing Smad3 protein were treated with TGFβ1.After transfected with pGL2F1900,the Fluc/Rluc ratio in MsCs transfected with Smad3 is 2.3 folds high than that in control cells,and it could be effectively inhibited by RNA interference specific to Smad3.Conclusions MsCs clones overexpressed Smad3 were successfully established. Increase of ColⅣand FN synthesis and MMP-2/TIMP-2 expression were identified in these clones with TGFβ1 stimulation.The promoter activity of FN was upregulated in MsCs overexpressed Smad3 and downregulated in MsCs transfected with Smad3 RNAi plasmid.PartⅡThe effect of Smad3 RNAi plasmid transfection on ECM metabolism in cultured rat MsC Objective To explore the effect of Smad3 RNAi plasmid transfection on the synthesis of ColⅣ,FN,and the expression of MMP-2/TIMP-2 in cultured rat MsCMethods Recombinant plasmid of pSilencerl.0-Smad3 plasmid were constructed and the MsCs stably-expressing Smad3,with TGF-β1 treatment,were transfected with the effective plasmid.The expression of ColⅣ,FN,MMP-2/TIMP-2 mRNAs and proteins were evaluated by real-time RT-PCR and western blot analysis, respectively.Results The recombinant plasmid of pSilencer1.0-Smad3 can effectively decrease the level of Smad3 mRNA and protein expression.MsC stably-expressing Smad3 with transfection of RNAi plasmid showed decreased synthesis of ColⅣand FN (fold induction:mRNA 44%and 54%,protein 32%and 45%respectively)(P＜0.05), and decreased expression and secretion of MMP-2 and TIMP-2(fold induction: mRNA 47%and 53%,protein 52%and 47%,respectively)(P<0.05).Conclusions Smad3 RNAi can effectively downregulate the synthesis of ColⅣand FN and the expression of MMP-2/TIMP-2.PartⅢEffect of IFN-γon the expression of TGF-β/Smad signal pathway, MMP-2 and TIMP-2 of rat cultured mesangial cellObjective To study the effect of interferon-γ(IFN-γ)on the proliferation of mesangial cells(MsC),the mRNA and protein expression of TGF-β1/Smad signal pathway,MMP-2 and TIMP-2,to provide experimental basis for the mechanism of IFN-γtreating renal fibrosis.Methods Cultured MsC were treated with IFN-γat different concentrations,the proliferation of MsC was examined by MTT.The MsC were collected at oh,0.5h,1h, 2h,4h,6h,12h,24h after treated by 100 IU/mL IFN-γ,RNA and protein of MsC were extracted from the samples.The mRNA and protein of Smad3,Smad7,MMP-2 and TIMP-2 were examined by real-time RT-PCR and western blot respectively. Results The proliferation of MsC were not influenced by IFN-γ.After treatment by IFN-γ,the expression of Smad7 mRNA and protein was promptly elevated at 0.5h,and maitained in 6h.The expression of Smad3,MMP2 mRNA and proteins increased after 6h.However,the expression of TIMP-2 mRNA and protein decreased after 6h.Conclusions The treating effect of IFN-γon renal fibrosis could be mediated by TGF-β/smads signal pathway,presenting the upregulation of MMP-2 expression, and downregulation of TIMP-2 expression.Conclusions1.MsCs clones overexpressed Smad3 were successfully established.Increase of ColⅣand FN synthesis and MMP-2/TIMP-2 expression were identified in these clones with TGFβ1 stimulation.2.Smad3 RNAi can effectively downregulate the synthesis of ColⅣand FN and the expression of MMP-2 and TIMP-2.3.Smad3 overexpression can upregulate the promoter activity of FN and the expression of FN in MsCs.4.The treating effect of IFN-γon renal fibrosis could be mediated by TGF-β1/smads signal pathway,presenting the upregulation of MMP-2 expression,and downregulation of TIMP-2 expression.