The Study Of PP2Ac Promotes Renal Interstitial Fibrosis By Dephosphorylating Smad3Linker Region

ObjectiveIn order to study the function of PP2Ac in tubulointersitial firosis,we established HK-2cells modle with TGF-β1stimulated in vitro.1.Object the effect of PP2Ac in tubulointersitial firosis through PP2Ac overexpression plasmid and PP2Ac siRNA transfected with HK-2cells.2. Whether PP2A promote renal fibrosis is mediated by regulating the dephosphorylation of Smad3linker region.Methods1. Human kidney proximal tubular epithelial cell line(HK-2)cells were incubated with serum-free DMEM for24hours to synchronize the cell growth, then the cells were divided into3groups:control group (only FBS-free DMEM/F12), TGF-β1group(treated with TGF-β15ng/ml for24h) and plasmid transfection+TGF-β1group (treated with TGF-β15ng/ml for24h after HK-2cells transfected with PP2Ac over expression or si-RNA plasmid).To detect the mRNA and protein expression of PP2Ac, fibronectin, collagen I, a-SMA and E-cadherin throuth RT-PCR and Western blot.2. HK-2cells were incubated and randomly divided into3groups:control group,TGF-β1group (treated with TGF-β15ng/ml for1h) and plasmid transfection+TGF-β1group (treated with TGF-β15ng/ml for1h after HK-2cells transfected with PP2Ac over expression or si-RNA plasmid).Detect the expression of PP2Ac,Smad3and Smad3-L S204,Smad3-L S208in cytoplasm and nucleus.were tested by RT-PCR.Results1. RT-PCR and Western blot showed that in TGF-β1stimulated cells, the expression of PP2Ac mRNA and protein were significantly increased, fibronectin, collagen I and a-SMA mRNA and protein expression were also increased while the E-cadherin mRNA and protein expression were decreased. A further increase of PP2Ac expression caused by incubation with TGF-β1after transfection of PP2Ac over expression plasmid, mRNA and protein of fibronectin, collagen I and a-SMA were more up-regulated and E-cadherin down-regulated than TGF-β1group. moreover, after transfected with the PP2Ac si-RNA plasmid,fibronectin, collagen I and α-SMAexpression reducted and E-cadherin increased.2.Western blot analysis showed that after TGF-β1stimulating HK-2cells,the best time point of the expression of PP2Ac and pSmad3-L Ser208is1hour. So in the following experiment we selectsed the time point that TGF-β1stimulated HK cells1h to research whether PP2Ac promotes renal fibrosis mediated by phosphorylating Smad3link region.3.Western blot analysis showed that after TGF-β1stimulating HK-2cells, the expression of Smad3L Ser204and Smad3-L Ser208in total and nucleus were both elevated, both of which were down-regulated than TGF-β1group when transfected with PP2Ac over expression plasmid and Smad3-L Ser204and Smad3-L Ser208were further upregulated when transfected with PP2Ac si-RNA plasmid.Conclusions1. The expresses of PP2Ac showed positive correlation with FN, Col-I and a-SMA and inverse relationship with Ecadherin, which means that PP2Ac promoted the formation of extracellular matrix and epithelial-mesenchymal transition(EMT).2.PP2Ac may promote renal fibrosis by regulating the dephosphorylation of Smad3linker region.