| Epithelial-to-mesenchymal transition (EMT) is one of the key mechanisms underlying tubulointerstitial fibrosis (TIF), which is the common pathological pathway through which chronic kidney diseases progress to end-stage renal failure.This phenotype has been shown to be a complicated multistep process instead of simply a morphological transition, accompanied by a decreased expression of epithelial markers E-cadherin and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers a-SMA, fibronectin, and vimentin. The dysregulation of E-cadherin/p-catenin complex, which is the core of epithelial cell-to-cell adhesion, play a critical role in EMT process. TGF-β1 is probably the key inducer of EMT because TGF-β1 signaling is sufficient to induce EMT in cultured epithelial cells. The underlying signaling mechanism of TGF-β1-induced EMT has received much attention in recent years, but still remains not well understood. It has been reported that signaling molecules, including Smad Phosphatidylinositol 3-kinase-Akt (PI3K-Akt), integrinlinked kinase(ILK), Rho-A, mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK), p38 MAPK, and c-Jun N-terminal kinases (JNK) are involved in the regulation of TGF-β1-induced EMT. It is emerging that protein kinase D1(PKD1) is associated with the TGF-β1-induced EMT, which is implicated in the regulation of a variety of cellular functions, including signal transduction, membrane trafficking, protein transport, and cell survival, migration, differentiation, and proliferation. Activation of PKD1 modulates E-cadherin/p-catenin activity associated with subcellular redistribution ofβ-catenin. In this study, we examine the activation of PKD in TGF-β1-induced tubular EMT. Then, we demonstrate that the activity of PKD1—ERK1/2 pathway induced by TGF-β1 is involved in the regulation of E-cadherin/β-catenin binding.Part I Construction of Eukaryotic Expression Vectors of Wide and Y654 mutants ofβ-catenin and their Expression in Human Proximal Tubular Epithelial cellsObjective To construct eukaryotic expression vectors of two mutants ofβ-catenin and study their expressions in human human proximal tubular epithelial cells (HKC).Methods Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Tyr654(TAT) inβ-catenin into Glu(GAA) and Phe(TTT) in pEGFP-Cl-β-catenin to obtain single-site-mutated vector pEGFP-β-catenin-Y654E and pEGFP-β-catenin-Y654F, which mimics the phosphorylation and dyphosphorylation ofβ-catenin, perspectively. After lipofectin-mediated transformation of with WT, Y654E,Y654F plasmids, the stably transfected cell lines were selected. Protein were determined using RT-PCR and Western blotting.Results DNA sequence analysis demonstrated success of the site-directed mutagenesis and the plasmid pEGFP-C1/WT-β-catenin, pEGFP-C1/Y654E-β-catenin and pEGFP-C1/ Y654F-β-catenin were obtained.Conclusion The plasmid pEGFP-C1/WT-β-catenin, pEGFP-Cl/Y654E-β-catenin and pEGFP-C1/Y654F-β-catenin have been successfully constructed with defficient expression in HKC cells. PARTⅡProtein Kinase D1 is Involved in the TGF-β1-induced Tubular Epithelial-mesenchymal TransformationObjective:To set up the model of TGF-β1-induced tubular EMT. Study the involvment of PKD1in renal fibrosis by western blotting, immunocytochemistry and siRNA.Methods:Compared the changes of HKC cells morphology and expression of E-cadherin, Vimentin, PKD1, p-PKD1 between groups of 10.0ng/ml TGF-β1, 10.0ng/mlEGF and 10.0ng/ml TGF-β1+10.0ng/mlEGF.Tranfected HKC cells by specific siRNA of PKD1 and observed the changes of HKC mophorlogy and expression of E-cadherin, Vimentin.Results:10.0ng/ml TGF-β1 induced the morphological changes of HKC cells from round to spindle and the expression of E-cadherin was reduced and Vimentin was increased.. p-PKD1 was also increase by TGF-β1. 10.0ng/ml TGF-β1+10.0ng/mlEGF had a more obviously effect on HKC while 10.0ng/mlEGF did not induce tubular EMT. HKC transfected by siPKD1 had no obvious changes by TGF-β1+EGF.Conclusion:EGF and TGF-β1 has coordinate action on inducing tubular EMT, which may be related with activity of PKD1.PART III Protein kinase D1 is Involved in the Regulation of E-cadherin/β-catenin Activity in TGF-β1-induced tubular Epithelial-mesenchymal TransformationObjective:Set up the model of TGF-β1-induced tubular EMT to study the regulation of E-cadherin/β-catenin activity by PKD1 in tubular EMT,by western blotting, immunocyto-chemistry and siRNA.Methods:Detected the expression of PKD1, p-PKD1, ERK1/2, p-ERK1/2, E-cadherin, β-catenin and PY20 in TGF-β1-induced tubular EMT. Tranfected HKC cells by specific siRNA of PKD1 to supress the expression of PKD1. And inhibited the expression of ERK1/2 by PD98059.Examined the effect of EMT induction by TGF-β1 with each of the two methods. And the phosphorylation of ERK1/2 was reduced by siPKDl. Observed whether the translocation ofβ-catenin induced by TGF-β1 in WTβ-catenin cells was changed by siPKD1 or PD98059.Results:10.0ng/ml TGF-β1 induced the phosphorylation of PKD1 and Erkl/2 and the changes of p-PKD1 was earlier than p-ERK1/2. Tyrosin phosphorylation of P-catenin was increased significantly while the totalβ-catenin had no obviously changes. siPKD1 or PD98059 supressed the TGF-β1-induced tubular EMT, respectively. siPKDl reduced the phosphorylation of Erkl/2 while PD98059 had no obvious effect on the phosphorylation of PKD1.Both of the methods inhibited the translocation ofβ-catenin from membrane to nulear induced by TGF-β1 in WTβ-catenin cells.Conclusion:PKD1-ERK1/2 is invovled in the regulation of E-cadherin/β-catenin activity during TGF-β1-induced EMT.
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