目的观察骨架调节蛋白CIP4（Cdc42 interacting protein 4）对转化生长因子β1（TGF—β1）诱导的人肾小管上皮细胞-间充质转分化（EMT）的影响，并探讨其产生的机制。方法10μg／L TGF—β1刺激72h诱导人近端肾小管上皮细胞（HK-2细胞）向间充质转分化。Western印迹法检测各组细胞内E—cadherin和α-SMA蛋白的表达。倒置显微镜观察细胞形态的变化。根据GenBank人CIP4的完全cDNA序列，设计1条特异性干扰CIP4表达的RNA片段（CIP4-siRNA）和含野生型CIP4的重组真核表达质粒（pcDNA3．1-hCIP4），利用lipofactamine2000将其转染HK-2细胞。Western印迹法检测对照组、TGF—B1刺激组、CIP4-siRNA转染组、pcDNA3．1-CIP4转染组细胞内CIP4、E—cadhefin和α-SMA蛋白的表达，共聚焦显微镜观察E—cadhefin和仪．SMA蛋白的分布改变；用P13K—Akt特异性抑制剂渥曼青霉素（wortmannin）1panol／L干预TGF—β1刺激的HK-2细胞48h，Western印迹法检测对照组和干预组CIP4表达的变化。结果TGF一[31干预后HK一2细胞E—cadhefin蛋白表达显著减少（P〈0．05），α—SMA蛋白表达显著增多（P〈0．05），细胞形态由典型的上皮细胞向肌成纤维细胞转变，表明TGF-β1诱导。肾小管上皮细胞EMT模型成功。CIP4-siRNA抑制TGF-β1诱导的HK-2细胞表达CIP4后，E—cadhenn蛋白表达显著增多（P〈0．05），α—SMA蛋白表达显著减少（P〈0．05），部分逆转了上述TGF—β1诱导的肾小管上皮细胞EMT。pcDNA3．1-hCIP4转染使CIP4高表达后，HK-2细胞E—cadhefin蛋白表达显著减少（P〈0．05），α-SMA蛋白表达显著增多（P〈0．05），诱导了肾小管上皮细胞EMT。用渥曼青霉素干预TGF—β1刺激的HK-2细胞48h，CIP4可能蛋白表达显著减少（P〈0．05）。结论TGF—β1通过P13K—Akt途径上调CIP4表达，CIP4可能进一步参与TGF—β1诱导的肾小管上皮细胞EMT过程。
Recently,phosphatase and tensin homolog deleted on chromosome 10(PTEN) is suggested as a new agent in the fighting against fibrogenesis.In tumor,DJ-1 is identified as a negative regulator of PTEN.But the expression of DJ-1 and the regulation of PTEN in fibrosis are unclear.Renal fibrosis was induced in 5/6 subtotal nephrectomy rat model.Human proximal tubular epithelial cells(HKC) were treated with transforming growth factor-beta 1(TGF-β1),or transfected with DJ-1 or PTEN.Confocal microscope was used to investigate the localization of DJ-1 and PTEN.The selective phosphoinositide-3 kinase(PI3K) inhibitor,LY294002,was administered to inhibit PI3K pathway.The DJ-1 and PTEN expression,markers of epithelial-mesenchymal transition(EMT) and Akt phosphorylation were measured by RT-PCR,Western blotting or immunocytochemistry.In vitro,after HKC cells were stimulated with 10 ng/mL TGF-β1 for 72 h,the expression of DJ-1 was increased,and that of PTEN was decreased.In vivo,the same results were identified in 5/6-nephrectomized rats.In normal HKC cells,most of DJ-1 protein localized in cytoplasm,and little in nucleus.TGF-β1 upregulated DJ-1 expression in both cytoplasma and nuclei.In contrary,TGF-β1 emptied cytoplasmic PTEN protein into nucleus.Overexpression of DJ-1 decreased the expression of PTEN,promoted the activation of Akt and the expression of vimentin,and also led to the loss of cytoplasmic PTEN.Contrarily,overexpression of PTEN protected HKC cells from TGF-β1-induced EMT.In conclusion,DJ-1 is upregulated in renal fibrosis and DJ-1 mediates EMT by suppressing cytoplasmic PTEN expression and Akt activation.
Erbin, a member of Leucine-rich repeat and PDZ-containing protein family, was found to inhibit TGF-β-induced epithelial-mesenchymal transition(EMT) in our previous study. However, the mechanism of Erbin in regulating EMT is unclear. Semaphorin protein Sema4C, with PDZ binding site at C-terminal has been recognized as a positive regulator of EMT. Here, we aimed to examine the interaction between Erbin and Sema4C. HK2 cells were treated with TGF-β1, or transfected with Erbin and(or) Sema4C. Interaction of Erbin and Sema4C was identified by immunoprecipitation. RT-PCR was used to detect the expression of Erbin and Sema4C at mRNA level after transfection. The expression levels of Erbin, Sema4C, and markers of EMT were measured by using Western blotting or ELISA. After HK2 cells were stimulated with 10 ng/mL TGF-β1 for 72 h, the protein expression levels of Erbin and Sema4C were both up-regulated, and immunoprecipitation results showed Erbin interacted with Sema4C in HK2 cells both at endogenous and exogenous levels. Furthermore, overexpression of Sema4C suppressed E-cadherin, induced vimentin and promoted fibronectin secretion, indicating Sema4C promotes the process of EMT. However, HK2 cells overexpressing Erbin were resistant to Sema4C-induced EMT. In contrast, Erbin specific siRNA promoted EMT induced by Sema4C. Taken together, these results suggest that Erbin can interact with Sema4C, and co-expression of Erbin blocks the process of Sema4C-induced EMT.
