肝细胞生长因子下调Smurf2拮抗肾小管上皮细胞-间充质细胞转分化

目的 通过观察肝细胞生长因子( HGF)对正常大鼠肾上皮细胞(NRK-52E)转化生长因子β1( TGF-β1)诱导的Smad泛素化调节因子2(Smurf2)表达的影响,探讨HGF拮抗肾小管上皮细胞-间充质细胞转分化(EMT)的分子机制.方法 以NRK-52E细胞为研究对象,给予TGF-β1(5μg/L)处理0～24 h；或部分细胞经HGF(20 μg/L)预处理30 min后,再予TGF-β1(5μg/L)处理1h或48 h；另外部分细胞予以Smurf2质粒表达载体或Smurf2 siRNA 转染24 h后,再予HGF处理24 h.应用Western印迹及间接免疫荧光染色方法检测Smurf2、SnoN、E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)和纤连蛋白(FN)的表达.结果 与对照组相比,TGF-β1处理后迅速上调NRK-52E细胞中Smurf2蛋白表达(P＜0.01)；同时显著诱导FN和α-SMA蛋白表达,并下调E-cadherin表达.而予HGF预处理细胞后,可快速抑制TGF-β1诱导的Smurf2表达上调(P＜0.01)；并逆转TGF-β1介导的SnoN(P＜0.01)、E-cadherin(P＜0.05)、α-SMA(P＜0.01)和FN(P＜0.01)表达变化.此外,在NRK-52E细胞中过表达Smurf2蛋白可部分抑制HGF诱导的SnoN蛋白上调；而抑制Smurf2表达则可进一步促进HGF诱导的SnoN蛋白表达.结论 在肾小管上皮细胞中,HGF可能通过下调Smurf2表达抑制SnoN蛋白发生泛素-蛋白酶体依赖性降解,进而拮抗TGF-β1介导EMT形成.

Hepatocyte growth factor surpresses epithelial-mesenchymal transition through down-regulating Smurf2 expression in rat NRK-52E cells

Objective To investigate the possible mechanism that hepatocyte growth factor （HGF） inhibits renal tubular epithelial-mesenchymal transition (EMT), and to determine whether Smurf2 expression induced by TGF-β1 can be reversed by HGF in normal rat kidney epithelial cells (NRK-52E). Methods Using rat NRK-52E cell line as an in vitro system, NRK-52E cells were incubated with 5 μg/L TGF-β1 for 0-24 h. Part of cells were pretreated with 20 μg/L HGF for 30 min or not, then incubated with or without 5 μg/L TGF-β1 for 1 h or 48 h. The other cells were transfected with pFlag-Smurf2 or Smurf2 siRNA for 24 h, then treated with or without 20 μg/L HGF for 24 h. The expressions of Smurf2, SnoN, E-cadherin, alpha-smooth muscle actin (α-SMA) and fibronectin (FN) were detected by Western blotting and indirect immunofluorescence staining assays. Results Compared to normal control, TGF-β1 could rapidly induce Smurf2 protein expression in a short time (P<0.01). Meanwhile, the expressions of FN and α-SMA were significantly induced, and the expression of E-cadherin was reduced in NRK-52E cells by TGF-β1. In contrast, in the NRK-52E cells pretreated with HGF, HGF could obviously inhibit Smurf2 expression induced by TGF-β1, and reversed the down-regulation of SnoN (P<0.01) and E-cadherin(P<0.05), the up-regulation of α-SMA (P<0.01) and FN (P<0.01) induced by TGF-β1. Moreover, overexpression of Smurf2 in NRK-52E cells could partly inhibit the up-regulation of SnoN protein by HGF, while down-regulation of Smurf2 could up-regulate the expression of SnoN induced by HGF. Conclusions HGF can abolish EMT induced by TGF-β1 in renal tubular epithelial cells through down-regulating Smurf2 expression and suppressing ubiquitin-proteasome dependent degradation of SnoN.