热休克蛋白72肽结合区对肾小管上皮间质转分化的影响

目的 探讨热休克蛋白72肽结合区在肾小管上皮间质转分化(EMT)过程中的作用和可能机制.方法 应用质粒转染方法分别诱导热休克蛋白72(HSP72)野生型、肽结合区缺失型(HSP72-△PBD)和肽结合区(PBD)的表达.用转化生长因子β1(TGF-β1)刺激大鼠肾小管上皮细胞(NRK-52E)48 h,Western印迹和免疫荧光染色检测细胞E-钙黏蛋白(cadherin),α-平滑肌肌动蛋白(SMA),HSP72和Smad3/磷酸化(p)-Smad3蛋白表达.结果 TGF-β1(10 μg/L)刺激NRK-52E细胞48 h后上调α-SMA和下调E-cadherin蛋白表达水平.Western印迹及细胞免疫荧光显示,过表达HSP72和PBD能明显减轻TGF-β1诱导的NRK-52E细胞E-cadherin蛋白表达下调和α-SMA蛋白表达上调,而过表达HSP72-△PBD不能改变上述蛋白的表达.此外,过表达HSP72和PBD显著抑制Smad3的磷酸化.结论 HSP72抑制Smad3活化和EMT的发生可能与PBD的功能有关.

Impact of peptide binding domain of heat shock protein 72 on epithelial to mesenchymal transition

Objective To investigate the effects of peptide-binding domain (PBD) of heat shock protein (HSP) 72 on epithelial to mesenchymal transition (EMT) in rat renal tubular epithelial cells. Methods The expressions of wild-type HSP72, mutant of HSP72 lacking peptide binding domain (HSP72 -△PBD) and HSP72-PBD were induced by plasmid transfection. NRK-52E cells were stimulated by TGF-β1 for 48 h. The expressions of α-smooth muscle actin (α-SMA), E-cadherin, HSP72 and Smad3/p-Smad3 were detected by Western blot and immunofluorescence. Results After NRK-52E cells were stimulated by TGF-β 1 (10 μg/L) for 48 h, the expression of α-SMA was increased and the protein level of E-cadherin was decreased. Western blotting and immunofluorescence showed that over-expression of both HSP72 and PBD inhibited TGF-β1-induced up-regulation of protein α-SMA expression, down-regulation of protein E-cadherin. However, over-expression of HSP72-△PBD did not change the protein level of E-cadherin and α-SMA. In addition, over-expression of HSP72 and PBD significantly inhibited the phosphorylation of Smad3. Conclusion Inhibition of Smad3 activation and EMT by HSP72 is associated with the function of PBD.