Molecular chaperon HSP regulates apoptosis signaling

本研究用热休克蛋白90的特异性抑制剂格尔达那霉素(GDM)研究了热休克蛋白90在抗Fas抗体(CH-11)诱导HSG细胞凋亡中的作用。GDM本身引起HSG细胞的凋亡，并且增强了CH-11诱导的细胞凋亡。RT-PCR和westernblotting显示GDM上调HSP90的表达，提示GDM对HSP90的功能抑制后，HSP90的表达立即发生互补。此外，将HSP90蛋白导入HSG细胞可显著抑制GDM对CH-11诱导的HSG细胞凋亡的促进作用。免疫沉淀-Western nblotting分析表明FliP、Caspase 8和RIP是HSP90的靶蛋白。经GDM处理后，Flip基因表达水平显著降低。这些结果表明，HSP90通过其对显性负性Caspase 8-Flip的伴侣作用来负调控Fas诱导的细胞凋亡。此外，我们还检测了携带热休克元件的荧光素酶报告基因转染HSG细胞后的转录激活情况。与未处理的细胞相比，GDM处理和热休克处理提高了荧光素酶的活性。这一结果表明，GDM通过将热休克因子从HSPs中解离出来，进而激活HSP90基因，并将其移位到细胞核内，进而激活HSP90基因，从而增加HSP90的表达。

In this study, the involvement of heat shock protein 90 in the anti-Fas antibody (CH-11)-induced apoptosis in HSG cells was investigated using geldanamycin (GDM), the specific inhibitor of HSP90. GDM itself caused the apoptosis in HSG cells, and moreover, it enhanced CH-11-induced apoptosis. RT-PCR and Westernblotting revealed that the expression of HSP90 was up-regulated by GDM, suggesting that the immediate complementation of HSP90 occurs after the functional inhibition of HSP90 by GDM. Further, the transfection of HSP90 protein into HSG cells significantly inhibited the enhancement of CH-11-induced apoptosis by GDM. Immunoprecipitation-Westernblotting analysis revealed FLIP, Caspase 8 and RIP as target proteins of HSP90. FLIP mRNA level was significantly decreased by GDM treatment. From these results, it was revealed that HSP90 negatively regulates Fas-induced apoptosis through its chaperoning effect on FLIP, the dominant-negative Caspase 8. Furthermore, we examined the transcription activation from the heat shock element-harboring luciferase reporter gene that was transfected into HSG cells. Treatment of the transfected cells with GDM, as well as heat shock treatment, increased the luciferase activity as compared with the untreated cells. This result suggested that GDM increases the expression of HSP90 through the dissociation of heat shock factor from HSPs and its following activation, translocation into nuclei, and then transcription activation of HSP90 gene.