【摘要】 目的探讨热休克蛋白90对氧化应激激活的细胞外信号调节激酶的作用。方法取SD雄性大鼠胸主动脉做原代平滑肌细胞培养。用1μmol/L可溶性鸟苷酰环化酶抑制剂6-Anilinoquinoline-5,8-quinolinedione(LY83583)处理4~12代大鼠血管平滑肌细胞不同时间,蛋白免疫印迹检测细胞内热休克蛋白90、磷酸化和未磷酸化细胞外信号调节激酶1/2。用格尔德霉素(热休克蛋白90的特异性阻断剂)预处理细胞30 min,再用LY83583处理120 min,免疫共沉淀和蛋白免疫印迹检测热休克蛋白90与细胞外信号调节激酶及磷酸化的细胞外信号调节激酶的结合,免疫荧光检测细胞核内磷酸化的细胞外信号调节激酶。结果LY83583处理120 min时细胞内热休克蛋白90表达达高峰,较0 min时增加9倍,与细胞外信号调节激酶1/2的第二个活化高峰一致。LY83583处理血管平滑肌细胞120 min后,热休克蛋白90与磷酸化的细胞外信号调节激酶1/2结合较对照组升高了5.5倍(P<0.01),磷酸化的细胞外信号调节激酶1/2的量增加了6.1倍(P<0.01),而细胞核内的磷酸化的细胞外信号调节激酶1/2也明显增加;5μmol/L格尔德霉素预处理后,LY83583的这些效应则被阻断。结论氧化应激增加大鼠血管平滑肌细胞内热休克蛋白90,并增加其与细胞外信号调节激酶1/2及磷酸化的细胞外信号调节激酶1/2的结合,促进磷酸化的细胞外信号调节激酶1/2核转位。这可能是氧化应激激活大鼠中膜血管平滑肌细胞内细胞外信号调节激酶1/2信号通路活性的重要机制之一,为抗氧化应激引起的血管中膜平滑肌细胞增殖提供新的分子靶点。更多还原

【Abstract】 Aim Phosphorylation and nuclear translocation of extracellular signalregulated kinases(ERK1/2) are most important for proliferation of oxidativestressed vascular smooth muscle cells(VSMC) and heat shock protein 90(HSP90) is involved in this process.We investigate whether heat shock protein 90 participated in extracellular signal-regulated kinases 1/2 pathway as a molecular chaperon. Methods Exposure vascular smooth muscle cells to LY83583(6-Anilinoquinoline-5,8-quinolinedione,produce reactive oxygen species,1 μmol/L) for different time,then heat shock protein 90,extracellular signal-regulated kinases 1/2 and phospho-extracellular signal-regulated kinases 1/2 in cell lysates were measured by western blot.Vascular smooth muscle cells were incubated with geldanamycin (a special inhibitor of heat shock protein 90,5 μmol/L) or vehicle for 30 min,then with LY83583(1 μmol/L) for 120 min,heat shock protein 90 binding with extracellular signal-regulated kinases 1/2 and phospho-extracellular signal-regulated kinases 1/2 were quatified by immunoprecipitation and western blot.The nuclear translocation of phospho-extracellular signal-regulated kinases 1/2 were measured by immunofluorenscense. Results Heat shock protein 90 increased in a time-dependent manner.It got the peak at 120 min which corresponded to the second peak of phospho-extracellular signal-regulated kinases 1/2.Immunoprecipitation and western blot analysis showed that LY83583 increased the complex of heat shock protein 90-phospho-extracellular signal-regulated kinases 1/2 about 5.5 times(P<0.01) vs control,and phospho-extracellular signal-regulated kinases 1/2 in total cell lysis(about 6.1 times vs control,P<0.01) and nuclear increased too.Geldanamycin attenuated the effect of LY83583. Conclusions Heat shock protein 90 bound with phospho-extracellular signal-regulated kinases 1/2 and promoted their nuclear translocation in oxidative-stressed vascular smooth muscle cells.更多还原