曲古菌素A抑制血清诱导血管平滑肌细胞p27kip1表达的降解

Trichostatin A inhibits serum-induced p27kip1 protein degradation in vascular smooth muscle cells

目的:探讨组蛋白去乙酰化酶抑制剂曲古菌素A(TSA)对血管平滑肌细胞(VSMCs)p27kip1表达的影响和调控机制。方法:半定量逆转录聚合酶链反应(RT-PCR)检测p27kip1 mRNA水平,蛋白印迹测定p27kip1和skp2蛋白表达,荧光分光光度法测定20S蛋白酶体活性。结果:100μg/LTSA不影响VSMCs中p27kip1的mRNA水平。100μg/LTSA显著抑制血清诱导的p27kip1蛋白下调,并延长p27kip1蛋白的半衰期。100μg/LTSA抑制血清诱导的skp2表达上调,且skp2表达与相应时点p27kip1蛋白呈负相关。100μg/LTSA对20S蛋白酶体活性物无影响。结论:TSA对VSMCs的p27kip1表达调控不是在转录水平上,而是通过翻译后机制抑制血清诱导VSMCs的p27kip1蛋白降解,其机制可能与TSA抑制泛素连接酶亚单位skp2表达有关。

AIM： To investigate the effect of trichostatin A （TSA） on p27kip1 gene expression in vascular smooth muscle cells. METHODS： Reverse transcription - polymerase chain reaction （ RT - PCR） was used to measure the level of p27kip1 mRNA. The protein levels of p27kip1 and S - phase kinase - associated protein - 2 （ skp2 ） were determined by Western blotting. 20S proteasome activity was quantified by using a fluorogenic proteasome - specific substrate. RESULTS： TSA did not affect mRNA level of p27kip1 in VSMCs, but attenuated serum - induced downregulation of p27kipl through stabilizing p27kip1 turnover. In addition, TSA decreased the expression of skp2, an F- box protein that targets p27kip1 for degradation, but had no effect on proteasome activity. CONCLUSION： TSA regulates p27kip1 expression at the post -translational level in VSMCs.