E1A激活基因阻遏子(CREG)过表达通过抑制p38/JNK信号分子活化对抗人血管平滑肌细胞凋亡

Overexpression of The Cellular Repressor of E1A-stimulated Genes Inhibits The Apoptosis of Human Vascular Smooth Muscle Cells via Blocking p38/JNK MAP Kinase Activation

血管平滑肌细胞(VSMCs)凋亡参与了动脉粥样硬化(AS)及冠状动脉介入治疗(PCI)术后再狭窄(RS)等心血管疾病的发生发展过程.E1A激活基因阻遏子(CREG)是新近发现的一种分泌型糖蛋白,在维持细胞和组织稳态方面发挥重要作用.前期研究发现CREG蛋白过表达能够对抗血清饥饿诱导的人血管平滑肌细胞(hVSMCs)凋亡,进一步探讨CREG对hVSMCs凋亡的调控作用及相关的分子机制.以逆转录病毒稳定转染的CREG过表达及表达抑制的hVSMCs为模型,应用两种药物Staurosporine(STS)和Etoposide(VP-16)诱导细胞发生凋亡,检测细胞凋亡和相关信号通路变化.结果显示,在药物干预后,CREG表达抑制时细胞凋亡明显增多,而CREG过表达明显抑制hVSMCs凋亡.同时也发现,CREG表达抑制时p38及JNK活性明显增强,而CREG过表达时p38和JNK活性被抑制.经腺病毒转染和药物干预抑制p38表达后,细胞凋亡均受到抑制,而且在p38活性被抑制的同时,JNK活化也受到抑制.说明p38和JNK表现为协同作用.结果也显示,VSMCs分化指标SMα-actin和SM MHC与CREG表达呈一致趋势,而细胞外基质蛋白Fibronection与CREG表达呈负相关.以上结果提示,CREG在维持VSMCs表型转换方面发挥重要作用,并且通过p38和JNK信号转导通路对hVSMCs凋亡进行调控.CREG可能对于AS和PCI术后RS的防治具有重要价值.

The cellular repressor of E1A-stimulated genes （CREG） is a recently described glycoprotein which plays a critical role in keeping cells or tissue in homeostasis states. The current study examined whether and how CREG may regulate VSMC apoptosis. Both loss-of-function （CREG-DW by retrovirus expressing CREG shRNAs） and gain-of-function （CREG-UP by retroviral infection with vector pLNCX-CREG） studies were performed to support this notion. Western blot showed that CREG knockdown stimulated with STS or VP-16 was identified to increase cleaved caspase-3, a marker for apoptosis. It was also observed that the number of cells undergoing apoptosis remarkably increased in CREG-DW cells by annexin V/PI dual-color flow cytometry and TUNEL assays. Moreover, p38 and JNK mitogen-activated protein kinases were significantly upregulated in CREG-DW and significantly reduced in CREG-UP VSMCs. More importantly, CREG-DW-induced VSMC apoptosis was blocked by a p38-specific inhibitor （SB203580）, or by overexpression of a dominant negative p38a （p38α AGF）. The data also showed that inactivation of p38 decreased the amount of phosphorylated JNK, indicated that the p38 fusion proteins are functionally active in regulating JNK activity and induced JNK phosphorylation contributes positively to VSMC apoptosis. In addition, CREG-UP increased expression of the VSMC differentiation markers SM α-actin and SM-MHC, and reduced cell-associated fibronectin in cultured VSMCs. These results demonstrate for the first time that CREG plays a key role in modulating VSMC apoptosis by p38/JNK signaling transduction pathway in vitro. Accordingly, CREG might have potential applicational perspective in attenuating the progression of atherosclerotic plaques and restenosis after percutaneous coronary intervention.