穹窿主体蛋白通过上调干扰素调节因子2抑制动脉内皮细胞凋亡

Major vault protein inhibits apoptosis of aortic endothelial cells through upregulating interferon regulatory factor 2

目的:探究穹窿主体蛋白(major vault protein,MVP)对动脉内皮细胞(endothelial cell,EC)增殖的作用,揭示MVP对动脉EC发挥保护作用的潜在机制。方法:以人主动脉EC(human aortic EC,HAEC)为细胞模型,感染慢病毒以抑制或过表达MVP。使用CCK-8实验和流式细胞术检测细胞增殖活性和死亡。使用凋亡抑制剂Z-VAD和坏死性凋亡抑制剂Nec-1确定细胞死亡方式。以流式细胞术检测Annexin V结合阳性率和Caspase 3活性,以Western blot检测Caspase剪切体蛋白表达以评价细胞凋亡。以荧光定量PCR和Western blot技术鉴定靶分子,并明确MVP与靶分子之间的调控关系。结果:敲降MVP抑制HAEC增殖,促进HAEC死亡,过表达MVP结果则相反。Z-VAD逆转MVP敲降引起的死亡,而Nec-1无此作用。过表达MVP抑制TNF-α诱导的HAEC凋亡,敲降MVP时作用相反。MVP通过上调干扰素调节因子2(interferon regulatory factor 2,IRF2)促进凋亡抑制蛋白1(cellular inhibitor of apoptosis protein 1,cIAP1)的转录。敲降IRF2逆转MVP过表达引起的cIAP1表达增多和凋亡抑制。结论:MVP通过上调IRF2蛋白促进cIAP1转录表达从而抑制TNF-α诱导的HAEC凋亡,发挥对EC的保护作用。

Objective:To investigate the effects and the underlying mechanism of major vault protein(MVP)on the proliferation of arterial endothelial cells.Methods:Human aortic endothelial cell(HAEC)were infected with lentivirus to inhibit or overexpress MVP.Cell proliferation and death were detected with CCK-8 assay and flow cytometry.Apoptosis inhibitor Z-VAD and necroptosis inhibitor Nec-1 were used to distinguish the mode of cell death.Annexin V binding and Caspase 3 activity were detected by flow cytometry,and cleaved Caspase was examined by Western blot.Real time PCR and Western blot were performed to investigate target molecules and the regulatory relationship.Results:Knockdown of MVP inhibited the proliferation activity of HAEC and promoted the HAEC cell death.Overexpression of MVP resulted in the opposite results.Treatment with Z-VAD reversed HAEC death caused by MVP knockdown,while Nec-1 did not.Consistently,TNF-α-induced HAEC apoptosis was inhibited by MVP overexpression and exaggerated by MVP knocked down.MVP promoted the transcriptional expression of cellular inhibitor of apoptosis proteins1(cIAP1)by up-regulating interferon regulatory factor 2(IRF2)protein expression.IRF2 knockdown reversed the increase in cIAP1 expression and the decrease in apoptosis caused by MVP overexpression.Conclusion:MVP promoted cIAP1 transcription by up-regulating IRF2 protein expression,thereby inhibiting TNF-α-induced apoptosis and promoting the proliferation of arterial EC.