Vps15 is critical to mediate autophagy in AngII treated HUVECs probably by PDK1/PKC signaling pathway.
Vps15 is critical to mediate autophagy in AngII treated HUVECs probably by PDK1/PKC signaling pathway.
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DOI：
10.1016/j.lfs.2019.116701
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发表时间：
2019-07
期刊：
影响因子：
6.1
通讯作者：
H. Shan;Yini He;Sijia Hao;Bofu Wang;Ming Xu;Huimeng Qi;Shen Liu;Yanmei Du;Xiaosong Yu
中科院分区：
文献类型：
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作者：
H. Shan;Yini He;Sijia Hao;Bofu Wang;Ming Xu;Huimeng Qi;Shen Liu;Yanmei Du;Xiaosong Yu
AimsVps 15是自噬诱导中III类PI 3 K活性的重要调节因子。AngII通过自噬对内皮细胞的早期保护起积极作用。本研究利用特异性shRNA敲低Vps 15的表达，探讨Vps 15对AngII刺激的HUVECs自噬、衰老和凋亡的影响。材料与方法MDC染色显示自噬小体。用β-半乳糖苷酶染色检测细胞衰老。流式细胞仪Annexin V-FITC/PI染色检测细胞凋亡。Western blot检测转染Vps 15-shRNA的HUVECs中LC 3-II/I的比值及相关细胞信号通路的激活情况。拯救实验表明，Vps 15的表达激活了细胞自噬，抑制了细胞的衰老和凋亡。此外，PDK 1和PKC底物的磷酸化在AngII处理后增加，而Vps 15敲低则降低。用PDK 1或PKC抑制剂预处理细胞，可减弱AngII刺激后的细胞自噬，但可促进细胞衰老和凋亡。用PDK 1抑制剂预处理的细胞中，PDK 1和PKC的磷酸化均受到抑制。提示Vps 15在AngII诱导的HUVECs保护性自噬过程中起重要作用，可能与PDK 1/PKC信号通路有关。
AimsVps15 is an important regulator on the activity of class III PI3K in autophagy induction. AngII plays a positive role of autophagy in the early protection of endothelial cells. In this study, the expression of Vps15 was knocked down using the specific shRNA to investigate the effects of Vps15 on cell autophagy, senescence and apoptosis in HUVECs stimulated by AngII. The associated cell signaling pathway was also explored.Materials and methodsMDC staining was applied to show autophagic bodies. Cell senescence was detected using β-galactosidase staining. Cell apoptosis was examined by flow cytometry using Annexin V-FITC/PI staining. And western blot was used to evaluate the ratio of LC3-II/I and the activation of associated cell signaling pathway.Key findingsCell autophagy induced by AngII was inhibited in HUVECs transfected with Vps15-shRNA, while cell senescence and apoptosis were enhanced. Rescue experiment revealed that cell autophagy was activated after Vps15 reexpression, while cell senescence and apoptosis were inhibited. Moreover, the phosphorylations of PDK1 and PKC substrates were increased after AngII treatment, which were decreased by Vps15 knockdown. Pretreatment of cells with the inhibitor for PDK1 or PKC attenuated cell autophagy after AngII stimulation, yet promoted cell senescence and apoptosis. The phosphorylations of both PDK1 and PKC were inhibited in cells pretreated with PDK1 inhibitor. Only the activation of PKC was inhibited when the inhibitor for pan-PKC was used.SignificanceThese results suggested that Vps15 was critical to the protective autophagy in HUVECs induced by AngII, and PDK1/PKC signaling pathway was probably involved.