FUN14结构域蛋白2经Akt/Foxo3a信号通路和线粒体自噬拮抗血管紧张素Ⅱ诱导的内皮细胞衰老

FUN14 domain containing 2 antagonizes angiotensin Ⅱ-induced endothelial cells aging by mitophagy via Akt/Foxo3a signaling pathway

目的FUN14结构域蛋白2(FUN14 domain containing 2,FUNDC2)是一种线粒体外膜蛋白,调控缺氧条件下血小板的寿命。血管紧张素Ⅱ(angiotensin Ⅱ,AngⅡ)是血管内皮细胞衰老的重要始动因子。本研究目标在于探讨FUNDC2对于人静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)线粒体自噬的作用和其下游可能的信号通路。方法带有GFP-LC3B标签的活细胞用Mito-Tracker染色以标记线粒体自噬。AngⅡ刺激细胞诱导细胞衰老,采用β-galactosidase染色显示细胞衰老。采用免疫沉淀和Western blot检测FUNDC2蛋白的拟素化修饰和Akt/foxo3a信号通路的活化情况。结果FUNDC2表达上调增加HUVECs细胞的线粒体自噬水平,并伴有Akt/foxo3a信号通路的活化。在此过程中FUNDC2发生由神经前体细胞表达的发育下调蛋白8(neuronal precursor cell-expressed developmentally down-regulated protein 8,NEDD8)介导的拟素化修饰,NEDD8的27位赖氨酸位点是其与FUNDC2结合必不可少的。AngⅡ可抑制FUNDC2介导的线粒体自噬,与内皮细胞衰老密切相关。结论FUNDC2发生拟素化修饰对于内皮细胞线粒体自噬非常关键。AngⅡ可能由于干扰FUNDC2介导的线粒体自噬从而诱导内皮细胞衰老。

Objective FUN14 domain containing 2(FUNDC2)is an outer mitochondrial membrane protein and regulates lifespan of platelet under hypoxic stress.Angiotensin Ⅱ(AngⅡ)is a critical initiating factor of endothelial cells aging.This study is to investigate the function of FUNDC2 on mitophagy and its possible downstream signaling in human umbilical vein endothelial cells(HUVECs),and to explore the effect of Ang Ⅱ on FUNDC2 mediated mitophagy.Methods Live HUVECs with GFP-LC3B were stained with Mito-Tracker red to show mitophagy bodies.Cell senescence was detected usingβ-galactosidase staining.Immunoprecipitation and western blot were used to detect the neddylation of FUNDC2 and to evaluate the activation of Akt/Foxo3a signaling pathway.Cells were stimulated with Ang Ⅱ when indicated.Results Our results showed that mitophagy of HUVECs was promoted after FUNDC2 upregulation,along with the activation of Akt/Foxo3a signaling pathway.FUNDC2 was neddylated by NEDD8 in the process of mitophagy.The K27 of NEDD8 was indispensable for this modification.Ang Ⅱ inhibited FUNDC2 mediated mitophagy,which was associated with endothelial cells aging.Conclusion Our results indicate that FUNDC2 neddylation is considerably critical for mitophagy of endothelial cells.Ang Ⅱ may induce endothelial cells aging probably by interfering with FUNDC2 mediated mitophagy.