Xia Weiwei, Yin Jie, Zhang Shuping, Guo Chuchu, Li Yuanyuan, Zhang Yue, Zhang Aihua, Jia Zhanjun, Chen Hongbing
Department of Clinical Laboratory, Children's Hospital of Nanjing Medical University, Nanjing, China.
Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China.
Cell Physiol Biochem. 2018;49(5):2022-2034. doi: 10.1159/000493713. Epub 2018 Sep 21.
BACKGROUND/AIMS: Although a number of reports documented the important role of parkin in mitophagy, emerging evidence also indicated additional functions of parkin besides mitophagy. The present study was undertaken to investigate the role of parkin in the regulation of ERRα/eNOS pathway in endothelial cells (ECs).
Mouse aortic endothelial cells (MAECs) and cardiac muscle HL-1 cells were transfected with parkin plasmid or siRNA. ERRα inhibitor XCT-790, autophagy inhibitor 3-MA and Bafilomycin A1, and caspase inhibitor Z-VAD-FMK were used to block autophagy or apoptosis. Western blotting was performed to examine the protein levels. Flow cytometry was applied to determine the cell apoptosis and ROS production. Mitochondrial membrane potential was measured using JC-1 and TMRM. Immunoprecipitation was performed to confirm the parkin effect on ERRα ubiquitination.
Overexpression of parkin resulted in a significant reduction of total-eNOS and p-eNOS in parallel with the downregulation of ERRα (a regulator of eNOS) protein and the enhancement of ERRα ubiquitination. To test the role of ERRα in regulating eNOS in this experimental setting, we treated ECs with ERRα inhibitor and found a decrement of total-eNOS and p-eNOS. On the contrary, overexpression of ERRα increased the levels of total-eNOS and p-eNOS. Meanwhile, parkin overexpression induced mitochondrial dysfunction and cell apoptosis in both ECs and HL-1 cells. Finally, we confirmed that the parkin effect on the regulation of eNOS was independent of the autophagy and apoptosis.
These findings suggested that parkin overexpression downregulated eNOS possibly through the ubiquitination of ERRα in endothelial cells.
背景/目的：尽管许多报告记录了帕金蛋白在线粒体自噬中的重要作用，但新出现的证据也表明帕金蛋白除了线粒体自噬外还有其他功能。本研究旨在探讨帕金蛋白在内皮细胞（ECs）中对雌激素相关受体α/内皮型一氧化氮合酶（ERRα/eNOS）信号通路的调节作用。
用帕金质粒或小干扰RNA（siRNA）转染小鼠主动脉内皮细胞（MAECs）和心肌HL-1细胞。使用ERRα抑制剂XCT-790、自噬抑制剂3-甲基腺嘌呤（3-MA）和巴弗洛霉素A1以及半胱天冬酶抑制剂Z-VAD-FMK来阻断自噬或凋亡。采用蛋白质免疫印迹法检测蛋白水平。应用流式细胞术测定细胞凋亡和活性氧（ROS）生成。使用JC-1和四甲基罗丹明甲酯（TMRM）测量线粒体膜电位。进行免疫沉淀以证实帕金蛋白对ERRα泛素化的作用。
帕金蛋白过表达导致总内皮型一氧化氮合酶（total-eNOS）和磷酸化内皮型一氧化氮合酶（p-eNOS）显著减少，同时雌激素相关受体α（eNOS的调节因子）蛋白下调以及ERRα泛素化增强。为了在该实验环境中测试ERRα对eNOS调节的作用，我们用ERRα抑制剂处理内皮细胞，发现总-eNOS和p-eNOS减少。相反，ERRα过表达增加了总-eNOS和p-eNOS水平。同时帕金蛋白过表达在ECs和HL-1细胞中均诱导线粒体功能障碍和细胞凋亡。最后，我们证实帕金蛋白对eNOS的调节作用独立于自噬和凋亡。
这些发现表明，帕金蛋白过表达可能通过在内皮细胞中使ERRα泛素化而下调eNOS。
