Parkin Modulates ERRalpha/eNOS Signaling Pathway in Endothelial Cells.
Parkin Modulates ERRalpha/eNOS Signaling Pathway in Endothelial Cells.
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Parkin 调节内皮细胞中的 ERRalpha/eNOS 信号通路。
DOI：
10.1159/000493713
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发表时间：
2018
影响因子：
--
通讯作者：
Chen Hongbing
中科院分区：
文献类型：
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作者：
Xia Weiwei;Yin Jie;Zhang Shuping;Guo Chuchu;Li Yuanyuan;Zhang Yue;Zhang Aihua;Jia Zhanjun;Chen Hongbing
背景/目的虽然一些报道证明了parkin在吞丝分裂中的重要作用，但新出现的证据也表明parkin除了吞丝分裂外还有其他功能。目的探讨parkin基因在血管内皮细胞ERRα/eNOS信号转导通路中的作用。ERRα抑制剂XCT-790、自噬抑制剂3-MA和巴菲罗星A1、半胱氨酸天冬氨酸氨基转移酶抑制剂Z-VAD-FMK用于阻断自噬或细胞凋亡。Western blotting检测蛋白质水平。流式细胞仪检测细胞凋亡率和ROS生成量。用JC-1和TMRM测定线粒体膜电位。免疫沉淀法证实parkin对ERRα泛素化的影响。结果parkin过表达导致总eNOS和p eNOS表达显著减少，同时eNOS调节因子ERRα蛋白表达下调，ERRα泛素化增强。在本实验中，为了测试ERRα对eNOS的调节作用，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的过表达增加了总eNOS和p-eNOS的水平。同时，parkin过表达诱导内皮细胞和HL-1细胞线粒体功能障碍和细胞凋亡。结论Parkin过表达可能通过ERRα在内皮细胞中的泛素化而下调eNOS的表达。
Background/AimsAlthough 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).MethodsMouse 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.ResultsOverexpression 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.ConclusionThese findings suggested that parkin overexpression downregulated eNOS possibly through the ubiquitination of ERRα in endothelial cells.