ClC-3 promotes angiotensin II-induced reactive oxygen species production in endothelial cells by facilitating Nox2 NADPH oxidase complex formation

Authors: Guo-zheng Liang1, Li-min Cheng1, Xing-feng Chen1, Yue-jiao Li1, Xiao-long Li1, Yong-yuan Guan1, Yan-hua Du1
1 Department of Pharmacology, Cardiac & Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China
Correspondence to: Yan-hua Du:,
DOI: 10.1038/s41401-018-0072-0
Received: 22 March 2018
Accepted: 10 June 2018
Advance online: 5 July 2018


Recent evidence suggests that ClC-3, a member of the ClC family of Cl− channels or Cl−/H+ antiporters, plays a critical role in NADPH oxidase-derived reactive oxygen species (ROS) generation. However, the underling mechanisms remain unclear. In this study we investigated the effects and mechanisms of ClC-3 on NADPH oxidase activation and ROS generation in endothelial cells. Treatment with angiotensin II (Ang II, 1 μmol/L) significantly elevated ClC-3 expression in cultured human umbilical vein endothelial cells (HUVECs). Furthermore, Ang II treatment increased ROS production and NADPH oxidase activity, an effect that could be significantly inhibited by knockdown of ClC-3, and further enhanced by overexpression of ClC-3. SA-β-galactosidase staining showed that ClC-3 silencing abolished Ang II-induced HUVEC senescence, whereas ClC-3 overexpression caused the opposite effects. We further showed that Ang II treatment increased the translocation of p47phox and p67phox from the cytosol to membrane, accompanied by elevated Nox2 and p22phox expression, which was significantly attenuated by knockdown of ClC-3 and potentiated by overexpression of ClC-3. Moreover, overexpression of ClC-3 increased Ang II-induced phosphorylation of p47phox and p38 MAPK in HUVECs. Pretreatment with a p38 inhibitor SB203580 abolished ClC-3 overexpression-induced increase in p47phox phosphorylation, as well as NADPH oxidase activity and ROS generation. Our results demonstrate that ClC-3 acts as a positive regulator of Ang II-induced NADPH oxidase activation and ROS production in endothelial cells, possibly via promoting both Nox2/p22phox expression and
Keywords: ClC-3; angiotensin II; oxidative stress; NADPH oxidase; endothelial cell

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