Department of Pharmacology, Cardiac & Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 510080, China.
Acta Pharmacol Sin. 2018 Nov;39(11):1725-1734. doi: 10.1038/s41401-018-0072-0. Epub 2018 Jul 5.
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 p38 MAPK-dependent p47phox/p67phox membrane translocation, then increasing Nox2 NADPH oxidase complex formation.
最近的证据表明，ClC-3 是 ClC 家族氯离子通道或 Cl/H 反向转运体的成员，在 NADPH 氧化酶衍生的活性氧 (ROS) 生成中发挥关键作用。然而，潜在的机制仍不清楚。在这项研究中，我们研究了 ClC-3 对内皮细胞 NADPH 氧化酶激活和 ROS 生成的影响和机制。用血管紧张素 II（Ang II，1 μmol/L）处理可显著增加培养的人脐静脉内皮细胞（HUVEC）中 ClC-3 的表达。此外，Ang II 处理可增加 ROS 产生和 NADPH 氧化酶活性，这种作用可被 ClC-3 的敲低显著抑制，并可被 ClC-3 的过表达进一步增强。SA-β-半乳糖苷酶染色显示，ClC-3 沉默可消除 Ang II 诱导的 HUVEC 衰老，而 ClC-3 过表达则产生相反的效果。我们进一步表明，Ang II 处理可增加 p47phox 和 p67phox 从细胞质向膜的易位，同时伴有 Nox2 和 p22phox 表达的增加，ClC-3 的敲低可显著减弱这种作用，而 ClC-3 的过表达则可增强这种作用。此外，ClC-3 的过表达可增加 Ang II 诱导的 HUVEC 中 p47phox 和 p38 MAPK 的磷酸化。用 p38 抑制剂 SB203580 预处理可消除 ClC-3 过表达诱导的 p47phox 磷酸化增加以及 NADPH 氧化酶活性和 ROS 生成。我们的结果表明，ClC-3 是内皮细胞中 Ang II 诱导的 NADPH 氧化酶激活和 ROS 生成的正调节剂，可能通过促进 Nox2/p22phox 表达和 p38 MAPK 依赖性 p47phox/p67phox 膜易位，从而增加 Nox2 NADPH 氧化酶复合物的形成。
