Departments of aAnesthesiology, University of Illinois at Chicago, Chicago, IL 60612.
Pharmacology, University of Illinois at Chicago, Chicago, IL 60612.
Mol Biol Cell. 2018 May 15;29(10):1190-1202. doi: 10.1091/mbc.E17-01-0049. Epub 2018 Mar 22.
We hypothesized that the maintenance of vascular homeostasis is critically dependent on the expression and reciprocal regulation of caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs). Skeletal muscle biopsies from subjects with type 2 diabetes showed 50% less Cav-1 and eNOS than those from lean healthy controls. The Cav-1:eNOS expression ratio was 200:1 in primary culture human ECs. Cav-1 small interfering RNA (siRNA) reduced eNOS protein and gene expression in association with a twofold increase in eNOS phosphorylation and nitrate production per molecule of eNOS, which was reversed in cells overexpressing Adv-Cav-1-GFP. Upon addition of the Ca ionophore A23187 to activate eNOS, we observed eNOS Ser1177 phosphorylation, its translocation to β-catenin-positive cell-cell junctions, and increased colocalization of eNOS and Cav-1 within 5 min. We also observed Cav-1 S-nitrosylation and destabilization of Cav-1 oligomers in cells treated with A23187 as well as insulin or albumin, and this could be blocked by L-NAME, PP2, or eNOS siRNA. Finally, caveola-mediated endocytosis of albumin or insulin was reduced by Cav-1 or eNOS siRNA, and the effect of Cav-1 siRNA was rescued by Adv-Cav-1-GFP. Thus, Cav-1 stabilizes eNOS expression and regulates its activity, whereas eNOS-derived NO promotes caveola-mediated endocytosis.
我们假设血管稳态的维持取决于内皮细胞（ECs）中 caveolin-1（Cav-1）和内皮型一氧化氮合酶（eNOS）的表达和相互调节。2 型糖尿病患者的骨骼肌活检显示，Cav-1 和 eNOS 的表达量比瘦体健康对照组少 50%。原代培养的人 ECs 中 Cav-1:eNOS 的表达比例为 200:1。Cav-1 小干扰 RNA（siRNA）降低了 eNOS 蛋白和基因表达，同时 eNOS 磷酸化和每分子 eNOS 产生的硝酸盐增加了两倍，而在过表达 Adv-Cav-1-GFP 的细胞中则相反。当加入 Ca2+载体 A23187 激活 eNOS 时，我们观察到 eNOS Ser1177 磷酸化，其向β-连环蛋白阳性细胞-细胞连接处转移，以及 eNOS 和 Cav-1 之间的共定位在 5 分钟内增加。我们还观察到 A23187 处理以及胰岛素或白蛋白处理后 Cav-1 的 S-亚硝基化和寡聚体的不稳定性，这可以被 L-NAME、PP2 或 eNOS siRNA 阻断。最后，Cav-1 或 eNOS siRNA 降低了白蛋白或胰岛素的 caveola 介导的内吞作用，而 Cav-1 siRNA 的作用可以被 Adv-Cav-1-GFP 挽救。因此，Cav-1 稳定了 eNOS 的表达并调节其活性，而 eNOS 衍生的 NO 促进了 caveola 介导的内吞作用。
