Reciprocal regulation of eNOS and caveolin-1 functions in endothelial cells.
Reciprocal regulation of eNOS and caveolin-1 functions in endothelial cells.
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DOI：
10.1091/mbc.e17-01-0049
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发表时间：
2018-05-15
影响因子：
3.3
通讯作者：
Minshall RD
中科院分区：
文献类型：
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作者：
Chen Z;D S Oliveira S;Zimnicka AM;Jiang Y;Sharma T;Chen S;Lazarov O;Bonini MG;Haus JM;Minshall RD
我们假设血管稳态的维持严重依赖于内皮细胞（ECs）中小窝蛋白-1 （Cav-1）和内皮一氧化氮合酶（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比瘦肉健康对照组低50%。Cav-1:eNOS在原代培养人内皮细胞中的表达比为200:1。Cav-1小干扰RNA （siRNA）降低eNOS蛋白和基因表达，eNOS磷酸化和eNOS每分子硝酸盐产量增加两倍，而在过表达Adv-Cav-1-GFP的细胞中，这种情况被逆转。在加入Ca2+离子载体A23187激活eNOS后，我们观察到eNOS Ser1177磷酸化，其易位到β-catenin阳性的细胞-细胞连接处，并在5分钟内增加eNOS和Cav-1的共定位。我们还观察到，在A23187、胰岛素或白蛋白处理的细胞中，Cav-1的s -亚硝基化和Cav-1低聚物的不稳定，这可以被L-NAME、PP2或eNOS siRNA阻断。最后，Cav-1或eNOS siRNA可减少小窝介导的白蛋白或胰岛素的内吞作用，并通过Adv-Cav-1-GFP恢复Cav-1 siRNA的作用。因此，Cav-1稳定eNOS表达并调节其活性，而eNOS衍生的NO促进小泡介导的内吞作用。
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 Ca2+ 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.