Reciprocal regulation of eNOS and caveolin-1 functions in endothelial cells

• Published in: Molecular biology of the cell Vol. 29; no. 10; pp. 1190 - 1202
• Main Authors: Chen, Zhenlong, D. S. Oliveira, Suellen, Zimnicka, Adriana M., Jiang, Ying, Sharma, Tiffany, Chen, Stone, Lazarov, Orly, Bonini, Marcelo G., Haus, Jacob M., Minshall, Richard D.
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 15.05.2018
• Subjects: Adult; Albumins - metabolism; Biopsy; Calcimycin - pharmacology; Calcium - metabolism; Case-Control Studies; Caveolin 1 - metabolism; Cell Membrane - metabolism; Diabetes Mellitus, Type 2 - metabolism; Diabetes Mellitus, Type 2 - pathology; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Endothelial Cells - pathology; HEK293 Cells; Human Umbilical Vein Endothelial Cells - drug effects; Human Umbilical Vein Endothelial Cells - metabolism; Humans; Insulin - metabolism; Intercellular Junctions - drug effects; Intercellular Junctions - metabolism; Ionophores - pharmacology; Middle Aged; Molecular Weight; Nitric Oxide - metabolism; Nitric Oxide Synthase Type III - metabolism; Nitrosation; Phosphorylation - drug effects; Protein Transport - drug effects; RNA, Small Interfering - metabolism; src-Family Kinases - metabolism
• ISSN: 1059-1524; 1939-4586; 1939-4586
• DOI: 10.1091/mbc.E17-01-0049

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 2+ 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.