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

Mol Biol Cell. 2018 May 15;29(10):1190-1202. doi: 10.1091/mbc.E17-01-0049. Epub 2018 Mar 22.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Albumins
  • Caveolin 1
  • Insulin
  • Ionophores
  • RNA, Small Interfering
  • Nitric Oxide
  • Calcimycin
  • NOS3 protein, human
  • Nitric Oxide Synthase Type III
  • src-Family Kinases
  • Calcium