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

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.

Cardiomyoblast caveolin expression: effects of simulated diabetes, α-linolenic acid, and cell signaling pathways

“…likely contributes to these changes: insulin up-regulated caveolinwithout influencing caveolin-1), an effect countered by HG and palmitate; while palmitate reduced caveolin-3 (without influencing caveolin-1), negating the stimulatory effects of insulin and glucose.Data are also consistent with the suggestion depression of caveolin-1 by MEK/ERK signaling (11), known to be up-regulated with T2D (53), requires a combination of multiple metabolic features of T2D, with hyperglycemia alone insufficient to induce changes. We are unaware of prior data on myocardial caveolin-1 in T2D, and while the decline in caveolin-1 with simulated T2D here contrasts elevations in models of hyperglycemia/T1D (56, 57), this agrees with T2D dependent caveolin-1 down-regulation in skeletal(8,50) and vascular smooth muscle (5), and human and rodent brain (2).Increased ERK1/2 signaling may underlie caveolin-1 repression (11), with evidence T2D increases ERK1/2 signaling in myocardium (84) and other tissue(53).…”

Mechanisms underlying selective coupling of endothelial Ca2+ signals with eNOS vs. IK/SK channels in systemic and pulmonary arteries

“…The activity of TRPV4 EC channels in PAs is probably controlled by other scaffolding proteins. In this regard, caveolin-1 (Cav-1), a crucial protein in the pulmonary circulation, has been shown to interact with both TRPV4 channels and eNOS in ECs (Saliez et al 2008;Goedicke-Fritz et al 2015); (Ju et al 1997;Bernatchez et al 2005;Chen et al 2018). The possibility that Cav-1-enriched membrane invaginations provide a signalling scaffold for TRPV4 EC -eNOS signalling in PAs remains to be tested.…”

Novel Anti-inflammatory Effects of Canagliflozin Involving Hexokinase II in Lipopolysaccharide-Stimulated Human Coronary Artery Endothelial Cells

“…Transfection with Small Interfering RNA (siRNA) for HKII Knockdown of HKII in HCAECs was performed as previously described [38,39]. In short, cells in 6-wells plates with confluency between 50 and 80% were transfected with 20-nM siRNA for HKII (Art# 4390824, ID# S6560, lot# ASO2DUJU, Thermo Fisher Scientifics) or negative control (AM4611, Ambion by Thermo Fischer Scientifics) for 24 h using Lipofectamine RNAiMax (Invitrogen by Thermo Fischer Scientifics, Waltham, MA, USA) and in antibioticand antimycotic-free medium.…”