Abstract 12: GRK2 Regulates Enos Level in Endothelial Cells Through a Proteasome-Dependent Mechanism
eNOS is fundamental to regulate vascular functions such vasodilation, permeability and it has been demonstrated that overexpression of the Kinase of G protein coupled receptor 2 (GRK2) in endothelial cell (EC) reduces eNOS activity through AKT. However potential implication of GRK2 inhibition/removal from EC has not been explored yet. For this aim, we used vascular cell isolated from aorta of transgenic mice with GRK2 gene flanked by p-lox sequence (GRK2flox/flox). CRE recombinase, introduced in these cells by means of adenovirus infection (AD-CRE), cut and legates at p-lox sequences producing GRK2 gene removal. We observed that GRK2 deletion by AD-CRE produces reduction of eNOS levels as compared to AD-GFP used as control (AD-CRE:0.5±0.01 vs AD-GFP:3.2±0.2, p<0.05). To evaluate if GRK2 deletion produces reduced transcription of eNOS gene, we performed RT-PCR which did not show significant differences between AD-CRE and AD-GFP treated cells in eNOS mRNA level. We then incubated cells with a proteasome inhibitor, MG-132 (10-5 M, 30’), to explore an involvement of Ubiquitin dependent degradation, and observed a recover of the eNOS level (AD-CRE:3.5±0.11 vs AD-GFP:3.4±0.3, ns). To evaluate if GRK2 regulates eNOS ubiquitination, we pulled down equal amount of eNOS from bovine aortic endothelial cells (BAEC) transfected with a plasmid codifying for GRK2 or a control plasmid, and evaluated levels of ubiquitinated eNOS by western blot. We observed that GRK2 overexpression reduces level of eNOS ubiquitination as compared to control, which is also accompanied by increased level of eNOS. Morever, we transfected BAEC with plasmids expressing GRK2 wild type (GRK2 wt) and GRK2 kinase defective mutant (GRK2K220R). We found that both GRK2 wt and K220R are able to induce eNOS accumulation in BAEC (GRK2: 4.5±0.3, GRK2K220R: 4.8±0.4 vs Control: 2.1±0.1, p<0.05) indicating a kinase independent mechanism of eNOS ubiquitination and degradation. More experiments are needed to evaluate mechanisms through which GRK2 protects eNOS from Ubiquitin mediated degradation, nevertheless, these data indicate a novel mechanism of regulation of eNOS levels that can have important implications in the overall cellular physiology and pathology.
- © 2012 by American Heart Association, Inc.