High Density Lipoprotein-induced Endothelial Nitric-oxide Synthase Activation Is Mediated by Akt and MAP Kinases (original) (raw)

2003, Journal of Biological Chemistry

High density lipoprotein (HDL) activates endothelial nitric oxide synthase (eNOS), leading to increased production of the antiatherogenic molecule NO. A variety of stimuli regulate eNOS activity through signaling pathways involving Akt kinase and/or MAP kinase. In the present study we investigated the role of kinase cascades in HDL-induced eNOS stimulation in cultured endothelial cells and COS M6 cells transfected with eNOS and the HDL receptor, scavenger receptor B-I (SR-BI). HDL (10-50 µg/ml, 20 min) caused eNOS phosphorylation at Ser-1179, and dominant negative Akt inhibited both HDL-mediated phosphorylation and activation of the enzyme. PI3 kinase inhibition or dominant negative PI3 kinase also blocked the phosphorylation and activation of eNOS by HDL. Studies with genistein and PP2 showed that the non-receptor tyrosine kinase, src, is an upstream stimulator of the PI3 kinase-Akt pathway in this paradigm. In addition, HDL activated MAP kinase through PI3 kinase, and MEK inhibition fully attenuated eNOS stimulation by HDL without affecting Akt or eNOS Ser-1179 phosphorylation. Conversely, dominant negative Akt did not alter HDL-induced MAP kinase activation. These results indicate that HDL stimulates eNOS through common upstream, src-mediated signaling which leads to parallel activation of Akt and MAP kinases and their resultant independent modulation of the enzyme. by guest on June 4, 2016 http://www.jbc.org/ Downloaded from Recently Li and colleagues also reported that HDL binding to SR-BI activates eNOS (5). The HDL-induced increase in NO production may be critical to the atheroprotective features of HDL since diminished bioavailablity of endotheliumderived NO has a key role in the early pathogenesis of hypercholesterolemiainduced vascular disease and atherosclerosis (6-8). However, the mechanisms by which HDL activates eNOS are yet to be clarified. eNOS is one of three isoenzymes which convert L-arginine to L-citrulline plus NO. The activity of eNOS is regulated by complex signal transduction pathways that involve various phosphorylation events and protein-protein interactions. Many stimuli modulate eNOS activity by activating kinases which alter the phosphorylation of the enzyme (9-15). Akt kinase (also known as PKB) by guest on June 4, 2016 http://www.jbc.org/ Downloaded from activates eNOS by directly phosphorylating the enzyme at serine-1179 (Ser-1179) (19). Akt itself is phosphorylated and activated by phosphoinositide 3kinase (PI3 kinase), which in turn is activated by a tyrosine kinase (TK). Both receptor TK and non-receptor TK are involved in PI3 kinase-Akt mediated eNOS activation by various agonists . In contrast to Ser-1179, phosphorylation of threonine-497 (Thr-497) of eNOS attenuates enzyme activity . eNOS is also modulated by MAP kinases (23,24) and unlike Akt, the effect of MAP kinases on eNOS activity can be either positive or negative (9,25-27). The role of kinase cascades in signaling by HDL from SR-BI to eNOS is entirely unknown.