Abstract
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 mitogen-activated protein (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. HDL (10-50 microg/ml, 20 min) caused eNOS phosphorylation at Ser-1179, and dominant negative Akt inhibited both HDL-mediated phosphorylation and activation of the enzyme. Phosphoinositide 3-kinase (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 nonreceptor 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 mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 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.
Highlights
The risk for cardiovascular disease from atherosclerosis is inversely proportional to serum levels of high density lipoprotein (HDL)1 [1, 2]
Using pharmacological inhibition or dominant negative mutant forms of selective kinases in endothelial cells (EC) or COS M6 cells transfected with endothelial nitric-oxide synthase (eNOS) and the HDL receptor, SR-BI, we investigated the involvement of tyrosine kinases, PI3 kinase, Akt, and MAP kinases in HDL-mediated eNOS activation
HDL Stimulation of eNOS Requires Phosphorylation at Ser1179 —In order to determine whether HDL stimulation of eNOS requires the phosphorylation of eNOS at Ser-1179, primary ECs were incubated with HDL (10 or 50 g/ml) for 20 min or with VEGF (100 ng/ml) for 5 min, serving as a positive control
Summary
Vol 278, No 11, Issue of March 14, pp. 9142–9149, 2003 Printed in U.S.A. High Density Lipoprotein-induced Endothelial Nitric-oxide Synthase Activation Is Mediated by Akt and MAP Kinases*. Dominant negative Akt did not alter HDLinduced 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. Using pharmacological inhibition or dominant negative mutant forms of selective kinases in EC or COS M6 cells transfected with eNOS and the HDL receptor, SR-BI, we investigated the involvement of tyrosine kinases, PI3 kinase, Akt, and MAP kinases in HDL-mediated eNOS activation. In addition to improving our specific understanding of eNOS modulation, the elucidation of the signaling cascade(s) coupling SR-BI to the enzyme provides important clues about multiple additional potential target of HDL action in EC.
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