Membrane Estrogen Receptor Engagement Activates Endothelial Nitric Oxide Synthase via the PI3-Kinase–Akt Pathway in Human Endothelial Cells

Abstract

Abstract-17␤-Estradiol (E 2 ) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E 2 -stimulated endothelial NO release can occur without an increase in cytosolic Ca 2ϩ . Here we demonstrate for the first time, to our knowledge, that E 2 rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase-Akt pathway. E 2 treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182,780. E 2 stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E 2 -activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca 2ϩ levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E 2 -stimulated NO release. The membrane-impermeant E 2 BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase-Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca 2ϩ fluxes and describes an important pathway relevant to cardiovascular pathophysiology. Key Words: estrogen Ⅲ endothelial nitric oxide synthase Ⅲ Akt Ⅲ membrane receptor E ndogenous and exogenous estrogen in premenopausal and postmenopausal women, respectively, is protective against the development of atherosclerotic cardiovascular disease. 1,2 The relevant biological effects of estrogen are numerous and include improvements in lipid and lipoprotein profiles as well as endothelial-dependent vasodilation stimulated by estrogen administered at physiological concentrations. Reports have described significant estrogen-stimulated increases in bioavailable NO. [3][4][5] Because the antiatherogenic properties of NO are emerging, it has been proposed that the cardiovascular protective effect of estrogen is mediated through augmentation of endothelial NO production. Using a human endothelial cell (EC) in vitro model, we have previously shown that 17␤-estradiol (E 2 ) induces endothelial NO release within minutes, is estrogen receptor (ER)-dependent and gene transcription-independent, and is the result of activation of endothelial nitric oxide synthase (eNOS). 6 The regulation of eNOS activity is multifaceted. This includes regulated palmitoylation and myristoylation, which are required for eNOS partitioning into membrane caveolae and consequent function. 7-10 A variety of cofactors are required for enzymatic function, including Ca 2ϩ , calmodulin, and tetrahydrobiopterin. 11,12 Recently, heat shock p...

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