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...

Estrogen induces both rapid and delayed effects on the cardiovascular system. The early effects take place within minutes (e.g., changes in vasomotor tone) and are mediated through rapid intracellular signaling pathways; whereas the delayed effects (e.g., remodeling or lipid alterations) require hours to days to occur and require transcriptional effects with subsequent modulation of protein expression. To study the acute effects of 17␤-estradiol (E2) treatment on vascular function, we have investigated the rapid (on the order of minutes) effects of E2 treatment on intracellular signaling in human endothelial cells (EC). Our previous data have shown that E2 induces rapid release of NO from and activation of guanylate cyclase in human EC. In this study, we demonstrate that E2 also activates mitogen-activated protein kinase (extracellular signal-related kinase) signaling within minutes in EC. We hypothesized that this effect might be mediated by estrogen receptors (ER) localized to the cell surface. Our data show that membraneimpermeant forms of E2 also activate EC mitogen-activated protein kinase as well as stimulate cGMP production and NO release. The ER antagonist ICI 182,780 blocks this effect. Using confocal microscopy and flow cytometric analysis, we demonstrate that EC contain surface binding sites for E2, detectable by cell-impermeant ligand binding and equally with an anti-ER␣ antibody. Immunoreactive bands of 66 and 45 kDa are detectable with an anti-ER␣ mAb in human EC, and their individual presence correlates functionally with E2-stimulated genomic and rapid nongenomic responses, respectively. Membrane ERs may provide key molecular switches in these novel, rapid signaling pathways induced by E2 in EC.

17␤-Estradiol activates endothelial nitric oxide synthase (eNOS),The cardioprotective effects of estrogen are diverse, including both rapid non-genomic and delayed genomic effects on the blood vessel wall (reviewed in Ref. 1). Specific, rapid vascular effects, such as moderation of vasomotor tone, have been linked to an estrogen-stimulated increase in bioavailable nitric oxide (NO) 1 (2-4). 17␤-estradiol (E2) treatment of human endothelial cells (EC) induces rapid release of NO by estrogen receptor (ER)-dependent activation of endothelial nitric oxide synthase (eNOS) (5). Many factors regulate eNOS enzyme activity, including fatty acid modification, subcellular localization, and binding to numerous proteins and cofactors, including calmodulin, caveolin-1, the 90-kDa heat shock protein (HSP90), and tetrahydrobiopterin (see Ref. 6 for review). eNOS is a Ca 2ϩ / calmodulin-dependent enzyme, the activity of which is also regulated by phosphorylation. Specific phosphorylation of eNOS by the serine/threonine kinase Akt renders the enzyme more active at much lower Ca 2ϩ concentrations (7,8). We demonstrated previously that the ER-dependent activation of eNOS occurs at resting Ca 2ϩ concentrations and requires activation of the phosphatidylinositol-3-OH kinase (PI3-kinase)/ Akt pathway (9). The regulatory subunit of PI3-kinase, P85, acts to stabilize and inhibit the catalytic activity of PI3-kinase. Recently, ER was shown to specifically bind to P85 in vitro (10). The E2-induced association correlated with increases in PI3-kinase activity in EC. However, the specific mechanism for E2 activation of PI3-kinase is not known.Evidence is emerging that membrane forms of steroid hormone receptors exist and participate in signaling pathways (11)(12)(13)(14). The activity of E2 at the cell membrane has been shown in EC, neurons, and breast cancer cell lines. We previously determined that rapid E2 activation of eNOS and MAP kinase occurs through a membrane-associated ER (9, 12). The EC line EAhy.926 used in these experiments exhibits rapid E2-induced signaling but is unable to stimulate ER-dependent gene transactivation. Additionally, EAhy.926 cells do not express the traditional 66-kDa ER␣ or ER␤ but express a 46-kDa protein immunoreactive with C-terminal ER antibodies. Recently, a protein of similar size reactive with E2 and anti-ER antibodies was found to be associated with the plasma membrane in MCF-7 cells (13,14). Additionally, a 46-kDa putative ER, reactive with anti-ER antibodies, was found in wild-type and in the initial ER␣ knockout mice. This form of the receptor was thought to be responsible for E2 enhancement of basal NO production in the initial ER␣ knockout mice, because this E2 effect was lost in the complete ER␣ knockout mouse (15). In human ECs expressing both the 66-and the 46-kDa receptor, both rapid signaling to MAP kinase and gene transactivation of estrogen-responsive element-luciferase reporter was stimulated with E2 treatment (12). As previously indicated, the specific mechanism of membrane-associated ER ...

Mouse kidney proximal tubular epithelial (MK-PT) cells die by apoptosis over 7-10 days when deprived of all survival factors. We show here that withdrawal of all survival factors from MK-PT cells is associated with a progressive increase in the activity of extracellular signal-regulated kinase-1 and -2 (ERK1/2) and a progressive decrease in phosphorylated Akt, a kinase critical to cell survival. Pharmacological inhibition of MEK1/2, the immediate upstream kinase for ERK1/2, not only prevented the decrease in phosphorylated Akt, but also prolonged MK-PT cell survival. Inhibition of ERK1/2, by itself, in the absence of any other known survival factors, was as potent as epidermal growth factor in maintaining MK-PT cell viability. ERK1/2 co-immunoprecipitated with Akt in a multimolecular assembly of signaling molecules, containing at a minimum ERK1/2, Akt, Rsk, and 3-phosphoinositide dependent kinase 1 (PDK1). We hypothesize that the kinase Rsk, whose activation requires phosphorylation by both ERK1/2 and PDK1, acts as a bridge bringing ERK1/2 into proximity with PDK1-associated Akt. Although a number of interactions between the Raf-MEK-ERK and PI3K-Akt signaling pathways have been described, our results are the first to show modulation of Akt activity by signaling events originating with ERK1/2. Spontaneous activation of ERK1/2 occurs via MEK1/2 and appears to depend on oxidant stress, accompanying induction of the default pathway of apoptosis. Together, these data suggest that the spontaneous activation of ERK1/2, in the absence of known extracellular stimuli, represents a previously unrecognized major regulatory pathway determining the fate of cells destined to die by the default pathway of apoptosis.