Vascular Endothelial Cells in the Akt-dependent Activation of Endothelial Nitric-oxide Synthase-Estrogen

Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17β-estradiol (E2), but not 17α-E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17β-E2 stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the 17β-E2-induced activation of Akt. 17β-E2-induced Akt activation was also inhibited by ICI-182,780, but not by actinomycin D. Either treatment with wortmannin or exogenous expression of a dominant negative Akt in TRLECs decreased the 17β-E2-induced eNOS activation. Moreover, 17β-E2-induced Akt activation actually enhances the phosphorylation of eNOS. 17β-E2-induced Akt activation was dependent on both extracellular and intracellular Ca2+. We further examined the 17β-E2-induced Akt activity in Chinese hamster ovary (CHO) cells transiently transfected with cDNAs for estrogen receptor α (ERα) or estrogen receptor β (ERβ). 17β-E2 stimulated the activation of Akt in CHO cells expressing ERα but not in CHO cells expressing ERβ. Our findings suggest that 17β-E2 induced eNOS activation through an Akt-dependent mechanism, which is mediated by ERα via a nongenomic mechanism. The inhibitory effect of estrogen on the development of atherosclerosis has been suggested by abundant human epidemiological and animal experimental data (1-9). The incidence of atherosclerotic diseases is lower in premenopausal women than in men, steeply rises in postmenopausal women, and is reduced to premenopausal levels in postmenopausal women who receive estrogen therapy (10-12). Until recently, the atheroprotective effects of estrogen were attributed principally to the effects on serum lipid concentrations. However, estrogen-induced alterations in serum lipids account for only approximately one-third of the observed clinical benefits of estrogen (1214). Recent evidence suggests that the direct actions of estrogen on blood vessels contribute to the cardioprotective effects of estrogen (13, 15). There are many kinds of direct effects of estrogen on blood vessels, such as estrogen-induced increases of vasodilatation and inhibition of the response of blood vessels to injury and the development of atherosclerosis. However, the molecular mechanism underlying the estrogen-induced vasodilatation has not yet been determined. Several studies suggest that a key mediator of this vasodilator response could be the endothelium-derived relaxing factor nitric oxide (NO), and that brief treatment with estrogen increases basal NO release in endothelial cells without elevation of eNOS mRNA or protein (16). Estrogen activates endothelial nitric oxide synthase (eNOS) without altering expression of the eNOS gene in vascular endothelium (17-20). However, the details of the mechanism of the estrogen-induced eNOS activation are not yet well understood The serine/threonine kinase termed Akt or protein kinase B (PKB)1 is an important regulator of various cellular processes, including glucose metabolism and cell survival (21, 22). Activation of receptor tyrosine kinases and G-protein-coupled receptors, and stimulation of cells by mechanical force, can lead to the phosphorylation and activation of Akt (23-25). Akt was identified as a downstream component of survival signaling through phosphatidylinositol 3-kinase (PI3K) (26-30). Akt may be regulated by both phosphorylation and the direct binding of PI3K lipid products to the Akt pleckstrin homology domain. Akt can then phosphorylate substrates such as glycogen synthase kinase-3, 6-phosphofructo-2-kinase, and BAD. More recently, it was found that eNOS is also an Akt substrate and is activated by Akt-dependent phosphorylation to release NO in endothelial cells (31-34). The actions of estrogen can be mediated by the classical nuclear receptors, ERα and ERβ (35, 36) or through other putative membrane receptors. By definition, rapid effects of estrogen that involve nongenomic mechanisms are independent of transcriptional activation by the nuclear ERs. These rapid effects are believed to be mediated by receptors located in or close to the plasma membrane (37, 38). Estrogen-induced vasodilatation occurs 5–20 min after estrogen administration (39, 40) and is not dependent on changes in gene expression; this action of estrogen is sometimes referred to as “nongenomic.” Therefore, we sought to determine whether the estrogen-induced eNOS activation is mediated by Akt activation and which type of ER is involved in this effect using both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs) (41). ing to the method of Jaffeet al. (42), plated in gelatin-coated tissue culture wells, and grown in M199 medium containing 20% fetal bovine serum and 50 μg/ml endothelial cell growth supplement (Clonetics Corp., San Diego, CA). HUVECs were used at passage 2 or 3. Constructs The vectors encoding the various HA-tagged forms of Akt, wild-type Akt (HA-Akt), kinase-inactive Akt (HA-AktK179M), and constitutively active Akt (HA-mΔ4–129Akt) used in this study have been described previously (26, 43, 45-47). The vectors encoding the wild-type eNOS and mutant eNOS of serine 1179 to alanine (S1179A eNOS) was a kind gift from Dr. W. C. Sessa (Yale University, New Haven, CT) (31). The human estrogen receptor α (ERα) expression vector, pSG5-HEGO, was a kind gift from Dr. P. Chambon (Institut de Chimie Biologique, Strasbourg, France) (48). The plasmid pSG5-mERβ encoding nucleotides 12–1469 of ERβ (35) was kindly provided Dr. E. R. Levin (University of California, Irvine, CA) via Dr. K. S. Korach (National Institutes of Health, Research Triangle Park, NC). Assay of eNOS Activity Cells were serum-starved overnight in phenol red-free medium before eNOS activity measurements. eNOS activity was determined as the conversion of radiolabeledL-arginine to L-citrulline by a method described previously (50, 51) with a minor modification. Briefly, 10 μl of a sample was incubated for 10 min at 37 °C in a solution consisting of 50 mM HEPES, 1 mM dithiothreitol, 1 mM CaCl2, 0.1 mMtetrahydro-L-biopterin, 1 mM NADPH, 10 μg/ml calmodulin, 10 μM FAD, and 1.55 μM L-[guanidino-14C]arginine (pH 7.8), in a final volume of 100 μl. The reaction was terminated by the addition of 200 μl of buffer A (100 mM HEPES, 10 mM EDTA, pH 5.2). The whole reaction mixture was then applied to a 0.3-ml Dowex 50WX column (Na+ form, 200–400 mesh) that had been equilibrated with buffer A. Citrulline was eluted with 0.5 ml of buffer A, and then radioactivity was measured with a liquid scintillation counter. Assay of eNOS Activity Using a Transient Expression System TRLECs cultured in 100-mm dishes were transfected with 1 μg of CMV-6, 1 μg of CMV-6 containing the gene for HA-AktK179M, 1 μg of pSG5, 1 μg of ERα expression vector (pSG5-HEGO), or 1 μg of ERβ expression vector (pSG5-mERβ) using LipofectAMINE plus (Life Technologies, Inc.) as described previously (52, 53). Seventy-two hours after transfection, serum-deprived cells were incubated with 10−7 M 17β-E2 for 15 min, and the eNOS activity was measured as described above. Assay of Akt Kinase Activity Cells were serum-starved overnight in phenol red-free medium and then treated with various materials. They were then washed twice with phosphate-buffered saline and lysed in ice-cold lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, and 1 mMphenylmethylsulfonyl fluoride). The extracts were centrifuged to remove cellular debris, and the protein content of the supernatants was determined using the Bio-Rad protein assay reagent. 250 μg of protein from the lysate samples was incubated with gentle rocking at 4 °C overnight with immobilized anti-Akt antibody cross-linked to agarose hydrazide beads. After Akt was selectively immunoprecipitated from the cell lysates, the immunoprecipitated products were washed twice in lysis buffer and twice in kinase assay buffer (25 mM Tris, pH 7.5, 10 mM MgCl2, 5 mMβ-glycerolphosphate, 0.1 mM sodium orthovanadate, and 2 mM dithiothreitol), and the samples were resuspended in 40 μl of kinase assay buffer containing 200 μM ATP and 1 μg of GSK-3α fusion protein. The kinase reaction was allowed to proceed at 30 °C for 30 min and stopped by the addition of Laemmli SDS sample buffer (54). Reaction products were resolved by 15% SDS-PAGE followed by Western blotting (55) with an anti-phospho-GSK-3α/β antibody as described previously (47). Assay of Akt Activity Using a Transient Expression System CHO cells cultured in 100-mm dishes were transfected with 1 μg of pSG5, 1 μg of ERα expression vector (pSG5-HEGO), or 1 μg of ERβ expression vector (pSG5-mERβ) using LipofectAMINE plus as described previously (52, 53). Seventy-two hours after transfection, serum-deprived cells were incubated with 10−7 M 17β-E2 for 15 min, and the Akt activity was measured as described above. Preparation of Partially Purified eNOS Human eNOS was overexpressed in Sf-21 cells, which had been infected with baculovirus carrying human eNOS cDNA (56). Human eNOS was partially purified by chromatography on 2′,5′-ADP-Sepharose gel, and its specificity was determined as described previously (57). Assay of eNOS Phosphorylation TRLECs cultured in 100-mm dishes were treated with 10−7 M 17β-E2 for 15 min. Cell lysates were subjected to immunoprecipitation with anti-Akt antibody. For assay using a transient expression system, TRLECs cultured in 100-mm dishes were transfected with 1 μg of HA-Akt, 1 μg of HA-AktK179M, or 1 μg of HA-mΔ4–129