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(Circulation Research. 2000;87:677.)
© 2000 American Heart Association, Inc.

Cellular Biology

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

M. Page Haynes, Diviya Sinha, Kerry Strong Russell, Mark Collinge, David Fulton, Manuel Morales-Ruiz, William C. Sessa, Jeffrey R. Bender

From the Division of Cardiovascular Medicine and Molecular Cardiobiology, Boyer Center for Molecular Medicine (M.P.H., D.S., K.S.R., M.C., J.R.B.) and Department of Pharmacology (D.F., M.M.-R., W.C.S.), Yale University School of Medicine, New Haven, Conn.

Correspondence to Jeffrey R. Bender, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Ave, New Haven, CT 06536-0812. E-mail jeffrey.bender{at}yale.edu

	Abstract

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Abstract—17ß-Estradiol (E₂) 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 E₂-stimulated endothelial NO release can occur without an increase in cytosolic Ca²⁺. Here we demonstrate for the first time, to our knowledge, that E₂ rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase–Akt pathway. E₂ 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. E₂ stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E₂-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca²⁺ levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E₂-stimulated NO release. The membrane-impermeant E₂BSA 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 Ca²⁺ fluxes and describes an important pathway relevant to cardiovascular pathophysiology.

Key Words: estrogen • endothelial nitric oxide synthase • Akt • membrane receptor

	Introduction

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Endogenous 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 17ß-estradiol (E₂) 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 8 9 10} A variety of cofactors are required for enzymatic function, including Ca²⁺, calmodulin, and tetrahydrobiopterin.^{11 12} Recently, heat shock protein 90 (Hsp90) was demonstrated through its inducible association with eNOS to be a positive regulator of enzyme activity.¹³ We have previously shown that rapid activation by E₂ of eNOS in human EC occurs in the absence of cytosolic Ca²⁺ increases. E₂ and shear stress are two of the few agonists that can activate eNOS in this Ca²⁺ flux–independent manner. However, the precise mechanism by which estrogen stimulates NO release is not known. As has been shown for other eNOS agonists, we have recently demonstrated that estrogen rapidly promotes an eNOS-Hsp90 association.¹⁴ Inhibition experiments with the Hsp90-specific agent geldanamycin confirmed that Hsp90 is required for E₂-stimulated NO release. However, this has not elucidated the diminished requirement for Ca²⁺ in the setting of estrogen.

Recently, our laboratory¹⁵ and other studies^{16 17} demonstrated that the serine/threonine kinase Akt, a downstream effector of phosphatidylinositol-3-OH kinase (PI3-kinase), phosphorylates human eNOS on serine 1177 in response to varied stimuli, such as vascular endothelial growth factor (VEGF) and shear stress. This phosphorylation not only activates eNOS, but also seems to increase the efficiency of activation by Ca²⁺/calmodulin.¹⁸ Given the similarities between shear stress–mediated and estrogen-mediated activation of eNOS, we evaluated the ability of estrogen to activate Akt in human ECs. We now show that E₂ does indeed activate eNOS through a PI3-kinase–dependent pathway. Because membrane impermeant estrogens trigger the same events, we discuss the activation pathway, from a putative, novel membrane estrogen receptor to distal components of the cascade, and its relevance in cardiovascular pathophysiology.

	Materials and Methods

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Materials
17ß-estradiol, ß-estradiol-17-hemisuccinate:BSA (E₂BSA) (30 mol E₂:molBSA) and ionomycin were purchased from Sigma. E₂ stocks were prepared in ethanol with final ethanol concentrations 0.01%. LY294002 was purchased from Calbiochem. ICI 182,780 was purchased from Zeneca Pharmaceuticals. Stocks of LY294002 and ICI 182,780 were prepared in Me₂SO with final concentrations of Me₂SO 0.1%. Anti-eNOS (N30020) was from Transduction Laboratories, and antiphosphorylated Akt (pAKT), anti-Akt, and antiphosphorylated eNOS (peNOS) were from New England Biolabs. All other reagents were purchased from Sigma.

Cell Culture
The permanently established EA.hy926 endothelial cell line¹⁹ was provided by CJS Edgell (University of North Carolina). Cells were maintained in 10% FBS, which was DMEM supplemented with 5 mmol/L hypoxanthine, 0.8 mmol/L thymidine, and 20 µmol/L aminopterin. Human umbilical vein endothelial cells (HUVECs) were isolated and maintained, as previously described.⁶ Before E₂ stimulation, cells were cultured in E₂-free medium, which was phenol-free DMEM with gelding horse serum (<1.0 pg/mL estradiol). After the initial E₂ deprivation period, some cells were additionally serum-starved for 4 to 16 hours for detection of phosphorylated eNOS or phosphorylated Akt in DMEM containing 0.1% fatty acid–free BSA.

NO Release Assays
NO-specific chemiluminescence, using potassium iodide and acetic acid reflux, was used to determine the amount of NO₂^- released from cell monolayers, as previously described.¹⁵ Cells were switched to E₂-free medium for 48 hours, after when the medium changed to HBSS supplemented with CaCl₂ (1.2 mmol/L), MgSO₄ (0.6 mmol/L), and L-arginine (100 µmol/L) for 30 minutes before agonist stimulation. Cells were stimulated with E₂, E₂BSA, or ionomycin for 1 hour at 37°C, and supernatants were collected for NO₂^- analysis. To address the requirements for ER engagement and the PI3-kinase pathway, some cells were treated with ICI 182,780 or LY294002, respectively, for 1 hour before agonist stimulation. LY294002, when used in the 3- to 50-µmol/L concentration range, has been shown to be highly PI3-kinase specific.^{20 21}

Immunoprecipitation and Western Blotting
Cell monolayers were stimulated as described in the figure legends. Phosphorylated Akt was detected in cell lysates after 48-hour E₂ deprivation and overnight serum starvation. pAkt immunoblots were stripped and blotted for total Akt. Phosphorylated eNOS was detected after 48-hour E₂ deprivation and 4-hour serum starvation from cell lysates in solution containing (in mmol/L) Tris-HCl 20 (pH 7.4), EDTA 2.4, Triton X-100 1%, sodium deoxycholate 1%, SDS 0.1%, NaCl 100, NaF 10, Na₃V0₄ 1, NaPiPO₄ 1, and protease inhibitor cocktail (Roche). Lysates were incubated with anti-eNOS antibody overnight at 4°C immunoprecipitated with protein A:protein G. Immunoprecipitates were immunoblotted with anti-peNOS (1:100) and anti-eNOS (1:2500) antibodies. Immunoblots were probed with species-specific secondary antibodies coupled to horseradish peroxidase and visualized by enhanced chemiluminescence.

Adenoviral Infection of Cells
The recombinant adenoviruses expressing control ß-galactosidase (ß-gal) and an Akt kinase–inactive mutant (AA-Akt) were obtained from K. Walsh (St. Elizabeth’s Medical Center, Boston, Mass) and were previously described.^{15 22} Monolayers were incubated with recombinant adenovirus at a multiplicity of infection of 100. After infection, E₂-free medium was added for the cell recovery period followed by serum starvation in 0.1% fatty acid–free BSA in phenol-free DMEM. Preliminary experiments with the ß-gal encoding virus were used to established conditions optimal for >95% cell culture transduction.

	Results

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PI3-Kinase Inhibitor Blocks E₂-Dependent NO Release
Previous studies from our laboratory¹⁵ and other studies¹⁷ have demonstrated that VEGF and shear stress can activate eNOS through a PI3-kinase/Akt-dependent pathway. After agonist stimulation of PI3-kinase, cytosolic Akt translocates to the plasma membrane where it is activated by serine and threonine phosphorylation. This membrane-activated form of Akt has been shown to specifically phosphorylate human eNOS at serine 1177, resulting in enhanced eNOS activity and increased NO release.^{15 17} To investigate the ability of E₂ to activate the PI3-kinase pathway in EC, EA.hy926 cells were pretreated with 10 µmol/L LY294002 or vehicle for 1 hour before agonist stimulation. Cells were then stimulated with E₂, ionomycin, or vehicle for 30 minutes, and the medium was collected for NO₂^- analysis. As shown in Figure 1, E₂ treatment of EA.hy926 cells results in a >4-fold induction of NO release. This augmentation was completely abrogated by pretreatment with LY294002 (Figure 1), which had no effect on basal control levels of NO (data not shown). As demonstrated here and in previous endothelial studies,^{6 14} E₂-induced increased NO production in EA.hy926 is an ER-dependent process that is completely inhibitable by pretreatment with the pure ER antagonist ICI 182,780. This demonstrates that E₂ stimulation of EC can dramatically augment NO release in a PI3-kinase–dependent manner through an ER-mediated pathway.

View larger version (10K): [in this window] [in a new window]   Figure 1. Effect of a PI3-kinase inhibitor on estrogen-induced NO release. Confluent EA.hy.926 monolayers were incubated with LY294002 (10 µmol/L), ICI 182,780 (10 µmol/L), or vehicle control for 1 hour at 37°C. Monolayers were then stimulated with vehicle, E2 (10 ng/mL), or ionomycin (2 µmol/L), and the medium was collected and NO2- content was determined by chemiluminescence. Data are mean±SD. Results are representative of 3 separate experiments. *P<0.01, where E2 is compared with control, E2+ICI, and E2+LY.

E₂ Stimulates Downstream Targets of the PI3-Kinase Pathway
One of the downstream targets of the PI3-kinase pathway is the serine/threonine kinase Akt/PKB. Known Akt substrates include eNOS as well as several proteins involved in cell survival and glucose metabolism.^{23 24} Because E₂-stimulated NO release is largely PI3-kinase–dependent, we addressed whether E₂ treatment could also result in Akt activation. To test this, both EA.hy926 and HUVEC cell lysates were prepared from E₂-stimulated or vehicle control–stimulated cells. When using an antibody directed at the serine 473–phosphorylated, activated form of Akt, phosphorylation was detected within 5 minutes of E₂ treatment in both cell types and persisted for at least 30 minutes (Figure 2). Dose-response experiments demonstrated induced endothelial Akt activation with as little as 100 pg/mL E₂ (data not shown). These results and the PI3-kinase inhibition data demonstrate that E₂ can activate endothelial PI3-kinase, resulting in the presumed translocation of Akt to the plasma membrane, where it is activated by phosphorylation.

View larger version (29K): [in this window] [in a new window]   Figure 2. Effect of 17ß-estradiol on Akt phosphorylation. Confluent monolayers of either HUVECs (A) or EA.hy.926 cells (B) were incubated in the presence or absence of E2 (10 ng/mL) for the indicated time periods at 37°C. Lysates were subjected to SDS-PAGE, transferred to nitrocellulose, probed with antiphospho Akt antibody, and reprobed with anti-Akt antibody. Densitometric analysis of HUVEC pAkt demonstrated a total Akt-normalized 3.2-fold increase at 15 minutes, relative to control.

The kinetics of E₂-stimulated EC Akt phosphorylation correlate with the rapid, nongenomic activation of eNOS we have reported previously.^{6 14} eNOS has been classically described as a Ca²⁺/calmodulin-dependent enzyme.¹² However, we have demonstrated that the E₂-induced increase in EC-NO release occurs without a cytosolic Ca²⁺ increase and does not alter the amount of eNOS-associated calmodulin.¹⁴ Recently, our laboratories¹⁵ and other studies¹⁷ have demonstrated that the phosphorylation of human eNOS serine 1177 (serine 1179 in bovine eNOS) by Akt lowers the apparent Ca²⁺/calmodulin requirements of the enzyme. Thus, using an antibody specific for this Akt substrate, phosphorylated eNOS site, Western blot analyses were performed to determine whether E₂ stimulation does, in fact, result in EC eNOS phosphorylation. ECs were E₂-deprived and serum-starved before stimulation with E₂ or vehicle control. As shown in Figure 3A, specifically phosphorylated eNOS was easily detected within 5 minutes of E₂ exposure. Phosphorylated eNOS persists for at least 30 minutes and is phosphorylated to the same extent as endothelial cells treated with VEGF (Figure 3B), a known inducer of bovine eNOS phosphorylation.¹⁵ Thus, the kinetics of E₂-stimulated Akt phosphorylation, eNOS phosphorylation, and EC-NO release are all consistent.

View larger version (26K): [in this window] [in a new window]   Figure 3. Effect of 17ß-estradiol on eNOS phosphorylation. Confluent EA.hy.926 monolayers were stimulated with E2 (10 ng/mL) for the indicated time periods (A) or with E2 (10 ng/mL, 30 minutes) or VEGF (50 ng/mL, 5 minutes) (B), after when eNOS was immunoprecipitated. Immunoblots were probed with antiphospho-eNOS antibody and reprobed with anti-eNOS antibody. Results are representative of 3 separate experiments.

Dominant-Negative Form of Akt Blocks E₂-Stimulated NO Release
To directly establish that the E₂-triggered signal transduction pathway resulting in NO release proceeds through Akt, EA.hy926 cells were transduced with adenoviral vectors encoding an AA-Akt or control ß-gal. AA-Akt has been shown to block VEGF-stimulated NO release.¹⁵ Additionally, kinase-inactive Akt is unable to phosphorylate eNOS in response to shear stress and thus is unable to enhance NO release in that setting.¹⁷ Cells were infected with the adenovirus for 2 hours, incubated for 24 hours in E₂-deficient serum, and kept serum-free for an additional 24 hours. As has been described with other ECs, the transduction efficiency was very high, with positive ß-gal staining in >95% of ß-gal–transduced cells (data not shown). Cells infected with control (ß-gal) exhibited a 2.25-fold increase in E₂-stimulated NO release similar to the 2.5-fold induction seen in noninfected cells (Figure 4), whereas AA-Akt expression largely abrogated E₂-stimulated NO production. This demonstrates that functionally active Akt is required for E₂-stimulated NO release.

View larger version (11K): [in this window] [in a new window]   Figure 4. Effect of dominant-negative Akt on estrogen-induced NO release. EA.hy.926 monolayers were transduced with ß-gal, dominant-negative kinase-deficient AA-Akt or mock infected cells (control). Monolayers were stimulated with E2 (10 ng/mL), the medium collected, and NO2- content determined by chemiluminescence. Data are mean±SD. Results are representative of 3 separate experiments. *P<0.03, where E2 is compared with control; ß-gal+E2 with ß-gal; and ß-gal+E2 with AA-Akt+E2.

Cell-Impermeant E₂ Activates the PI3-Kinase/Akt Pathway
The existence of cell-surface steroid hormone receptors has been debated. Recently, membrane progesterone receptors have been detected and shown to be physiologically active.²⁵ In addition, overexpression of ER and ß in Chinese hamster ovary cells results in the detection of a few surface ERs that can respond to E₂.²⁶ E₂ conjugated to BSA is membrane-impermeant and has been used to detect surface binding sites for E₂.^{14 27 28} We have described an apparent EC-membrane ER capable of rapidly inducing NO release and activating mitogen-activated protein (MAP) kinase (ERK 1/2).²⁹ E₂BSA was thus used to determine whether the PI3-kinase/Akt pathway can be activated by engagement of a surface ER. Cell monolayers were stimulated with E₂BSA or vehicle in the presence of excess fatty acid–free BSA. Figure 5 demonstrates that E₂BSA does trigger Akt phosphorylation. Similar to that seen with cell-permeant E₂, E₂BSA-stimulated Akt phosphorylation occurs within 5 minutes (Figure 5) and persists for at least 30 minutes (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 5. Effect of membrane-impermeant E2 on Akt phosphorylation. Confluent EA.hy.926 monolayers were incubated in the presence or absence of E2BSA (equivalent to 10 ng/mL free E2 on a calculated molar basis) or serum (positive control) for 5 minutes at 37°C. Lysates were subjected to SDS-PAGE, transferred to nitrocellulose, probed with antiphospho Akt antibody, and reprobed with anti-Akt antibody. Results are representative of 3 separate experiments.

To again determine whether this Akt activation is required for NO release, ECs were AA-Akt–transduced or ß-gal–transduced with the respective adenovirus before stimulation with E₂BSA or vehicle control. Figure 6 demonstrates that cells overexpressing kinase-inactive Akt were unable to generate any E₂-stimulated NO release, whereas control ß-gal–transduced cells maintained an excellent response to E₂BSA. This indicates that E₂ can stimulate an increase in NO via a surface receptor that signals through the PI3-kinase/Akt pathway. As expected, E₂BSA triggered the critical, Akt-induced phosphorylation on eNOS serine 1177 within 5 minutes (Figure 7). Thus, all the activation events induced by free E₂ can be reproduced by engagement of a surface ER.

View larger version (9K): [in this window] [in a new window]   Figure 6. Effect of dominant-negative Akt on membrane-impermeant estrogen-induced NO release. EA.hy.926 monolayers were transduced with ß-gal–expressing adenovirus or dominant-negative, kinase-deficient Akt-expressing adenovirus. Monolayers were stimulated with E2BSA (equivalent to 10 ng/mL free E2 on a calculated molar basis), the medium collected, and NO2- content determined by chemiluminescence. Data are mean±SD. Results are representative of 2 separate experiments. *P<0.01, where ß-gal+E2BSA is compared with ß-gal or with AA-Akt+E2BSA.

View larger version (20K): [in this window] [in a new window]   Figure 7. Effect of membrane-impermeant estrogen stimulation on eNOS phosphorylation. Confluent EA.hy.926 monolayers were stimulated with E2BSA (equivalent to 10 ng/mL free E2 on a calculated molar basis) for the indicated time periods, after when eNOS was immunoprecipitated. Immunoblots were probed with anti–phospho-eNOS antibody and reprobed with anti–eNOS antibody. Results are representative of 3 separate experiments.

	Discussion

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The ability of estrogen to induce a rapid, endothelial- and NO-dependent vasodilatory response has been demonstrated in several systems,^{6 14 30} but the precise activating, molecular mechanism remains largely unknown. We have previously shown that E₂ can rapidly induce human umbilical vein EC-NO release without requiring increases in cytosolic Ca²⁺ (ie, occurring at resting EC Ca²⁺ levels).⁶ Here, we demonstrate that E₂ activates the PI3-kinase/Akt pathway in human EC. The relevance of this activation is confirmed by abrogation of E₂-stimulated NO release with pharmacological inhibition of PI3-kinase as well as recombinant expression of an activation-deficient, dominant-negative Akt. Additional evidence for the E₂-triggered activation includes rapid phosphorylation of the critical serine 473 Akt residue and consequent activating phosphorylation of serine 1177 on eNOS.

eNOS is a Ca²⁺ and calmodulin-dependent enzyme, and its regulated activation by agonists such as histamine and acetylcholine seems to require a stimulated rise in intracellular Ca²⁺.³¹ However, recent evidence supports that several stimuli, including fluid shear stress,³² insulin,³³ and estrogen,⁶ can activate eNOS and augment NO release in the absence of identifiable Ca²⁺ fluxes and in the setting of Ca²⁺ chelation. This apparent paradox has been difficult to reconcile and, in the context of estrogen, has been debated.³⁴ Recently, our laboratories¹⁵ and other studies^{16 17} have shown that Akt phosphorylates human eNOS on a critical serine 1177 (1179 in bovine eNOS). Altering the charge on this residue, either through Akt-induced phosphorylation or by mutagenesis-based substitution with aspartate, is associated with an increase in NO production and enzyme activation at much lower Ca²⁺ and calmodulin concentrations.^{15 16 17} That is, Akt-induced eNOS phosphorylation greatly increases the activity of eNOS at resting Ca²⁺ concentrations. This is based, in part, on a faster rate of electron flux through the eNOS reductase domain and a reduced calmodulin dissociation from eNOS when Ca²⁺ levels are low.¹⁸ Phosphorylation may confer a conformational alteration on the interaction of calmodulin with the 45 amino acid autoinhibitory flavin mononucleotide insert domain or in the carboxy tail, thereby effecting disinhibition. Our results clearly demonstrate that E₂ stimulates human endothelial Akt activation and eNOS phosphorylation. The findings that PI3-kinase–dependent Akt activation is not Ca²⁺-dependent and that this specific serine 1177 phosphorylation enhances eNOS Ca²⁺ sensitivity are consistent with our previous data that estrogen-stimulated endothelial NO release does not require a rise in free intracellular Ca²⁺ and provide the first mechanistic explanation of how this may occur.

The signaling cascade leading to PI3-kinase/Akt activation by E₂ is presently unknown. There are many examples of growth factors stimulating hormone-independent estrogen receptor–mediated events through phosphorylation of the critical ER transactivation function domain-1.^{35 36} However, details of the converse have not been defined. That is, the PI3-kinase/Akt activation by estrogen demonstrated here, and inhibited by conventional ER antagonists, depicts rapid growth factor receptor–type responses induced by a steroid hormone. In the mouse uterus, estrogen has been shown to trigger the tyrosine phosphorylation of insulin-like growth factor receptor-1 (IGF-1R), as well as tyrosine phosphorylation of the docking substrate IRS-1 and consequent IGF-1R/IRS-1/PI3-kinase p85 complex formation.^{37 38} This induced recruitment of the regulatory unit of PI3-kinase is sufficient to initiate enzyme activation. Much of the estrogen-induced p85-IGF-1R association was absent in IGF-1–deficient mice, suggesting that a majority of this pathway activation is a consequence of E₂-stimulated IGF-1 synthesis. However, residual induced p85-IGF-1R complexes remained in the absence of IGF-1. Estrogen-induced recruitment of p85 to tyrosine kinase growth factor receptors could be responsible for the signaling events described in this study.

Other potential cascades should be considered on the basis of several analogies between fluid shear stress–stimulated and estrogen-stimulated eNOS activation, including the induction of relative Ca²⁺ independence. Shear stress activates Akt in a PI3-kinase–dependent fashion, and consequent NO production is inhibited by the PI3-kinase inhibitor wortmannin.¹⁷ Shear stress induces a clustering of the VEGF receptor Flk-1 and its rapid tyrosine phosphorylation and association with the adapter protein Shc, which can result in Ras activation and subsequent MAP kinase induction through the assembly of Shc/Grb2/SOS complexes.³⁹ Our laboratories¹⁴ and other studies⁴⁰ have demonstrated a rapid MAP kinase activation induced by E₂ in ECs. In addition to its role in the MAP kinase pathway, GTP-bound Ras can also activate PI3-kinase.⁴¹ This is another potential signaling pathway by which E₂ could trigger EC-NO release.

We recently demonstrated that Hsp90 is rapidly recruited to eNOS on endothelial treatment with E₂.¹⁴ Geldanamycin, which binds to the ATP-binding site of Hsp90 and inhibits its function, abrogates E₂-stimulated EC-NO release. The relationship between Hsp90 recruitment and Akt activation, both triggered by E₂ and required for stimulated eNOS activation, remains unclear. Herbimycin A, in the same pharmacological class as geldanamycin, does not block shear stress–induced Akt activation. Considering the analogy to estrogen responses, this suggests either that functional Hsp90-eNOS association is a critical downstream mediator of eNOS activation or that the pathways by which Akt and Hsp90 regulate eNOS activity, as induced by estrogen, are both required but independent. We are presently investigating the interplay between estrogen-induced Hsp90-eNOS association and modulation of eNOS by Akt.

Finally, one must consider the most proximal signal transduction event in response to E₂: that of ER engagement. The events described occur rapidly, are inhibited by conventional ER antagonists, and are consistent with rapid transmembrane signaling. Our results demonstrate for the first time that PI3-kinase and Akt activation can be triggered with the cell impermeant E₂BSA conjugate. We have used this conjugate extensively, including after charcoal extraction of any free E₂, and determined E₂BSA binding sites on human EC membranes that are competitively blocked by conventional ER antagonists and free E₂.²⁹ As opposed to uterine and breast cells, in which estrogen has been shown to activate PI3-kinase,^{38 42} EA.hy926 ECs, used in these experiments, do not express the traditional 66 kDa ER (or ERß) nor are they able to undergo the normal E₂-stimulated, ER-dependent gene transactivation (Reference 29²⁹ and unpublished data, July 2000). However, they do express a 45-kDa protein, immunoreactive with an anti–C-terminal ER antibody, which seems to correlate with intact rapid signaling responses.²⁹ Experiments to identify this molecule are presently underway.

In summary, we have found that the mechanism by which estrogen stimulates endothelial NO production involves a rapid, PI3-kinase–dependent activation of Akt and consequent serine phosphorylation of eNOS. These activation events occur after engagement of membrane receptors for estrogen. This explains the emerging paradigm relating rapid, estrogen-stimulated endothelial signaling, enhanced eNOS sensitivity to cytosolic Ca²⁺, and unconventional estrogen receptors. Additional definition of these receptors and the proximal signaling events leading to Akt activation will provide opportunities for specialized therapeutic interventions in cardiovascular disease.

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL61782 (to J.R.B) and HL61371 and HL64793 (to W.C.S.). We thank Lynn O’Donnell for technical assistance, Dana Brenckle for manuscript preparation, and all those who provided valuable reagents, including Dr Ken Walsh for generously providing the adenoviral constructs.

Received August 7, 2000; revision received September 1, 2000; accepted September 6, 2000.

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