Endocrine Reviews 23 (5): 665-686
Copyright © 2002 by The Endocrine Society
Estrogen Modulation of Endothelial Nitric Oxide Synthase
Ken L. Chambliss and
Philip W. Shaul
Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390
Correspondence: Address all correspondence and requests for reprints to: Philip W. Shaul, M.D., Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas Texas 75390. E-mail: pshaul{at}mednet.swmed.edu
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Abstract
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Over the past decade, clinical and basic research has demonstrated that estrogen has a dramatic impact on the response to vascular injury and the development of atherosclerosis. Further work has indicated that this is at least partially mediated by an enhancement in nitric oxide (NO) production by the endothelial isoform of NO synthase (eNOS) due to increases in both eNOS expression and level of activation. The effects on eNOS abundance are primarily mediated at the level of gene transcription, and they are dependent on estrogen receptors (ERs), which classically serve as transcription factors, but they are independent of estrogen response element action. Estrogen also has potent nongenomic effects on eNOS activity mediated by a subpopulation of ER
localized to caveolae in endothelial cells, where they are coupled to eNOS in a functional signaling module. These observations, which emphasize dependence on cell surface-associated receptors, provide evidence for the existence of a steroid receptor fast-action complex, or SRFC, in caveolae. Estrogen binding to ER on the SRFC in caveolae leads to Gi activation, which mediates downstream events. The downstream signaling includes activation of tyrosine kinase-MAPK and Akt/protein kinase B signaling, stimulation of heat shock protein 90 binding to eNOS, and perturbation of the local calcium environment, leading to eNOS phosphorylation and calmodulin-mediated eNOS stimulation. These unique genomic and nongenomic processes are critical to the vasoprotective and atheroprotective characteristics of estrogen. In addition, they serve as excellent paradigms for further elucidation of novel mechanisms of steroid hormone action.
I. Introduction
II. Estrogen, Nitric Oxide, and Vascular Function and Disease in Humans
A. Estrogen and human vascular disease
B. Sources of estrogens in humans
C. Long-term effects of estrogen on eNOS function
D. Short-term effects of estrogen on eNOS function
E. Role of estrogen receptors
III. Estrogen, NO, and Vascular Function and Disease in Animal Models
A. Estrogen, NO, and animal models of vascular disease
B. Role of ER in animal models of vascular disease
C. Long-term effects of estrogen on eNOS in animal models
D. Short-term effects of estrogen on eNOS in animal models
IV. Genomic Mechanisms of eNOS Regulation by Estrogen
A. Genomic regulation of eNOS expression by estrogen
B. Basis for eNOS up-regulation by estrogen
C. Other genomic mechanisms modifying eNOS function
V. Nongenomic Regulation of eNOS by Estrogen
A. Estrogen and eNOS activation
B. Localization of eNOS activation by estrogen to plasma membrane
C. Localization of ER-eNOS interaction to caveolae
D. Role of ERß in nongenomic eNOS activation
E. Coupling of plasma membrane ER to downstream signaling events
VI. Summary and Future Directions
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I. Introduction
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ESTROGEN IS AN important atheroprotective molecule with marked effects on the vasculature that are mediated, at least in part, by increased availability of the signaling molecule nitric oxide (NO) (1, 2). The endothelial isoform of NO synthase (eNOS) is the principal source of NO in the vascular wall (3). This article will review our current knowledge of estrogen modulation of eNOS expression and activity in vascular endothelium. The role of estrogen and NO in vascular function and disease in humans will first be summarized. Important insights obtained in animal models will also be addressed. Recent investigations in cell culture revealing that the actions of estrogen on eNOS are both genomic and nongenomic in nature will be discussed. In addition, the roles of the two known estrogen receptor (ER) isoforms, ER and ERß, will be described. Finally, the cellular and molecular mechanisms underlying the genomic and nongenomic regulation of eNOS by estrogen will be considered in depth. Further knowledge about the complex impact of estrogen on endothelial cell biology is needed to understand the normal actions of the hormone in the cardiovascular system, and to guide future considerations of hormone therapy to prevent or treat cardiovascular disease (4).
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II. Estrogen, Nitric Oxide, and Vascular Function and Disease in Humans
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A. Estrogen and human vascular disease
The dramatic impact of estrogen on cardiovascular health is apparent from population-based studies, which indicate that premenopausal women have very little coronary artery disease compared with men, that the incidence of the disease rises markedly after menopause, and that hormone replacement therapy (HRT) under certain conditions reduces the risk to premenopausal levels (5, 6, 7, 8). In addition, women who undergo surgical menopause without estrogen replacement have two times the risk of coronary artery disease than other premenopausal women (9). Whereas the mechanisms underlying these findings are not completely understood, considerable emphasis has been placed previously on the capacity of the hormone to modify circulating lipid levels (10). However, only approximately one third of the clinical advantages of estrogen can be attributed to effects on lipid balance (6, 11, 12, 13). Instead, recent evidence indicates that estrogen acts directly on the blood vessel wall to provide a major atheroprotective effect (14).
B. Sources of estrogens in humans
The estrogen compounds to which the vascular endothelium may be exposed in women are multiple, and they arise from both endogenous and exogenous sources. Before menopause, the major endogenously derived circulating estrogen compound is 17ß-estradiol (E2) produced by the ovary. Other endogenous estrogens and estrogen metabolites have also been described. After menses, when circulating E2 levels increase and begin to cycle, levels range from less than 0.36 nM during the follicular phase to 2.8 nM during midcycle. During pregnancy, estrogen levels rise to 70 nM due to placental production, which increases near term and further with the onset of parturition. After menopause, E2 concentrations fall to levels that are equivalent to those in males (0.04–0.21 nM). During menopause, the primary form of endogenous circulating estrogen is estrone derived peripherally from androstenedione by conversion in adipose tissue (15). Endothelial cells in both women and men may also be exposed to estrogens derived from the local conversion of testosterone or 
4-testosterone to E2 by aromatase (16). Aromatase, E2-hydroxysteroid dehydrogenase, and 17-ketoreductase enzyme activities were first demonstrated in rat arterial vascular smooth muscle (VSM) cells in cell culture (17), and more recently aromatase expression was noted in human VSM by in situ hybridization (18). Only trace levels were detected in samples from infants, and greater aromatase mRNA and activity were found in adult samples. In addition, studies in cultured human VSM demonstrated up-regulation of aromatase mRNA in response to cAMP, phorbol ester, or dexamethasone (18). Thus, there is evidence that aromatase expression in VSM is developmentally regulated and also modulated by multiple signal transduction pathways and by glucocorticoids. However, the relative impacts of circulating vs. locally derived estrogens on endothelial cell function have not yet been clearly elucidated.
In addition to endogenously derived estrogens, there are often important exogenous sources in humans. Contraception is frequently accomplished using a combination of ethinyl E2 and levonorgostrel or norethindrone. HRT is also often provided to postmenopausal women. This is frequently in the form of conjugated equine estrogens, and other oral and transdermal forms are also often considered. HRT frequently entails conjugated equine estrogens in combination with medroxyprogesterone acetate. In addition, selective estrogen receptor modulators, or SERMs, such as raloxifene are used for the treatment of osteoporosis, and it is likely that vascular-specific SERMS will also soon be available. Furthermore, phytoestrogens are a class of compounds found in certain plant-derived beverages and foods, and they have both estrogenic and antiestrogenic effects (14, 19, 20). As such, the endothelium may be exposed to a variety of forms and levels of estrogen compounds depending on the sex and age of the individual, his/her diet, and therapies that he/she may be receiving.
C. Long-term effects of estrogen on eNOS function
The onset of menopause provides a natural model of estrogen deprivation in which the effects of endogenous levels of the hormone on vascular function can be revealed. In studies of changes in brachial artery diameter after reactive hyperemia, which provides an assessment of flow-mediated, endothelium-dependent, and NO-dependent vasodilation, responses were greater in premenpausal vs. postmenopausal women (21). Similarly, determinations of forearm blood flow constrictor responses to NOS antagonism with L-NG-monomethyl arginine (L-NMMA), which also evaluate vascular NO activity, revealed greater constriction in premenopausal vs. postmenopausal women. Importantly, blood flow responses to the NO donor glyceryl trinitrate (GTN) were similar in the two study groups, indicating comparable VSM responses to NO. As might be predicted, the responses in postmenopausal women were comparable to those observed in men (Fig. 1
and Ref. 22).
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Figure 1. Menopause is associated with a decline in endothelium-dependent, NO-dependent vasodilation. Constrictor responses to brachial artery infusion of L-NMMA (A) and vasodilator responses to brachial artery infusion of GTN (B) were assessed by measurements of forearm blood flow (FBF) in 15 premenopausal women (diamonds), 12 postmenopausal women (squares), and 14 men (triangles). L-NMMA responses were increased in premenopausal women vs. postmenopausal women and men, P < 0.05. In contrast, vasodilator responses to GTN were similar between groups. Values are mean ± SEM. [Reprinted with permission from: N. G. Majmudar et al.: J Clin Endocrinol Metab 85:1577–1583, 2000 (22 ). © The Endocrine Society.]
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The menstrual cycle certainly provides another natural model of varying estrogen status. In studies of healthy young women, the capacity for endothelium-dependent vasodilation in the brachial artery paralleled serum E2 levels, and, additionally, there was evidence of progesterone antagonism of this effect (23, 24). More recently, endothelium-dependent responses to bradykinin in resistance vessels have also been observed to be enhanced at midcycle during the period of greatest serum E2 levels, whereas responses to GTN are unchanged (Fig. 2 and Ref. 25). In agreement with these findings, sex hormone deprivation after ovariectomy for uterine leiomyoma is associated with a decline in endothelium-dependent vasodilation, whereas the response to the NO donor sodium nitroprusside is unaltered (26). Although the exact sources of NO are undetermined, the enhanced endothelium-dependent vasodilation observed during periods of greater estrogen abundance during the menstrual cycle correlates with higher serum levels of the NO metabolites nitrate and nitrite and greater exhaled NO (23, 27, 28). Thus, although many other parameters may vary in parallel with the changes in estrogen status that accompany menopause or surgical ovariectomy or the menstrual cycle, there is a strong correlation between endogenous estrogen levels and the capacity for endothelial NO production in women.
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Figure 2. The capacity for endothelium-dependent vasodilation varies during the menstrual cycle. Forearm blood flow response to bradykinin (A) and to GTN (B) at midcycle (MC) and during the early menstrual phase (EMP) were evaluated in healthy women. The response to bradykinin was greater during MC vs. EMP, whereas the response to GTN was similar, P < 0.05. Data are mean ± SEM, n = 15 subjects, and statistical analysis was performed on area under the curve values. [Reprinted with permission from N. N. Chan et al.: J Clin Endocrinol Metab 86:2499–2504, 2001 (25 ). © The Endocrine Society.]
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The effects of HRT also provide insights into NOS regulation by estrogen. In landmark studies reported in 1994 it was demonstrated that endothelium-dependent vasodilation of the brachial and coronary arteries is enhanced after estrogen replacement therapy in postmenopausal women (29, 30). Estrogen replacement therapy also causes elevations in plasma NO and NO metabolites (31, 32). In addition, there is evidence that forearm blood flow constrictor responses to NOS antagonism are greater after estrogen replacement therapy, whereas responses to glyceryl trinitrate are unchanged, indicating augmented basal NO availability (22). Furthermore, flow-mediated, endothelium-dependent vasodilation of the brachial artery is enhanced similarly by HRT with estrogen and by raloxifene therapy, whereas endothelium-independent responses are not altered (Fig. 3). In addition, the interventions yield comparable increases in plasma NO levels (33). Interestingly, the inclusion of progestin in postmenopausal HRT appears to blunt the effects of estrogen on endothelial NO production (21), perhaps paralleling the findings noted above for variations in endothelial function during the menstrual cycle (24). Premenopausal women receiving estrogen replacement after ovariectomy also have enhanced endothelial function after therapy, and similar effects have been documented in young women receiving oral contraceptives (34). Long-term estrogen administration also improves vascular function in males, and this is at least partially mediated through endothelium-derived NO (35, 36). As such, prolonged exposure to exogenously derived estrogen has positive effects on endothelium-derived NO availability, which parallel those noted with altered endogenous levels and provides further evidence of potential long-term modulation of eNOS function by the hormone in humans.
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Figure 3. Flow-mediated vasodilation is increased after chronic HRT or raloxifene treatment in humans. Flow-mediated endothelium-dependent vasodilation (A) and nitroglycerin-mediated endothelium-independent vasodilation (B) were examined in postmenopausal women treated for 6 months with placebo, raloxifene, or HRT. #, P < 0.05 vs. placebo; *, P < 0.05 vs. before treatment. Values are mean ± SD, n = 30 per group. [Reprinted with permission from: A. Saitta et al.: Arterioscler Thromb Vasc Biol 21:1512–1519, 2001 (33 ).]
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D. Short-term effects of estrogen on eNOS function
There is also evidence of short-term effects of estrogen on eNOS function in humans. In key early studies reported in 1994, iv ethinyl E2 was found to cause a direct decrease in coronary vasomotor tone within 15 min in postmenopausal women (Fig. 4). In addition, ethinyl E2 caused attenuation of vasoconstrictor responses to acetylcholine (Ach) when given 15 min before Ach (37). Experiments with intracoronary E2 administered in a manner yielding premenopausal levels did not demonstrate changes in basal coronary vasomotor tone, but there was greater vasodilatory response to Ach when E2 and Ach were given simultaneously. In these studies, E2 had no effect on vasodilation with sodium nitroprusside, suggesting that the hormone rapidly modifies the availability of endothelium-derived NO (38). Findings were similar when E2 effects on forearm vascular responses were assessed in postmenopausal women, with the exception that endothelium-independent vasodilation was also enhanced (39). In further studies in the coronary circulation of older women, the capacity of E2 to cause augmented Ach-induced responses was prevented by NOS antagonism, providing additional evidence that the underlying mechanism involves enhanced bioavailability of NO (40). In contrast to the observations made in postmenopausal women, intracoronary E2 administration to similarly aged men caused neither a change in basal vasomotor tone nor greater vasodilation with Ach tested 20 min later (41). However, the provision of iv conjugated estrogens 15 min earlier caused greater Ach-induced coronary blood flow and less cold-induced coronary vasoconstriction in men (42, 43). In a similar manner, phytoestrogens cause direct, rapid, NO-dependent dilation of the forearm vasculature of both men and women, which is comparable in degree to that observed with E2 (Fig. 5). Lower concentrations of phytoestrogen also potentiate the vasodilatory response to Ach. It is important to note that the threshold concentration of phytoestrogen yielding these effects does not greatly exceed those found in East Asian subjects in whom dietary phytoestrogen intake has been suggested to contribute to an extremely low incidence of atherosclerosis and coronary artery disease (44). These cumulative observations indicate that, in addition to the long-term effects of estrogen and estrogen-like compounds on eNOS action, there are short-term effects on the bioavailability of NO. Importantly, these effects may generally be comparable in men and women, and the estrogen formulation is a critical variable.
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Figure 4. Ethinyl E2 causes rapid coronary vasodilation in humans. Changes in coronary blood flow (CBF), coronary vascular resistance (CVR), and epicardial coronary cross-sectional area (CSA) were determined within 15 min of iv ethinyl E2 (E2) administration in postmenopausal women. Values are mean ± SEM, n = 11 and 22 subjects for placebo control (C) and ethinyl E2, respectively, *, P < 0.05 vs. control. [Reprinted with permission from: S. E. Reis et al.: Circulation 89:52–60, 1994 (37 ).]
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Figure 5. Both estrogen and the phytoestrogen genistein cause rapid, NO-dependent vasodilation in humans. A, Increase above baseline in forearm blood flow (forearm blood flow) after brachial artery infusion of genistein/daidzein vehicle (open circles, n = 6), daidzein (open diamonds, n = 6), and genistein in men (closed squares, n = 9) and premenopausal women (open squares, n = 6). *, P < 0.0001 compared with vehicle by ANOVA for all doses. B, Increase above baseline in forearm blood flow (forearm blood flow) during coinfusion of genistein with saline (closed circles) and then after coinfusion with L-NMMA (8 µmol/min, open circles) on the same occasion (n = 6). *, P < 0.002 for comparison of genistein plus saline vs. genistein plus L-NMMA by ANOVA for all doses. C, Vasodilator effects of genistein and 17ß-estradiol (AUC) during coinfusion of saline (open bar) and L-NMMA (closed bar, 8 µmol/min, n = 5). *, P < 0.01 for L-NMMA vs. saline. [Reprinted with permission from: H.A. Walker et al.: Circulation 103:258–262, 2001 (44 ).]
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E. Role of estrogen receptors
Little experimental data are available in humans to reveal the role of ER in vascular function and health in the intact state. However, a young adult male with a disruptive mutation in the ER gene was found to have premature coronary artery disease at age 31 yr (45, 46). Flow-mediated vasodilation of the patient’s brachial artery was also attenuated, whereas vasodilation with sublingual nitroglycerin was intact, suggesting a diminished capacity for endothelial NO production. Interestingly, rapid vasodilation in response to sublingual E2 was normal (46). These observations may be explained by recent studies demonstrating that either ER or ERß can mediate rapid eNOS activation in cell culture. These findings are presented below in Section V.D.
More direct studies of ER involvement in endothelial NO production have been performed in isolated human mammary artery segments. In precontracted rings obtained from men at the time of coronary artery bypass surgery, E2 at concentrations of 100 nM or greater caused relaxation that was prevented by either endothelium removal or NOS antagonism. The response to E2 was also attenuated 71% by the ER antagonist tamoxifen. Although issues relating to the concentration of E2 required for this in vitro response may be raised, these findings suggest that the short-term effects of E2 on endothelial NO bioavailability in humans are mediated by endothelial ER (47).
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III. Estrogen, NO, and Vascular Function and Disease in Animal Models
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A. Estrogen, NO, and animal models of vascular disease
The impact of estrogen on the response to vascular injury has been vigorously investigated in rodent models. In studies of carotid artery injury in rats, E2 attenuated the resulting intimal and medial hypertrophy in females but not in males, and the addition of medroxyprogesterone acetate blocked the protective effects of estrogen (48, 49, 50). Estrogen also enhances endothelial recovery after vascular injury, as indicated by greater reendothelialization and greater endothelium-dependent relaxation in previously injured arterial segments. In these studies the augmented recovery was associated with increased capacity for NO production, but it was not determined whether the effect of estrogen is mediated by an up-regulation of eNOS abundance or activity (51, 52). It has also been noted that estrogen reduces neointimal formation in mice deficient in the inducible isoform of NOS to an extent that is similar to that seen in wild-type mice. Since the inducible form of NOS and eNOS are the principal forms of the enzyme found in the vasculature, these observations suggest that eNOS is the primary isoform involved in estrogen-mediated protection (53). An additional explanation is that estrogen may primarily modify vascular injury by promoting angiogenesis (54).
Animal models have also been employed to demonstrate that estrogen has an important impact on the function of atherosclerotic arteries. In pioneering studies by Williams and colleagues (55, 56), both long-term (26 months) and brief (20 min) E2 treatment caused enhanced Ach-mediated vasodilation in atherosclerotic coronary arteries of female cynomologus monkeys. Similarly, long-term E2 administration in hypercholesterolemic swine led to the preservation of endothelium-dependent relaxation (57). Perhaps more importantly, there is considerable animal evidence that estrogen modifies the development of atherosclerosis. This includes studies in cholesterol-fed rabbits in which both ethinyl E2 and conjugated equine estrogen reduced atherosclerosis by 35% in the aortic arch and by 75–80% in the thoracic and abdominal aorta (58). Similarly, E2 administration to apolipoprotein E (apoE)-deficient mice decreased the atherosclerotic lesion area in both males and ovariectomized females. Importantly, neither the less biologically active 17-estradiol nor tamoxifen affected lesion development in ovariectomized females in the apoE deficiency paradigm, and the beneficial effects of 17ß-estradiol were only partially explained by changes in plasma lipoprotein levels (59). The latter variable has been elegantly controlled in studies of cholesterol-clamped rabbits in which the antiatherogenic properties of estrogen remain apparent. In addition, in the cholesterol-clamped rabbits the capacity of estrogen to prevent cholesterol accumulation in the vascular wall was abolished by balloon catheter injury, suggesting that an intact endothelium is required (Fig. 6) (60, 61). Furthermore, in the same model NOS inhibition reduced the atheroprotection afforded by estrogen, and comparable observations were made with the SERM levormeloxifene (Fig. 6). Moreover, the antiatherogenic effect of the hormone was preceded by an increase in NO production that was followed by diminished mononuclear-endothelial cell binding. It was noted additionally that the effect of E2 was to increase the basal release of NO, whereas NO release by Ach and NOS abundance was unaltered (62, 63). These cumulative observations suggest that the antiatherosclerotic characteristics of estrogen involve direct effects on the vascular endothelium, and that these effects may be primarily mediated by enhanced NO production unrelated to genomic effects of the hormone on eNOS expression.
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Figure 6. Estrogen attenuates aortic cholesterol accumulation in cholesterol-clamped rabbits. Studies were performed with or without NOS antagonism with nitro-L-arginine methyl ester (L-NAME). Estrogen had an antiatherogenic effect in the undamaged aorta (P < 0.05 vs. placebo, no L-NAME by ANOVA). In contrast, this effect was abolished by balloon catheter injury (bottom panel). Similar findings, although not as pronounced, were obtained for levormeloxifene (P < 0.05 vs. placebo, no L-NAME by ANOVA). The atheroprotective effects of estrogen and levormeloxifene were attenuated by simultaneous NOS antagonism (P < 0.05 for yes L-NAME vs. no L-NAME). Values are mean ± SEM, n = 13–18 per group. Solid bars, Without L-NAME; hatched bars, with L-NAME. [Reprinted with permission from: P. Holm et al.: J Clin Invest 100:821–828, 1997 (62 ).]
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B. Role of ER in animal models of vascular disease
The role of ER in the effects of estrogen on the response to vascular injury has been addressed using nonselective ER antagonism or genetically engineered mice. In models of carotid artery injury in wild-type mice, physiological levels of E2 inhibited the increases in vascular medial area and smooth muscle cell proliferation that occur with injury (48, 64). Comparable findings were also obtained in ER-deficient and ERß-deficient mice (64, 65). Such observations suggested that either receptor isoform may be sufficient to mediate the effects of estrogen or that the effects are through a non-receptor-mediated process or through another unidentified ER. The first of these possibilities was addressed in recent studies of ER,ß double knockout mice. In the double knockouts, E2 did not prevent the injury-induced increase in vascular medial area. However, VSM cell proliferation continued to be attenuated by the hormone (66). In contrast, in experiments in rats employing nonselective pharmacological ER antagonism, it has been observed that the vasoprotective effects of E2 are completely inhibited (67). The role of ER in the antiatherogenic features of estrogen has also been addressed in a recent report. In mice deficient in apoE alone, E2 reduced the size of atherosclerotic lesions and also their histological complexity, as previously observed (59). Plasma cholesterol levels were decreased by E2, but the degree of the effects of the hormone on atherogenesis could not be explained solely on the basis of changes in the plasma lipid profile. In contrast, in mice with disrupted apoE and disrupted ER, E2 had a more modest effect on lesion size and total plasma cholesterol was unaltered. However, the complexity of the plaques was diminished by E2 despite the absence of ER (68). As such, certain mechanisms whereby estrogen alters the vascular response to injury and the progression of atherosclerosis are evidently mediated by ER or ERß. However, the bases for multiple other processes are yet to be determined. Furthermore, in contrast to the evidence that strongly implicates an important role for eNOS in the hormonal modulation of atherogenesis, studies are yet to be performed to clarify the specific contribution of eNOS to E2-induced changes in vascular injury responses.
C. Long-term effects of estrogen on eNOS in animal models
There is considerable evidence from investigations in intact animal models that long-term estrogen exposure enhances the capacity for endothelial NO production. For example, E2 treatment of rabbits for 4 d caused an increase in endothelium-dependent relaxation in femoral artery rings (69). Using NOS antagonism, it has also been shown that bioassayable endothelium-derived NO is higher in thoracic aortas isolated from female vs. male rats (70). The same group of investigators employed ER knockout mice to demonstrate that there is a significant association between the abundance of ER and the basal release of NO (71). In 1994 it was reported that steady-state eNOS mRNA levels are increased in guinea pig skeletal muscle during pregnancy and after E2 treatment. This may have been related to the induction of eNOS in endothelium, but it is notable that the cell specificity of eNOS up-regulation was not determined, and eNOS is expressed in skeletal myocytes (72, 73). The long-term effects of estrogen on eNOS expression have been demonstrated in studies of the uterine circulation in sheep. It was reported in 1996 that after 3 d of systemic E2 treatment, there was enhanced NO-dependent, endothelium-dependent relaxation, which was related to greater NOS enzymatic activity (74). It has since been shown that eNOS abundance is up-regulated in uterine artery endothelium after both prolonged (days) and brief (2 h) E2 treatment (75, 76). Although E2-related effects on eNOS do not occur in certain systemic arteries in the sheep (75), studies in rats have demonstrated dramatic eNOS up-regulation in cerebral microvessels after chronic E2 treatment. Interestingly, the eNOS responses to E2 in the cerebral microvessels were similar in female and male rats (77). In addition, studies with knockout mice have demonstrated that the E2-induced up-regulation in eNOS expression in the cerebral microvessels is mediated by ER, and that parallel changes occur in the abundance of cyclooxygenase type 1, which is the rate-limiting enzyme in prostacyclin production (Fig. 7) (78). Furthermore, work in porcine coronary arteries has shown that the effects of E2 are direct and not mediated by changes in blood flow, since isolated ring segments displayed greater NO-dependent relaxation after 18–22 h exposure to E2 in vitro (79).
The capacities to up-regulate eNOS and to reverse endothelial dysfunction are not limited to endogenous forms of estrogen. In studies in sheep, the SERM raloxifene caused more than a 10-fold increase in uterine blood flow over 6 h and a 22% rise in coronary blood flow over 24 h, and these effects were at least partially NO dependent (80). Raloxifene also prevented the decrease in NOS expression which occurs with ovariectomy in rats (81). Furthermore, in rats it has been shown that the long-term administration of the phytoestogen -zearalenol causes enhanced endothelial NO production, and the effect is fully prevented by the ER antagonist ICI 182,780, indicating that it is mediated by ER binding (82). Chronic exposure to phytoestrogen also improves endothelial dysfunction induced by ovariectomy in rats (83).
D. Short-term effects of estrogen on eNOS in animal models
Experiments in isolated segments of rabbit coronary artery, rat superior mesenteric artery, and rat aorta from female animals have demonstrated that physiological levels of E2 cause rapid, NO-dependent, endothelium-dependent relaxation or rapid, NO-dependent inhibition of platelet aggregation (84, 85, 86). However, a considerable number of additional works have alternatively demonstrated NO-independent, endothelium-independent E2-mediated relaxation (87, 88, 89). The varying results related to endothelial NO production may be due to differences in the species or vascular beds under study, as well as varying usage of precontraction. In addition, the estrogen status of the animal is critically important. Collins and colleagues (84) demonstrated that coronary artery rings from oophorectomized, estrogen-treated, and acutely estrogen-withdrawn rabbits display robust endothelium-dependent, NO-dependent relaxation to E2, whereas rings from oophorectomized, untreated or oophorectomized, estrogen-maintained rabbits display more modest responses (Fig. 8). Interestingly, the SERM raloxifene caused rapid NO-dependent, endothelium-dependent relaxation of coronary artery rings from both male and female rabbits studied under conditions that yielded only NO-independent responses to E2 (88). In addition, 30-min in vitro exposure to phytoestrogen restored NO-mediated relaxation in pulmonary arteries isolated from chronically hypoxic rats (90). Thus, paralleling the findings related to endothelial NO-mediated responses in humans, studies in animals indicate that estrogen causes both long-term up-regulation of eNOS expression and rapid enhancement of eNOS enzyme activation.
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Figure 8. Estrogen causes rapid, NO-dependent, endothelium-dependent vasodilation in rabbit coronary artery rings after estrogen withdrawal. The effects of E2 (10-9 to 5 x 10-5 M) on contractile responses to prostaglandin F2 (3 x 10-5 M) were examined in coronary arterial rings from oophorectomized, untreated (sham-operated) female rabbits (n = 5; open circles); oophorectomized, estrogen-maintained female rabbits (n = 5; open triangles); and female rabbits oophorectomized, estrogen replaced, and then deprived for 48 h (n = 6; closed circles). Values are mean ± SEM; *, P < 0.01 vs. untreated and estrogen-maintained groups (ANOVA). [Reprinted with permission from P. Collins et al: Circulation 90:1964–1968, 1994 (84 ).]
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IV. Genomic Mechanisms of eNOS Regulation by Estrogen
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A. Genomic regulation of eNOS expression by estrogen
The mechanisms by which long-term E2 exposure increases eNOS abundance have been delineated in experiments in cultured endothelial cells. Studies reported in 1995 demonstrated that the capacity for NO production by human aortic endothelium is greater after 8 h or more of E2 treatment at concentrations of approximately 10 nM or greater. eNOS protein levels were also increased (91). Additional work in human umbilical vein endothelial cells (HUVEC) demonstrated that both tamoxifen and ICI 182,780 prevent the increase in eNOS. Immunocytochemical analysis revealed ER expression in early-passage HUVEC and diminished levels of expression with additional passage (92). Further studies in ovine endothelial cells demonstrated that E2 induces increases in NOS enzymatic activity and eNOS protein abundance at concentrations of 0.1 nM E2 or greater, and that these changes are accompanied by increases in eNOS mRNA levels. Transient transfection assays with a specific estrogen-responsive reporter system also demonstrated that endothelial ERs are capable of estrogen-induced transcriptional transactivation (93). Similar observations have also been made in human osteoblast-like cells, consistent with other data suggesting that NO may mediate estrogen action in bone (94). eNOS expression in neonatal and adult cardiac myocytes is also up-regulated by E2 in an ER-dependent manner, potentially explaining some of the cardioprotective effects of the hormone (95). In addition, estrogen treatment caused an increase in eNOS mRNA abundance in LNCaP human prostate carcinoma cells within 2–4 h of exposure. Interestingly, estrone sulfate, the most abundant circulating estrogen that may serve as a prehormone for the terminal biologically active estrogen E2 in men, also up-regulated eNOS mRNA in LNCap cells, and the response required 48 h, suggesting conversion to E2 (96).
B. Basis for eNOS up-regulation by estrogen
Work in human endothelial EA.hy 926 cells indicates that E2 or the more stable 17-ethinyl E2 enhances eNOS protein and mRNA abundance in the absence of changes in the stability of eNOS mRNA. Nuclear run-on assays showed that the increase in eNOS mRNA abundance is related to greater eNOS gene transcription. The impact of 17-ethinyl E2 on eNOS gene transcription was further evaluated in transient transfection studies using a 1.6-kb human eNOS promoter fragment, which lacks classical estrogen response elements (EREs). Promoter activity was enhanced to a degree comparable to the increase in eNOS mRNA levels, and EMSAs suggested greater DNA-protein complex formation involving a putative Sp1 binding element after estrogen treatment. However, immunodepletion or supershift analyses were not performed to specifically identify the nuclear protein that is involved (97). The role of ER subtypes in eNOS gene activation by E2 has been elucidated in neonatal rat cardiac myocytes. eNOS up-regulation by E2 was fully prevented by the ERß-specific antagonist RR-tetrahydrochrysene (THC), indicating a primary role for that subtype (Fig. 9) (98). Further work in cultured endothelial cells indicated that eNOS down-regulation by TNF, which is due to enhanced eNOS mRNA degradation, is prevented by E2, and that this mechanism is also ER dependent (99). Thus, there is transcriptional regulation of eNOS expression by E2 through mechanisms that do not involve classical ERE-mediated processes, and additional nontranscriptional mechanisms may be important under certain conditions.
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Figure 9. ERß mediates E2-stimulated eNOS expression in neonatal rat cardiac myocytes. Neonatal rat cardiac myocytes were cultured in serum-free defined medium in the absence or presence of E2 (10-9 M). A representative immunoblot is shown in the upper panel, and cumulative results for three independent experiments are displayed in the lower panel. The level of expression of eNOS protein is low in the absence of E2 (control, lane 1), and it increases markedly in cells exposed to E2 for 24 h (lane 2). Coincubation with the ERß-selective antagonist RR-THC (10-5 M) completely inhibited E2-induced expression of eNOS (lane 3). Cotreatment with ICI 182,780 (10-8 M) for 24 h also inhibited the estrogen-mediated increase in eNOS expression (lane 4). RR-THC (lane 5) and ICI 182,780 (lane 6) alone had no effect on protein expression. Values are mean ± SEM. [Reprinted with permission from S. Nuedling et al.: FEBS Lett 502:103–108, 2001 (98 ).]
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C. Other genomic mechanisms modifying eNOS function
The eNOS enzyme is localized to endothelial cell caveolae, which are lipid-ordered domains on the plasma membrane that compartmentalize signal transduction molecules. Within caveolae, there are multiple mechanisms that modify eNOS activity, including protein-protein interaction between the enzyme and the structural and regulatory protein caveolin, which leads to attenuated eNOS activity (100). Studies in rat cerebral arterioles have demonstrated that chronic estrogen depletion (ovariectomy) causes a complete loss of Ach-induced, endothelium-dependent relaxation, and this defect is associated with not only a decrease in eNOS expression, but also an up-regulation in caveolin abundance. In addition, these changes were mimicked by simultaneous interventions that decreased eNOS and increased caveolin abundance in animals with intact ovaries, and estrogen replacement normalized the Ach responses in the ovariectomized females at the same time that it up-regulated eNOS and down-regulated caveolin (101, 102). In recent work in bovine aortic endothelial cells, eNOS expression was increased after 24 h of E2 exposure, as seen in the intact animal; however, caveolin expression rose after 48 h of treatment. In addition, the increase in caveolin expression was prevented by ER antagonism. After E2, NO production was elevated at 24 h but not at 48 h, which would be consistent with greater caveolin-related attenuation in eNOS activity at the later time point (103). These cumulative findings suggest that the effects of E2 on caveolin expression may have an important impact on eNOS function, but they also emphasize that factors such as the identity of the endothelial cell target, the cellular and tissue context of the experiment, and the timing of hormone exposure must be considered.
In addition to the relative level of eNOS enzymatic activity, the ultimate bioavailability of NO as a signaling molecule is modified by the degree of scavenging by reactive oxygen species such as superoxide. In human endothelial cells, superoxide levels are dictated by the production of the radical by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and its degradation by superoxide dismutase (SOD). In recent studies in HUVEC, E2 caused a 60% decrease in the expression of the protein and mRNA for the NADPH oxidase subunit gp91phox. There was also a less dramatic, but significant, E2-induced decline in mRNA abundance for p22phox, which, together with gp91phox, constitute the membrane-bound cytochrome b558 portion of the NADPH oxidase complex. The two cytosolic activating factors of the complex, p47phox and p67phox, were also down-regulated by E2, and the net effect was an approximately 50% diminution in the capacity to produce superoxide. In addition, the effects of E2 on NADPH oxidase subunit expression and superoxide production were prevented by ER antagonism. Heat shock protein 90 (HSP90) and eNOS expression were also up-regulated by E2. In contrast, levels of expression of the reactive oxygen species-metabolizing enzymes glutathione-peroxidase, copper/zinc SOD, manganese SOD, and catalase were not affected by E2 (Fig. 10 and Ref. 104). Thus, in addition to up-regulating eNOS abundance, E2 inhibits the expression of NADPH oxidase in endothelial cells, thereby potentially increasing the bioavailability of NO through more than one process.
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Figure 10. Estrogen inhibits NADPH oxidase expression in endothelial cells. Time- and concentration-dependent effect of cyclodextrin-encapsulated E2 on NADPH oxidase subunit gp91phox, HSP-90, eNOS, glutathione peroxidase (GSH-Px), SOD, and catalase mRNA expression were evaluated in cultured HUVEC. A, Typical RT-PCR analyses with the relative intensities (%), as judged by densitometry, are indicated at the top. The cells were incubated with E2 (1 and 100 nmol/liter) for the indicated periods. Elongation factor-1 (EF-1) RT-PCR was performed as a control. Results are representative of two separate experiments with different batches of cells. B, Summary data are shown for HUVEC incubated for 8 h with E2 (1 and 100 nmol/liter), calculated as percentage of expression in the presence of cyclodextrin alone (basal). Values are mean ± SEM, n = 4–8, *, P < 0.05 vs. basal. [Reprinted with permission from A. J. Wagner et al.: FASEB J 15:2121–2130, 2001 (104 ).]
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V. Nongenomic Regulation of eNOS by Estrogen
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A. Estrogen and eNOS activation
The bases for rapid effects of E2 on eNOS function have been elucidated in studies of cultured endothelial cells. It was shown initially that estrogen acutely (5–10 min) stimulates eNOS activity, that the response is attenuated by ER antagonism but not by inhibition of gene transcription, and that ER is expressed in cultured endothelial cells (Fig. 11 and Refs. 105, 106, 107). It has also been shown that the overexpression of ER in endothelial cells causes enhancement of the acute response to E2 that is blocked by ER antagonism and specific to E2 vs. other agonists (Fig. 12). In addition, the enhanced response is dependent on the ER hormone binding domain. In COS-7 cells, which do not constitutively express ER or eNOS, the acute stimulation of eNOS by E2 can be demonstrated after the cotransfection of ER and eNOS cDNAs (107). Raloxifene also activates endothelial cell eNOS within 10 min, and the process is ER dependent since it is abolished by ICI 182,780 (108). eNOS activation by E2 has also been observed in human bronchiolar epithelial cells, which also constitutively express the enzyme and ER, suggesting that mechanisms similar to those delineated in the vasculature may play a role in hormonal modulation of airway function (109).
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Figure 11. Estrogen causes rapid activation of eNOS in endothelial cells. A, Effect of E2 on eNOS activity in intact cells. 3H-L-Arginine conversion to 3H-L-citrulline was measured over 5–15 min in the presence of 10-8 M E2. B, Effect of actinomycin D (Act D) on the rapid activation of eNOS. After 120 min preincubation in the absence or presence of 25 µg/ml Act D, 15-min incubations were done with or without continued Act D and either 10-8 M E2 or the calcium ionophore A23187 (10-5 M). C, Effect of tamoxifen (Tam) on E2-stimulated eNOS activity. Fifteen-minute incubations were performed in the absence or presence of 10-8 M E2, with or without 10-6 Tam added simultaneously. Partial inhibition (50–70%) was also noted with 10-8 M Tam (13 ). D, Effect of ICI 182,780 on E2-stimulated eNOS activity. Fifteen-minute incubations were performed in the absence or presence of 10-8 M E2, with or without 10-5 M ICI 182,780 added simultaneously. Full inhibition was also observed with 10-6 M ICI 182,780 (13 ). Values are mean ± SEM; n = 4–6, *, P < 0.05 vs. basal. [Reprinted with permission from Z. Chen et al.: J Clin Invest 103:401–406, 1999 (107 ).]
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Figure 12. Effect of ER overexpression on transcriptional transactivation and on basal and estrogen-stimulated eNOS activity. A, Effect of ER overexpression on ERE-mediated gene transcription in endothelial cells. Transient transfections were performed with either the estrogen-responsive reporter plasmid ERE-luciferase (Luc) or the control plasmid thymidine kinase luciferase (TK-Luc), in combination with either sham plasmid or ER cDNA. Reporter activity was then determined in control cells (upper panel) and cells exposed to 10-8 M E2 for 48 h (lower panel). Reporter activity is expressed as luciferase activity/ß galactosidase activity (LUC/ß-gal). *, P < 0.05 vs. TK-Luc;  , P < 0.05 vs. control cells;  , P < 0.05 vs. sham. Similar findings were obtained in three independent experiments. B, Effect of ER overexpression on basal eNOS activity in endothelial cells. Cells were transfected with sham plasmid or ER cDNA and placed under estrogen-free conditions, and 72 h later 3H-L-arginine conversion to 3H-L-citrulline was measured over 15 min in nonstimulated, intact cells. C, Effect of ER overexpression on acute eNOS activation by E2. Endothelial cells were transiently transfected with sham plasmid or ER cDNA as in panel B, and 72 h later 3H-L-arginine conversion to 3H-L-citrulline was measured in intact cells over 15 min in the absence or presence of 10-8 M E2, with or without 10-5 M ICI 182,780 added simultaneously. Values are mean ± SEM; n = 4–6. *, P < 0.05 vs. basal; , P < 0.05 vs. sham. [Reprinted with permission from Z. Chen et al.: J Clin Invest 103:401–406, 1999 (107 ).]
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The signal transduction mechanisms by which E2 activates eNOS have also been delineated. In experiments performed in ovine endothelial cells, tyrosine kinase inhibition completely prevented the response to E2. In addition, the specific MEK inhibitor PD98059 also fully negated eNOS stimulation by E2. Furthermore, E2 caused a rapid increase in MAPK activity, and this effect was prevented by both tamoxifen and ICI 182,780 (Fig. 13). These data indicate that the acute stimulation of eNOS by E2 and ER entails the activation of tyrosine kinase/MAPK (107). There is also evidence of a role for the recruitment of the PI3-kinase-protein kinase B/Akt pathway. In studies of human endothelial cells, E2-induced eNOS activation was prevented by the PI3-kinase inhibitor, LY294002, and E2 caused rapid Akt phosphorylation on serine 473. E2 also caused eNOS phosphorylation on serine 1177, which is a critical residue for eNOS activation and modification of the sensitivity to cellular calcium levels (Fig. 14). In addition, expression of a kinase-deficient, dominant-negative Akt prevented E2-stimulated NO production (110, 111, 112). Additional work suggests that E2-induced PI3-kinase/Akt signaling is mediated exclusively by ER and not ERß, and that it entails direct interaction between ER and the p85 subunit of PI3-kinase (111, 112). Recent examinations of the signal transduction events by which raloxifene stimulates eNOS have also implicated involvement of MAPK and PI3-kinase/Akt signaling that is mediated by ER (113).
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Figure 13. Role of tyrosine kinase-MAPK signaling pathway in eNOS activation by estrogen. A, Role of tyrosine kinase in acute eNOS activation by E2. 3H-L-Arginine conversion to 3H-L-citrulline was measured over 15 min in intact endothelial cells in the absence or presence of 10-8 M E2, with or without treatment with the tyrosine kinase inhibitor (TKI) genistein (50 µM). Identical findings were obtained with herbimycin A. B, Role of MEK in acute eNOS activation by E2. eNOS activity was measured in the absence or presence of 10-8 M E2, with or without treatment with the MEK inhibitor (MEKI) PD98059 (50 µM). Values in panels A and B are mean ± SEM, n = 4–6; *, P < 0.05 vs. basal. C, Effect of E2 on MAPK activity in endothelial cells. Cells were treated for 5 min with 10-8 M E2 in the absence or presence of 10-5 M tamoxifen (Tam) or 10-5 M ICI 182,780, or with serum to serve as a positive control. Endogenous kinase was immunoprecipitated with anti-ERK2 antibody, and protein kinase activity was measured by evaluating the capacity to phosphorylate myelin basic protein. Quantification by phosphoimager yielded values of 1, 2.6, 1, 1, 1, and 7.5, respectively, relative to untreated cells. Results shown are representative of five independent experiments. [Reprinted with permission from Z. Chen et al.: J Clin Invest 103:401–406, 1999 (107 ).]
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Figure 14. PI3-kinase/Akt signaling plays a role in eNOS activation by estrogen. A, Effect of a PI3-kinase inhibitor on estrogen-induced NO release. Confluent EA.hy.926 monolayers were incubated with LY294002 (10 µM), ICI 182,780 (10 µM), or vehicle control for 1 h at 37 C. Monolayers were then stimulated with vehicle, E2 (10 ng/ml), or ionomycin (2 µM), and the medium was collected and NO2 content was determined by chemiluminescence. Data are mean ± SD. Results are representative of three separate experiments. *, P < 0.01 for E2 vs. control, E2+ICI, or E2+LY. B, Effect of E2 on Akt phosphorylation. Confluent monolayers of either HUVECs (upper panel) or EA.hy.926 cells (lower panel) 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 3.2-fold increase at 15 min, relative to control. C, Effect of E2 on eNOS phosphorylation. Confluent EA.hy.926 monolayers were stimulated with E2 (10 ng/ml) for the indicated time periods (upper panel) or with E2 (10 ng/ml, 30 min) or VEGF (50 ng/ml, 5 min; lower panel), and eNOS was immunoprecipitated. Immunoblots were probed with antiphospho-eNOS antibody and reprobed with anti-eNOS antibody. Results are representative of three separate experiments. VEGF, Vascular endothelial growth factor; pAKT, phosphorylated AKT; peNOS, phosphorylated eNOS. [Reprinted with permission from M. P. Haynes et al.: Circ Res 87:677–682, 2000 (110 ).]
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The role of calcium in E2-induced activation of eNOS has also been ascertained. In the two initial reports of eNOS stimulation in cultured endothelial cells, it was unclear whether calcium is involved. It was noted in studies in ovine endothelial cells that the removal of extracellular calcium completely prevented the response (105), yet changes in cytosolic calcium levels were not detected in parallel with eNOS activation by E2 in HUVEC (106). Later work in bovine aortic endothelial cells and human arterial endothelial cells indicated that there is a transient rise in intracellular calcium concentration upon E2 exposure (114, 115). Both E2-induced Akt activation and eNOS translocation from the plasma membrane upon E2 stimulation have been found to be calcium dependent (112, 114). These cumulative findings suggest that E2 activation of eNOS is a calcium-dependent process, but global increases in intracellular calcium levels may not be required.
Additional mechanisms of eNOS regulation in addition to those involving protein kinase-mediated phosphorylation events and changes in calcium homeostasis have also been investigated. In studies of HUVEC, inhibitors of HSP90 function prevented E2-stimulated NO release and cGMP production. E2 also was found to induce HSP90-eNOS association, and this event was prevented by ER antagonism, providing further evidence for a role for HSP90 in eNOS activation by E2 (116). Thus, the short-term effects of estrogen on eNOS that are central to cardiovascular physiology are mediated by ER functioning in a novel, nongenomic manner, and multiple signal transduction events are likely to be involved.
B. Localization of eNOS activation by estrogen to plasma membrane
Studies employing immunoidentification or conjugated estrogen have indicated that a subpopulation of ER may be associated with the cell surface in certain cell types (117, 118). In fact, immunofluorescence experiments suggest that the ligand binding domain of the ER may actually reside on the extracellular face of the plasma membrane (119). As mentioned above, there is strong evidence that eNOS is targeted to the endothelial plasma membrane, particularly to caveolae, which are specialized, cholesterol-rich lipid-ordered domains that compartmentalize signal transduction. eNOS trafficking to caveolae is dependent upon the myristoylation and palmitoylation of the protein (100, 120, 121, 122). Initial determinations of the subcellular site of interaction between ER and eNOS involved studies of plasma membranes isolated from ovine endothelial cells, in which eNOS stimulation was evaluated by measuring 3H-L-arginine conversion to 3H-L-citrulline (123). In the absence of added calcium, calmodulin, or cofactors, 10-8M E2 (15 min) caused a 92% increase in NOS activity compared with basal levels, whereas 17-estradiol had no effect. Maximal NOS activity was assessed by replacing E2 with a mixture of calcium, calmodulin, and cofactors, yielding a 170% rise in activity compared with no additives; E2 (10-8M) did not enhance this activity (Fig. 15A). These observations indicated that all of the signal transduction machinery necessary for eNOS stimulation by E2 is associated with the plasma membrane. Since E2 alone activated the enzyme to approximately half of maximal levels, the response is quite robust. Time course experiments with E2 alone further revealed a progressive, linear increase in NOS activity during the first 30 min of incubation, followed by a plateau (Fig. 15B). This contrasted with NOS activity with added calcium, calmodulin, and cofactors, which displayed linearity with time for at least 120 min (123). These data suggest that the availability of one or more of these molecules is limited in the isolated membranes; in contrast, in intact cells there are most likely mechanisms that replenish these factors in the locale of the plasma membrane.
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Figure 15. Estrogen activates eNOS in isolated endothelial cell plasma membranes. A, Effect of E2 on NOS activity. 3H-L-Arginine conversion to 3H-L-citrulline was measured in isolated plasma membranes in the absence (basal) or presence of 10-8 M E2, and in the absence or presence of exogenous eNOS cofactors, Ca2+ and calmodulin (CM). B, Time course of the effect of E2 on NOS activity. Incubations were performed without added cofactors, Ca2+ and CM, in the absence (basal) or presence of 10-8 M E2 for 15–60 min. C, Effect of ER antagonism on response to E2. NOS activation was measured without added cofactors, Ca2+ or CM, in the absence (basal) or presence of 10-8 M E2, with or without 10-5 M ICI 182,780 added. D, Effect of ER antibody (ERAb) on response to E2. Incubations were performed without added cofactors, Ca2+ or CM, in the absence (basal) or presence of 10-8 M E2 with or without antibody to ER (TE111) or unrelated IgG added. Values are mean ± SEM, n = 4–6, *, P < 0.05 vs. basal; , P < 0.05 vs. E2 alone. [Reprinted with permission from K. L. Chambliss et al.: Circ Res 87:E44–E52, 2000 (123 ).]
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The role of ER in E2 activation of plasma membrane eNOS was examined using the ER antagonist ICI 182,780, and the agent was found to completely prevent E2-stimulated NOS activity (Fig. 15C
). In addition, antibody to the ligand binding domain of ER (TE111) blocked E2-stimulated NOS activation, whereas unrelated IgG had no effect (Fig. 15D). These observations indicated that the response to E2 is mediated by an ER or ER-like protein associated with the endothelial cell plasma membrane. Immunoidentification experiments, which compared the plasma membrane ER with cytosolic and nuclear ER using antibodies directed against three different ER epitopes, were then performed. Antibodies directed against amino acids 495–595 (AER320), 302–553 (TE111), or 120–170 of human ER (AER304) all detected a single 67-kDa protein species in endothelial cell plasma membranes that was identical in size to the protein detected in nuclear and cytosolic fractions (Fig. 16A). Confirmatory studies were done involving the localization of epitope-tagged ER (ER-myc) transiently transfected into COS-7 cells. Whereas antibody to ER revealed no signal in sham-transfected cells, there was positive signal for ER of comparable size in nucleus, cytosol, and plasma membrane from cells transfected with ER-myc. In parallel, immunoblot analysis with antibody to the myc tag revealed no signal in sham-transfected cells, but a protein of similar size was detected in the nucleus, cytosol, and plasma membranes of cells expressing the tagged receptor (Fig. 16B). These cumulative findings indicate that E2-stimulated eNOS activity is mediated by a subpopulation of ER that is associated with the endothelial plasma membrane (123). Independent investigations demonstrating eNOS activation by membrane-impermeant E2 conjugated to BSA have provided additional evidence of eNOS signaling by cell surface ER (110, 115).
The importance of plasma membrane colocalization for ER-stimulated eNOS activity was evaluated in a reconstitution paradigm in COS-7 cells. Plasma membranes from cells expressing eNOS and ER displayed rapid ER-mediated NOS stimulation, whereas membranes from cells expressing eNOS alone or ER plus myristoylation-deficient mutant eNOS were insensitive. In fact, membranes from cells expressing myristoylation-deficient mutant eNOS and ER displayed a decline in NOS activity with E2 that was partially reversed by ICI 182,780. A myristoylation-deficient mutant eNOS is minimally directed to the plasma membrane but unaltered in enzymatic activity (120), these findings indicate that both normal plasma membrane targeting of eNOS and localization of ER to that site are required for eNOS activation by E2 (123).
C. Localization of ER-eNOS interaction to caveolae
Since plasma membrane eNOS is exclusively localized to caveolae (120), further experiments were done to determine whether ER protein is also associated with this subfraction of endothelial cell plasma membranes. ER protein was detected in caveolae, and it was also detected, but to a lesser extent, in the noncaveolae fraction of the plasma membrane (Fig. 17A). Experiments were then performed to evaluate the capacity of E2 to activate eNOS in isolated caveolae and noncaveolae fractions. In the absence of added calcium, calmodulin, or cofactors, there was no measurable NOS activity in the noncaveolae fraction under basal conditions or with E2 added. Basal NOS activity was also below detection limits in caveolae membranes. However, 10-8 M E2 caused robust activation of NOS in caveolae membranes, and this effect was prevented by ICI 182,780 (Fig. 17B). These data strongly indicate that ER and all of the additional molecular machinery necessary for E2-mediated activation of eNOS exist in a functional signaling module in endothelial caveolae. Since ER was found in both caveolae and noncaveolae fractions and eNOS is solely in caveolae (120), the specificity of ER coupling to eNOS to caveolae is evidently due to the localization of the effector, and not the receptor, in the microdomain. Furthermore, the effect of E2 on eNOS in caveolae is prevented by calcium chelation (123), suggesting that ER activation in caveolae modifies the local calcium environment.
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Figure 17. Localization of ER-eNOS interaction to caveolae. A, Immunoblot analysis for ER and caveolin-1 in noncaveolae membranes (NCM) and caveolae membranes (CAV) obtained from endothelial cell whole-plasma membranes. Results are representative of three independent experiments. B, E2-mediated activation of eNOS in endothelial cell caveolae membranes. 3H-L-Arginine conversion to 3H-L-citrulline was measured in noncaveolae and caveolae membranes obtained from endothelial cell plasma membranes. Membrane incubations were performed without added eNOS cofactors, Ca2+ or calmodulin, in the absence (basal, B) or presence of 10-8 M E2 with or without 10-5 M ICI 182,780 added. NOS | |
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Abstract
I. Introduction
