ERβ Has Nongenomic Action in Caveolae (original) (raw)

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1Departments of Pediatrics (K.L.C., I.S.Y., P.W.S.), Dallas, Texas 75390

*Address all correspondence and requests for reprints to: Ken L. Chambliss, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9063.

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1Departments of Pediatrics (K.L.C., I.S.Y., P.W.S.), Dallas, Texas 75390

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2Cell Biology (R.G.W.A.), University of Texas Southwestern Medical Center, Dallas, Texas 75390

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3Molecular Cardiology Research Institute (M.E.M.), New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111

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1Departments of Pediatrics (K.L.C., I.S.Y., P.W.S.), Dallas, Texas 75390

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Received:

05 October 2001

Accepted:

09 January 2002

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Ken L. Chambliss, Ivan S. Yuhanna, Richard G. W. Anderson, Michael E. Mendelsohn, Philip W. Shaul, ERβ Has Nongenomic Action in Caveolae, Molecular Endocrinology, Volume 16, Issue 5, 1 May 2002, Pages 938–946, https://doi.org/10.1210/mend.16.5.0827
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Abstract

ERα and ERβ serve classically as transcription factors, and ERα also mediates nongenomic responses to E2 such as the activation of endothelial nitric oxide synthase (eNOS). In contrast, the nongenomic capacities of endogenous ERβ are poorly understood. We evaluated eNOS activation by E2 in cultured endothelial cells that express endogenous ERβ to determine whether the ERβ isoform has nongenomic action and to reveal the subcellular locale of that function. A subpopulation of ERβ was localized to the endothelial cell plasma membrane, overexpression of ERβ enhanced rapid eNOS stimulation by E2, and the response to endogenous ER activation was inhibited by the ERβ-selective antagonist RR-tetrahydrochrysene (THC). eNOS activation through ERβ was reconstituted and shown to occur independent of ERα in COS-7 cells, and ERβ protein in COS-7 was directed to the plasma membrane. THC also blunted E2 activation of eNOS in isolated endothelial cell plasma membranes. Furthermore, ERβ protein was detected and THC attenuated E2 stimulation of eNOS in isolated endothelial cell caveolae, and functional ERβ-eNOS coupling was recapitulated in caveolae from transfected COS-7 cells. These findings in the ER-eNOS signaling paradigm indicate that endogenous ERβ has nongenomic action in caveolae.

ALTHOUGH MEMBERS OF the steroid hormone receptor (SHR) superfamily serve classically as transcription factors (1–3), there is mounting evidence that SHR also act in a nongenomic manner to regulate signal transduction events (4–13). The capacity for SHR nongenomic action has been elucidated to a considerable degree in studies of nongenomic estrogen receptor function in a number of cell types, including osteoblasts, neurons, and vascular endothelium (4, 5, 7, 8, 10, 13, 14). We and others have demonstrated that 17β-E2 causes nongenomic activation of endothelial nitric oxide synthase (eNOS) (4, 5, 7, 8, 13), thus explaining an important mode of atheroprotection mediated by the hormone (15). We have also shown that overexpression of ERα in cultured endothelial cells causes an increase in eNOS activation by estrogen, and that a subpopulation of ERα colocalizes with eNOS in caveolae where they are coupled to the enzyme in a functional signaling module (4, 5). Caveolae are specialized lipid ordered domains, which contain numerous signaling molecules including protein tyrosine kinases, MAPKs ERK1 and ERK2, G protein-coupled receptors, and ras and src family members (16).

Similar to ERα, the more recently discovered ERβ subtype is known to function as a transcription factor (17). The two ER isoforms share significant homology in their DNA binding and ligand binding domains (95% and 55%, respectively), but they vary greatly in their N-terminal (A/B) domains that contain the AF-1 transcriptional activation site. ERα and ERβ have overlapping but nonidentical tissue distribution and expression levels, suggesting functionally distinct biological roles. In addition, the two receptor subtypes have opposite effects on the activation of estrogen responsive promoters containing an AP1 site, with ERα activating transcription and ERβ attenuating transcription (18). Whereas we have considerable knowledge of ERβ as a transcription factor, it is currently unknown whether ERβ has nongenomic action when present at constitutive levels.

To determine if endogenous ERβ has nongenomic function, we assessed the capacity of the receptor to mediate E2-stimulated eNOS activity in primary ovine endothelial cell lines in which ERα and ERβ are endogenously coexpressed (18A ). Using these cells, we tested the hypothesis that ERβ mediates nongenomic eNOS stimulation. In addition, studies were performed to answer the following questions: 1) Does nongenomic ERβ action require ERα? 2) Does nongenomic ERβ action occur in the endothelial cell plasma membrane? and 3) More specifically, does nongenomic ERβ action occur in endothelial caveolae?

RESULTS

Localization of ERβ in Endothelial Plasma Membranes

To determine whether ERβ is localized to the endothelial cell plasma membrane, immunoblot analyses were performed in subcellular fractions. As demonstrated before, ERα protein was present in the nucleus, cytoplasm, and plasma membrane (Fig. 1A, upper panel) (4). Similarly, ERβ protein was detected in the nucleus, cytoplasm and plasma membrane (Fig. 1A, lower panel). In contrast to ERα, less ERβ was localized in the endothelial cell nucleus vs. cytoplasm. To generate a reconstitution paradigm in which plasma membrane-associated ER function can be tested, COS-7 cells were transfected with ERα or ERβ cDNA, and receptor distribution was assessed in cell subfractions. As previously seen, ERα protein was detected in the nucleus, cytoplasm and plasma membrane (Fig. 1B, upper panel) (4). ERβ protein was also detected in the nucleus, cytoplasm, and plasma membrane (Fig. 1B, lower panel).

Figure 1.

Endogenous ERα and ERβ Are Both Localized to Endothelial Cell Plasma Membranes Immunoblot analyses were performed on equal amounts of endothelial cell nucleus (Nuc), cytoplasm (Cyt), and plasma membrane (PM) using antibodies derived against amino acids 302–553 and 467–485, respectively, of ERα and ERβ (A). Parallel studies were performed using subfractions of COS-7 cells transfected with either ERα or ERβ cDNA (B). The findings shown are representative of three independent experiments.

Nongenomic Action of ERβ

ERβ nongenomic action was first examined by transient transfection of ERβ cDNA into endothelial cells followed by assessment of NOS activation in intact cells. Parallel studies were performed with cells transfected with ERα cDNA. Primary endothelial cell isolates displaying minimal responses to endogenous receptor activation were chosen for use in these studies, thereby optimizing the capacity to evaluate the effects of exogenously introduced receptors. To first confirm the overexpression of equivalent amount of functional receptors, E2-induced transcriptional transactivation was assessed by cotransfection of the estrogen-responsive reporter plasmid estrogen response element (ERE)-luciferase with either sham plasmid, ERα cDNA, or ERβ cDNA. Overexpression of ERα caused a 431% ± 48% increase in reporter activity upon 48 h exposure to 10−8m E2 as compared with sham transfected cells (mean ± sem, n = 5, P < 0.05). Similarly, overexpression of ERβ resulted in a 445% ± 34% increase in reporter activity (mean ± sem, n = 5, P < 0.05 vs. sham, no difference vs. ERα overexpression). Experiments were then done to compare the effects of enhanced ERα vs. ERβ expression on nongenomic eNOS activation by E2. Endothelial cell transfection was performed, the cells were placed in estrogen-free conditions, and acute experiments were done 48 h later. 3H-l-arginine conversion to 3H-l-citrulline was examined in intact cells during 15-min incubations in the presence or absence of hormone with or without receptor antagonist added. In contrast to sham-transfected cells (Fig. 2A), E2 treatment for 15 min caused a 230% increase in eNOS activity in cells overexpressing ERα (Fig. 2B), and this effect was reversed by simultaneous ER antagonism with ICI 182,780. Overexpression of ERβ yielded similar findings, with E2 causing a 220% increase in eNOS activity during a 15-min incubation which was attenuated by ICI 182,780.

Figure 2.

Effect of ERα vs. ERβ Overexpression on Nongenomic E2 Action in Endothelial Cells Cells with negligible responses to endogenous receptor activation were transfected with sham plasmid (A), ERα cDNA (B), or ERβ cDNA (C), and 48 h later 3H-l-arginine conversion to 3H-l-citrulline was measured over 15 min in intact cells, in the absence (basal) or presence of 10−8m E2, or E2 plus 10−5m ICI 182,780. Basal NOS activity ranged from 2–6 fmol l-3H-citrulline/well·15 min in individual experiments, and it was similar in the three study groups. Values are mean ± sem; n = 3. *, P < 0.05 vs. basal; †, P < 0.05 vs. E2 alone.

Because the endothelial cells studied express both ERα and ERβ endogenously, the observed response after ERβ overexpression may entail ERα-ERβ interaction (19). To determine whether ERβ has nongenomic action independent of ERα, eNOS activation was examined in COS-7 cells transfected with either ERα or ERβ and eNOS cDNA. COS-7 cells do not constitutively express ERs, and they do not display E2-stimulated eNOS activity when transfected with eNOS alone (5). COS-7 cell transfection was performed, the cells were placed in estrogen-free conditions for 48 h, and 3H-l-arginine conversion to 3H-l-citrulline was then measured during 15-min incubations in the presence or absence of hormone with or without receptor antagonist added. As has previously been shown (5), COS-7 cells expressing eNOS alone were not responsive to E2 treatment (Fig. 3A), whereas cells expressing eNOS and ERα exhibited a 238% increase in NOS activity with 15-min E2 treatment that was prevented by ER antagonism (Fig. 3B). COS-7 cells expressing eNOS and ERβ displayed a similar (228%) increase in eNOS activity upon rapid stimulation with E2, which was inhibited by the ER antagonist ICI 182,780 (Fig. 3C).

Figure 3.

Capacity of ERα and ERβ to Independently Mediate Nongenomic E2 Responses COS-7 cells were transfected with eNOS cDNA and either sham plasmid (A), ERα cDNA (B), or ERβ cDNA (C), and 48 h later 3H-l-arginine conversion to 3H-l-citrulline was measured over 15 min in intact cells, in the absence (basal) or presence of 10−8m E2 or E2 plus 10−5m ICI 182,780. Basal NOS activity ranged from 4–6 fmol l-3H-citrulline/well·15 min in separate experiments, and it was comparable in the three study groups. Values are mean ± sem; n = 3. *, P < 0.05 vs. basal; †, P < 0.05 vs. E2 alone.

To assess nongenomic ERβ action in an entirely independent manner, the effect of the selective ERβ antagonist RR-tetrahydrochrysene (THC) on E2-stimulated eNOS activity was evaluated (20). The differential effect of THC on the nongenomic actions of ERα and ERβ was first determined in studies of COS-7 cells transfected 48 h earlier with eNOS and either ERα or ERβ cDNAs. 3H-l-arginine conversion to 3H-l-citrulline was examined in intact cells during 15-min incubations in the presence or absence of E2 with or without receptor antagonist added. Whereas the nonselective ER antagonist ICI 182,780 completely inhibited E2-mediated eNOS activation occurring through ERα over 15 min, THC had no effect (Fig. 4A). In COS-7 cells expressing ERβ and eNOS, ICI 182,780 blunted E2-stimulated activity to levels similar to unstimulated activity, and THC caused 78% inhibition (Fig. 4B). Studies were then performed with THC in endothelial cells (Fig. 4C). ICI 182,780 inhibited E2-mediated NOS activity by 86%, to levels that were similar to basal activity. THC blunted E2-stimulated NOS activation by 72%, to levels that were indistinguishable from those obtained with ICI 182,780.

Figure 4.

Effect of Selective ERβ Antagonism on Nongenomic E2 Responses 3H-l-arginine conversion to 3H-l-citrulline was measured over 15 min in cells exposed to 10−8m E2, or E2 plus either ICI 182,780 (10−5m) or THC (10−7m). A, eNOS activation in COS-7 cells expressing eNOS and ERα. B, eNOS activation in COS-7 cells expressing eNOS and ERβ. C, eNOS activation in endothelial cells. In cotransfected COS-7 cells and endothelial cells, stimulated activity levels ranged from 4–14 fmol 3H-l-citrulline/well above basal values in separate experiments. Results are expressed as percent of activity above basal levels stimulated by E2 alone. Values are mean ± sem; n = 3. *, P < 0.05 vs. E2-stimulated activity.

Localization of ERβ Nongenomic Action

To localize nongenomic ERβ action, the stimulation of eNOS in isolated endothelial cell plasma membranes was studied. 3H-l-arginine conversion to 3H-l-citrulline was examined in the isolated membranes during 60-min incubations in the presence or absence of hormone with or without receptor antagonist added. We first determined if THC modifies E2-mediated activation of eNOS. As shown in Fig. 5A, ICI 182,780 inhibited 80% of estrogen stimulated NOS activity in the isolated membranes, and THC yielded 94% inhibition.

Figure 5.

ERβ Are Functional in Endothelial Cell Caveolae A, Effect of ICI 182,780 (10−5m) and THC (10−7m) on E2 (10−8m)-mediated eNOS activation in isolated endothelial cell plasma membrane. 3H-l-arginine conversion to 3H-l-citrulline was measured over 60 min. Basal NOS activity ranged from 5.2–7.2 pmol citrulline/mg protein·min, and E2-stimulated NOS activity ranged from 5.6–8.4 pmol citrulline/mg protein·min above basal levels in separate experiments. Results are expressed as percent of activity induced by E2. Values are mean ± sem, n = 3. *, P < 0.05 vs. E2 alone. B, Immunoblot analysis for ERα and ERβ in endothelial cell noncaveolae (NCM) and caveolae (CAV) membrane fractions. Caveolin-1 and Rack1 protein abundance were also determined to assess fraction separation. Results shown are representative of three independent studies. C, Effect of ICI 182,780 and THC on E2-mediated eNOS activation in isolated endothelial cell caveolae membranes. 3H-l-arginine conversion to 3H-l-citrulline was measured over 60 min. NOS activity was not detected under any conditions in noncaveolae membranes (4 ). In caveolae, basal NOS activity was undetectable and stimulated NOS activity ranged from 0.5–0.9 pmol citrulline/mg protein·min in separate studies. Results are expressed as percent of activity stimulated by E2. Values are mean ± sem; n = 3. *, P < 0.05 vs. E2 alone. D, E2 stimulated eNOS activity in noncaveolae and caveolae membranes from COS-7 cells expressing ERβ and eNOS. Experiments were performed as described in panel C. E2-stimulated NOS activity was not detected in caveolae from cells expressing eNOS alone (data not shown). Values are mean ± sem; n = 3.*, P < 0.05 vs. noncaveolae.

The distribution of ERβ protein within the endothelial plasma membrane was then assessed by immunoblot analysis (Fig. 5B). Antibodies to the caveolae marker caveolin-1 and the noncaveolae marker receptors for activated C-kinase 1 (Rack1) demonstrated successful isolation of the two fractions. As previously observed, ERα was detected in both the caveolae and noncaveolae fractions of the plasma membrane at equivalent levels of abundance (4). ERβ protein was also detected in both fractions with greater amounts in the noncaveolae compared with caveolae membranes.

To determine whether endogenous ERβ are functional in caveolae, the stimulation of eNOS in isolated endothelial cell caveolae membranes was studied. E2-mediated activation of eNOS was assessed during 60-min incubations of purified caveolae membranes in the absence or presence of ICI 182,780 or THC. ICI 182,780 inhibited 70% of estrogen-stimulated NOS activity in caveolae, and THC caused 92% inhibition (Fig. 5C).

Further evaluation of the capacity of ERβ for signaling action in caveolae was accomplished by reconstitution in subfractions of plasma membranes from COS-7 cells transfected with eNOS and ERβ cDNAs (Fig. 5D). Membranes were isolated 48 h after cell transfection, and eNOS activation by E2 was assessed during 60-min incubations of either caveolae or noncaveolae membranes. E2-stimulated NOS activity in noncaveolae membranes was similar to background levels. In contrast, E2-stimulated enzymatic activity was readily demonstrable in caveolae membranes.

DISCUSSION

There is now evidence in multiple paradigms that E2 initiates nongenomic signaling events at the cell surface (4, 7, 11, 14, 21). Considerable focus has been placed on the role of plasma membrane-associated subpopulations of ERα. In the present study, we reveal that ERβ mediates nongenomic eNOS activation in both intact endothelial cells and isolated plasma membrane and caveolae membrane fractions from them. The current work is the first to demonstrate the localization of a subpopulation of endogenous ERβ to plasma membrane and to caveolae, and a role for endogenous ERβ in nongenomic estrogen signaling.

The first evidence of a potential role for endogenous ERβ on the cell surface came from immunoblot analyses, which revealed that ERβ protein is present on the endothelial cell plasma membrane as well as in the nucleus and cytoplasm. Detection of ERβ in the plasma membrane fraction prompted further studies of its potential nongenomic actions. In an attempt to first do so in a cell system in which ERα has nongenomic function, overexpression studies were performed in primary endothelial cells. The nongenomic activation of eNOS by E2 was increased similarly after overexpression of ERβ or ERα in a manner that yielded comparable enhancement of classical, nuclear ER action. This finding suggested that ERβ is capable of nongenomic action, but because endothelial cells express endogenous ERα and ERβ and there is evidence of ERα-ERβ interaction modifying their nuclear function (19), it remained unknown whether ERα is required for nongenomic ERβ action. To approach this question, studies of independent ERα or ERβ function were performed in COS-7 cells that do not express endogenous ER and are not responsive to E2. Cotransfection of ERβ and eNOS yielded E2-stimulated increases in enzyme activity that were similar to those observed with ERα and eNOS coexpression. Thus, nongenomic ERβ action does not require ERα.

A second, entirely independent approach was used to determine the participation of ERβ in signaling events in endothelial cells expressing endogenous levels of both ER subtypes. The ER ligand THC selectively inhibits ERβ-mediated transcriptional transactivation of an ERE-containing promoter (20). Employing reconstitution in COS-7 cells, we first demonstrated that THC has no effect on ERα-mediated eNOS activation, whereas ERβ-mediated stimulation of the enzyme is markedly attenuated by the agent. When tested in endothelial cells, THC blunted E2-stimulated eNOS activation to levels approaching those obtained with ICI 182,780. These data suggest that endogenous ERβ mediate a large portion of nongenomic signaling leading to eNOS activation by E2 in the model employed. The experiments exploring ER overexpression in the endothelial cells (Fig. 2) suggest that the relative contribution of ERα and ERβ to nongenomic E2 responses may depend on the relative abundance of each receptor subtype. However, in contrast to the present findings for THC blockade of eNOS activation by endogenous ER, recent studies of the impact of THC on the transcriptional transactivation of an estrogen-responsive gene in these cells has revealed an equivalent contribution of the two receptor isoforms (Ihionkhan, C., K. L. Chambliss, L. L. Gibson, and P. W. Shaul, manuscript in preparation). Thus, there are potentially important disparities in the relative roles of endogenous ERα and ERβ in nongenomic vs. genomic estrogen actions in the same cell. The current concept that ERα is the primary endogenous mediator of nongenomic estrogen action is evidently oversimplified (22).

To localize ERβ nongenomic action, we first assessed the effect of THC on eNOS activation in isolated endothelial cell plasma membranes. Treatment with THC resulted in a marked reduction in E2 stimulated enzyme activity, similar to the level of inhibition obtained with ICI 182,780 and consistent with the results observed in intact endothelial cells. These findings suggest that a subpopulation of endogenous ERβ reside in a signaling module in the plasma membrane. Further evaluation of ERβ localization and nongenomic action was performed in isolated endothelial cell caveolae membranes. Immunoblot analysis demonstrated the presence of ERβ protein in caveolae as well as in noncaveolae membrane fractions, as we have previously reported for ERα (4). More importantly, THC caused inhibition of E2-mediated eNOS activation in endothelial cell caveolae membranes, indicating that functional endogenous ERβ are localized to this microdomain. The requirement for caveolae membrane localization for ER action on the cell surface was also assessed by reconstituting the interaction of the receptor and eNOS in COS-7 cells expressing both the receptor and the enzyme. COS-7 cell caveolae membranes displayed dramatic E2-mediated NOS activation, whereas noncaveolae membranes were insensitive to E2. Thus, all of the molecules needed for E2-mediated cell surface signaling through ERβ are localized in caveolae.

The present findings provide two important new dimensions to recent observations about rapid, nongenomic actions of ERβ in transfected cells. Razandi et al. (11) showed that a subpopulation of ERβ transfected into CHO cells was membrane bound and capable of activating Gαq and Gαs and of stimulating inositol 1,4,5-triphosphate production. They also demonstrated that overexpressed ERβ mediates the activation of the MAPKs ERK and c-Jun kinase, whereas ERα does not mediate c-Jun kinase activation. Others have shown an ability of overexpressed ERβ to activate src kinase (23). Kousteni et al. (14) further demonstrated that the activation of src via overexpressed ERβ inhibits apoptosis. However, it is critical to note that all previous studies of nongenomic ERβ action have been performed in overexpression experiments, and that the specific localization of nongenomic ERβ action has been entirely unknown. The present studies provide the first evidence of nongenomic ERβ action when the receptor is present at endogenous levels, and they also localize this function to caveolae.

The signal transduction pathways involved in nongenomic ER actions are beginning to be dissected in the eNOS paradigm. We have previously shown that tyrosine kinase and MAPK inhibitors prevent eNOS stimulation by E2 (5). Those observations are supported by more recent reports of src:ERβ interaction (23) and rapid src/Shc/ERK pathway activation by E2 (14). Two groups have also described the phosphorylation of eNOS by E2-activated Akt kinase, resulting in increased eNOS activity (13, 24). However, they both showed that ERα, and not ERβ mediate this process. When considered along with the current findings, these cumulative observations indicate that more than one signaling pathway may lead to eNOS stimulation after ER activation, as recently suggested by Mendelsohn (22). Further experiments are warranted to better understand both common and disparate signaling events initiated by cell surface ERα and ERβ. It should now be possible to employ isolated membrane fractions to identify and characterize all of the molecules required for ERα and ERβ coupling to resident signaling cascades in caveolae, thereby greatly facilitating such endeavors.

The mechanisms by which subpopulations of ERβ and ERα are associated with the plasma membrane are yet to be determined. ERα and ERβ have no likely sites for acylation, prenylation, or other modification known to direct proteins to the plasma membrane (25). Because only a small fraction of total cell ERα or ERβ localize to the plasma membrane, processes must exist that regulate the relative amount of receptor targeted to the cell surface while leaving the bulk of the receptor populations in the cytoplasm and nucleus. It is likely that ERα and ERβ would employ similar mechanisms of membrane localization, and that homologous regions of the two receptors would be involved. The DNA binding domain is the region of highest homology between ERα and ERβ (95%), and there is also considerable homology within the ligand binding domains (55%). Kousteni et al. (14) recently reported that an ERα mutant consisting of only the ligand binding domain displayed antiapoptotic activity in an overexpression system that was comparable to that observed with a chimera of the ligand binding domain directed to membranes via palmitoylation. These observations suggest that residues involved in membrane targeting and action of ERs may be present within the ligand binding domain. However, detailed studies employing mutagenesis of both ERα and ERβ and determinations of membrane localization and function are now indicated. Such work will enhance both our specific understanding of nongenomic responses to estrogen in a variety of cell types, and our overall knowledge of the multiple nongenomic actions of steroid hormones.

MATERIALS AND METHODS

Cell Culture and Transfection

Primary ovine endothelial cells were obtained from the intrapulmonary arteries of fetal lambs at 125–135 d gestation (term = 144 d) by collagenase digestion, and were propagated as previously described. Animal care and euthanasia procedures were approved by the Institutional Animal Care and Research Advisory Committee. Near-confluent primary cells were studied at passage 4–6. For studies of endothelial cell subfractions, an immortalized cell line derived from the primary cells was employed at passage 16–26. The primary and immortalized endothelial cells display similar levels of eNOS activation by E2 (4, 26). For reconstitution experiments, COS-7 cells (American Type Culture Collection, Manassas, VA) were grown in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated FBS plus 200 U/ml penicillin and 200 μg/ml streptomycin. COS-7 cells were employed because they do not constitutively express ERs, and they do not display E2-stimulated eNOS activity when transfected with eNOS alone (5).

The capacity of ERβ to activate signal transduction leading to eNOS activation was first tested by employing transient transfection to overexpress the receptor in a primary endothelial cell line with minimal eNOS stimulation by endogenous ER. Parallel studies were done in cells transfected with either sham plasmid or ERα cDNA. For these studies, human ERα cDNA or mouse ERβ cDNA was inserted into the _Eco_RI site of pCDNA3.1 (Invitrogen, San Diego, CA). Comparable overexpression of functional ERα and ERβ was confirmed in additional experiments evaluating estrogen-induced transcriptional transactivation by cotransfection with a luciferase reporter plasmid that contains three copies of the Xenopus vitellogenin ERE, ERE-luciferase, as previously reported (28). After transfection, cells were placed in either phenol red-free, estrogen-free media, or phenol red-free media containing 10−8m E2 for 48 h, and reporter activity was measured (28). Cells were cotransfected with a plasmid containing SV40-driven β-galactosidase to normalize for transfection efficiency.

To further evaluate ERβ-mediated signal transduction and to determine whether the process occurs independent of ERα, E2-mediated eNOS activation was reconstituted in COS-7 cells transfected with bovine eNOS cDNA (29) and either ERα cDNA, ERβ cDNA, or empty vector. In both transfected primary endothelial cells and transfected COS-7 cells, successful ER expression was confirmed by immunoblot analysis. Coimmunofluorescence experiments revealed that transfection efficiency was approximately 20% for either ER or eNOS, and that the majority of transfected cells expressed both the receptor and enzyme.

Subcellular Fractionation

To study the subcellular distribution of ERβ and to localize its function, subfractionation was performed on immortalized endothelial cells or transfected COS-7 cells as previously described (4). The purity of the plasma membrane fraction obtained has been previously confirmed by measurements of alkaline phosphatase (plasma membrane), galactosyl transferase (Golgi), and NADPH cytochrome C reductase (endoplasmic reticulum) activity (30). All fractionation steps were done in the absence of exogenous calcium. Successful separation of the plasma membrane into the caveolae subfraction and the noncaveolae bulk of the plasma membrane was confirmed by immunoblot analyses for the caveolae marker protein caveolin-1 and the noncaveolae protein, Rack1 (31). The protein contents of all samples were determined with the method of Bradford (32).

Immunoblot Analyses

Immunoblot analyses were performed using standard procedures to evaluate the abundance and distribution of ERα, ERβ, eNOS, caveolin-1 and Rack1 (33). Equivalence of protein loads was confirmed by amido black staining (Sigma, St. Louis, MO). The analyses used a mouse monoclonal antibody directed against amino acids 495–595 of ERα (2.5 μg/ml, AER320; Labvision, Freemont, CA,) or polyclonal antiserum to amino acids 467–485 of ERβ (1 μg/ml; Affinity BioReagents, Inc., Golden, CO). Monoclonal antiserum to eNOS (0.25 μg/ml) and polyclonal antiserum to caveolin-1 (0.05 μg/ml) or Rack1 (0.1 μg/ml) were from Transduction Laboratories, Inc. (Lexington, KY). In preliminary studies, the abundance of ERα was similar in primary vs. immortalized endothelial cells, and levels of ERβ were also comparable in the two cell types.

NOS Activation

eNOS activation was assessed in intact endothelial or COS-7 cells by measuring 3H-l-arginine conversion to 3H-l-citrulline, using previously reported methods (9). Adherent cells grown in six-well plates were placed in l-arginine-deficient, serum-free endothelial-SFM growth media (Life Technologies, Inc.) for 2 h, and then preincubated in PBS (pH 7.4) containing 120 mm NaCl, 4.2 mm KCl, 2.5 mm CaCl2, 1.3 mm MgSO4, 7.5 mm glucose, 10 mm HEPES, 1.2 mm Na2HPO4, and 0.37 mm KH2PO4 for 15 min at 37 C. The ensuing 15-min incubation for eNOS activity was initiated by replacing the preincubate with PBS containing 1.5 μCi/ml 3H-l-arginine. After 15 min, the reaction was stopped by adding 1 n trichloroacetic acid, the cells were freeze-fractured in liquid nitrogen and scraped with a rubber spatula, the contents of each well were ether extracted, and the 3H-l-citrulline generated was isolated using Dowex AG50WX-8 columns and quantified by liquid scintillation spectroscopy.

To evaluate the nongenomic effects of estrogen on eNOS in either endothelial or COS-7 cells, 3H-l-arginine conversion to 3H-l-citrulline was measured in intact cells either under basal conditions or in the presence of 10−8m E2 during 15-min incubations. In previous experiments, the effect of E2 was demonstrable within 5 min, the maximal response was obtained at 10−8m, and the threshold concentration was 10−10m (5). The role of ERs in eNOS activation was evaluated by the addition of either ICI 182,780 (10−5m) to antagonize both ERα and ERβ, or THC (10−7m), which is an antagonist of ERβ and an activator of ERα-mediated gene transcription in some paradigms (20). ER antagonism was implemented solely during the 15-min incubation for NOS activity. In studies employing ERα or ERβ overexpression in endothelial cells, basal NOS activity was not altered by receptor overexpression, as previously observed for ERα (5). Both basal and stimulated NOS activity were completely inhibited by 2.0 mm nitro-l-arginine methyl ester. NOS activity was expressed relative to basal levels in the same six-well plate. All findings were confirmed in at least three independent studies.

eNOS activation was also assessed in purified whole plasma membranes or noncaveolae or caveolae subfractions of plasma membrane reconstituted in 50 mm Tris HCl buffer (pH 7.4) with 0.1 mm EDTA, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml Nα-p-tosyl-l-lysine chloromethyl ketone, 10 nm phenylmethylsulfonyl fluoride, 3 mm dithiothreitol, and 10 mm 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Membranes (10 μg plasma membrane or 2 μg noncaveolae or caveolae membrane) were incubated with 2.0 μCi/ml of 3H-l-arginine and 2 μm cold l-arginine, and citrulline generation was evaluated over 60 min at 37 C. The assay was terminated by the addition of 400 μl of 40 mm HEPES buffer, pH 5.5, with 2 mm EDTA and 2 mm EGTA. 3H-l-citrulline generated was isolated and quantified as described for the intact cell experiments. All observations were confirmed in a minimum of three separate studies.

To determine the effect of estrogen on eNOS in the isolated membranes, incubations for activity were performed in the absence or presence of 10−8m E2β. To reveal the role of membrane-associated ERα or ERβ in E2 responses, studies were performed in the absence or presence of the ER antagonist ICI 182,780 (10−5m); selected experiments were also performed in the presence of THC to reveal the role of membrane-associated ERβ exclusively. NOS activity in all membrane samples was fully inhibited by the addition of 2 mm nitro-l-arginine methyl ester.

Statistical Analysis

Differences in observations between treatment groups were evaluated by ANOVA after establishing equivalence of variances and normal distribution of data. The level of significance of differences between mean values was assessed by the Student-Newman-Keuls method. Nonparametric analysis was used when indicated. Values shown are mean ± sem, with n = 3 or more independent experiments. Significance was accepted at the 0.05 level of probability.

Acknowledgments

This work was supported by NIH Grants HL-58888, HL-53546, and HD-30276 (to P.W.S.), GM-52016 (to R.G.W.A.), and HL-63494, HL-55309, and HL-56069 (M.E.M.). This project was supported in part by the Lowe Foundation and the Perot Family Foundation.

THC was the kind gift of John and Benita Katzenellenbogen (University of Illinois at Chicago, Chicago, IL).

Abbreviations:

• eNOS,
  Endothelial nitric oxide synthase;
• ERE,
  estrogen-response element;
• Rack1,
  receptors for activated C-kinase;
• SHR,
  steroid hormone receptor;
• THC,

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Copyright © 2002 by The Endocrine Society

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