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Endocrinology Vol. 143, No. 10 3785-3795
Copyright © 2002 by The Endocrine Society

ARTICLE

Estrogen Receptors {alpha} and ß Have Similar Activities in Multiple Endothelial Cell Pathways

Mark J. Evans, Heather A. Harris, Chris P. Miller, Sotirios K. Karathanasis1 and Steven J. Adelman

Women’s Health Research Institute (M.J.E., H.A.H., S.K.K., S.J.A.) and Department of Chemical Sciences, Wyeth Research (C.P.M.), Collegeville, Pennsylvania 19426

Address all correspondence and requests for reprints to: Dr. Mark J. Evans, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426. E-mail: evansm{at}wyeth.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The presence of both estrogen receptor {alpha} (ER{alpha}) and ERß in vascular cells has greatly increased the complexity of potential estrogen regulatory pathways in the cardiovascular system. Here, human umbilical vein endothelial cells were engineered using adenovirus vectors to express either ER{alpha} or ERß. The activities of ER{alpha} and ERß were compared in three distinct gene regulatory pathways, including inhibition of IL-1ß induction of E-selectin expression, inhibition of basal endothelin-1 production, and the ability to induce two matrix-stabilizing enzymes: tissue transglutaminase and a novel member of the lysyl oxidase family. Both ERs were active on these end points, although ERß was typically less efficacious than ER{alpha}. As no class of gene regulation could differentiate ER{alpha} from ERß activity, we characterized a novel steroid (7{alpha}-thiophenyl-E2) that bound with similar affinities to ER{alpha} and ERß, but functioned as an ER{alpha} agonist and ERß antagonist for all of these endothelial responses. This pattern of receptor subtype-selective activity was not unique to endothelial cells, but was also seen in metabolically active HepG2 cells, suggesting potential in vivo utility. The panel of endothelial responses coupled with a selective modulator should provide a means to characterize the roles of ER{alpha} and ERß in endothelial cells in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARDIOVASCULAR disease is by far the leading cause of mortality of women in the developed world, accounting for approximately one third of female deaths (1). The incidence of cardiovascular disease in women is extremely low during the premenopausal years; however, after menopause the incidence of cardiovascular disease increases dramatically. Numerous early observational studies summarized in a meta-analysis (2) have suggested a protective effect of hormone replacement therapy (HRT). These findings have recently been confirmed in a single large study, the Nurses Health Study, in which either prior or current use of hormone replacement therapy reduced the relative risk of myocardial infarction or death from cardiovascular disease by 30–60% (3). One recent exception to these favorable results occurred in the Heart and Estrogen/Progestin Replacement Study trials, in which no beneficial effect was seen when elderly postmenopausal women with preexisting cardiovascular disease were treated with a combination of estrogens plus medroxyprogesterone (4).

The role of estrogens in cardiovascular disease prevention is complex. HRT has been shown in numerous studies to significantly lower low density lipoprotein levels (5, 6, 7, 8). Furthermore, low levels of high density lipoprotein (HDL) are now known to be a significant risk factor for the development of cardiovascular disease (9, 10, 11), and estrogens increase HDL levels by approximately 10–20% (5, 6, 7). Finally, estrogens decrease levels of lipoprotein(a) in women (12, 13), another suggested risk factor for the development of cardiovascular disease (13, 14, 15). The mechanism of action for these effects probably resides in the liver, as transdermal administration of estrogens fails to either elevate HDL levels or decrease lipoprotein(a) levels (16, 17). Comparison of the magnitude of estrogen-mediated lipid changes with the degree of reduction in mortality has suggested that these lipid changes account for only a minority of the beneficial effects (18). As much as 75% of the beneficial effects of HRT are thus currently believed to be independent of lipid changes.

Development of the atherosclerotic lesion is now recognized as an inflammatory process. One of the key early steps in development of the lesion is transmigration of monocytes from the circulation into the subendothelial space, a process dependent upon the expression of adhesion molecules such as E-selectin, vascular cellular adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 by endothelial cells through a nuclear factor-{kappa}B (NF-{kappa}B)-dependent up-regulation of gene expression (19, 20). HRT decreases levels of soluble E-selectin and VCAM-1 in women (21, 22, 23, 24), suggesting that endothelial expression of these molecules is decreased in vivo. In addition, estrogen treatment of ovariectomized mice results in the inhibition of diet-induced VCAM-1 expression (25), suggesting that down-regulation of VCAM-1 may be a significant component of estrogen inhibition of atherosclerotic lesion development (26). These effects are probably due to the direct influence of estrogens on endothelial cell gene expression. Indeed, it has been recently shown that treatment of cultured human umbilical vein endothelial cells (HUVEC) with 17ß-estradiol inhibits IL-1ß induction of these adhesion molecules (27). Furthermore, in addition to lesion size, lesion complexity/stability are major determinants of clinical coronary events, which can be precipitated by either endothelial denudation or plaque rupture (28), two processes facilitated by elevated blood pressure. In women, estrogens are known to decrease levels of endothelin-1 (ET-1) (29, 30, 31), a major determinant of blood pressure and a potent chemoattractant for monocytes (32). In HUVEC, estrogen regulates transcriptional expression of ET-1 through the transcription factor activating protein-1 (AP-1) (33).

The concept of direct vascular therapeutic effects of estrogen is further supported by experimental findings using rabbits in which the cholesterol levels were maintained constant by varying the animals’ diet. Treatment of these rabbits with estrogen greatly decreased aortic cholesterol accumulation (34). In contrast, if the endothelium was removed by balloon catheter injury, estrogen no longer had any protective effect (34). Thus, endothelial cells appear to be significantly involved in estrogen protective activities. This model has been reinforced by the finding of both estrogen receptor {alpha} (ER{alpha}) and ERß in vessel endothelial cells, with the levels of ERß increasing up to 40-fold after vessel injury (35, 36). Notably, estrogen treatment reduces vascular injury in wild-type, ER{alpha} knockout (ER{alpha}KO), and ERßKO mice (37, 38, 39), but does not reduce injury in ER{alpha}ERß double KO mice (40), suggesting that ER{alpha} and ERß may play redundant roles in vascular protection.

In cotransfection studies, ER{alpha} and ERß behave similarly on synthetic estrogen response element (ERE)-driven reporters in response to various ligands, including 17ß-estradiol (41, 42). In contrast, in cotransfection studies, 17ß-estradiol activates AP-1-dependent transcription through ER{alpha}, but inhibits AP-1-dependent transcription through ERß (43, 44). Thus, 17ß-estradiol produces similar results for ER{alpha} and ERß in one transcriptional pathway (regulation of ERE activity), but has opposite effects on ER{alpha} and ERß in a second transcriptional pathway (regulation of AP-1 activity). Little is known regarding the comparative abilities of ER{alpha} and ERß to regulate gene expression mediated by complex promoters within a chromosomal context, especially in endothelial cells. Here we demonstrate that ER{alpha} and ERß are both able to mediate estrogen activity in three distinct processes: inhibition of IL-1ß induction of expression of E-selectin, inhibition of basal ET-1 production, and induction of matrix-modifying enzymes. However, a novel estrogen derivative, 7{alpha}-thiophenyl-E2, functioned as an ER{alpha} agonist, but an ERß antagonist, for all of these responses. The combination of knowledge of endothelial cell functions mediated by ERs coupled with a pharmacological tool to differentially regulate these receptors should allow a clearer understanding of which receptor is active in endothelial cells in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
Human aortic endothelial cells and HUVEC were obtained as frozen stocks from Clonetics (San Diego, CA) at passage 2 or 3. The cells were maintained in EGM [endothelial cell basal medium (EBM) medium supplemented with 10 ng/ml human epidermal growth factor, 1 µg/ml hydrocortisone, 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, 3 mg/ml bovine brain extract, and 2% fetal bovine serum]. The cells were used for experiments between passages 4 and 7.

ER Western blotting
On d 1, 100-mm dishes were seeded with 2 x 106 human aortic endothelial cells in EGM. On d 2, the cells were refed deficient EGM [EGM prepared using phenol red-free EBM and 2% charcoal/dextran-treated fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT)] containing replication-defective adenovirus (45) expressing human full length wild-type ER{alpha} (Ad5ER{alpha}) or ERß (Ad5ERß) at an approximate multiplicity of infection (MOI) of 50 or 500, respectively. After allowing the virus to adsorb to the cells for 1 h at 37 C, the cells were washed once with deficient EGM and refed deficient EGM. Six hours later the cells were refed deficient EGM containing 100 nM 17ß-estradiol. On d 3, nuclear extracts were prepared as previously described (46). Twenty-five micrograms of nuclear extract protein were subjected to SDS-PAGE on an 8% gel, transferred to nitrocellulose, and probed using rabbit polyclonal antibodies specific for ER{alpha} or ERß. Bound primary antibody was detected using peroxidase-coupled goat antirabbit IgG (Zymed Laboratories, Inc., San Francisco, CA), followed by chemiluminescent (ECL, Amersham, Arlington Heights, IL) detection of peroxidase activity.

Binding assays
For in vitro binding assays, the ligand-binding domains of human ER{alpha} (with an N-terminal His tag) or ERß (with an N-terminal His tag and a C-terminal flag epitope) were expressed in Escherichia coli strain BL21(DE3) by isopropyl-ß-D-1-thiogalactopyranoside induction for 2 h at 25 C. A crude lysate was prepared in 50 mM Tris-Cl and 150 mM NaCl (pH 7.4) using a French press, and insoluble material was removed by centrifugation. Binding reactions were performed in Wallac high binding, cross-talk-free, 96-well plates containing 2 nM [3H]17ß-estradiol (NEN Life Science Products, Boston, MA), unlabeled compounds and 1 µg crude lysate in Dulbecco’s PBS supplemented with 1 mM EDTA. After incubation at room temperature for 5–18 h, unbound material was removed by rinsing, and bound disintegrations per minute were determined by liquid scintillation counting.

For whole cell binding assays, HUVEC were plated on d 1 in 24-well plates at 1 x 105 cells/well in EGM medium. On d 2, the cells were refed deficient EGM containing Ad5ER{alpha} or Ad5ERß at an approximate MOI of 30. After allowing the virus to adsorb to the cells for 1 h at 37 C, the cells were refed deficient EGM. On d 3, the cells were refed medium B [phenol red-free EBM (Clonetics) supplemented with 0.25% BSA (Sigma, St. Louis, MO) and 0.1% antibiotic-antimycotic (Life Technologies, Inc., Gaithersburg, MD)] containing 0.8 nM [3H]17ß-estradiol plus various concentrations of competitor. After incubation at 37 C for 1 h, the cells were washed four times with ice-cold PBS containing 0.1% BSA and twice with ice-cold HBSS supplemented with 7.5 mM EDTA plus 0.1% methylcellulose. The cells were treated with 100% ethanol for 15 min. The ethanol extract was added to Optiphase scintillant (Wallac, Inc., Turku, Finland) and counted using a 1450 MicroBeta scintillation counter (Wallac, Inc.). Nonspecific binding was determined by the addition of 1 µM unlabeled 17ß-estradiol to the binding reactions. Fifty percent inhibitory concentration values were determined by fitting a four-parameter equation to a plot of specific bound counts vs. competitor concentration.

E-selectin assay
On d 1, HUVEC were plated in EGM at 7000 cells/well in 96-well plates. On d 2, the cells were infected with Ad5ER{alpha} or Ad5ERß at an approximate MOI of 300. After infection, the cells were washed and refed with deficient EGM. After 6 h, the cells were refed medium B containing compounds. On d 3, the cells were refed medium B containing compounds plus 30 U/ml recombinant human IL-1ß (Roche Molecular Biochemicals, Indianapolis, IN). After 5 h of incubation at 37 C, the cells were washed three times with ice-cold PBS and fixed with PBS containing 2% paraformaldehyde for 30 min. The cells were again washed three times with PBS. E-selectin expression was determined by an ELISA using 2 µg/ml anti-CD62E monoclonal antibody (PharMingen, San Diego, CA), followed by 1 µg/ml peroxidase-labeled goat antimouse IgG+A+M (Zymed Laboratories, Inc.). Antibody binding was quantified by using o-phenylenediamine dihydrochloride as the substrate and measuring end-point absorbance at 490 nm.

ET-1 assay
On the morning of d 1, HUVEC were plated in EGM at 14,000 cells/well in 96-well plates. Approximately 6 h later, the cells were infected with Ad5ER{alpha} or Ad5ERß at an approximate MOI of 300. After allowing the virus to adsorb for 1 h at 37 C, the cells were washed and refed deficient EGM. On d 2, the cells were refed medium B containing compounds. After 5 h of incubation at 37 C, the medium was removed and assayed by ET-1 ELISA (R\|[amp ]\|D Systems, Inc., Minneapolis, MN).

Isolation of endothelial estrogen-regulated gene-7 (EER-7)
EER-7 was identified by differential display using HUVEC RNA. On d 1, HUVEC were plated at 3 x 106 cells/150-mm plate. On d 2, the cells were infected as described above. Five hours after treatment with either vehicle or 100 nM 17ß-estradiol, the cells were harvested using TRIzol (Life Technologies, Inc.). RNA was prepared according to the manufacturer’s protocol, treated with 10 U deoxyribonuclease I (Life Technologies, Inc.) at 37 C for 1 h, and repurified by RNeasy spin column (QIAGEN, Chatsworth, CA).

After deoxyribonuclease I treatment, 6 µg total RNA were incubated with 1x reverse transcriptase buffer [25 mM Tris-Cl (pH 8.3), 37.6 mM KCl, 3 mM MgCl2, and 5 mM dithiothreitol], 20 µM deoxy-NTPs, and 0.2 µM HT11C oligonucleotide (AAGCTTTTTTTTTTTC) in a final volume of 600 µl. This reaction mixture was incubated at 65 C for 5 min to denature secondary structures, followed by a 10-min incubation at 37 C. Superscript reverse transcriptase (30 µl; 200 U/µl; Life Technologies, Inc.) was then added to the reaction, and incubation was continued for 1 h at 37 C. The enzyme was inactivated by heating at 75 C for 5 min. An aliquot of this reaction was then used for the second strand synthesis in PCR buffer [10 mM Tris-Cl (pH 8.4), 100 mM KCl, 1.5 mM MgCl2, and 0.001% gelatin] containing 2 µM deoxy-NTPs, 15 nM [33P]deoxy-ATP (NEN Life Science Products), 1 U AmpliTaq (Perkin-Elmer, Norwalk, CT), and 1 µM arbitrary primer 5'-AAGCTTGCCATGG-3'. This reaction was amplified using 40 cycles of 92 C for 15 sec, 40 C for 2 min, and 72 C for 30 sec. The PCR products were separated on a 6% denaturing polyacrylamide gel for 3 h at 2000 V. The gel was transferred to filter paper, dried under vacuum at 80 C for 1 h, and exposed to x-ray film for 24 h. The developed film was then superimposed over the dried gel, and the band of interest was identified. The corresponding gel slice was excised and boiled in water for 15 min. After centrifugation, the supernatant was removed, and recovered PCR product was precipitated using glycogen as a carrier at -20 C overnight with sodium acetate/ethanol. The resulting pellet was used in a reamplification PCR in the presence of 1x PCR buffer, 20 mM deoxy-NTPs, 0.2 mM arbitrary primer, 0.2 mM HT11C oligonucleotide, and 2 U AmpliTaq, with the same conditions as those described above.

One band (no. 7), approximately 320 bp in size, showed a very strong induction by 17ß-estradiol treatment and was designated endothelial EER-7. The EER-7 fragment was cloned by TA cloning into the pCRII vector (Invitrogen, San Diego, CA). Sequencing of the insert revealed it to be a novel clone. Other bands identified in a similar manner corresponded to known genes, such as transglutaminase.

To isolate the complete EER-7 cDNA, a PCR screening approach (OriGene Rapid-Screen) was used with a human placenta library. Oligonucleotide primers derived from the EER-7 insert sequence (5'-TTTGCTCAGCTGAGCTCCT-3' and 5'-TAAGATAAAGGTAAGGACACTA-3') were used to screen the master plate. Subplates corresponding to each positive well were rescreened in multiple rounds to identify positive individual colonies. One clone contained a cDNA insert corresponding to the predicted EER-7 transcript size determined by Northern analysis and was sequenced in its entirety. Blast analysis of the NCBI UniGene database identified this sequence as Hs.306814.

Transglutaminase protein expression and activity
On d 1, HUVEC were plated in EGM at 7000 cells/well in 96-well plates. On d 2, the cells were infected with Ad5ßGal, Ad5ER{alpha}, or Ad5ERß at an approximate MOI of 300. After infection, the cells were washed and refed with deficient EGM. After 6 h, the cells were refed medium B containing compounds. For determination of transglutaminase protein, cell lysates were analyzed by Western blot using a monoclonal antibody directed against human transglutaminase type II (MS-300-P1, Lab Vision, Fremont, CA) at a 1:500 dilution, followed by a goat antimouse peroxidase-conjugated secondary antibody (Zymed Laboratories, Inc.) with chemiluminescent detection (ECL, Amersham Pharmacia Biotech). For measurement of transglutaminase activity, cells were incubated with [3H]putrescine at 37 C followed by trichloroacetic acid precipitation of total cellular protein essentially as described previously (47).

Northern analysis
On d 1, HUVEC were plated in EGM at 3 x 106 cells/150-mm dish. On d 2, the cells were infected with Ad5ER{alpha} or Ad5ERß at an approximate MOI of 300, washed, and refed medium B. Five hours later, the cells were refed medium B containing compounds. After incubation at 37 C for 20 h, total RNA was prepared from the cells by RNeasy method (QIAGEN). RNA was fractionated by electrophoresis on 1% agarose, transferred to nylon membranes, and hybridized to 32P-labeled probes prepared from EER-7 and GAPDH cDNAs using an oligonucleotide labeling kit (Amersham Pharmacia Biotech) as previously described (38).

HepG2 transfection
HepG2 cells (1 x 107) were transfected with 50 µg NF-{kappa}B luciferase reporter plasmid, 20 µg Rous sarcoma virus-ß-galactosidase, and either 25 µg cDNA3 ER{alpha} or cDNA3 ERß expression plasmids by electroporation at 100 V/cm, 1700 µF, 72 {Omega} (BTX, San Diego, CA). Transfected cells were allowed to recover for 20 min, resuspended in deficient growth medium (DMEM supplemented with 1% MEM nonessential amino acids, 10% heat-inactivated charcoal-stripped fetal bovine serum, 10 U/ml penicillin, and 100 µg/ml streptomycin) and aliquoted at 1 x 105 cells/well in 96-well plates. On d 2, the cells were refed deficient growth medium supplemented with 100 U/ml IL-1ß and the indicated compounds. Luciferase and ß-galactosidase activities were assayed 6 h later using commercial luciferase (Promega Corp., Madison, WI) and ß-galactosidase (Tropix, Bedford, MA) reagents. The NF-{kappa}B luciferase reporter plasmid contains three copies of the human MHC class I promoter NF-{kappa}B-binding site upstream of the thymidine kinase promoter truncated at position -105.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ER expression system
In vivo endothelial cells express both ER{alpha} and ERß (35, 36). However, the expression of ER varies dramatically with culture conditions and passage number of primary endothelial cells. Treatment of HUVEC with high doses of 17ß-estradiol maintains the expression of ER{alpha} (27), but the use of ligand to maintain the expression of ER confounds further analysis. We therefore employed an adenovirus infection system (45) to selectively express either ER{alpha} or ERß in endothelial cells. Western blotting of nuclear extracts prepared from cultured human aortic endothelial cells or HUVEC did not detect significant levels of either ER{alpha} or ERß (Fig. 1Go). In contrast, high levels of ER{alpha} were detected in nuclear extract prepared from cells infected with replication-defective adenovirus designed to express ER{alpha}. No induction of ERß was seen in these cells. Similarly, high levels of ERß were detected in cells infected with adenovirus expressing ERß, with no ER{alpha} detectable in these cells. Both ER{alpha} and ERß were present in nuclear extracts prepared from cells infected with both adenoviruses. Staining of cells infected with an adenovirus expressing ß-galactosidase demonstrated that under these conditions greater than 90% of cells were infected, allowing analysis of endogenous gene regulations.



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  		Figure 1. Expression of ER{alpha} and ERß in endothelial cells. Human aortic endothelial cells were mock infected (lane 1) or infected with adenovirus expressing ER{alpha} (Ad5 ER{alpha}; lanes 2 and 3) or ERß (Ad5 ERß; lanes 3 and 4). After treating the cells for 16 h with 100 nM 17ß-estradiol, nuclear extracts were prepared. Nuclear extract protein (25 µg) was analyzed by Western blot using a mixture of antibodies specific for ER{alpha} or ERß. The identities of the ER{alpha} and ERß bands denoted by arrows were confirmed by Western blotting using either the ER{alpha} or ERß primary antibody individually.

 
ER{alpha} and ERß activity in diverse gene regulatory pathways
Having established a system for selective expression of ER in cultured endothelial cells, we analyzed the ability of these receptors to mediate various activities of 17ß-estradiol. Treatment of endothelial cells with estrogen inhibits IL-1ß induction of adhesion molecules such as E-selectin (27), likely through the known ability of ER{alpha} to inhibit NF-{kappa}B signaling (48). Following stimulation with IL-1ß, HUVEC expressing either ER{alpha} or ERß showed a 5-fold increase in cell surface E-selectin levels in the absence of receptor ligand. Addition of increasing concentrations of 17ß-estradiol inhibited E-selectin induction by up to 80% in cells expressing ER{alpha} and up to 50% in cells expressing ERß (Fig. 2Go). No effect of 17ß-estradiol on E-selectin induction was found in uninfected HUVEC or in HUVEC infected with adenovirus expressing ß-galactosidase (not shown). The maximal inhibitory activity of both receptors was achieved with addition of 1 nM 17ß-estradiol, a physiologically relevant concentration. As expected, ICI-182780 was able to completely block the inhibitory activity of both receptors. In fact, the expression of E-selectin was greater in the presence of ICI-182780 than in cells treated with vehicle alone, likely due to residual estrogenic activity present in the "depleted" medium.



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  		Figure 2. Regulation of E-selectin expression by ER{alpha} and ERß. For agonist activity characterization, HUVEC infected with adenovirus expressing ER{alpha} (A) or ERß (B) were treated with 17ß-estradiol ({bullet}), or 7{alpha}-TP-E2 ({circ}). After 16 h of incubation, the cells were stimulated by the addition of 30 U/ml IL-1ß. Five hours later, the cells were fixed, and relative surface E-selectin levels were determined by ELISA. In each experiment the fold induction of E-selectin expression in the absence of compound was defined as 100%. Values are the mean relative induction of E-selectin expression in the presence of the indicated molar concentration of compounds. Results are the mean determinations from four independent experiments, with each concentration assayed in triplicate in each experiment. Error bars denote the SE. *, P < 0.01 for comparison to the fold induction in the absence of compound. For antagonist activity characterization, HUVEC expressing ER{alpha} (C) or ERß (D) were treated with 2 nM 17ß-estradiol plus the indicated molar concentration of ICI-182780 ({blacktriangleup}) or 7{alpha}-TP-E2 ({triangleup}). *, P < 0.01 for comparison to fold induction in the presence of 2 nM 17ß-estradiol alone.

 
Estrogen has been shown to decrease plasma levels of ET-1 in postmenopausal women and in animals, primarily due to a decrease in the rate of ET-1 production (49). Treatment of HUVEC expressing either ER{alpha} or ERß with increasing concentrations of 17ß-estradiol reduced ET-1 production by up to 50% and 30%, respectively (Fig. 3Go), a degree of inhibition comparable with the magnitude of the in vivo decreases. These inhibitions were again seen at low concentrations of ligand, with 1 nM 17ß-estradiol producing maximal activity through ER{alpha} and 10 nM 17ß-estradiol producing maximal activity through ERß. The addition of increasing concentrations of ICI-182780 completely blocked the inhibition of ET production by 2 nM 17ß-estradiol in HUVEC expressing either ER{alpha} or ERß (Fig. 3Go).



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  		Figure 3. Regulation of ET-1 expression by ER{alpha} and ERß. For agonist activity characterization, HUVEC infected with adenovirus expressing ER{alpha} (A) or ERß (B) were treated with 17ß-estradiol ({bullet}) or 7{alpha}-TP-E2 ({circ}). After 6 h of incubation, the medium was removed and assayed for ET-1 content by ELISA. In each experiment the production of ET-1 in the absence of compound was defined as 100% (values ranged from 61–126 pg/ml). Values are the mean relative ET-1 levels in the presence of the indicated molar concentration of compounds and were derived from two or three independent experiments, with each concentration assayed in triplicate in each experiment. Error bars denote the SE. *, P < 0.05 for comparison to ET-1 levels produced in the absence of compound. For antagonist activity characterization, HUVEC expressing ER{alpha} (C) or ERß (D) were treated with 2 nM 17ß-estradiol plus the indicated molar concentration of ICI-182780 ({blacktriangleup}) or 7{alpha}-TP-E2 ({triangleup}). {square}, Relative production of ET-1 in the presence of 2 nM 17ß-estradiol only. *, P < 0.05 for comparison to fold induction in the presence of 2 nM 17ß-estradiol alone.

 
To examine the ability of ER{alpha} and ERß to induce gene expression in endothelial cells, it was first necessary to identify genes induced by estrogen treatment of endothelial cells. Differential expression analysis of HUVEC expressing ER (data not shown) revealed a novel gene, designated here EER-7, which was induced up to 100-fold by treatment with 17ß-estradiol. EER-7 is highly homologous to the recently identified mouse LOXC gene (50) in both primary amino acid sequence and gene structure (Fig. 4Go). Human aorta was among the tissues with the highest level of expression of EER-7 (Fig. 5Go), suggesting that endothelial cells may be a primary site of expression of EER-7 in vivo. The promoter of the human EER-7 gene contains a very strong match (AGGGTCATTAAGACCTG at -1638) to the canonical ERE 5'-C(A/G)GGTCAnnnTGACC(T/C)G-3' (51) (underline denotes core consensus sequence). The mouse LOXC promoter also contains two near-perfect EREs (TCTGTCACGTTGACCTC at -2501 and ATGGTCAAGGTGACCTG at -1010), whereas the corresponding rat promoter also contains a strong ERE (ATGGTCAGGGCGACCTG at -956). The conservation of near-perfect ERE throughout evolution suggests the EER-7 promoter is directly activated by ERs. As for the E-selectin and ET-1 responses, both ER{alpha} and ERß functioned similarly to stimulate expression of EER-7 at the mRNA level in the presence of 17ß-estradiol, but were inactive in the presence of ICI-182780 (Fig. 6Go).



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  		Figure 4. A novel member of the lysyl oxidase family induced by 17ß-estradiol. A, The protein sequence of EER-7 compared with that of LOXC, a recently described novel member of the mouse lysyl oxidase gene family. Amino acids identical between the human EER-7 and mouse LOXC sequence are overlined. B, The human EER-7 and mouse LOXC gene structures were identified from the respective complete genomes (Celera Genomics, Rockville, MD). A portion of the rat promoter was also identified in the rat genome (NIH). Exons are denoted by numbers. The translation start codon is located in exon 2, denoted by an arrow. The human exon 1 containing the 5'-noncoding region sequence was identified by comparison with a human cDNA, whereas the mouse exon 1A containing the 5'-noncoding region sequence was identified by comparison with a mouse cDNA. Exon 1A is conserved in the human genome, suggesting that alternative exons 1 may be used in the human. The positions of putative EREs are indicated in each promoter.

 


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  		Figure 5. A human RNA dot blot was hybridized with a labeled probe derived from the 3'-noncoding region of EER-7 to minimize cross-reactivity with other members of the lysyl oxidase family. The tissues assayed were whole brain (Br), amygdala (Am), caudate nucleus (CN), cerebellum (Cb), cerebral cortex (CC), frontal lobe (FL), hippocampus (Hc), medulla oblongata (MO), occipital lobe (OL), putamen (Pu), substantia nigra (SN), temporal lobe (TL), thalamus (Th), acumens (Ac), spinal cord (SC), heart (H), aorta (Ao), skeletal muscle (SM), colon (C), bladder (B), uterus (U), prostate (Pr), stomach (S), testis (T), ovary (O), pancreas (Pa), pituitary gland (Pi), adrenal gland (Ad), thyroid gland (Ty), salivary gland (Sa), mammary gland (M), kidney (K), liver (Li), small intestine (SI), spleen (Sp), thymus (Tm), leukocytes (Le), lymph node (Ly), bone marrow (BM), appendix (Ap), lung (Lu), trachea (Tr), placenta (Pl), fetal brain (fB), fetal heart (fH), fetal kidney (fK), fetal liver (fL), fetal spleen (fS), fetal thymus (fT), and fetal lung (fLu).

 


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  		Figure 6. Activation of EER-7 gene expression by ER{alpha} and ERß. HUVEC cells expressing either ER{alpha} (top panels) or ERß (lower panels) were treated with 300 nM of the indicated compounds (left panels) or 3 nM 17ß-estradiol plus 1 µM of the indicated compounds (right panels). After 18 h of incubation, total RNA was prepared and analyzed by Northern blotting to determine EER-7 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) message levels. The Northern blots shown are from one of two independent experiments performed with similar results.

 
A second gene identified by differential expression analysis as being induced by 17ß-estradiol treatment was tissue type transglutaminase (TGM2; Hs.8265). The proximal human tissue transglutaminase promoter has an 8 of 10 match (GCGGTCAAGGCTACCTG) to the canonical ERE, which may mediate this induction. Treatment of HUVEC expressing either ER{alpha} or ERß with 17ß-estradiol resulted in approximately a 2-fold increase in cellular transglutaminase activity (Fig. 7Go). Similarly, the levels of tissue transglutaminase protein were increased by 17ß-estradiol treatment of cells expressing either ER{alpha} or ERß, but not in cells infected with a control ß-galactosidase adenovirus or in uninfected cells. ICI-182780 was able to block the 17ß-estradiol inductions.



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  		Figure 7. Increase in transglutaminase by ER{alpha} and ERß. Uninfected HUVEC or HUVEC infected with adenoviruses expressing the indicated receptors or ß-galactosidase were treated with vehicle, 30 nM 17ß-estradiol, or 1 µM ICI-182780 for approximately 24 h. A, Transglutaminase activity was determined by monitoring the incorporation of [3H]putrescine into total cellular protein. B, Transglutaminase protein levels were determined by Western blot analysis using a monoclonal antibody specific for transglutaminase type II.

 
Pharmacological dissociation of ER{alpha} and ERß responses in HUVEC
None of the above-characterized responses can provide a mechanism to differentiate ER{alpha} from ERß activity in vivo, as both receptors had similar potentials to interact with multiple endogenous signaling pathways. However, these responses have allowed identification of a compound [7{alpha}-thiophenyl-17ß-estradiol (7{alpha}-TP-E2)] that functions as an ER{alpha} agonist and ERß antagonist in each of these diverse pathways. 7{alpha}-TP-E2 binds in vitro with high affinity to both ER{alpha} and ERß bacterially expressed ligand binding domains (Table 1Go) and to full-length ER{alpha} and ERß expressed in HUVEC (Fig. 8Go). In both systems 17ß-estradiol had a similar potency for ER{alpha} and ERß, whereas 7{alpha}-TP-E2 had a few-fold higher potency for ERß than for ER{alpha}. This difference was more prominent in the HUVEC whole cell binding assay, possibly attributable to the use of the full-length receptor in HUVEC compared with use of only the ligand binding domain of the ER in the in vitro assays.


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  		Table 1. In vitro binding of ligands to ER{alpha} and ERß

 


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  		Figure 8. Binding of 7{alpha}-TP-E2 to ER{alpha} and ERß. HUVEC infected with adenovirus expressing either human ER{alpha} ({bullet}) or ERß ({circ}) were treated with 0.8 nM [3H]17ß-estradiol with increasing concentrations of unlabeled 17ß-estradiol (A) or 7{alpha}-TP-E2 (B). The binding of [3H]17ß-estradiol in the absence of competitor was defined as 100% in each experiment. Values are the mean relative [3H]17ß-estradiol binding in the presence of the indicated molar concentration of competitor. Triplicate determinations were made for each concentration of compound, with the SD shown for each value. Data shown are from a representative experiment of multiple experiments performed. Fifty percent inhibitory concentration (IC50) values were determined using a four-parameter regression model. *, P < 0.05 for comparison of ER{alpha} and ERß IC50 values by ANOVA. C, The structure of 7{alpha}-TP-E2.

 
Surprisingly, 7{alpha}-TP-E2 functioned as an ER{alpha} agonist, but ERß antagonist, for inhibition of E-selectin induction by IL-1ß (Fig. 2AGo), basal ET-1 production (Fig. 3AGo), and induction of EER-7 mRNA (Fig. 6Go). In each system, cells expressing ER{alpha} had a similar maximal response to treatment with either 17ß-estradiol or 7{alpha}-TP-E2. 7{alpha}-TP-E2 was approximately 100-fold less potent than 17ß-estradiol for regulation of E-selectin expression (Fig. 2Go) and 30-fold less potent for inhibition of ET-1 production (Fig. 3Go), which corresponds well with the relative in vivo binding affinities of 17ß-estradiol and 7{alpha}-TP-E2 (Fig. 8Go). 7{alpha}-TP-E2 had no agonist activity in HUVEC expressing ERß for any of these regulations. In contrast, 7{alpha}-TP-E2 was able to inhibit 17ß-estradiol activation of ERß activity to the same extent as ICI-182780 for regulation of E-selectin expression (Fig. 2DGo), ET-1 production (Fig. 3DGo), and EER-7 mRNA induction.

To ascertain whether the activity of 7{alpha}-TP-E2 was unique to endothelial cells, HepG2 cells were cotransfected with ER expression plasmids along with an NF-{kappa}B-driven luciferase reporter plasmid. Both ER{alpha} and ERß were able to inhibit this NF-{kappa}B-driven reporter response (Fig. 9AGo), consistent with the results obtained on E-selectin expression in HUVEC. 7{alpha}-TP-E2 also inhibited NF-{kappa}B promoter activation in cells expressing ER{alpha}, but not in cells expressing ERß. Rather, 7{alpha}-TP-E2 functioned similar to ICI-182780 in cells expressing ERß and inhibited ERß activity (Fig. 9BGo). Thus, 7{alpha}-TP-E2 activity as an ER{alpha} agonist and ERß antagonist is not specific for either a particular estrogen response pathway or a particular cell type.



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  		Figure 9. Activity of 7{alpha}-TP-E2 in HepG2 cells. HepG2 cells were cotransfected with a luciferase reporter gene under transcriptional control of a synthetic NF{kappa}B-dependent promoter along with an ER{alpha} or ERß expression plasmid as indicated. For agonist activity determinations (A), the following day the cells were treated with 100 U/ml IL-1ß plus 100 nM 17ß-estradiol, 1 µM ICI-182780, or 2 µM 7{alpha}-TP-E2. For antagonist activity determinations (B), the cells were treated for 6 h with 100 U/ml IL-1ß plus 10 nM 17ß-estradiol, or 10 nM 17ß-estradiol plus either 1 µM ICI-182780 or 2 µM 7{alpha}-TP-E2. Values are the mean ± SE from two independent experiments, with each value determined in triplicate. The normalized luciferase activity produced in the vehicle-treated samples was defined as 100 for each series. *, P < 0.01 for comparison with the absence of compound (A) or the presence of 10 nM 17ß-estradiol only (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although endothelial cells express both ER{alpha} and ERß, their contributions to specific endothelial cell functional end points in response to ER agonists has not been thoroughly investigated. By using adenoviruses selectively expressing either ER{alpha} or ERß we demonstrate here that both receptors can inhibit IL-1ß induction of E-selectin expression (Fig. 2Go) and VCAM-1 expression (not shown). Maximal receptor activity required the presence of approximately 1 nM 17ß-estradiol, within the physiological range in humans (52). Induction of E-selectin promoter activity depends upon both NF-{kappa}B and ATF2 pathways (53, 54). Glucocorticoid receptor inhibits the induction of E-selectin expression by selectively blocking the NF-{kappa}B pathway (55). The ability of ER{alpha} and ERß to inhibit expression from an NF-{kappa}B-driven reporter plasmid (Fig. 9Go) suggests that this may also be the mechanism for ER inhibition of E-selectin induction.

Recently, ERßKO, but not ER{alpha}KO, mice have been shown to develop systemic and diastolic hypertension due to a defect in smooth muscle function (56). In humans, polymorphisms at both the ER{alpha} and ERß loci have been associated with the development of hypertension (57, 58). One potential explanation for these observations is the ability of estrogen treatment to lower plasma levels of ET-1. ET-1 levels are elevated within the atherosclerotic lesion, where it is produced by endothelial cells, macrophages, and smooth muscle cells (32). Both ER{alpha} and ERß lowered basal production of ET-1 in HUVEC to a magnitude similar to that seen in women.

In contrast to the known inhibitory effects of estrogens on endothelial cells, little is known about the ability of estrogens to induce gene expression in endothelial cells. By differential gene expression analysis we identified several genes induced in HUVEC expressing ER. Notable among these were two genes involved in the maturation of the extracellular matrix. First, ER{alpha} and ERß both induced the expression of a novel gene, designated here EER-7, by as much as 100-fold. The EER-7 protein is greater than 90% identical in amino acid sequence to the recently described LOXC (50) protein. Further, the human EER-7 and murine LOXC genes have an identical exon structure, and both contain strong ERE elements within their promoters, suggesting that they are true orthologs. The LOXC gene has been demonstrated to be a novel member of the lysyl oxidase family, capable of cross-linking type I and type II collagen. A human multiple tissue RNA blot demonstrated that EER-7 expression was very high in the aorta, suggesting that this gene may, in fact, be important in the maintenance of vascular wall structure. A second gene regulated by ER in HUVEC at the RNA, protein, and activity levels is tissue transglutaminase. Tissue transglutaminase is also involved in the cross-linking of extracellular matrix proteins, including collagen. Potentially, estrogen up-regulation of matrix cross-linking proteins, such as EER-7 and tissue transglutaminase, contributes to the low incidence of aortic aneurysms seen in women compared with men (59). In regard to atherosclerosis, transglutaminase is required for activation of latent TGFß (60), and tamoxifen elevation of TGFß levels correlates with its protection against lesion development in the mouse (61). ER induction of transglutaminase in endothelial cells may explain this correlation.

The ability of ER{alpha} and ERß to regulate the same genes in HUVEC is consistent with the expression of both receptors in endothelial cells and the apparent redundancy of ER{alpha} and ERß in certain animal models, but it does not imply that both receptors necessarily mediate all these effects in vivo. For this conclusion to be drawn, a pharmacological dissociation between ER{alpha} and ERß in these pathways is necessary. 7{alpha}-TP-E2, a 7{alpha}-thio-substituted 17ß-estradiol (62), binds to ER{alpha} and ERß with similar potencies, but acts as an ER{alpha} agonist and an ERß antagonist for E-selectin inhibition, ET-1 inhibition, and EER-7 induction. Recently, two other compounds, R,R-THC, a cis-diethyl-substituted tetrahydrochrysene (63), and 2,2-bis-(p-hydroxy-phenyl)-1,1,1-trichloroethane (64) have been shown to act as ER{alpha} agonists and ERß antagonists in cotransfection assays measuring ER ability to stimulate gene expression. It is unclear that these compounds would maintain this pattern of activity on endogenous promoters in multiple stimulatory and inhibitory pathways. For example, we have recently found that in the mouse liver raloxifene and genistein can function as ER{alpha} agonists for inhibition of inflammatory gene induction, but are inactive for stimulation of gene expression (65). Further, receptor cross-reactivity will be an important parameter for this class of compounds. In fact, 2,2-bis-(p-hydroxy-phenyl)-1,1,1-trichloroethane has recently shown to be an antagonist of androgen receptor activity (66). As 7{alpha}-TP-E2 is a derivative of 17ß-estradiol, and steroidal estrogens typically do not interact strongly with other nuclear hormone receptors, it may retain a high degree of receptor selectivity.

The similarity of the binding potency of 7{alpha}-TP-E2 for ER{alpha} compared with ERß along with the high degree of conservation between ER{alpha} and ERß of ligand-interacting amino acids suggest that 7{alpha}-TP-E2 may make similar contacts to each receptor. However, the overall resulting protein structure is likely to be very different: ER{alpha} folds into a conformation capable of interacting productively with coactivators, whereas ERß fails to do so. The crystal structure of ER{alpha} with 17ß-estradiol revealed a large unoccupied cavity opposite the {alpha} face of the B ring (67). The 7{alpha}-TP substituent of 7{alpha}-TP-E2 could potentially be accommodated in this cavity in ER{alpha}. Alteration of this cavity in ERß might allow 7{alpha}-TP-E2 to still bind, but the resulting overall structure of the ERß protein could be altered to a form unable to interact with necessary cofactors. Similarly, ICI-182780 is comprised of 17ß-estradiol with a very large side-chain at the 7{alpha} position (68). However, this side-chain may be sufficiently large so as to be unable to be accommodated by either the ER{alpha} or ERß ligand-binding domains without distorting the receptor into a conformation unable to productively interact with coactivators. Structure-activity studies of compounds similar to 7{alpha}-TP-E2 indicate that the composition of the 7{alpha} group is indeed a major factor in establishing the ER{alpha} agonist/ERß antagonist activity pattern (data not shown). The structures of R,R-THC bound to both ER{alpha} and ERß have recently been determined (69). As expected, helix 12 in ER{alpha} bound to R,R-THC is in the same position as when E2 is bound. Surprisingly, helix 12 in ERß bound to R,R-THC is in a position similar to that seen with the agonist genistein and is not in a position similar to the helix 12 orientation observed when the antagonists raloxifene and ICI-182780 are bound. Whether this "passive antagonism" would also occur with 7{alpha}-TP-E2 is unknown.

In summary, the results in this study suggest that ER{alpha} and ERß can play redundant roles in at least three distinct regulatory pathways: inhibition of inflammatory cytokine-mediated endothelial cell activation, inhibition of basal ET-1 production, and induction of matrix-stabilizing enzymes. In addition, the results in this study document a unique profile of novel steroidal ER ligand, 7{alpha}-TP-E2, that functions as a dual ER{alpha} agonist and ERß antagonist. This compound may be useful in elucidating the contributions of these receptors to endothelial cell function in vivo.


   Acknowledgments
 
We thank A. Bapat, L. Hsu, R. Mastroeni, and M. Scicchitano for technical assistance.


   Footnotes
 
1 Current address: Pfizer, Inc., Global Research and Development, Cardiovascular Pharmacology, Ann Arbor, Michigan 48105. Back

Abbreviations: AP-1, Activating protein-1; EBM, endothelial cell basal medium; EER-7, endothelial estrogen-regulated gene-7; ER, estrogen receptor; ERE, estrogen response element; ET-1, endothelin-1; HDL, high density lipoprotein; HRT, hormone replacement therapy; HUVEC, human umbilical vein endothelial cells; KO, knockout; MOI, multiplicity of infection; NF-{kappa}B, nuclear factor-{kappa}B; RR-THC, cis-diethyl-substituted tetrahydrochrysene; 7{alpha}-TP-E2, 7{alpha}-thiophenyl-17ß-estradiol; VCAM-1, vascular cellular adhesion molecule 1.

Received March 28, 2002.

Accepted for publication June 4, 2002.


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