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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 5 2373-2381
Copyright © 2004 by The Endocrine Society

Differential Regulation of Estrogen Receptor Subtypes {alpha} and ß in Human Aortic Smooth Muscle Cells by Oligonucleotides and Estradiol

Federica Barchiesi, Edwin K. Jackson, Bruno Imthurn, Juergen Fingerle, Delbert G. Gillespie and Raghvendra K. Dubey

Department of Obstetrics and Gynecology (F.B., B.I., R.K.D.), Clinic for Endocrinology, University Hospital Zurich, 8091 Zurich, Switzerland; Center for Clinical Pharmacology (E.K.J., D.G.G., R.K.D.), Departments of Medicine and Pharmacology (E.K.J.), University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213-2582; and Preclinical Pharma Research 68/209 (J.F.), F. Hoffmann La-Roche, CH-4070 Basel, Switzerland

Address all correspondence and requests for reprints to: Dr. Raghvendra K. Dubey, Department of Obstetrics and Gynecology, Clinic for Endocrinology D217, NORD-1 Frauenklinik University Hospital Zurich, CH-8091 Zurich, Switzerland. E-mail: raghvendra.dubey{at}usz.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the mechanisms regulating estrogen receptor (ER) expression in human aortic smooth muscle cells (HASMCs) and the mechanisms by which estradiol inhibits HASMC growth. The autologous down-regulation pathway involves binding of liganded ER to the ER gene, thus suppressing transcription. Blockade of this pathway with sense and AS-OLIGOs to ERs up-regulated the expression of ER{alpha} but not ERß. Activation of the autologous down-regulation pathway with ER agonists down-regulated the expression of ER{alpha} but not ERß. The proteasomal degradation pathway entails ubiquination of liganded ER, followed by proteasome-mediated degradation. Blockade of the proteasomal degradation pathway increased the expression of ERß. Up-regulation of ER{alpha} by AS-OLIGOs did not increase the antimitogenic effects of estradiol on HASMCs; the estradiol metabolites 2-hydroxyestradiol and 2-methoxyestradiol were more potent inhibitors of HASMC growth, compared with estradiol; and blockade of metabolism of estradiol to hydroxyestradiols and methoxyestradiols abrogated the inhibitory effects of estradiol on HASMC growth. We conclude that, in HASMCs: 1) the expression of ER{alpha} is regulated by the autologous downregulation pathway; 2) the expression of ERß is governed by the proteasomal degradation pathway; and 3) the antigrowth effects of estradiol are not mediated by ER{alpha}, but rather by metabolism of estradiol to methoxyestradiols.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANIMAL STUDIES SUGGEST that estrogens protect blood vessels from disease (1); however, recent randomized, placebo-controlled primary and secondary prevention clinical trials of conjugated equine estrogens with progestin do not support this hypothesis (2, 3). The reasons for these discordant findings are unclear. To correctly interpret the results of animal and clinical studies, it is critical to elucidate the mechanisms by which estrogens influence the vessel wall and to identify the independent variables that may influence the vascular actions of estrogens.

An important variable that may govern the vascular actions of estrogens is the vascular expression of estrogen receptors (ERs). There are two isoforms of ERs, ER{alpha} and ERß, and evidence suggests that ER{alpha} and ERß may perform distinct functions in the vessel wall. Thus the regulation of the expression of these two ER subtypes in the vessel wall may importantly modulate the actions of estrogens in the vascular system. For example, one study in ER{alpha} knockout mice suggests that ER{alpha} may inhibit vascular injury-induced lesion formation (4). Another study reports increases in arterial blood pressure in ERß knockout mice, (5); and in Japanese postmenopausal women, a specific polymorphism in the ERß gene is associated with hypertension (6). Together, these studies suggest that ER{alpha} and ERß play a critical role in modulating vascular injury and regulating vascular tone, respectively.

Studies in several tissues and cell types indicate that two mechanisms, i.e. autologous down-regulation (7) and proteosome-mediated degradation (8, 9, 10, 11), regulate the expression of ERs. The autologous down-regulation pathway involves binding of liganded ER to ER genes, thus suppressing transcription of ER genes (7, 12). By this mechanism, estrogens induce a decline in both ER protein and mRNA (12). It is important to note that antisense (AS) and sense (S), but not scrambled, oligonucleotides (OLIGOs) block the autologous down-regulation pathway and up-regulate ER expression by serving as decoys that bind liganded ERs and thus prevent binding of liganded ERs to ER genes (13). The proteosome-mediated degradation pathway involves ubiquination of liganded ERs followed by proteasome-mediated degradation (8, 9, 10, 11), and this pathway can be inhibited by a number of agents, including cycloheximide (7, 8), lactacystin (9), and MG132 (8, 9, 10, 11), as well as by inhibition of the MAPK cascade using inhibitors of MAPK kinase (MEK) such as PD98059 (14, 15).

Similar to other estrogen-sensitive tissues, ER expression in the vasculature is also highly regulated. In the vena cava, both estradiol and tamoxifen cause autologous down-regulation of ER{alpha} gene expression (16). In contrast, in cerebral microvessels, deprivation of estradiol is associated with a significant decrease in the expression of ER{alpha} and ERß (17), and estradiol replacement up-regulates ER{alpha} but does not influence ERß expression (17). Together these findings suggest that the expression of ERs {alpha} and ß may be regulated by different mechanisms that vary among different types of blood vessels (18).

Studies of ER expression in whole blood vessels do not distinguish between changes in the expression of ERs in endothelial cells vs. vascular smooth muscle cells (SMCs). In this regard, several studies describe the regulation of ERs in vascular endothelial cells. In human uterine arterial endothelial cells, estradiol down-regulates both ER{alpha} and ERß expression, and these effects are blocked by the partial ER antagonist tamoxifen (8). Proteolysis inhibitors also selectively abrogate estradiol-induced down-regulation of ER{alpha}, but not ERß, suggesting that estradiol-induced down-regulation of ER{alpha} is mediated by the proteasomal degradation pathway, whereas down-regulation of ERß is not (8). In ovine intrapulmonary artery endothelial cells, short-term (2 h) treatment with estradiol down-regulates ER{alpha}, but not ERß, expression, whereas long-term (24 h) treatment up-regulates ER{alpha} and down-regulates ERß expression (19). Also, in ovine intrapulmonary artery endothelial cells, ER antagonists, such as ICI182780, block the effects of estradiol on ER expression (19); whereas in other tissues, the effects of estradiol on ER down-regulation is not blocked by ICI182780 (9, 20).

ER{alpha} and ERß expression is also clearly present in isolated vascular SMCs (21) and is regulated by estradiol (22). However, little is known regarding the mechanisms that govern the expression of ER isoforms in vascular SMCs. Therefore, one goal of the present study was to investigate the mechanisms regulating ER{alpha} and ERß expression in human aortic vascular SMCs (HASMCs).

A second goal of this study was to determine whether ER{alpha} participates in the inhibitory effects of estradiol on growth of HASMCs. In this regard, we discovered that blockade of the autologous down-regulation pathway with decoy OLIGOs markedly increases the expression of ER{alpha} in HASMCs, and we employed this method to determine whether increasing ER{alpha} expression would enhance the ability of estradiol to inhibit growth of HASMCs.

The results of this study show, for the first time, that in HASMCs, the expression of ER{alpha} is regulated by the autologous down-regulation pathway, whereas the expression of ERß is governed by the proteosomal degradation pathway. This study also provides additional evidence that the antigrowth effects of estradiol are not mediated by ER{alpha}, but rather by metabolism of estradiol to methoxyestradiols.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

Human female aortic HASMCs between 6–8th passage (Cascade Biologics, Inc., Portland, OR) were cultured under standard tissue culture conditions in M231 culture medium (Cascade Biologics, Inc.) supplemented with smooth muscle growth supplement (Cascade Biologics, Inc.) and as described previously (9). MCF-7 cells in 8th passage (ATCC, Rockville, MD) were cultured under standard tissue culture conditions in DMEM/F12 (Life Technologies AG, Basel, Switzerland) supplemented with 10% fetal calf serum (Hyclone, Perbio Science Switzerland SA, Lausanne, Switzerland).

OLIGOs

GenBank and NCBI sequence viewer were used to obtain the human ER{alpha} and ERß cDNA sequences, and MacVector 4.1 was used to select the sequences complementary to ER{alpha} and ERß mRNA. OLIGOs were commercially synthesized and purified (Microsynth GmbH, Balgach, Switzerland). The phosphorothioated sequences of the various OLIGOs used are shown in Table 1Go.


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  		TABLE 1. Phosphorothioated sequences of the various OLIGOs used

 
Treatments

HASMCs were grown to subconfluency in the presence of optimal growth medium. Subsequently, monolayers were washed and treated for 48 h with DMEM/F12 (phenol red free) supplemented with 1% (vol/vol) FuGENE 6 (Roche Diagnostic AG, Rotkreuz, Switzerland) and containing or lacking 10 µmol/liter phosphorothioated OLIGOs. In some experiments, cells were treated with OLIGOs in the presence of 25 ng/ml PDGF-BB (Sigma, Buchs, Switzerland), or 10 µmol/liter of the protein synthesis inhibitor cycloheximide (Sigma), the MEK inhibitor PD98059 (23) (Sigma), or the proteasome inhibitors lactacystin (Calbiochem, Juro Supply GmbH, Lucerne, Switzerland) or MG132 (Sigma). At the end of the experiment, cells were lysed, and the expressions of ER{alpha} and ERß were evaluated in whole-cell lysates by Western blot analysis.

To study the effects of estradiol on ER expression, subconfluent HASMCs or MCF-7 cells in 60-mm dishes were treated for 48 h with complete growth medium containing or lacking various concentrations (0–1000 nmol/liter) of estradiol (Steraloids, Newport, RI) or vehicle (0.1% dimethylsulfoxide). To study the role of ERs in mediating the effects of estradiol on ER{alpha} and ERß expression, we evaluated the effects of ICI82780 (ER{alpha}/ß antagonist; 1 µmol/liter), tamoxifen (agonist via ER{alpha} and antagonist via ERß; 1 µmol/liter) and 4,4'-[4-methyl-5-[4-[2-(1-piperidnyl) ethoxy]-phenyl-1H-pyrazole]-1,3 diyl]-bis-phenol HCl (5 µmol/liter; MPP, selective ER{alpha} antagonist; Obiter research, Urbana, IL) (24). We also used agonists selective for ER{alpha} and ERß. In these experiments, we tested the effects of propyl pyrazole triol (PPT) (25), a selective ER{alpha} agonist (100 nmol/liter; Tocris, Cookson Ltd., Bristol, UK) and 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) (26), a selective ERß agonist (10 nmol/liter; Tocris) by treating the cells for 48 h and subsequently analyzing the levels of ER{alpha} and ERß. To study whether the effects of estradiol on ER levels are influenced by OLIGOs, the cells were preincubated for 2 h with AS OLIGOs before the treatment with estradiol. After 48 h, ER{alpha} and ERß expression in whole-cell lysates were evaluated by Western blot analysis.

Analysis of ER{alpha} and ERß by Western blotting

Cells were lysed in 70 µl lysis buffer [50 mmol/liter Tris/HCl, pH 7.5–8; 150 mmol/liter NaCl; 1% deoxycholate; 0.2% sodium dodecyl sulfate (SDS); 1% Triton-X; 10 µg/ml aprotinin; 10 µg/ml leupeptin; 0.287 mmol/liter phenylmethylsulfonylfluoride], and 20 µg protein from whole-cell lysates were diluted in 5x loading buffer (0.313 mmol/liter Tris/HCl, pH 6.8; 10% SDS; 0.05% bromphenol blue; 50% glycerol, Fermentas, Inc., Hanover, MD). After addition of 0.1 mol/liter dithiothreitol and 2.5% 2-mercaptoethanol, the samples were denaturated by boiling at 95 C for 5 min and then loaded onto a 10% SDS polyacrylamide gel.

After electrophoretic migration, the proteins were transferred to a nitrocellulose membrane (Schleicher und Schuell, Bottmingen, Switzerland), and the membrane was probed with specific antibodies against ER{alpha} (Alexis, 210–201-C050, no cross-reactivity with ERß) or ERß (Alexis, 210–180-C050, no cross reactivity with ER{alpha}). The immunoreactive proteins were subsequently detected after incubation with a peroxidase-conjugated secondary antibody (antirabbit IgG, Sigma) through ECL reaction (Pierce Biotechnology Inc., Rockford, IL) and exposure to X-OMAT LS films (Kodak, Rochester, NY).

To test the specificity of the ER{alpha} and ERß antibodies used, the Western blots of the cell lysates were probed with the respective antibodies in the presence and absence of the respective neutralizing ER{alpha} or ERß peptides. Briefly, to neutralize the ERs, 0.5 µg/ml ER{alpha} or ERß antibodies were incubated overnight at 4 C with 15 µg/ml of their respective immunizing peptides in PBS containing 0.1 mg/ml BSA.

Growth studies

All growth studies were conducted using phenol red free culture medium and steroid free FCS. To evaluate the role of ER{alpha} in mediating the growth inhibitory effects of estradiol in HASMCs, we assayed the inhibitory effects of estradiol on PDGF-BB (25 ng/ml)-induced DNA synthesis, collagen synthesis, and cell proliferation in HASMCs pretreated with or without OLIGOs. Briefly, subconfluent monolayers of HASMCs were growth-arrested by feeding DMEM containing 0.25% albumin plus 1% (vol/vol) FuGENE 6 (Roche Diagnostics AG, Schweiz) for 48 h in the presence or absence of 10 µmol/liter AS OLIGOs. Growth was induced for 24 h by treating HASMCs in the presence of fresh OLIGOs with PDGF-BB, and DNA synthesis was evaluated after 24 h. For cell proliferation studies, cells were treated in the presence of fresh OLIGOs with PDGF-BB, every 48 h for 6 d, and changes in cell number were assayed. For collagen synthesis, confluent HASMCs were treated for 48 h.

To evaluate whether the inhibitory effects of estradiol were ER-independent and mediated by the sequential conversion of estradiol to methoxyestradiol, we studied the effects of estradiol on PDGF-BB-induced HASMC growth (DNA synthesis and cell number) in the presence and absence of 1-aminobenzotriazole (a broad-spectrum CYP450 inhibitor that inhibits the conversion of estradiol to the methoxyestradiol precursor hydroxyestradiol) (27), OR486 [a catechol-O-methyltransferase (COMT) inhibitor that inhibits the conversion of estradiol/hydroxyestradiol to methoxyestradiol and does not bind to ERs] (27), and ICI182780 (an ER antagonist).

As previously described by us (23), 3H-thymidine and 3H-L-proline incorporation were used to assay DNA and collagen synthesis, respectively, and cell counting was performed for cell proliferation studies. Each experiment was conducted in triplicate and with three separate cultures of HASMCs. To assure that the inhibitory effects of the experimental agents on collagen synthesis were not due to changes in cell number, the experiments were conducted in confluent monolayers of cells in which changes in cell number were precluded. Additionally, cell counting was performed in cells treated in parallel with the cells used for the collagen synthesis studies, and the data were normalized to cell number.

Statistics

Data were analyzed using ANOVA and statistical significance (P < 0.05) calculated using Fisher’s LSD test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To assess the specificity of our assay system for ER{alpha} and ERß expression in HASMCs, Western blots were probed with the specific antibodies in the presence and absence of the respective neutralizing/blocking peptides. As shown in Fig. 1AGo, the ER{alpha} and ERß bands were expressed in HASMCs. It is important to note that the ER{alpha} and ERß bands disappeared in membranes incubated with preblocked ER{alpha} and ERß antibodies. To assess the influence of OLIGOs on ER{alpha} and ERß expression, cells were treated with specific OLIGOs in the presence of the transfection reagent FuGENE 6, which is known to increase OLIGO uptake by cells (28). As shown in Fig. 1BGo, treatment of cells with FuGENE 6 alone did not influence the expression of ER{alpha} or ERß, indicating that FuGENE 6 did not interfere with the Western blot method and did not influence ER expression. It is important that FuGENE 6 did not cause cell injury as assessed by trypan blue uptake and morphological analysis (data not shown).



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  		FIG. 1. Representative Western blots depicting the expression of ER{alpha} and ERß in HASMCs. A, Specificity of the antibodies used and the expression of ER{alpha} and ERß bands in the presence and absence of the neutralizing peptides. B, Presence of both ER isoforms in HASMCs and the influence of FuGENE 6, the transfection agent used, on ER{alpha} and ERß expression. Similar results were obtained in three separate experiments.

 
Treatment of HASMCs with 10 µmol/liter AS-OLIGO{alpha}1 to ER{alpha} up-regulated the expression of ER{alpha} by 184% compared with untreated HASMCs. In contrast to ER{alpha}, the expression of ERß was unchanged by AS-OLIGO{alpha}1 to ER{alpha} (Fig. 2Go). In HASMCs treated with 10 µmol/liter AS-OLIGOß1 to ERß, the expression of ER{alpha} was also up-regulated by 208% compared with untreated HASMCs, whereas the expression of ERß did not change (Fig. 2Go). Sense (S)-OLIGOs, either S-OLIGO{alpha}1 to ER{alpha} or S-OLIGOß1 to ERß, also increased the levels of ER{alpha}, but not ERß (Fig. 2Go).



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  		FIG. 2. Western blots depicting the modulatory effects of 10 µmol/liter AS- and S-OLIGOs to ER{alpha} (OLIGO{alpha}1) and ERß (OLIGOß1) on the expression of ER{alpha} and ERß in total cell lysates of HASMCs. The bar graphs show the densitometric analysis of the changes observed, normalized to the internal standard, ß-actin. The results are presented as mean ± SEM (n = 6). *, P < 0.05 vs. control cells (C).

 
To ascertain whether the stimulatory effects of OLIGOs were confined to certain specific regions of the ER mRNA, we analyzed and compared the effects of various AS-OLIGOs against 3'–regions as well as 5'–regions of ER{alpha} and ERß mRNA. As shown in Fig. 3AGo, all OLIGOs against ER{alpha} and ERß mRNA induced ER{alpha} expression regardless of their specificity for the 3'–region or 5'–region of ER{alpha} or ERß mRNA. In contrast to AS- and S-OLIGOs, expression of ER{alpha} and ERß was not influenced by scrambled (Scr) OLIGOs to ER{alpha} or ERß (Fig. 3BGo). Moreover, the expression of ERß was not altered by Scr-OLIGO to ERß (Fig. 3BGo).



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  		FIG. 3. A, Bar graphs showing the effects of various antisense AS-OLIGOs (10 µmol/liter) against 3' and 5' regions of ER{alpha} and ERß. The values depict the densitometric analysis of the ER{alpha} and ERß levels by Western blots and normalized to the internal standard, ß-actin. B, Western blots depicting the modulatory effects of Scr-OLIGOs to ER{alpha} and ERß on the expression of ER{alpha} and ERß. The results are presented as mean ± SEM (n = 3). *, P < 0.05 vs. control cells (C). hER, Human ER.

 
As shown in Fig. 4Go, the stimulatory effect of AS-OLIGO{alpha}1 or AS-OLIGOß1 on ER{alpha} expression was blocked by the protein synthesis inhibitor cycloheximide (Fig. 4AGo). In the presence of AS-OLIGOß1, cycloheximide significantly increased the expression of ERß (Fig. 4BGo). To assess whether the stimulatory effects of cycloheximide were related to its inhibitory effects on proteasome and MAPK activation, we assessed the effects of proteasome inhibitors and MEK inhibitors on ERß expression. The stimulatory actions of cycloheximide on ERß expression in the presence of AS-OLIGOß1 were mimicked by the proteasome inhibitors lactacystin and MG132 (Fig. 5AGo) and by the MEK inhibitor PD98059 (Fig. 5BGo). In cells treated with cycloheximide under basal conditions, the up-regulation of ERß expression was accompanied with a simultaneous down-regulation of phosphorylated ERK1/ERK2 (Fig. 5Go, C and D).



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  		FIG. 4. Western blots showing the modulatory effects of 10 µmol/liter AS-OLIGOs to ER{alpha} (AS-{alpha}1) and ERß (AS-ß1) on the expression of ER{alpha} (A) and ERß (B) in HASMCs in the presence and absence of the protein synthesis inhibitor cycloheximide (CY, 10 µmol/liter). ER{alpha} and ERß expression was measured in total cell lysates. The bar graphs show the densitometric analysis of the changes observed and normalized to the internal standard, ß-actin. The results are presented as mean ± SEM (n = 3). *, P < 0.05 vs. control cells (C).

 


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  		FIG. 5. A, Western blots showing the modulatory effects of 10 µmol/liter AS-OLIGOs to ERß (AS-ß1) on the expression of ERß in HASMCs in the presence and absence of the proteasome inhibitors lactacystin (LAC, 10 µmol/liter), MG132 (MG, 10 µmol/liter), and cycloheximide (CY; 10 µmol/liter). B, Western blots showing the modulatory effects of 10 µmol/liter AS-OLIGOs to ERß (AS-ß1) on the expression of ER{alpha} and ERß in HASMCs in the presence and absence of the MAPK inhibitor PD98059 (PD; 10 µmol/liter). C and D, Simultaneous changes in ERß and phosphorylated ERK1/ERK2 expression in HASMCs treated with cycloheximide (CY, 10 µmol/liter). The bar graphs show the densitometric analysis of the changes observed and normalized to the internal standard, ß-actin. The results are presented as mean ± SEM (n = 3). *, P < 0.05 vs. control cells (C).

 
As shown in Fig. 6Go, treatment of HASMCs with estradiol concentration-dependently reduced the expression of ER{alpha}, but not ERß. In contrast to ER{alpha}, a modest, but significant, increase in the expression of ERß was observed in HASMCs treated with high (nonphysiological) concentrations of estradiol. A maximal decrease of 65% in the expression of ER{alpha} was observed in HASMCs treated with 1000 nmol/liter estradiol. Similar to HASMCs, estradiol concentration-dependently decreased the expression of ER{alpha} in MCF-7 cells. However, in contrast to HASMCs, estradiol down-regulated the expression of ERß in the MCF-7 cells.



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  		FIG. 6. Representative Western blots showing the effects of estradiol (E2, 0–1000 nmol/liter) on the expression of ER{alpha} and ERß in HASMCs (A) and MCF-7 cells (B). The bar graphs show the densitometric analysis of the changes observed and normalized to the internal standard, ß-actin. The results are presented as mean ± SEM (n = 3). *, P < 0.05 vs. control cells (C).

 
To assess the role of ERs {alpha} and ß in regulating ER expression, we tested the effects of ICI182780 (ER{alpha}/ß antagonist), tamoxifen (agonist via ER{alpha} and antagonist via ERß), MPP (selective ER{alpha} antagonists), PPT (selective ER{alpha} agonist), and DPN (selective ERß agonist). Treatment of HASMCs with ICI182780, tamoxifen, and MPP down-regulated the expression of both ER{alpha} and ERß (Fig. 7Go). In some tissues, ligands for ERs such as ICI182780 and tamoxifen are potent down-regulators of ER expression (9, 16, 20). Treatment of HASMCs with the ER{alpha} agonist, PPT, down-regulated the expression of ER{alpha} but had no effect on ERß expression (Fig. 8Go). Moreover, treatment of HASMCs with ERß agonist, DPN, up-regulated ERß expression but down-regulated ER{alpha} expression (Fig. 8Go).



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  		FIG. 7. Western blots showing the effects of ICI182780 (ICI; ER{alpha} and ß antagonist; 1 µmol/liter); tamoxifen (TAM; ER{alpha} agonist and ERß antagonist; 1 µmol/liter), and MPP (ER{alpha} antagonist; 5 µmol/liter) on the expression of ER{alpha} and ERß in HASMCs. The bar graphs show the densitometric analysis of the changes observed and normalized to the internal standard, ß-actin. The results are presented as mean ± SEM (n = 3). *, P < 0.05 vs. control cells (C).

 


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  		FIG. 8. Western blots showing the effects of PPT (ER{alpha} agonist; 100 nmol/liter), DPN (ERß agonist, 10 nmol/liter), and estradiol (E2, 10 nmol/liter) on the expression of ER{alpha} and ERß in HASMCs. The bar graphs show the densitometric analysis of the changes observed and normalized to the internal standard, ß-actin. The results are presented as mean ± SEM (n = 3). *, P < 0.05 vs. control cells (C).

 
In cancer cell lines, intracellular ERs inhibit the transcriptional activity of their own genes by interacting with negative hormone-responsive elements located within the gene coding sequence (7). Consequently, in cancer cell lines, AS and S-OLIGOs stimulate ER expression by serving as decoys that prevent ERs from binding to regions of their own genes (13). To test whether the stimulatory effect of AS-OLIGOs on ER{alpha} in HASMCs was due to this same mechanism, we determined whether AS-OLIGOs blocked the down-regulatory effects of estradiol on ER expression. As shown in Fig. 9Go, treatment of HASMCs with estradiol significantly down-regulated ER{alpha}, and this was reversed by AS-OLIGO to ER{alpha}.



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  		FIG. 9. Representative Western blots showing effects of E2 (100 nmol/liter)-induced down-regulation of ER{alpha} in the absence and presence of antisense (AS-{alpha}1) OLIGOs to ER{alpha}. The bar graph shows the densitometric analysis of the changes observed and normalized to the internal standard, ß-actin. The results are presented as mean ± SEM (n = 3). *, P < 0.05 vs. control cells (C); §, P < 0.05 vs. cells treated with E2 alone.

 
Treatment of HASMCs with 10 µmol/liter ER{alpha} or ERß AS-OLIGOs up-regulated ER{alpha} expression (Fig. 10Go) but did not alter the stimulatory effects of PDGF-BB on DNA synthesis, cell number, or collagen synthesis (data not shown). Estradiol inhibited PDGF-BB-induced DNA synthesis, cell proliferation, and collagen synthesis (Fig. 10Go). In HASMCs pretreated with 10 µmol/liter AS-OLIGOs to ER{alpha}, ERß, or both ER{alpha} and ERß, the inhibitory effects of estradiol on PDGF-BB-induced DNA synthesis, cell proliferation, and collagen synthesis were not increased (Fig. 10Go). Similar to AS-OLIGOs, the inhibitory effects of estradiol on HASMC growth were not altered in the presence of S-OLIGOs to ER{alpha} and ERß (data not shown).



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  		FIG. 10. Inhibitory effects of E2 (1–100 nmol/liter) on PDGF-BB-induced DNA synthesis (3H-thymidine incorporation), collagen synthesis (3H-L-proline incorporation), and cell number in HASMCs treated with 10 µmol/liter AS-OLIGOs to ER{alpha}, ERß, or ER{alpha} and ERß. Top panel, Expression of ERs in HASMCs treated with AS-OLIGOs to ER{alpha} and ERß. Values represent mean ± SEM from three separate experiments, each conducted in quadruplicate. Treatment with AS-OLIGOs alone did not alter the growth of SMC, in the presence or absence of PDGF-BB (±2% change). *, P < 0.05 vs. control cells (C).

 
Estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol inhibited PDGF-BB-induced cell number, with 2-methoxyestradiol being the most potent (Figs. 11AGo). Treatment with AS-OLIGO up-regulated ER{alpha} expression (Fig. 11BGo). The inhibitory effects of estradiol were not blocked by simultaneous treatment with the ER antagonist ICI182780 (Fig. 11Go, C and D). Moreover, cotreatment with the CYP450 and COMT inhibitors, 1-aminobenzotriazole and OR486, respectively, abrogated the inhibitory actions of 1 and 10 nmol/liter estradiol on PDGF-BB-induced HASMC growth (cell number; Fig. 11CGo) and collagen synthesis (Fig. 11DGo), respectively. Similar results were obtained for DNA synthesis and in HASMCs treated with S-OLIGOs (data not shown).



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  		FIG. 11. A, Comparison of the concentration-dependent inhibitory effects of E2 (1–1000 nmol/liter), 2-hydroxyestradiol (OE), and 2-methoxyestradiol (ME) on the PDGF-BB (25 ng/ml)-induced proliferation of HASMCs. B, Up-regulation of ER{alpha} in response to AS-OLIGO to ER{alpha}, whereas panels C and D show the modulatory effects of the ER antagonist ICI182780 (ICI, 10 µmol/liter), CYP450 inhibitor 1-aminobenzotriazole (ABT; 10 µmol/liter), and COMT inhibitor OR486 (OR; 10 µmol/liter) on the inhibitory effects of 1 and 10 nmol/liter estradiol on cell proliferation (cell number; treatment time 6 d; panel C) and collagen synthesis (3H-L-proline incorporation; D), respectively. Values represent mean ± SEM from three separate experiments, each conducted in quadruplicate. PDGF-BB induced HASMC growth, and estradiol inhibited PDGF-BB-induced SMC growth. The antimitogenic effects of estradiol were blocked by ABT and OR486, but not by ICI182780. Treatment with ICI, ABT, or OR alone did not alter the effects of PDGF-BB on HASMC growth (±2% change). * and §, P < 0.05; *, significant reversal of the inhibitory effects of estradiol; §, significantly different from HASMCs treated with PDGF-BB alone [control (C)]. Conc., Concentration.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Regulation of ER expression in HASMCs

A major goal of this study was to investigate the regulation of ERs in HASMCs. Our results are consistent with the conclusion that in HASMCs, ER{alpha} and ERß are differentially regulated. Specifically, ER{alpha} appears to be regulated in HASMCs primarily by autologous down-regulation; i.e. after binding with estradiol, ER{alpha} interacts with the ER gene and represses its transcription. In contrast, ERß appears to be regulated in HASMCs predominantly by ubiquination and proteasomal degradation of estradiol-ERß complexes.

Our conclusion that ER{alpha} is regulated in HASMCs primarily by autologous down-regulation is based on several considerations. First, this mechanism is known to exist in other cell types. For example, in MCF-7 cells, studies by Santagati et al. (13) and Williard et al. (29) demonstrate that OLIGOs complimentary to ER mRNA bind to ERs with affinities in the nanomolar range and thereby block ERs from binding to ER genes, thus preventing estradiol-induced down-regulation of ERs. Second, in the present study, treatment of HASMCs with antisense and sense, but not scrambled, OLIGOs to ER{alpha} induce an up-regulation of ER{alpha}, irrespective of the precise region targeted by the antisense and sense constructs. This result is highly consistent with the conclusion that ER{alpha} binds to the antisense and S-OLIGOs, thus preventing the interaction of ER{alpha} with the ER{alpha} gene. It is highly unlikely that the effects of antisense and S-OLIGOs are due to the specific interaction with ER{alpha} mRNA because S-OLIGOs would not be expected to engage in such an interaction and because such an interaction would decrease, rather than increase, the expression of ER{alpha}. Also, it is unlikely that the increased expression of ER{alpha} is secondary to nonspecific effects of the antisense and S-OLIGOs because the scrambled OLIGOs do not produce the same effect. Third, our observation that estradiol significantly down-regulates the expression of ER{alpha} in the absence, but not in the presence, of AS-OLIGOs is consistent with the conclusion that the major pathway governing the expression of ER{alpha} is autologous down-regulation. Fourth, our finding that the stimulatory effects of AS-OLIGOs on ER{alpha} expression are blocked by cycloheximide suggests that the increased levels of ER{alpha} are due to de novo synthesis of ER{alpha}. Stimulation of de novo synthesis is the expected result of blocking autologous down-regulation because the ER{alpha} gene is no longer repressed and new synthesis of ER{alpha} protein can occur. Fifth, antisense and S-OLIGOs to ERß also increase ER{alpha} expression. By binding ERß, antisense and S-OLIGOs to ERß may prevent the dimerization of ER{alpha} with ERß and thereby interfere with their cojoined binding to the ER{alpha} gene. Sixth, treatment of HASMCs with PPT, an ER{alpha} agonist, down-regulates the expression of ER{alpha}. Moreover, treatment of cells with DPN, an ERß agonist, also down-regulates ER{alpha}. These findings imply that the down-regulatory effects of estradiol on ER{alpha} expression may be via both ER{alpha} and ERß, a conclusion consistent with the observation that antisense and sense constructs to both ER{alpha} and ERß up-regulate the expression of ER{alpha}. Seventh, the expression of ER{alpha} is down-regulated by the ER antagonists ICI182780, tamoxifen, and MPP. It is well established that autologous down-regulation of ERs is defined by the conformation of the ER-ligand complex, which varies considerably among agonists and antagonists (30, 31). In this regard, conformational changes govern the recruitment of cofactors and transcriptional responses (30, 31, 32). Indeed conformational changes induced by ICI82780 render it more potent than estradiol in down-regulating ER expression (20, 9).

Our conclusion that ERß is regulated in HASMCs predominately by ubiquination and proteasomal degradation of estradiol-ERß complexes is also based on multiple lines of evidence. First, this mechanism is known to exist in other cell types. Proteolysis of liganded ERs by proteasomes plays a key role in ER down-regulation in pituitary lactotrope cells and transfected HeLa cells (10, 11), and differential regulation of proteasome-dependent ER{alpha} and ERß turnover occurs in uterine artery endothelial cells (8). Second, unlike ER{alpha}, antisense and sense constructs to either ER{alpha} or ERß do not increase ERß expression, a finding inconsistent with the hypothesis that autologous down-regulation significantly contributes to the regulation of ERß expression. Third, cycloheximide increases the levels of ERß. Because cycloheximide inhibits proteasomes, this observation is consistent with the involvement of proteasomal activity in the regulation of ERß. Fourth, the selective proteasomal inhibitors lactacystin and MG132 increase the expression of ERß. Fifth, the MEK inhibitor PD98059 also increases ERß expression. Phosphorylation of ERs via multiple extracellular signaling pathways, including MAPK, can modulate ER expression via the proteasome-dependent pathway (33). Also, activation of MAPK in MCF-7 cells is associated with decreased ER expression (34) most likely because proteolytic activity of proteasomes is stimulated by activation of MAPK (14). Sixth, ER receptor agonists such as estradiol, PPT, and DPN do not down-regulate, and in fact estradiol and DPN up-regulate, the expression of ERß. These findings rule-out an important role for autologous down-regulation inducible by ER agonists with regard to governing ERß expression in HASMCs. Also, our finding that ERß expression is up-regulated by a MEK inhibitor, together with the recent observation that in arterial SMCs, estradiol inhibits MAPK activity (35), suggests that estradiol up-regulates ERß expression by inhibiting MAPK; whereas, the down-regulatory effects of estradiol on ER{alpha} are largely MAPK-independent and mostly due to autologous down-regulation.

Overall, our findings indicate that whether the proteosomal or autologous down-regulation pathway predominates depends on several key factors, including the ER subtype, whether the ligand is an agonist or antagonist, and the cell type. In HASMCs, although the proteosomal pathway appears to be the major mechanism modulating ERß levels, the autologous down-regulation pathway may become important in the presence of ER antagonists. In this regard, the expression of ERß in HASMCs is markedly down-regulated by ER antagonists such as tamoxifen, ICI182,780, and MPP. Our finding that estradiol down-regulates the expression of ERß in MCF-7 cells, but not in HASMCs, suggests that the regulatory effects of estradiol on ERs differ in MCF-7 cells vs. HASMCs and that the regulation of ER gene expression differs across cell types. Because steroid receptors appear to be the limiting factor in the response of a cell to the steroidal signal (36, 37), the above finding may be of great importance in explaining the disparate biological effects of estradiol observed across cell types.

ERs and antimitogenic effects of estradiol in HASMCs

A second objective of the current study was to investigate the mechanism of estradiol-induced inhibition of vascular SMC growth. Estradiol is a potent inhibitor of vascular SMC growth; however, the mechanism is unknown. Because vascular SMCs express ERs, the antimitogenic effects of estradiol in vascular SMC may be ER-mediated. However, two recent findings challenge this concept. First, exogenous estradiol inhibits injury-induced SMC proliferation in vascular lesions of mice lacking ER{alpha} (38), lacking ERß (39), and in mice lacking both ER{alpha} and ERß (40). Second, administration of estradiol blocks neointima formation in gonadectomized, but not nongonadectomized, rats, even though both express ERs (41). However, a recent study concludes that ER{alpha} importantly contributes to vasoprotection induced by estradiol (4). Thus, whether the antimitogenic effects of estradiol are ER-dependent or ER-independent remains an open question.

Because 2-methoxyestradiol, an endogenous metabolite of estradiol with no affinity for ERs, has potent antiangiogenic and anticarcinogenic effects (27), we hypothesize that the inhibitory effects of estradiol on vascular SMC growth are mediated via an ER-independent pathway that involves the local conversion of estradiol to methoxyestradiols. This contention is supported by several observations made in this study. First, estradiol inhibits growth similarly in control HASMCs vs. HASMCs expressing almost 100% more ER{alpha}. The present discovery that AS-OLIGOs markedly up-regulate ER{alpha} expression in HASMCs affords the opportunity to examine the effects of estradiol in HASMCs in which ER{alpha} expression is up-regulated. The current experiments demonstrate that up-regulation of ER{alpha} expression does not affect the ability of estradiol to inhibit growth of HASMCs. The observation that the antimitogenic effects of estradiol are not enhanced by OLIGO-induced expression of ER{alpha} strongly indicates that the effects are ER{alpha}-independent. The possibility that, at the concentrations used, the effects of estradiol are maximal can be ruled out because estradiol inhibits HASMC growth in a concentration (1–1000 nM)-dependent manner, and even the effects of the lowest concentrations of estradiol are not enhanced by AS-OLIGOs. Second, the current studies also demonstrate that metabolites of estradiol that have little (2-hydroxyestradiol) or no (2-methoxyestradiol) affinity for ERs are more potent than estradiol in inhibiting growth of HASMCs. Third, the antimitogenic effects of estradiol, both in the presence and absence of OLIGOs up-regulating ER expression, are blocked by 1-aminobenzotriazole, a broad spectrum inhibitor of CYP450 (CYP450s are responsible for converting estradiol to hydroxyestradiols, the precursor of methoxyestradiols) (27). Fourth, OR486, a specific inhibitor of COMT (responsible for converting hydroxyestradiols to methoxyestradiols) (27), blocks the antimitogenic effects of estradiol. Fifth, ICI182780 is ineffective in inhibiting the antimitogenic effects of estradiol on HASMC growth. Moreover, the observation that estradiol induces ERß levels, yet the antimitogenic effects of estradiol are not blocked by ICI182780, argues against a role for ERß and suggests that the effects are ER-independent. It is important that the role of methoxyestradiols in mediating the inhibitory effects of estradiol are also supported by our recent finding that the antimitogenic effects of estradiol are lost in SMCs isolated from COMT (essential to convert estradiol to methoxyestradiol) knockout mice that express both ER{alpha} and ERß (42).

Although, our findings provide evidence that the antimitogenic effects of estradiol are mediated via its conversion to methoxyestradiols, this effect may only be limited to estrogens that are metabolized to methoxyestradiols, and ER-dependent mechanisms may still play an important role in mediating the growth regulatory effects of estrogens not metabolized to methoxyestradiols. In this regard, the effect of estradiol on NO synthesis is ER-dependent, and increased expression of ERs results in a parallel increase in NO and vascular endothelial growth factor production (43, 44). Similarly, the inhibitory effects of estradiol on endothelin-1 synthesis is enhanced with the increase in ER expression (43).

Conclusion

Our findings provide evidence that the levels of ER{alpha} and ERß in HASMCs are differentially regulated. In this regard, ER{alpha} appears to be regulated in HASMCs primarily by autologous down-regulation, whereas ERß appears to be regulated in HASMCs predominately by ubiquination and proteasomal degradation of estradiol-ERß complexes. Finally, our findings provide evidence that the antimitogenic effects of estradiol are unaltered in HASMCs expressing a 2-fold higher level of ER{alpha}, suggesting that the antimitogenic effects of estradiol are not ER{alpha}-mediated.


   Footnotes
 
This work was supported by Swiss National Science Foundation Grant 32-64040.00.

Abbreviations: AS, Antisense (OLIGO); COMT, catechol-O-methyltransferase; DPN, 2,3-bis(4-hydroxyphenyl)propionitrile; ER, estrogen receptor; HASMC, human aortic smooth muscle cell; MEK, MAPK kinase; MPP, 4,4'-[4-methyl-5-[4-[2-(1-piperidnyl) ethoxy]-phenyl-1H-pyrazole]-1,3 diyl]-bis-phenol HCl; OLIGO, oligonucleotide; PPT, propyl pyrazole triol; S, sense (OLIGO); Scr, scrambled (OLIGO); SDS, sodium dodecyl sulfate; SMC, smooth muscle cell.

Received May 12, 2003.

Accepted February 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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