Estrogens, by binding to and activating two estrogen receptors (ERα and ERβ), are critically involved in the development of the mammary gland and breast cancer. An isoform of ERβ, ERβ2 (also called ERβcx), with an altered COOH-terminal region, is coexpressed with ERα in many human breast cancers. In this study, we generated a stable cell line from MCF7 breast cancer cells expressing an inducible version of ERβ2, along with endogenous ERα, and examined the effects of ERβ2 on the ERα protein levels and function. We showed that ERβ2 inhibited ERα-mediated transactivation via estrogen response element and activator protein-1 sites of reporter constructs as well as the endogenous genes pS2 and MMP-1. Chromatin immunoprecipitation assays revealed that ERβ2 expression caused a significant reduction in the recruitment of ERα to both the pS2 and MMP-1 promoters. Furthermore, ERβ2 expression induced proteasome-dependent degradation of ERα. The inhibitory effects of ERβ2 on ERα activity were further confirmed in HEK293 cells that lack functional endogenous ERs. We also showed that ERβ2 can interact with ERα both in vitro and in mammalian cells, which is compatible with a model where ERβ2/ERα heterodimers are targeted to the proteasome. Finally, in human breast cancer samples, we observed that expression of ERβ2 significantly correlated with ERα-negative phenotype. Our data suggest that ERβ2 could influence ERα-mediated effects relevant for breast cancer development, including hormone responsiveness. [Cancer Res 2007;67(8):3955–62]

Estrogens bind to and activate two estrogen receptors (ERα and ERβ) and exert their effects through a complex array of signaling pathways that mediate genomic and non-genomic events (1, 2). The ERs are members of the nuclear receptor superfamily of ligand-regulated transcription factors (3). ERs regulate gene expression through distinct DNA response elements. The classic mechanism of estrogen signaling is through an estrogen response element (ERE). The molecular details of this process are well characterized. ER dimerizes and interacts with EREs in target gene promoters, followed by recruitment of a variety of coregulators to alter chromatin structure and facilitate recruitment of the RNA polymerase II transcriptional machinery (2, 4). Estrogen signaling also occurs through alternative mechanisms where liganded ERs are tethered to DNA via association with other transcription factor complexes, including Fos/Jun (activator protein-1 [AP-1]– responsive elements; ref. 5) or SP-1 (GC-rich SP-1 motifs; ref. 6). The mechanistic details of activation through these pathways are less clear. In addition to these ligand-induced transcriptional activities of ER, ligand-independent pathways to activate ERs have been described. Growth factor signaling or stimulation of other signaling pathways leads to activation of kinases that can phosphorylate and thereby activate ERs or associated coregulators in the absence of ligand (7). Furthermore, estrogen may elicit effects through non-genomic mechanisms where estrogen binds to the ER localized outside of the cell nucleus, in turn activating signal transduction pathways in the cytoplasm (8).

The role of ERs in breast cancer has been intensely investigated. ERβ is found in both ductal, lobular epithelial and stromal cells of the rodent mammary gland (9). ERα, on the other hand, is only found in the ductal and lobular epithelial cells but not in stroma (10). It is generally believed that breast tumors, at least initially, are dependent on the stimulatory effects of estrogens. However, many breast tumors eventually progress to an estrogen-independent growth phenotype. Tamoxifen and similar antiestrogens are currently the first-line therapy for treatment of hormone-dependent breast cancer (11). Various ER transcripts have been found in breast carcinomas (10), and data exist supporting protein expression for several of these isoforms (12). Normal and cancer tissues display a variety of profiles regarding ERα, ERβ, and ER splice variants at both mRNA and protein levels (13, 14). This heterogeneity in ER isoform profiles could influence estrogen signaling relevant for breast cancer risk, hormone responsiveness, and survival.

An isoform of ERβ, ERβ2 (also called ERβcx), encodes a protein of 495-amino-acid residues, with a molecular weight of 55.5 kDa. It uses an alternative exon 8, which encodes for an additional 26 amino acids due to alternative splicing. ERβ2 has undetectable affinity for E2 and cannot activate transcription of ERE-driven reporters. When ERβ2 is cotransfected with ERα, it inhibits ligand-induced ERα transcriptional activity on an ERE reporter gene (15). This intriguing property suggests that ERβ2 has an important function in neutralizing the effect of functional ERα. Expression of ERβ2 could also explain tamoxifen resistance in some ERα-positive breast cancer patients. Indeed, one study reported that expression of ERβ2 correlated with a poor response to antiestrogen (13). It has been suggested that expression of ERβ2 could have a prognostic value in breast and prostate cancers (13, 16).

In this study, we established stable transfectants of ERα-positive MCF7 breast cancer cells with tetracycline-regulated ERβ2 expression to investigate the influence of ERβ2 on ERα signaling. Collectively, our results indicate that proteasome-dependent degradation of ERα induced by ERβ2 in breast cancer cells may represent a possible molecular mechanism for the antagonistic effect of ERβ2 on ERα-mediated functions. The inhibitory effects of ERβ2 on ERα activity were further confirmed in HEK293 cells that lack functional endogenous ERs. Finally, we show that expression of ERβ2 correlated with ERα-negative phenotype in human breast cancer samples.

Cell culture. Modified MCF-7 human breast cancer cells and HEK293 cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 5% FCS and 1% penicillin/streptomycin (Invitrogen) at 37°C in a humidified atmosphere of 5% CO2 in air. For experiments to evaluate the effects of 4,4′,4″-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT), kindly provided by KaroBio AB (Stockholm, Sweden), DMEM without phenol red, and FCS treated with dextran-coated charcoal (DCC-FCS) were used.

Generation of stable MCF7 tet-off ERβ2 and HEK293 tet-on ERβ2 clones. MCF-7 cells stably transfected with tetracycline-regulated ERβ2 expression plasmid were generated in two steps. The cells were first transfected with pTet-tTAK (Life Technologies, Gaithersburg, MD) modified to support puromycin resistance using Lipofectin according to the manufacturer's instructions (Life Technologies). Selection was done with 0.5 μg/mL puromycin (Sigma, St. Louis, MO) in the presence of 1 μg/mL tetracycline (Sigma). A clone showing high levels of induction upon tetracycline withdrawal and low basal activity was selected using the pUHC13-3 control plasmid (Life Technologies). ERβ2 cDNA was fused to the flag tag and cloned into pBI-EGFP (Clontech, Palo Alto, CA). This construct was transfected into the highly inducible clone and isolated in step one, together with a neomycin resistance plasmid, and selection was done with 1,000 μg/mL G418 (Calbiochem, La Jolla, CA). For generating stable HEK293 tet-on ERβ2 clones, the pBI-EGFP-ERβ2 plasmid was transfected into HEK293 tet-on cells, which were obtained from BD Biosciences Clontech (Palo Alto, CA).

Transient transfection and luciferase assays. Transient transfection was done essentially as described previously (17). Briefly, cells were seeded in six-well plates and grown in phenol red–free DMEM supplemented with 5% DCC-FCS for 24 h before transfection. The cells were cotransfected with the reporter plasmid (ERE-TK-Luc or coll517-Luc containing 517 bp of the human collagenase gene promoter including a single AP-1 binding site) and/or ERα expression plasmid and pRL-TK control plasmid, which contains a Renilla luciferase gene, for normalizing transfection efficiency. Cells were transfected using LipofectAMINE 2000 (Invitrogen/Life Technologies, Carlsbad, CA). After 5 h of transfection, tetracycline was removed, or doxycycline was added 12 h before initiation of treatment with PPT to induce ERβ2 expression. Transfected cells were then treated with 10 nmol/L PPT or vehicle for 24 h before harvest and luciferase assay (Biothema, Dalarö, Sweden).

RNA isolation and real-time PCR. Cells were grown for 48 h in phenol red–free DMEM supplemented with 5% DCC-FCS serum. To express ERβ2, tetracycline was removed, or doxycycline was added 12 h before addition of 10 nmol/L PPT or vehicle. Real-time PCR was done as described previously (18). Taqman Universal Master Mix (PE Applied Biosystems, Foster City, CA) was used for amplifying MMP-1 gene; for pS2, QPCR Master Mix for Cybergreen (Medprobe, Minneapolis, MN) was used. The PCR primer pairs are as follows: pS2 mRNA, were 5′-CATCGACGTCCCTCCAGAAGAG-3′ and 5′-CTCTGGGACTAATCACCGTGCTG-3′; MMP-1 mRNA, 5′-TTGAAGCTGCTTACGAATTTGC-3′ and 5′-GTCCCTGAACAGCCCAGTACTT-3′. The probe sequence for MMP-1 was 5′-CAGAGATGAAGTCCGGTTTTTCAAAGGGAA-3′. All target gene transcripts were normalized to the β-glucuronidase mRNA (PE Applied Biosystems) content and to the time 0 sample. For measurement of expression levels of ERβ2 in breast tumor samples, real-time PCR was done using primers specific for ERβ2 as described previously (19).

Chromatin immunoprecipitation. MCF7 tet-off ERβ2 cells were seeded in 150-mm dishes and grown for 48 h in phenol red–free DMEM supplemented with 5% DCC-FCS serum. For expression of ERβ2, tetracycline was removed 12 h before initiation of treatment with ligands. Cells were then treated with 10 nmol/L PPT for the indicated times. Soluble, sonicated chromatin was prepared as previously described (20). Chromatin fractions were immunoprecipitated with 0.5 to 1 μg of the indicated antibodies, and the immune complexes were recovered using protein A/G-Sepharose (50% slurry; Pharmacia, Piscataway, NJ) and processed as described (20). The antibodies used were as follows: ERα, H-184 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse antihuman IgG (Santa Cruz Biotechnology), and anti-FLAG (M5; Sigma). The immunoprecipitated DNA was amplified by real-time PCR using Platinum SYBR green quantitative PCR supermix uracil DNA glycosylase (Invitrogen). The primer pairs used are as follows: pS2 promoter, 5′-CCGGCCATCTCTCACTATGAA-3′ and 5′-CCTCCCGCCAGGGTAAATAC-3′; MMP-1 promoter, 5′-TTGCAACACCAAGTGATTCCA-3′ and 5′-CCCAGCCTCTTGCTACTCCA-3′.

Western blotting. Cells were seeded in 100-mm dishes and grown for 48 h in phenol red–free DMEM supplemented with 5% DCC-FCS serum. For expression of ERβ2, tetracycline was removed, or doxycycline was added 12 h before initiation of treatment with ligands. Cells were then treated with 10 nmol/L PPT or vehicle for the indicated times, and nuclear extracts were prepared as described in ref. (21). To examine the effect of proteasome inhibition, we pretreated the cells for 2 h with 10 μmol/L MG132 (Sigma) before the removal of tetracycline or addition of doxycycline. After 12 h, cells were harvested, and nuclear extracts were prepared. ERα was detected using H-184 rabbit polyclonal antibody (Santa Cruz Biotechnology) at 1:10,000 dilution and ECL anti-rabbit IgG, horseradish peroxidase–linked (Amersham Biosciences, Arlington Heights, IL) at 1:100,000 dilution (20). The actin antibody (Sigma) was used at a 1:50,000 dilution. The Image J software (Research Services Branch, National Institute of Mental Health, Bethesda, MD) was used for densitometry of the autoradiographs.

Glutathione S-transferase pull-down assay. Glutathione S-transferase (GST) fusions of ERα-(309–595) and His-tagged ERβ2 LBD (R254 to Q495) were generated by cloning the appropriate DNA sequences into the pGEX2-TK vector (Amersham Pharmacia Biotech) and the pET15b vector (Novagen, Madison, WI), respectively. GST and GST-ERα proteins were purified on glutathione-Sepharose beads (Sigma) according to standard methods and incubated with partially purified His-tagged ERβ2 LBD, prepared as described previously (22), in pull-down buffer [50 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 1 mmol/L MgCl2, 10% glycerol, and 0.5% NP40] and 1.5% serum bovine albumin. Incubation and rotation were carried out for 2 h at 4°C. After extensive washing with pull-down buffer, the bound proteins were analyzed by SDS-PAGE followed by Western blotting using mouse monoclonal anti-His antibody (Clontech).

Coimmunoprecipitation. Total cell extracts from MCF7 tet-off ERβ2 cells were prepared by direct lysis of cells with buffer containing 20 mmol/L HEPES (pH 7.5), 180 mmol/L NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 10% glycerol, 0.5 mmol/L DTT, and 1 mmol/L phenylmethylsulfonyl fluoride. Protein concentrations were measured using Bio-Rad Protein Assay reagent. Cell lysates were incubated with anti-FLAG (M5) antibody at 4°C with rotation for 2 h. Thereafter, prewashed protein G-agarose beads (Amersham Biosciences) were added, and the incubation continued for another 2 h at 4°C followed by four washes with lysis buffer. Subsequently, the immune complex was boiled in electrophoresis sample buffer and analyzed on SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane and visualized using anti-FLAG M5 monoclonal antibody and ERα, H-184 antibody, respectively.

Human breast tumor samples. Primary breast tumor tissues from 40 patients with invasive ductal carcinoma and undergoing breast cancer surgery were provided by the Charing Cross Hospital, London. All of the samples were frozen in liquid nitrogen immediately after resection and stored at −80°C until use. The studies were approved by the ethical committee of the Karolinska Institute.

Immunohistochemistry. Expression of ERα and ERβ2 in breast tumor samples was measured by immunohistochemistry as previously described (13, 23). The primary antibodies used were ERα (1D5, 1:30) from DAKO (High Wycombe, United Kingdom) and a specific ERβ2 antibody (1:400) produced by us (13). For negative controls, the primary antibody was replaced with PBS alone or with primary antibody after absorption with the corresponding antigen. Sections were incubated in biotinylated goat anti-mouse immunoglobulin (1:200 dilution; Vector Laboratories, Inc., Burlingame, CA) for 2 h at room temperature followed by incubation in avidin-biotin-horseradish peroxidase (Vector Laboratories) for 1 h.

Statistics. Student's t test, Mann-Whitney U test, or Fisher's exact probability test was used to determine significance of differences between groups.

Expression of ERβ2 represses PPT-stimulated ERE and AP-1 activity. To create a system for studying the function of ERβ2, we generated stable transfectants of MCF7 breast cancer cells with a tetracycline-regulated vector for expression of ERβ2. Native estrogen-responsive MCF7 cells express predominantly ERα and only very low levels of endogenous ERβ2. A Western blot of ERβ2 protein with a Flag tag in response to tetracycline withdrawal is shown in Fig. 1A. No detectable Flag-ERβ2 protein was expressed in the presence of tetracycline, whereas high levels of Flag-ERβ2 protein were observed when the cells were cultured in the absence of tetracycline.

Figure 1.

ERβ2 expression inhibits ERα-mediated transactivation. A, MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells were cultured in the presence of tetracycline (+ tet) or absence of tetracycline (− tet) for 12 h. ERβ2 was expressed only in the absence of tetracycline as measured by anti-flag-tag Western blot. B, transfection of MCF7 tet-off FLAG-ERβ2 cells with an ERE-luciferase reporter or a collagenase luciferase reporter shows that the induction of ERβ2 by tetracycline withdrawal (+ERβ2, black columns) reduces both basal and PPT-induced ERE and AP-1 activity. Columns, mean; bars, SD. Representative results of three independent experiments. *, P < 0.05, significant difference between samples grown in the presence of tetracycline (−ERβ2, white columns) or absence of tetracycline (+ERβ2, black columns).

Figure 1.

ERβ2 expression inhibits ERα-mediated transactivation. A, MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells were cultured in the presence of tetracycline (+ tet) or absence of tetracycline (− tet) for 12 h. ERβ2 was expressed only in the absence of tetracycline as measured by anti-flag-tag Western blot. B, transfection of MCF7 tet-off FLAG-ERβ2 cells with an ERE-luciferase reporter or a collagenase luciferase reporter shows that the induction of ERβ2 by tetracycline withdrawal (+ERβ2, black columns) reduces both basal and PPT-induced ERE and AP-1 activity. Columns, mean; bars, SD. Representative results of three independent experiments. *, P < 0.05, significant difference between samples grown in the presence of tetracycline (−ERβ2, white columns) or absence of tetracycline (+ERβ2, black columns).

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To investigate the effects of ERβ2 on ERα-mediated transactivation through ERE- and AP-1–response elements, we transfected the MCF7 tet-off FLAG-ERβ2 cells with an ERE- or an AP-1-luciferase reporter construct. Cells were grown in the presence or absence of tetracycline, and reporter gene activity after treatment with vehicle or PPT, an agonist selective for ERα, was determined. The data shown in Fig. 1B support previous studies (15) and show that ERβ2 reduced basal as well as PPT-induced ERE activity. In addition, our results show that ERβ2 inhibited both the basal and PPT-induced AP-1 activity.

Expression of ERβ2 reduces mRNA levels of the endogenous estrogen-regulated genes and inhibits recruitment of ERα to estrogen-responsive promoters. MCF7 tet-off FLAG-ERβ2 cells were cultured in the presence or absence of tetracycline and treated with PPT to determine whether ERβ2 inhibits the endogenous expression of estrogen-responsive genes regulated by ERα. Determination of endogenous expression levels for the ERE-controlled gene pS2 and the AP-1–dependent gene MMP-1 was done by quantitative real-time PCR analysis. As shown in Fig. 2A, PPT stimulates pS2 and MMP-1 mRNA expression after 6 and 12 h of treatment, respectively. Expression of ERβ2 suppressed PPT induction of pS2 and MMP-1. These data extend our findings that ERβ2 antagonizes ERα-mediated transactivation from reporter genes to endogenous genes.

Figure 2.

Expression of ERβ2 inhibits PPT induction of pS2 and MMP-1 mRNA levels and recruitment of ERα to their promoters. A, real-time PCR results of the effect of ERβ2 on expression levels of pS2 and MMP-1. MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells were cultured in the presence of tetracycline (−ERβ2, white columns) or in the absence of tetracycline (+ERβ2, black columns) and treated with 10 nmol/L PPT for the indicated amount of time. Total RNA was isolated, reverse transcribed, and amplified with primers recognizing the mRNA form of pS2 and MMP-1. B, chromatin immunoprecipitation analysis of recruitment of ERα and ERβ2 to the pS2 and MMP-1 promoters. MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells were cultured in the presence or absence of tetracycline and treated with 10 nmol/L PPT for 0, 1, and 2 h. Chromatin immunoprecipitation assays were done as described in Materials and Methods with antibodies recognizing ERα and FLAG (for FLAG-ERβ2) in parallel with control IgG. Quantitative real-time PCR of 1 μL of purified immunoprecipitated DNA was done using SYBR Green and primer pairs that amplify the pS2 and MMP-1 promoter regions. Relative promoter enrichment compared with IgG at time 0 in the presence of tetracycline. Columns, mean; bars, SD. Representative results of three independent experiments. *, P < 0.05, significant difference between time-matched samples grown in the presence or absence of tetracycline.

Figure 2.

Expression of ERβ2 inhibits PPT induction of pS2 and MMP-1 mRNA levels and recruitment of ERα to their promoters. A, real-time PCR results of the effect of ERβ2 on expression levels of pS2 and MMP-1. MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells were cultured in the presence of tetracycline (−ERβ2, white columns) or in the absence of tetracycline (+ERβ2, black columns) and treated with 10 nmol/L PPT for the indicated amount of time. Total RNA was isolated, reverse transcribed, and amplified with primers recognizing the mRNA form of pS2 and MMP-1. B, chromatin immunoprecipitation analysis of recruitment of ERα and ERβ2 to the pS2 and MMP-1 promoters. MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells were cultured in the presence or absence of tetracycline and treated with 10 nmol/L PPT for 0, 1, and 2 h. Chromatin immunoprecipitation assays were done as described in Materials and Methods with antibodies recognizing ERα and FLAG (for FLAG-ERβ2) in parallel with control IgG. Quantitative real-time PCR of 1 μL of purified immunoprecipitated DNA was done using SYBR Green and primer pairs that amplify the pS2 and MMP-1 promoter regions. Relative promoter enrichment compared with IgG at time 0 in the presence of tetracycline. Columns, mean; bars, SD. Representative results of three independent experiments. *, P < 0.05, significant difference between time-matched samples grown in the presence or absence of tetracycline.

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The effect of ERβ2 expression on the binding of ERα to the pS2 and MMP-1 promoters was examined by chromatin immunoprecipitation. Cells were treated with PPT for 0, 1, and 2 h, after which chromatin was cross-linked, and protein-DNA complexes were immunoprecipitated with antibodies recognizing normal rabbit IgG, ERα, or FLAG ERβ2. Figure 2B shows that ERα was recruited to the pS2 and MMP-1 promoter regions. PPT induced significant recruitment of ERα to the pS2 promoter after 1 and 2 h of treatment, whereas significant recruitment of ERα to the MMP-1 promoter was observed after 2 h of treatment. In agreement with previous reports, ERα bound to the pS2 promoter and, to a lesser extent, to the MMP-1 promoter in the absence of ligand (24). Ligand-independent binding of ERβ2 to either promoter region was not observed under our assay conditions. The PPT-dependent recruitment of ERβ2 to the pS2 and MMP1 promoters was observed in the absence of tetracycline and not in its presence (Fig. 2B). The expression of ERβ2 significantly reduced the recruitment of ERα to both the pS2 and MMP-1 (apparent after 2 h of treatment) promoters, suggesting a plausible mechanism for the ERβ2 antagonism of ERα activity.

ERβ2 down-regulates ERα protein via the proteasome degradation pathway. The effects of ERβ2 expression on ERα protein levels were investigated in MCF7 tet-off FLAG-ERβ2 cells grown in the presence or absence of tetracycline. Cells were treated with vehicle or PPT for 0, 2, and 6 h. As shown in Fig. 3A, ERβ2 expression caused a decrease in immunoreactive ERα protein in cells treated with vehicle and PPT, whereas PPT treatment slightly increased ERα protein levels. These changes in ERα protein levels were not associated with changes in ERα mRNA levels (data not shown), suggesting that ERβ2 expression may affect ERα protein stability.

Figure 3.

Western blot analysis of the influence of ERβ2 on ERα protein levels. A, MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells were cultured in the presence of tetracycline (−ERβ2) or in the absence of tetracycline (+ERβ2) and treated with either vehicle or PPT for 0, 2, and 6 h. Western blots were done as described in Materials and Methods. B, MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells were cultured in the presence of tetracycline or in the absence of tetracycline and treated with either vehicle or proteasome inhibitor (10 μmol/L MG132). Equal amounts of protein extract were resolved by SDS-PAGE and transferred to Hybond-P nylon membrane, and the membranes were probed for ERα. Data from one experiment that was repeated three independent times.

Figure 3.

Western blot analysis of the influence of ERβ2 on ERα protein levels. A, MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells were cultured in the presence of tetracycline (−ERβ2) or in the absence of tetracycline (+ERβ2) and treated with either vehicle or PPT for 0, 2, and 6 h. Western blots were done as described in Materials and Methods. B, MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells were cultured in the presence of tetracycline or in the absence of tetracycline and treated with either vehicle or proteasome inhibitor (10 μmol/L MG132). Equal amounts of protein extract were resolved by SDS-PAGE and transferred to Hybond-P nylon membrane, and the membranes were probed for ERα. Data from one experiment that was repeated three independent times.

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Regulated proteolysis by the proteasome accounts for turnover of most short- and long-lived proteins, including at least some nuclear receptors. To examine if ERβ2 down-regulates ERα through a proteasome-dependent pathway, we tested whether the proteasome inhibitor MG132 (25) would block ERβ2-induced down-regulation of ERα. MCF7 tet-off FLAG-ERβ2 cells grown in the presence or absence of tetracycline were pretreated with vehicle or MG132. Nuclear fractions were prepared and analyzed by Western blotting. As expected, expression of ERβ2 in cells not treated with proteasome inhibitor reduced the level of ERα protein compared with cells that did not express ERβ2 (Fig. 3B, lane 1 versus 2). Importantly, MG132 blocked the ERβ2-induced down-regulation of the ERα protein levels (lane 3 versus 4). These results suggest that ERβ2-induced down-regulation of ERα protein levels proceeds through the proteasome.

Inhibitory effects of ERβ2 on ERα activity are not restricted to MCF7 cells. The inhibitory effects of ERβ2 on ERα activity were also examined in HEK293 cells that lack functional endogenous ERs (26, 27). Cell clones stably expressing ERβ2 were established in HEK293 tet-on cells. The induction of ERβ2 protein by doxycycline treatment was verified by Western blotting (Fig. 4A). To investigate the effects of ERβ2 expression on ERα-mediated transactivation and ERα protein levels, cells were cotransfected with an expression plasmid for ERα, an ERE-luciferase reporter construct, and a pRL-TK control plasmid for monitoring the transfection efficiency. Confirming the results seen with the MCF7 cells, ERβ2 inhibited both the basal and PPT-induced ERE activity in HEK293 cells (Fig. 4B). ERβ2 also suppressed PPT induction of the endogenous pS2 mRNA (data not shown). Furthermore, the level of ERα protein was reduced when ERβ2 was expressed (Fig. 4C, lane 1 versus 2); MG132 blocked the ERβ2-induced down-regulation of the ERα protein levels (lane 3 versus 4). Thus, our results confirmed that ERβ2 induced proteasome-mediated degradation of ERα in HEK293 cells.

Figure 4.

Inhibitory effects of ERβ2 on ERα activity in HEK293 cells. A, HEK293 tet-on FLAG-ERβ2 cells were cultured in the absence of doxycycline (− Dox) or presence of doxycycline (+ Dox) for 12 h. ERβ2 was expressed only in the presence of doxycycline as measured by anti-flag-tag Western blot. B, transfection of HEK293 tet-on FLAG-ERβ2 cells with an expression plasmid for ERα and an ERE-luciferase reporter shows that induction of ERβ2 by addition of doxycycline (+ERβ2, black columns) reduces both basal and PPT-induced ERE activity. Columns, mean; bars, SD. Representative results of three independent experiments. *, P < 0.05, significant difference between samples grown in the absence of doxycycline (−ERβ2, white columns) or presence of doxycycline (+ERβ2, black columns). C, HEK293 tet-on FLAG-ERβ2 cells were transiently transfected with an expression plasmid for ERα. The cells were then treated with either vehicle or proteasome inhibitor (10 μmol/L MG132) both in the absence of doxycycline or in the presence of doxycycline. Equal amounts of protein extract were resolved by SDS-PAGE and transferred to Hybond-P nylon membrane, and the membranes were probed for ERα. Data from one experiment that was repeated three independent times.

Figure 4.

Inhibitory effects of ERβ2 on ERα activity in HEK293 cells. A, HEK293 tet-on FLAG-ERβ2 cells were cultured in the absence of doxycycline (− Dox) or presence of doxycycline (+ Dox) for 12 h. ERβ2 was expressed only in the presence of doxycycline as measured by anti-flag-tag Western blot. B, transfection of HEK293 tet-on FLAG-ERβ2 cells with an expression plasmid for ERα and an ERE-luciferase reporter shows that induction of ERβ2 by addition of doxycycline (+ERβ2, black columns) reduces both basal and PPT-induced ERE activity. Columns, mean; bars, SD. Representative results of three independent experiments. *, P < 0.05, significant difference between samples grown in the absence of doxycycline (−ERβ2, white columns) or presence of doxycycline (+ERβ2, black columns). C, HEK293 tet-on FLAG-ERβ2 cells were transiently transfected with an expression plasmid for ERα. The cells were then treated with either vehicle or proteasome inhibitor (10 μmol/L MG132) both in the absence of doxycycline or in the presence of doxycycline. Equal amounts of protein extract were resolved by SDS-PAGE and transferred to Hybond-P nylon membrane, and the membranes were probed for ERα. Data from one experiment that was repeated three independent times.

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ERβ2 interacts with ERα in vitro and in mammalian cells. To test whether ERβ2 interacts with ERα, we did GST pull-down assays using His-ERβ2 LBD and GST-ERα LBD fusion proteins. Western blot analysis showed that ERβ2 specifically associated with GST-ERα, but not with GST alone (Fig. 5A), indicating a direct interaction between both ER subtypes.

Figure 5.

ERβ2 interacts with ERα in vitro and in mammalian cells. A, equal amounts of GST-ERα or GST alone were prepared and incubated with purified His-tagged ERβ2 LBD. ERα-associated proteins were analyzed by Western blotting using a His antibody. Aliquots of purified His-tagged ERβ2 LBD protein (3% of input) were also loaded directly onto the gels and analyzed by Western blotting (Input, lane 3). B, MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells grown in the presence or absence of tetracycline were lysed. Cell lysates were immunoprecipitated with the M5 monoclonal antibody to FLAG. Immunocomplexes were separated by SDS-PAGE, and proteins were transferred to polyvinylidene difluoride membranes. Blots were probed with an antibody to FLAG or ERα. In parallel, endogenous ERα was detected in nonprecipitated lysates (0.5% of the volume used in the immunoprecipitation assays) from the cells in both presence and absence of tetracycline (top). *, position of the heavy-chain IgG cross-reacting with the secondary antibody. Data from one experiment that was repeated three times.

Figure 5.

ERβ2 interacts with ERα in vitro and in mammalian cells. A, equal amounts of GST-ERα or GST alone were prepared and incubated with purified His-tagged ERβ2 LBD. ERα-associated proteins were analyzed by Western blotting using a His antibody. Aliquots of purified His-tagged ERβ2 LBD protein (3% of input) were also loaded directly onto the gels and analyzed by Western blotting (Input, lane 3). B, MCF7 tet-off FLAG-ERβ2 human breast carcinoma cells grown in the presence or absence of tetracycline were lysed. Cell lysates were immunoprecipitated with the M5 monoclonal antibody to FLAG. Immunocomplexes were separated by SDS-PAGE, and proteins were transferred to polyvinylidene difluoride membranes. Blots were probed with an antibody to FLAG or ERα. In parallel, endogenous ERα was detected in nonprecipitated lysates (0.5% of the volume used in the immunoprecipitation assays) from the cells in both presence and absence of tetracycline (top). *, position of the heavy-chain IgG cross-reacting with the secondary antibody. Data from one experiment that was repeated three times.

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To show interactions between ERβ2 and ERα in mammalian cells, we did coimmunoprecipitation assays using the ERα-expressing MCF7 cell line containing an inducible tet-off FLAG-ERβ2 (Fig. 5B). Cell lysates were immunoprecipitated with a mouse monoclonal antibody to the FLAG-tag and probed with a rabbit antibody to ERα. The anti-FLAG antibody did not precipitate proteins from lysates of cells grown in the presence of tetracycline, but it did precipitate FLAG-ERβ2 protein from lysates of cells grown in the absence of tetracycline. Endogenous ERα was found in FLAG immunoprecipitates from the lysates of cells grown in the absence of tetracycline, but not in the presence of tetracycline. Endogenous ERα was detected in nonprecipitated lysates from the cells both in the presence or absence of tetracycline. Incubation of cell lysates with beads alone or control IgG failed to immunoprecipitate either FLAG-reactive or ERα proteins (data not shown). These results indicate that ERβ2 associated with endogenous ERα in mammalian cells.

ERα-positive breast tumors express lower levels of ERβ2. To determine whether our findings that ERβ2 expression down-regulates ERα protein described above for the MCF7 human breast cancer cell line can be extended to breast cancer patient samples, we analyzed samples obtained from breast cancer surgery. A total of 37 individual human invasive ductal carcinoma samples were immunohistochemically analyzed for ERα protein expression (Fig. 6A and B). Of these, 18 samples were classified as ERα positive according to standard criteria (>10% of total cells were positive); the remaining 19 samples were considered as ERα negative. We then examined expression of ERβ2 in these breast tumor samples by a quantitative real-time PCR assay. The level of ERβ2 mRNA expression was found to be significantly lower in the ERα-positive group (48.4 ± 29.4) than in the ERα-negative group (116.2 ± 67.4; Mann-Whitney U test, P < 0.001). Next, 30 of these tumor samples were further stained with an ERβ2 antibody (Fig. 6C and D). Of these, 18 samples were evaluated as positive for ERβ2 using this assay, with 60% of cells positive as cutoff value (13).

Figure 6.

Representative immunohistochemical staining of ERα and ERβ2 in human breast cancer samples. In a sample in which ERα staining is positive (A), there is negative nuclear staining with the ERβ2 antibody (C). In a sample in which ERα staining is negative (B), there is positive nuclear staining with the ERβ2 antibody (D). The cutoff of 10% and 60% staining was used to define sections positive for ERα and ERβ2, respectively.

Figure 6.

Representative immunohistochemical staining of ERα and ERβ2 in human breast cancer samples. In a sample in which ERα staining is positive (A), there is negative nuclear staining with the ERβ2 antibody (C). In a sample in which ERα staining is negative (B), there is positive nuclear staining with the ERβ2 antibody (D). The cutoff of 10% and 60% staining was used to define sections positive for ERα and ERβ2, respectively.

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In the ERβ2 positive tumors, 14 of 18 were evaluated as ERα negative, whereas 10 of 12 were ERα positive in the ERβ2-negative group. Thus, consistent with the results above, ERβ2 protein staining was associated with absence of ERα protein staining (P < 0.05, Fisher's exact probability test).

Using a stable cell line derived from MCF7 breast cancer cells expressing ERβ2 in an inducible fashion, we show that ERβ2 interacts with ERα both in vitro and in mammalian cells, and that ERβ2 induces proteasome-dependent degradation of ERα. We propose that the ERβ2-induced proteasome-dependent degradation of ERα is caused by the formation of ERβ2/ERα heterodimers. We suggest that ERβ2-mediated degradation of ERα is at least one mechanism whereby expression of ERβ2 inhibits recruitment of ERα to the estrogen-responsive promoters, leading to suppression of ERα-regulated genes. We thus present a molecular mechanism by which ERβ2 could antagonize ERα activity in breast cancer cells.

Although the possible mechanisms have remained unclear, a few studies have shown that ERβ2 inhibits the ERα-mediated transactivation through the classic ERE-pathway in reporter systems (15, 28). Our work confirms these observations (Fig. 1B). We extend these studies and show that ERβ2 inhibits the expression of the endogenous pS2 gene that contains an ERE site within its promoter (29). Furthermore, we studied AP-1 sites. The MMP-1 gene is one of several hormone-responsive genes in breast cancer cells regulated by ERα/AP-1 (30), and this gene was used as a model to investigate the effect of ERβ2 on ERα transactivation through nonclassic AP-1–mediated pathway. Our results show that ERβ2 expression inhibits PPT-induced MMP-1 mRNA and reporter gene activity in cells transfected with AP-1-luciferase reporter constructs. The mechanism of the inhibitory effect of ERβ2 on ERα activity was further investigated by chromatin immunoprecipitation assay. Treatment with PPT induced a dramatic increase in the occupancy of the pS2 and MMP-1 gene promoters by ERα. However, the recruitment of ERβ2 to either promoter was much weaker than of ERα even when ERβ2 was overexpressed. This is presumably due to the much lower DNA binding ability of ERβ2 than ERα (28). We observed that the expression of ERβ2 significantly reduced the recruitment of ERα to both the pS2 and MMP-1 promoters. This suggests that ERβ2-mediated reduction of ERα-mediated transcriptional activity is related to the reduced recruitment of ERα to the estrogen-responsive regions of these promoters. Consistent with this, our laboratory has previously shown that wild-type ERβ modulates ERα activity by altering the recruitment of ERα, c-Fos, and c-Jun to estrogen-responsive promoters (20).

The ubiquitin proteasomal degradation multicomplex accounts for turnover of most short-lived proteins, including nuclear receptors (31, 32). Previous studies have shown that estradiol-mediated ERα degradation occurs through the 26 S proteasome pathway (33, 34). Our results show that ERβ2 decreases ERα protein levels in MCF7 cells, and that an inhibitor of proteasomal degradation (MG132) blocks ERβ2-induced down-regulation of ERα. This is consistent with the ERβ2-inducing proteasomal degradation of ERα. In our study, treatment up to 6 h with the ERα agonist PPT did not cause a decrease in ERα protein levels. The discrepancy between our findings and a report showing down-regulation of ERα following a 24-h treatment with estradiol (25) could be explained by differences in the duration of ligand treatment. The ERβ2 and ERα are coimmunoprecipitated in MCF7 cells (Fig. 5), suggesting a possible mechanism of ERβ2-induced proteasomal degradation of ERα that involves initial interaction of ERβ2 with ERα. Indeed, a mechanism in which protein-protein interactions activate the ubiquitin-proteasome pathway for degradation of one or both interacting proteins has been suggested by Wormke et al. (35).

We have shown a direct protein-protein interaction between ERβ2 and ERα in vitro and in mammalian cells. Similarly, previous studies showed that transient coexpression of wild-type ERβ and ERα leads to formation of heterodimers, binding to a synthetic ERE in vitro (36, 37). The major dimer interface between ERα and ERβ has been mapped to a conserved region of the hormone binding domain corresponding to helix 10. Indeed, the amino acid sequence of helix 10 is also conserved between wild-type ERβ and ERβ2. The last 61 amino acids of ERβ, which are replaced by a unique 26-amino-acid sequence in ERβ2, encode part of helix 11 and helix 12, but leaving helix 10 unchanged. It is therefore not surprising that ERβ2 forms heterodimers with ERα. A recent study indicated that coexpression of ERβ and ERα can uniquely regulate gene expression (38). During the process of breast cancer progression, ERα and ERβ2 coexist, and the ratio of ERα to ERβ2 changes (39), suggesting that ERβ2 may be of biological importance during breast cancer development.

The molecular mechanisms behind the inhibitory effect of ERβ2 on ERα-mediated transactivation may involve a number of different pathways. For example, after the heterodimerization between ERβ2 and ERα, the ERβ2/ERα complex may dissociate from the estrogen-responsive promoters, resulting in repression of ERα target gene expression. In addition, heterodimerization may hinder the recruitment of coactivators to the receptors (e.g., due to steric hindrance or because heterodimerization induces receptor conformations that are nonpermissive for transactivation). However, it is unlikely that ERβ2 and ERα act as a heterodimer on an ERα-responsive promoter because our findings show that ERβ2 was much less efficiently recruited to such promoters. Furthermore, it has previously been reported that heterodimerization between NRs sometimes inhibits receptor activity. For example, heterodimerization between ERRγ and ERRα was found to inhibit the transcriptional activities of both receptors (40). Results of the present study suggest another possible mechanism where ERβ2 induces proteasome-dependent degradation of ERα, resulting in limiting levels of this protein, thus leading to suppression of ERα transcriptional activity. This model is supported by other studies investigating nuclear receptor crosstalk with other signaling systems (35, 41). For instance, decreased ERα levels may contribute to the decreased expression of some E2-responsive genes in breast cancer cells cotreated with E2 plus TCDD.

Although the majority of human breast cancers express ERβ2, and the level of expression of ERβ2 often exceeds that of wild-type ERβ in these cancers (42, 43), the clinical significance of ERβ2 still remains to be defined. Clinical studies indicate that expression of ERβ2 in breast cancer correlates with a poor response to antiestrogen (13). In this regard, it is of interest that ERβ2 reduces ERα protein levels because the presence of ERα and progesterone receptors (PR) is predictive for response to endocrine therapy and improved disease-free survival (44). Approximately 50% to 60% of women with ERα-positive breast cancer benefit from endocrine therapy. In contrast, only a small minority of ERα/PR–negative patients respond to endocrine therapy (45). In this study, we show that high levels of ERβ2 were expressed in ERα-negative breast tumors, implying that the presence of ERβ2 in breast cancer might lead to tamoxifen resistance. Our findings are concordant with the observations that a decrease of ERβ2 is associated with the development of ERα-expressing breast cancer (46).

In summary, we have shown that ERβ2 binds directly to ERα and regulates ERα protein levels and transcriptional activity in a negative manner. Further studies are required to understand the distinct role of ERβ2 in estrogen-dependent cell proliferation and development of hormone-dependent tumors.

Potential conflict of interest: J-Å. Gustafsson is cofounder, shareholder, deputy board member, and consultant of KaroBio AB.

Grant support: Swedish Cancer Fund, KaroBio AB, and Susan Komen Foundation (G. Cheng).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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