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. 2008 Dec 23;150(5):2429–2435. doi: 10.1210/en.2008-1148

Estradiol and Tamoxifen Mediate Rescue of the Dominant-Negative Effects of Estrogen Response Element-Binding Protein in Vivo and in Vitro

Hong Chen 1, Thomas L Clemens 1, Martin Hewison 1, John S Adams 1
PMCID: PMC2671906  PMID: 19106221

Abstract

Biological responses to estrogens are dependent on the integrated actions of proteins, including the estrogen receptor (ER)-α, that regulate the transcription of estrogen response element (ERE)-containing target genes. We have identified a naturally occurring ERE antagonist, termed an ERE-binding protein (BP). To verify that ERE-BP can induce estradiol (E2) resistance in vivo, we generated transgenic mice that overexpress this protein in breast tissue. Female transgenic mice with high levels of ERE-BP were unable to lactate, and we hypothesized that this effect was dependent on the relative levels of ERE-BP and ERα ligand. To test this hypothesis, wild-type and ERE-BP-expressing female mice were implanted with capsules containing E2, the selective estrogen receptor modulator tamoxifen, or placebo. Histological analysis of nonlactating mammary glands showed a 4.5-fold increase in gland branch number and 3.7-fold increase in ducts in ERE-BP mice treated with E2 (7.5 mg, 21 d) compared with placebo-treated ERE-BP mice. Wild-type mice showed a 5.3-fold increase in branches and 1.4-fold increase in ducts under the same conditions. Similar results were obtained with tissue from lactating mice, in which tamoxifen also increased mammary gland branch number. Studies using ERE-BP-expressing MCF-7 breast cells showed that high doses of E2 (1000 nm) restored normal ERα-chromatin interaction in these cells, whereas tamoxifen was able to achieve this effect at a dose of 10 nm. These data highlight the importance of ERE-BP as an attenuator of normal ERα signaling in vivo and further suggest that ERE-BP is a novel target for modulation by selective estrogen receptor modulators.

The estrogen-response element binding protein suppresses estrogen activity; its effects in vivo can be rescued by high doses of estradiol or tamoxifen.

Target cell responses to steroid hormones are dependent on a variety of factors including the expression and regulation of cognate intracellular receptors (1,2,3), their associated accessory proteins (4), and prereceptor regulation of the hormones themselves (5,6). In previous studies we compared tissues from New World primates (NWPs) and Old World primates to identify two novel classes of proteins that are associated with vitamin D and estrogen signaling (7). The first of these, heat-shock proteins (hsps), are known to act as chaperones for steroid hormone receptors (8,9,10). However, we have shown that specific hsps abundantly expressed by NWPs also function as intracellular chaperones for the actual receptor ligands by acting as an alternative cytosolic binding site for vitamin D metabolites and estrogens (11,12,13). For example, an intracellular vitamin D binding protein (IDBP) purified from NWP cells was shown to be homologous with the human hsp70 (13), and subsequent studies confirmed that the constitutive form of hsp70, hsc70, is able to bind 25-hydroxyvitamin and 1,25-dihydroxyvitamin D with high affinity (14). Although hsc70 appears to be the human IDBP, functional analyses showing that overexpression of hsc70 enhances vitamin D metabolism and function (14,15) suggest that this protein is not the underlying cause of vitamin D resistance in NWPs. In contrast, an NWP intracellular estrogen binding protein (IEBP) with homology to human hsp27 has been shown to suppress estradiol (E2) signaling, supporting its role in the insensitivity to E2 that is characteristic of these animals (16).

In contrast to IDBP and IEBP, the second class of proteins shown to be overexpressed in steroid hormone-resistant NWPs do not appear to bind ligand or interact with nuclear receptors. Rather they were identified by their ability to bind to DNA and compete with nuclear receptors for access to hormone response elements in target gene promoters (17,18). Subsequent studies showed that these NWP response element binding proteins (REBiPs) are homologous to members of the human heterogeneous nuclear ribonucleoprotein (hnRNP) family: the vitamin D response element (VDRE) binding protein (BP) has 99.5% nucleotide homology with human hnRNP C1/C2 (19), whereas the estrogen response element binding protein (ERE) BP is similar to hnRNPC-like or hnRNP-D (18). Although the VDRE-BP and ERE-BP are overexpressed in NWP cells, they also appear to play a pivotal role in spatiotemporal organization of the transcriptional machinery associated with normal VDRE and ERE-mediated gene regulation (19,20). Thus, the VDRE-BP and ERE-BP appear to be integral members of the group of coregulator proteins known to be associated with steroid hormone signaling (4,21).

To assess the broader impact of the ERE-BP on estrogen signaling we developed a transgenic mouse model that overexpresses the NWP hnRNPC-like ERE-BP at varying levels under the control of the whey acidic acid gene promoter (22). The resulting animals were fertile and viable but showed specific inhibition of estrogen function in breast tissue, which resulted in aberrant mammary gland development and a complete lack of lactation after delivery of normal litters of pups (22). Here we have expanded these studies to assess the effects of selective estrogen receptor modulators (SERMs) in rescuing ERE-BP-induced estrogen insensitivity. Data indicate that both E2 and the estrogen receptor (ER)-α antagonist/agonist tamoxifen can compete out the effects of ERE-BP and restore normal breast development in the REBiP transgenic mice.

Materials and Methods

Cell culture and transfection of ERE-BP

MCF-7 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. Transient transfection of ERE-BP cDNA in MCF-7 cells was carried out using 5.0 μg pcDNA3.1/v5-His-TOPO ERE-BP plasmid [as described previously (20)] in lipo-TAXI solution (Stratagene, La Jolla, CA) for 5 h, followed by an equal volume of 20% FCS-supplemented medium. MCF-7 cells were grown to 80–90% confluence in a 12-well plate. Each well received 0.8 μg ERE-BP expression plasmid in Opti-MEM (Invitrogen, Carlsbad, CA) containing 4.0 μl Lipofectamine 2000 (Invitrogen) per 100 ml medium and incubated overnight. The next day, medium was replaced and treatments (vehicle, 1 or 1000 nm E2, or 1 or 1000 nm tamoxifen) added. After an additional 24 h at 37 C, cells were harvested for chromatin immunoprecipitation as described below.

ERE-BP-expressing transgenic mice

ERE-BP overexpressing mice used in the study were as described previously (22). Briefly these animals were generated using a full-length ERE-BP cDNA (18) ligated to the 3′ end of the rabbit ß-globin second intron by insertion into the EcoRI and XhoI sites of plasmid pKBPA (kindly provided by Dr. F. DeMayo, Baylor College of Medicine, Houston, TX). The 2.6-kb whey acidic protein (WAP) mammary gland-specific promoter transgene cassette provided by Dr. L. Hennighausen (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) (23) was cloned into T-Easy vector (Promega Madison, WI), digested with NotI, and inserted into the NotI site at the 5′ end of the rabbit ß-globin second-intron ERE-BP. The excised DNA fragment was sequenced and then microinjected into mouse (strain FVB/N) fertilized eggs. Transgenic offspring of founder animals were identified by PCR using a set of primers specific for the transgene, possessing the WAP-promoter forward primer 5′-CAAAGTCTTCCTCCTGTGGG-3′ and rabbit ß-globin second-intron reverse primer 5′-GGTGATACAAGGGACATCTTCC-3′. Five different transgenic lines were initially established with variable levels of transgene expression in the breast. In the current study, mice expressing the highest levels of ERE-BP were used for analysis of the effects of estrogen or tamoxifen treatment.

Implantation of SERMs

Four-week-old wild-type and ERE-BP overexpressing female mice were lightly anesthetized by isoflurane under a fume hood. One pellet per mouse was implanted sc in the flank of each mouse using a sterile 10-gauge trochar with either control pellet (vehicle) or pellets that release E2 (7.2 mg per 21 d, 0.72 mg per 60 d, 0.5 mg per 21 d) or tamoxifen (12.5 mg per 21 d or 6.5 mg per 21 d), respectively (Innovative Research of America, Sarasota, FL). After a 2-wk, drug-free hiatus after the expiration of the release pellets, sexually mature females in each group were mated with male to generate litters. Blood samples were collected by using a heparinized capillary tube at before implantation and d 7, 14, and 21 of implantation. For experiments with virgin females, mice were euthanized on completion of estradiol release. All mice were cared for and used in accordance with institutional animal care policies (Cedars-Sinai Medical Center Institutional Animal Care and Use Committee No. 1139).

Tissue collection and histological and immunohistochemical analysis

Mice were euthanized by CO2 asphyxiation. The thoracic mammary glands were immediately frozen on liquid nitrogen and then stored at −80 C until used for RNA or protein extraction. The inguinal mammary glands were excised and used for whole-mount slides and histochemistry. The mammary gland whole-mount slides and histochemistry slides were prepared as described as previously (23). Briefly, for whole-mount slides, the mammary glands were fixed in Carnoy’s solution for 4 h and stained overnight in carmine alum solution. Glands were then dehydrated. For histochemistry, paraffin-embedded slides were cut 4 μm and stained with hematoxylin/eosin (H&E). For whole tissue mounts and H&E sections, mammary gland branching was quantified using Photoshop Creative Suite 3 (CS3) tool (Adobe, San Jose, CA) to determine values for the area of branches or perimeter of ducts or as percent area occupied. Data are shown as arbitrary pixel numbers for branches or ducts. For whole tissue mounts, data are shown as the percent of fat pads occupied by mammary gland branches. Values obtained for histological evaluation are based on simple point counting of three microscopic fields for each slide in three mammary glands per group.

For immunostaining, deparaffinized slides were incubated overnight at 4 C with anti-ERE-BP antibody and/or antihuman ERα antibody and anti-WAP antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Specifically bound antibody was visualized using fluorescein thiocyanate and/or Texas Red-conjugated fluorescent secondary antibodies (duck antigoat and duck antirabbit, respectively; Santa Cruz Biotechnology) under an optical BX41 microscope (Olympus, Tokyo, Japan). Normal goat/rabbit IgG was used as negative control antiserum.

Chromatin immunoprecipitation (ChIP) assays

ChIP assays were performed as described previously (20). Briefly, after treatment, cells proteins-DNA were cross-linked with 1% formaldehyde at 37 C for 10 min. After quenching of cross-linking with 1.25 m glycine, cells were harvested, rinsed with PBS, and the resulting pellets resuspended in 1 ml of cell lysis buffer [5 mm 1,4-piperazine diethane sulfonic acid (pH 8.0); 85 mm KCl; 0.5% Nonidet P-40; 1 mm dithiothreitol; 0.25 mm phenylmethylsulfonyl fluoride; and 1 μg/ml each of pepstatin, leupeptin, and aprotinin]. Nuclei were collected and resuspended in 500 μl of nuclear lysis buffer [50 mm Tris-HCl (pH 8.1), 10 mm EDTA, 1% sodium dodecyl sulfate (SDS), 1 mm dithiothreitol, 2.5 mm phenylmethylsulfonyl fluoride; and 1 μg/ml each of pepstatin, leupeptin, and aprotinin, all from Sigma, St. Louis. MO]. The resulting chromatin samples were sonicated to yield sheared DNA fragments of sizes between 300 and 1000 bp. For each immunoprecipitation, sheared chromatin was diluted with immunoprecipitation dilution buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tis-HCl (pH 8.1), and 167 mm NaCl]. Chromatin was collected and incubated at 4 C overnight with 5 μg of anti-ER antibodies (Santa Cruz Biotechnology), anti-ERE-BP antibody. Rabbit IgG was used as a negative control. The immune complexes were precipitated with 60 μl of protein A-Sepharose beads (source) at 4 C for 1 h and then subjected to serial 1-ml washes of thereafter: IP dilution buffer; low-salt buffer [0.01% SDS, 1.1% Triton X-100, Tris-HCl (pH 8.1), 150 mm NaCl]; high-salt wash buffer [buffer 0.01% SDS, 1.1% Triton X-100, Tris-HCl (pH 8.1), 500 mm NaCl]; LiCl/detergent buffer [100 mm Tris-HCl (pH 8.1); 500 mm LiCl; 1% Nonidet P-40; 1% deoxycholic acid] and Tris-EDTA buffer. Antibody-protein-DNA immunocomplexes were then eluted with 1% SDS in 50 mm NaHCO3. Formaldehyde cross-linking was reversed by heating at 65 C overnight with the addition of 5 m NaCl to a final concentration of 200 mm. All samples were then digested at 45 C for 1 h with 20 μg proteinase K and the resulting DNA amplified by PCR using primers (5′-TATGAATCACTTCTGCAGTGAG, reverse 5′-GAGCGTTAGATAACATTTGCC) for DNA sequences within the promoter of the gene for pS2, which has been extensively studied for ERα-chromatin interactions (24).

Statistical analyses

Data were expressed as mean ± sd unless otherwise stated. Statistical evaluation of correlations for circulating vitamin D metabolites was by linear regression. Statistical analysis of treatment studies was carried out by one-way ANOVA with the Holm-Sidak method as a post hoc multiple comparison procedure applied to raw data. All statistical values were defined using Sigmaplot 9.0 software (Systat Inc., San Jose, CA).

Results

In previous studies, we have shown that the WAP-targeted ERE-BP overexpressing mice (ERE-BP+) exhibited failure of mammary gland ductal development and lactation (22). In NWP, similar elevated expression of ERE-BP occurs in the face of high circulating levels of E2 (18). In preliminary analyses, we showed that expression of the ERE-BP in mammary gland ductal epithelial cells from ERE-BP+ mice is coincident with ERα (Fig. 1). We therefore postulated that elevation of estrogen levels in these mice could act to normalize ERα responses by competing out the dominant-negative effects of ERE-BP. To test this hypothesis, wild-type (WT) and ERE-BP+ mice were administered varying doses of E2 or the SERM tamoxifen for different time periods. The animals were then assessed for changes in breast tissue histology.

Figure 1.

Figure 1

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ERE-BP is expressed in ERα-positive cells in mammary gland ductal cells from ERE-BP+ mice. Immunofluorescence analysis of ERE-BP and ERα protein in sections of mammary gland tissue from a nonlactating ERE-BP+ female mouse. Coincident expression of ERα and ERE-BP is shown as merged immunofluorescence. Control staining is shown as shown as immunofluorescence for IgG as primary antibody.

Implantation of a 7.5-mg E2 pellet in ERE-BP+ mice resulted in substantial rise in the circulating levels of this hormone to 4553.3 ± 3314 pg/ml at d 10 and 7953.3 ± 2578.8 ng/ml at d 21. These levels were much higher than placebo-treated animals (259 ± 150 pg/ml) but similar to the serum E2 concentrations in WT mice implanted with a 7.5-mg E2 pellet (Fig. 2). Tamoxifen (6.25 mg per 21d) had no significant effect on circulating levels of E2.

Figure 2.

Figure 2

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Serum E2 levels in female mice after treatment with SERMs. WT and ERE-BP transgenic mice (ERE-BP+) were implanted with 7.5 mg E2 slow-release pellets and blood sampled at different time points to assess serum levels of E2. Values shown are the mean of n = 5 mice for each treatment and time point. TAM, Tamoxifen.

The effects of E2 or tamoxifen on breast morphology in virgin WT and ERE-BP+ mice were studied by tissue histology of excised mammary glands. Whole-tissue mounts (Fig. 3A) and H&E stained tissue sections (Fig. 3B) showed that nonlactating virgin ERE-BP+ mice exhibited low levels of mammary gland branching compared with equivalent WT female mice (40 and 72% lower, respectively). In both types of mice, treatment with E2 pellets increased branch formation. Quantification of the percentage area of fat pads covered by branches showed a 2-fold increase in ERE-BP+ mice after exposure to low dose (0.5 mg) E2. A similar increase in WT mice was observed with higher dose E2 (7.5 mg) (Fig. 3C). In tissue stained by H&E, branch area increased 3-fold in WT mice and 8-fold in ERE-BP+ mice with exposure to 0.5 mg E2 (Fig. 3D). Both showed a further 2-fold increase in branching with a higher dose of E2 (7.5 mg). The net effect of this was that mammary gland branching was similar for WT and ERE-BP+ mice after treatment with 7.5 mg E2. Analysis of H&E-stained sections showed that tamoxifen also potently increased mammary gland branching, but the diffuse nature of the branching in tissues from these animals prevented accurate quantification (see supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

Figure 3.

Figure 3

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Effect of E2 on mouse mammary gland branching in virgin, nonlactating, female mice. Four-week-old virgin female WT and ERE-BP transgenic mice (ERE-BP+) were implanted with slow-release pellets for either E2 (0.5 mg or 7.5 mg). After a further 21 d, mice were euthanized, and whole tissue mounted and stained with carmine aluminum solution (A); breast tissue sections assessed histologically after H&E staining (B; magnification, ×40). Quantification of carmine aluminum and H&E staining is shown in C and D, respectively (n = 3 separate slides with three separate fields of view for each slide), based on arbitrary units for branch area quantified by Adobe CS3. Statistical analysis of data in C and D is shown as P values calculated by ANOVA.

Mammary gland tissue from the placebo-treated ERE-BP+ mice (Fig. 4B) exhibited no lobule formation and 50% fewer ducts compared with WT mice (Fig. 4A). Treatment with E2 increased duct formation in WT mice 1.4-fold (P = 0.024), whereas ERE-BP+ mice showed a 3.7-fold increase in duct number (P < 0.001) (Fig. 4C). As with the analysis of branching in Fig. 3D, there was no statistical difference in mammary gland duct levels between WT and ERE-BP+ mice after treatment with E2.

Figure 4.

Figure 4

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Effect of E2 on mammary gland ducts in virgin, nonlactating, female mice. Four-week-old female WT (A) and ERE-BP transgenic mice (ERE-BP+) (B) were implanted with slow-release pellets for either E2 (7.5 mg) or placebo. After a further 21 d, mice were euthanized and breast tissue sections assessed histologically after H&E staining. Inset on ×10 magnification images corresponds to field for ×40 magnification. Quantification of H&E staining is shown in C (n = 3 separate slides with three separate fields of view for each slide), based on arbitrary units for duct perimeter quantified by Adobe CS3. Statistical analysis of data in C is shown as P values calculated by ANOVA.

Further studies were then carried out to determine whether lactation altered estrogen responsiveness in breast tissue from ERE-BP+ mice. All WT and ERE-BP+ mice were reproductively fertile and delivered normal litters when implanted with the placebo pellet. Treatment with E2 at 7.5 mg was tolerated by female WT mice but these animals were completely infertile. By contrast, female WT mice implanted with a 0.5 mg E2 pellet showed no effect on fertility, and 25% of WT mice receiving tamoxifen (12.5 mg) produced pups when mated with normal WT male mice. When treated with E2 (7.5 mg), 40% of ERE-BP+ mice produced litters (n = 5 matings). Similar observations were also made for ERE-BP+ mice receiving 0.5 mg E2.

As shown in Fig. 5A, placebo-treated lactating ERE-BP+ mice exhibited mammary glands with impaired branch duct development compared with WT mice (Fig. 5B). Analysis of whole tissue mounts from ERE-BP+ females showed distended ducts but lubuloalveolar development was not extensive. After treatment with E2 or tamoxifen, ductal branches were transformed into alveolar or lobular structures, although normal mammary gland tissue showed greater density of lobule-alveolar formation. H&E staining also showed a lack of lobule formation in ERE-BP+ mice without E2 or tamoxifen treatment. The ERE-BP+ mice also exhibited only scattered ductal elements with far less acinar formation and progressively more prominent interlobular fat reminiscent of the immature pubertal, nonlactating alveoli formation. There was more enlargement of ducts containing the milk in the mice treated with E2. Quantification of branch area and duct perimeter in the lactating ERE-BP+ females confirmed that these features were much less abundant than in WT females (Fig. 5C). Branch numbers were 15-fold lower in ERE-BP+ mice compared with WT, with duct numbers bring 9-fold lower. Both of these parameters were significantly increased in mice treated with either low (0.72 mg per 60 d) or high (7.5 mg per 21 d) E2. However, the most pronounced improvement in ERE-BP+ mammary gland development was observed in those mice treated with tamoxifen (6.25 mg per 21 d). Branch number increased 10-fold and duct number increased 7-fold). Both parameters were significantly higher in tamoxifen-treated mice compared with equivalent E2-treated mice (both P < 0.001) but were also significantly lower than WT lactating mammary glands (Fig. 5B).

Figure 5.

Figure 5

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Effect of E2 or tamoxifen on mammary gland development in lactating female mice. A, One-month-old female ERE-BP transgenic mice (ERE-BP+) were implanted with slow-release pellets for E2 (0.72 or 7.5 mg), tamoxifen (TAM) (6.25 mg), or placebo. After expiration of the timed-release pellets (21 d for 7.5 mg E2 and 6.25 mg TAM or 60 d for 0.72 mg E2), mice underwent a 2-wk SERM-free hiatus. The resulting 9- or 14-wk-old females were then bred with ERE-BP+ male counterparts and breast tissue examined at d 3 of lactation after birth. Whole-mount breast tissue sections were stained with carmine aluminum solution (top panel) and breast tissue sections assessed histologically after H&E staining (middle and lower panels). In each case, insets show magnification values. B, Carmine aluminum and H&E staining in lactating mammary glands from WT mice. C, Quantification of H&E staining of mammary gland branches and ducts (n = 3 separate slides with three separate fields of view for each slide), based on arbitrary units for branch area and duct perimeter quantified by Adobe CSE. Statistical analysis of data in C is shown as P values calculated by ANOVA. *, Statistically different from placebo-treated ERE-BP+ mice P < 0.05; **, statistically different from placebo-treated ERE-BP+ mice P < 0.01; ***, statistically different from placebo-treated ERE-BP+ mice P < 0.001.

Analysis of the in vivo effects of E2 and tamoxifen in ERE-BP+ mice confirmed that the response element-binding protein confers resistance to the contraceptive and mammary gland development effects of E2. The effects of ERE-BP on sensitivity to endogenous E2 can be rescued with high doses of exogenous E2, suggesting that molecular mechanism of ERE-BP action is reversible. Finally, tamoxifen is also able to rescue the effects of ERE-BP on endogenous E2 sensitivity, despite the fact that this SERM is known to act as a partial antagonist for ERα. To clarify these observations, ChIP analyses were carried out using transfected MCF-7 breast cells to mimic the effects of overexpression of ERE-BP. Data for control MCF-7 cells indicated that in the absence of E2, the pS2 ERE was constitutively occupied by the ERE-BP with no apparent precipitation of ERα (Fig. 6A). In these cells, the addition of low dose (10 nm) E2 displaced ERE-BP from the ERE in favor of the liganded ERα. MCF-7 cells treated with high-dose E2 (1000 nm) or tamoxifen (10 or 1000 nm) also showed recruitment of ERα to the pS2 ERE, but this was coincident with the presence of the ERE-BP on the ERE.

Figure 6.

Figure 6

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Effect of E2 or tamoxifen (tam) on dysregulated ERα-chromatin interaction in WT and ERE-BP overexpressing MCF-7 cells. Nuclear chromatin from parental MCF-7 cells (A) and stable transfectant variants of MCF-7 overexpressing the ERE-BP (B; MCF-7 ERE-BP) were used in ChIP assays. Cells were treated with or without E2 (10 or 1000 nm) or tamoxifen (10 or 1000 nm) for 15 min. Occupancy of the ERE in the pS2 gene promoter by ERα, ERE-BP, or IgG (negative control) was determined by PCR amplification of the estrogen-responsive DNA region of the pS2 promoter after precipitation with antibodies to ERα (ERAb), ERE-BP (ERE-BPAb), or IgG (see Materials and Methods).

In MCF-7 cells overexpressing ERE-BP, both the ERE-BP and ERα were detected on the ERE in the absence of any added E2 or tamoxifen (Fig. 6B). ERE-BP overexpressing cells also showed a different pattern of ERE occupancy after treatment with E2 or tamoxifen. Low-dose E2 (10 nm) displaced ERE-BP from the ERE but also eliminated binding of ERα, whereas high-dose E2 (1000 nm) or tamoxifen (10 and 1000 nm) enhanced binding of ERα to the ERE, similar to the effects seen with low-dose E2 in wild-type MCF-7 cells. However, in contrast to the MCF-7 cells the ERE-BP overexpressing cells treated with high-dose E2 or tamoxifen showed displacement of the ERE-BP from the ERE.

Discussion

Comparative analysis of NWP endocrinology has greatly helped to shed light on the complex machinery that is required to mediate steroid hormone signaling. NWPs such as marmosets, tamarins, and squirrel monkeys exhibit significantly higher circulating levels of steroid hormones than their Old World primates counterparts (including humans), but they are also profoundly resistant to these hormones (25,26). In some instances, notably for glucocorticoids, end-organ resistance is associated with altered binding affinity of glucocorticoid receptors for its ligand cortisol (26), possibly because of differential interaction between glucocorticoid receptors and cochaperones such as the immunophilins FKBP52 and FKBP51 (27).

NWPs are also profoundly resistant to estrogens and vitamin D, and in previous studies of these hormones, we have shown that this involves two novel classes of proteins: 1) intracellular chaperones that actively bind and translocate steroid hormones; 2) REBiPs such as VDRE-BP and ERE-BP that bind to DNA and compete with liganded steroid hormone receptors for occupancy of cognate hormone response elements in target gene promoters (7,28). In the case of estrogen signaling, both types of protein may act to mediate resistance to E2. For example, the IEBP (hsp27) can compete with ERα for binding of E2, and this may abrogate normal ERα-mediated responses (16). However, in recent studies we have shown that IEBP/hsp27 also interacts directly with ERE-BP, playing a key role in the subcellular localization of the latter (20). In particular, we have shown that increased expression of IEBP/hsp27 results in aberrant association between the ERE-BP and target EREs, suggesting that REBiP function may be the rate-limiting step in estrogen resistance in NWPs. The aim of the current study was to investigate further the dynamics of ERE-BP function with respect to the regulation of estrogen signaling by using in vivo and in vitro models that recapitulate the hormone resistance characteristic of NWPs.

In previous studies we validated the use of tissue-specific induction of ERE-BP in the breast as an in vivo model for the physiological actions of this REBiP (22). In the current study, we used these mice to examine the relationship between ERE-BP and the hormone it suppresses, namely E2. Analysis of transgenic mice presented in the current study indicates that the ERE-BP is able to attenuate mammary gland responses to ERα in a similar fashion to recently reported effects for the putative tumor suppressor prohibitin (4). Prohibitin acts as an ERα corepressor protein, and many other corepressors are also known to induce resistance to SERMs in vitro and in vivo (29). In contrast to corepressor-induced estrogen resistance, the inhibitory effects of ERE-BP appear to be readily counteracted by high doses of SERMs both in vivo and in vitro. This is underlined by the fact that the circulating levels of E2 achieved in the transgenic animals after implantation with the E2 pellet were similar to those that occur during the normal estrus cycle or pregnancy in NWPs such as the marmoset (30,31). As yet, it is unclear whether overexpression of ERE-BP is associated with estrogen resistance in diseases such as breast cancer. However, in view of the ability of SERMs to compete out ERE-BP suppression in the mouse model presented here, this seems unlikely.

The ability of tamoxifen to mimic the effects of high-dose E2 in this in vivo model of estrogen resistance highlights an entirely novel facet of this SERM. Tamoxifen is a relatively weak ligand for ERα (32), but its metabolite 4-hydroxy-tamoxifen has a similar binding affinity as E2 itself (33). Thus, as with other SERMs such as raloxifene, tamoxifen is able to actively promote some aspects of liganded ERα signal transduction, such as nuclear translocation and receptor binding to EREs. The ability of tamoxifen to act as a gene-specific partial antagonist stems from more subtle effects on ERα that include conformational changes and differential recruitment of coactivators and corepressors (34,35). In vivo and in vitro data presented in this study indicate that in the context of the ERE-BP, tamoxifen is in fact more effective than E2 in counteracting ERE blockade. ChIP analyses show that a 10-nm dose of E2 or tamoxifen displaced the ERE-BP from target gene EREs in MCF-7 cells overexpressing the REBiP (see Fig. 6B). However, reciprocal binding of liganded ERα was achieved with 10 nm tamoxifen, whereas the dose of E2 required was 1000 nm. This suggests that the synthetic SERM is more effective in restoring full estrogen responsiveness in the face of high levels of ERE-BP. However, interpretation of these observations is complicated by the fact that occupancy of hormone response elements is likely to be transient. In previous studies we have shown a reciprocal cyclical occupancy of the VDRE by VDR and the VDRE-BP (19). Preliminary data suggest that there are similar spatiotemporal variations for occupancy of the ERE (see supplemental Fig. 2).

In vivo tamoxifen greatly enhanced mammary gland branching in ERE-BP+ mice suggesting that in these animals the SERM acts in a predominant estrogenic or agonist fashion. Previous studies using animal models of mammary gland development demonstrated that tamoxifen can act as an ERα agonist with respect to gland development, but this depended on the dose used and age of administration (36). It is also important to note that overexpression of ERE-BP has been shown to affect subcellular localization of the IEBP/hsp27, which is an active competitor for cytosolic E2 binding, but does not appear to bind tamoxifen (20). Thus, it is possible that some of the potency of tamoxifen in counteracting the effects of ERE-BP is due to its ability to circumvent dysregulated localization of the IEBP.

In summary, data presented here provide further evidence of a role for the ERE-BP as rheostatic attenuator of estrogen responses. Experiments in vivo and in vitro confirmed our original hypothesis that the suppressive effects of ERE-BP on ERα signaling can be rescued by increased doses of its ligand E2. The fact that we were also able to restore normal ERα function using similar dose levels of the partial ERα antagonist tamoxifen reveals another attribute of this synthetic steroid and also indicates that the ERE-BP affects ERα signaling in a manner, which is distinct from that observed with receptor corepressor proteins. Future studies to clarify the molecular interactions between ERE-BP and ERα agonists/antagonists may provide important information for the development of new generations of SERMs.

Supplementary Material

[Supplemental Data]
en.2008-1148_index.html (1.1KB, html)

Footnotes

This work was supported by National Institutes of Health Grants RO1DK055843 (to H.C.) and R01AR37399 (to J.S.A.).

Disclosure Summary: The authors have no conflict of interest to declare.

First Published Online December 23, 2008

Abbreviations: BP, Binding protein; ChIP, chromatin Immunoprecipitation; E2, estradiol; ER, estrogen receptor; ERE, estrogen response element; H&E, hematoxylin/eosin; hnRNP, heterogeneous nuclear ribonucleoprotein; hsc70, constitutive form of hsp70; hsp, heat-shock protein; IDBP, intracellular vitamin D binding protein; IEBP, intracellular estrogen binding protein; NWP, New World primate; REBiP, response element binding protein; SDS, sodium dodecyl sulfate; SERM, selective estrogen receptor modulator; VDRE, vitamin D response element; WAP, whey acidic protein; WT, wild type.

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