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The Human Estrogen Receptor- Isoform hER46 Antagonizes the Proliferative Influence of hER66 in MCF7 Breast Cancer Cells

Equipe d’Endocrinologie Moléculaire de la Reproduction, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6026, 35042 Rennes Cedex, France

Address all correspondence and requests for reprints to: Gilles Flouriot, Equipe d’Endocrinologie Moléculaire de la Reproduction, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6026, Université de Rennes I, 35042 Rennes Cedex, France. E-mail: gilles.flouriot{at}univ-rennes1.fr.

	Abstract

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The expression of two human estrogen receptor- (hER) isoforms has been characterized within estrogen receptor--positive breast cancer cell lines such as MCF7: the full-length hER66 and the N terminally deleted hER46, which is devoid of activation function (AF)-1. Although hER66 is known to mediate the mitogenic effects that estrogens have on MCF7 cells, the exact function of hER46 in these cells remains undefined. Here we show that, during MCF7 cell growth, hER46 is mainly expressed in the nucleus at relatively low levels, whereas hER66 accumulates in the nucleus. When cells reach confluence, the situation reverses, with hER46 accumulating within the nucleus. Although hER46 expression remains rather stable during an estrogen-induced cell cycle, its overexpression in proliferating MCF7 cells provokes a cell-cycle arrest in G₀/G₁ phases. To gain further details on the influence of hER46 on cell growth, we used PC12 estrogen receptor--negative cell line, in which stable transfection of hER66 but not hER46 allows estrogens to behave as mitogens. We next demonstrate that, in MCF7 cells, overexpression of hER46 inhibits the hER66-mediated estrogenic induction of all AF-1-sensitive reporters: c-fos and cyclin D1 as well as estrogen-responsive element-driven reporters. Our data indicate that this inhibition occurs likely through functional competitions between both isoforms. In summary, hER46 antagonizes the proliferative action of hER66 in MCF7 cells in part by inhibiting hER66 AF-1 activity.

	Introduction

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GROWTH AND DIFFERENTIATION of the female reproductive tracts are under the critical influence of estrogens such as 17ß-estradiol (E₂) (1, 2). It is well established that the mitogenic actions of these steroids also have critical influences on the etiology and progression of human breast and uterus cancers (3, 4). Normal and pathological growth-promoting effects of E₂ are achieved through stimulating cells in G₀ phase to enter the cell cycle and hastening the G₁ to S phase transition (5). Estrogens actions are exerted through specific receptors, the estrogens receptors (ER)- (NR3A1) and -ß (NR3A2) (6, 7, 8). Targeted disruption of ER and ERß genes clearly demonstrated that the postnatal development of uterus and mammary glands rely on ER rather than ERß (9). Furthermore, ER expression is intimately associated with breast cancer (10, 11). E₂ stimulates the proliferation of breast cancer cells that express ER, and ER-positive tumors are more differentiated and have less metastatic potential than ER-negative tumors. ER is therefore used as a prognosis factor and is targeted in therapies aiming to cure E₂-dependent cancers. The specific functions of ERß in breast cancers are not precisely known. However, this protein is detected in human breast cancer and, notably, exhibits a decreased expression in invasive breast tumors vs. normal tissues (12).

ER belongs to the nuclear receptor superfamily of transcription factors, structurally organized in six functional domains (A to F) (13). The C domain is necessary and sufficient for the specific binding of the receptor to DNA. The E domain allows hormone binding, an event that induces specific conformational changes within the receptor. This three-dimensional remodeling allows ER to modulate the transcriptional activity of target genes through two transactivation functions (AFs), AF-1 and AF-2, located in the B and E domains, respectively. The respective contribution that AF-1 and AF-2 make toward the activity of the full-length ER is both promoter and cell specific (13, 14, 15, 16). Accordingly, promoter and cell contexts can be defined as AF-1 or AF-2 permissive, depending on which AF is principally involved in ER activity. Transcriptional modulation of E₂-target genes involves recruitment of ER either directly through interaction with cognate DNA sequences [estrogen-responsive elements (EREs)], or protein/protein interaction with other transcriptional factors (17). ER-mediated transactivation is then achieved through an ordered sequence of interactions established between the AFs and coactivators such as: 1) members of the p160 subfamily (exemplified by steroid receptor coactivator-1 and transcription intermediary factor-2); 2) cAMP response element binding protein-binding protein/p300; 3) complexes of the Srb-Med coactivator complex/thyroid hormone receptor-associated proteins/vitamin D receptor- interacting proteins/activator recruited cofactor class; and 4) AF-1-specific coactivators such as p68 and p72 RNA helicases (18, 19, 20).

Corroborating the role that estrogens have as mitogen, the expression of genes involved in the control of cell proliferation such as cyclin D1 (21), c-fos, c-myc (22, 23), or growth factor genes (IGF-I) (24) are under ER control. Besides its transcriptional functions, ER also presents nongenomic actions. For instance, ER stimulates rapidly the Src kinase and MAPK pathways to trigger cell cycle progression (25).

An isoform of ER, 46 kDa in size [human estrogen receptor- (hER)46], encoded by an mRNA variant was identified in MCF7 human breast cancer cells in which it is coexpressed with the full-length ER (hER66) (26). The importance of this isoform is illustrated by the observation that 50% of ER mRNA encode hER46 in osteoblasts (27). Expression of the hER46 isoform was also reported in endothelial cells (28, 29). hER46 lacks the N-terminal A and B domains and is consequently devoid of AF-1 (26). Mechanistically, hER46 induces the transcription of an ERE-derived reporter gene construct only in AF-2-permissive cell contexts (26). In contrast, this naturally occurring truncated hER is unable to transactivate the same reporter gene construct in cellular contexts in which AF-1 is the primary AF involved in hER activity. Moreover, when both isoforms are coexpressed, hER46 efficiently suppresses the AF-1 activity of hER66 in a cell-specific context (26). Finally, unliganded hER46 efficiently represses the transcription of target genes, this effect being reversed after E₂ binding (30, 31).

To date, no information exists on the exact function of hER46 in epithelial breast cancer cells. Exhibiting functional properties different from those of hER66, we hypothesized that the hER46 may have a role to play in the control of ER-positive breast cancer cell proliferation.

	Materials and Methods

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Plasmids
The reporter plasmids ERE-TK-Luc, hC3-Luc, and pCMV-ß-Gal internal control have been previously described (32). The c-fos-Luc and cyclin D1-Luc reporter genes were obtained by inserting human genomic PCR products (–730/+41 and –205/+54, respectively) into pGL3-basic (Promega, Charbonnier, France). The reporter plasmid (E/GRE)₂-Luc was obtained by inserting two annealed oligonucleotides in the pGL3-promoter vector (Promega): [5'-CCGGGAAAGGGCAGACTGTTCTTGGATCCAAGGGCAGTCTGTTCTTTAAGCTTATA-3'] and [5'-GATCTATAAGCTTAAAGAACAGACTGCCCTTGGATCCAAGAACAGTCTGCCCTT-3']. Expression vectors pCR hER66, pCR hER46, and pCR hER66_GR were generated by cloning the coding region of hER66 (+228/+2030), hER46 (+727/+2030), and hER66_GR (HE82; generously provided by P. Chambon, IGBMC, Illkirch, France) into the pCR 3.1 vector (Invitrogen, Cergy-Pontoise, France). Inducible expression vectors pIND hER66 and pIND hER46 were prepared by cloning corresponding open reading frame into the pIND vector (Invitrogen). Ecdysone-mediated expression of these open reading frames was performed using the pVgRXR vector (Invitrogen).

Cell culture and transfections
Hela, HepG2, and MCF7 cells were maintained in DMEM (Invitrogen) supplemented with 5% fetal calf serum (FCS; Sigma, St. Quentin Fallavier, France), penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin (35 µg/ml) at 37 C in 5% CO₂. PC12 cells were cultivated in DMEM/F12 containing 7.5% charcoal dextran-treated FCS and 2.5% charcoal dextran-treated horse serum.

Stably transfected MCF7 clones, MCF7 pIND, MCF7 pIND hER66, and MCF7 pIND hER46, were obtained by transfecting MCF7 cells with pVgRXR plasmid and corresponding expression vectors with FuGENE 6 reagent (Roche, Meylan, France), and selection with 0.8 mg/ml G418 and 0.8 mg/ml zeocin (Invitrogen). Stably transfected PC12 cell lines, PC12 pCR3.1, PC12 hER66, and PC12 hER46, were obtained by transfecting PC12 cells with corresponding pCR3.1 expression vectors and selection with 0.8 mg/ml G418 (Invitrogen).

Transient transfections were performed with the FuGENE 6 transfection reagent (Roche) as previously described (33). After either 12 h (for ERE-controlled reporter gene analysis) or 48 h (for c-fos and cyclin D1-Luc reporter analysis), cells were washed and then treated for 36 h (ERE-controlled reporter) or 12 h (c-fos and cyclin D1-Luc reporters) with ethanol (vehicle control), 10 nM E₂, or 2 µM 4-hydroxytamoxifen (4-OHT). Luciferase and ß-galactosidase activities were assayed on cell extracts.

Flow cytometry analysis (FACS) and [³H]thymidine incorporation assay
Cells growing in 10-cm-diameter dishes were pulse labeled with 1 mM 5-bromo-2'-deoxyuridine (BrdU) for 3 h. After trypsinization, cells were collected in PBS containing 30% immunofunctional assay (IFA) buffer [10 mM HEPES (pH 7.4), 150 mM NaCl, 4% FCS, 0.1% NaN3], pelleted at 1000 rpm for 10 min, and fixed in 70% ethanol as previously described (34). Fixed cells were incubated in IFA buffer containing the -BrdU-fluorescein isothiocyanate antibody (CALTAG Laboratories, Burlingame, CA) for 1 h at 4 C and then washed in IFA buffer including 0.5% Tween 20. These steps were omitted in control untreated samples. Finally, fixed cells were incubated in IFA buffer containing 100 µg/ml RNase A for 15 min at 37 C, and 25 µg/ml propidium iodide were added before analysis with a FACScan equipment (Becton Dickinson, Le Pont de Claix, France).

When assaying [³H]thymidine incorporation, the cells were incubated with 0.6 µCi [³H]thymidine 12 h before harvesting. Cells were then frozen and thawed, and incorporated [³H]thymidine was collected on A filter papers using a 96-well harvester and quantified by ß-counting.

Protein extracts
Subcellular fractionation was performed as described in the current protocol. Briefly, cells were harvested and resuspended in lysis buffer [10 mM Tris-HCl (pH 7.4), 3 mM CaCl2, 2 mM MgCl2] with protease inhibitors (Roche). Cells were then pelleted and incubated in Nonidet P-40 (NP-40) lysis buffer [10 mM Tris-HCl (pH 7.4), 3 mM CaCl2, 2 mM MgCl2, 0.5% NP-40, protease inhibitors] during 15 min. After centrifugation, the supernatant (cytoplasmic extract) was recovered, whereas the pellet (nuclei) was resuspended in radioimmunoprecipitation assay-lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS] containing protease inhibitors and sonicated (nuclear extract).

Western blotting
Twenty micrograms of proteins extracts were resolved on 10% SDS-PAGE and electrotransferred onto nitrocellulose membranes as previously described (26). Blots were incubated with the polyclonal anti-hER HC20 (TEBU), the monoclonal anti-Lamin B Ab-1 (Oncogene, Boston, MA), or the monoclonal anti-ß-actin AC-15 (Sigma) in PBS containing 0.1% Tween 20 and 5% nonfat milk powder for 1.5 h at room temperature. After washings, the blots were incubated with either a peroxidase-conjugated goat antirabbit (Pierce, Rockford, IL) or a peroxidase-conjugated goat antimouse (Pierce) for 1 h. Membrane-bound secondary antibodies were detected using the SuperSignal West Dura kit (Pierce) according to the manufacturer’s instructions.

EMSA
In vitro transcription and translation were performed using the TNT-coupled reticulocyte lysate system as recommended by the manufacturer (Promega) with pCR 3.1, pCR hER66, and pCR hER46 used as templates. Translation efficiency was checked by Western blot. Four microliters of rabbit reticulocyte lysate expressing ER proteins were preincubated in gel shift assay buffer [10 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 100 mM KCl, 10% glycerol, 100 µg/ml BSA, 5 µg/ml of protease inhibitors, and 1 mM phenylmethylsulfonyl fluoride] with 2 µg of poly(dIdC) for 15 min at room temperature. The samples were then incubated for 15 min with decreasing concentrations (1–0.0625 ng) of radioactive oligonucleotide probe end labeled with [-³²P]ATP using T₄ polynucleotide kinase (Roche). Protein-DNA complexes were separated from free probes by nondenaturing electrophoresis on 5% polyacrylamide gels in 0.5x Tris-borate EDTA. The sequence of the 30-bp oligonucleotide used in these experiments is: 5'-ctgtgctcAGGTCAgacTGACCTtccatta-3', with the consensus ERE sequence shown in capital letters.

	Results

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hER46 is mainly located in the nucleus and its expression increases in confluent MCF7 cells
Aiming to further characterize functional differences between hER66 and -46 isoforms, we first analyzed their respective subcellular localization during MCF7 cells growth, from scattered to confluent cells. During this time lapse, cell growth was monitored through cell numeration. Flow cytometry analysis was also used to evaluate the relative proportion of cells being in each of the different cell cycle phases (Fig. 1A). The percentage of MCF7 cells in S phase reaches its highest level 3 d after cell seeding and then progressively decreases until cells achieve confluence between d 9 and 12 (Fig. 1A). In parallel, Western blots performed on nuclear and cytoplasmic protein extracts probed the relative expression of either hER isoforms in each compartment (Fig. 1, B and C). Antibodies against the Lamin B, a nuclear protein, controlled the efficiency of the fractionation, whereas ß-actin was used as a loading control. Results indicate that hER46 is almost totally localized in the nucleus and strongly accumulates in this compartment when cells reach confluency (Fig. 1B). In a few experiments, hER46 was weakly detected in the cytoplasmic fraction at confluence. In contrast, hER66 is localized in both the nucleus and cytoplasm, with a gradual accumulation observed during cell growth (until d 9, Fig. 1C).

View larger version (34K): [in this window] [in a new window]   FIG. 1. hER46 expression increases in hyperconfluent MCF7 cells. 10-cm-diameter dishes were seeded with 4 x 105 MCF7 cells in medium containing 5% FCS, and cells were harvested at different days of culture. A, Cell growth was monitored by cell numeration and DNA content determination through propidium iodide labeling and FACS analysis. Data represent the average ± SEM of three independent experiments. B, Western blot analysis of hER66 and hER46 levels in nuclear and cytoplasmic fractions of MCF7 cells harvested at the indicated days of culture. After subcellular fractionation, protein extracts (20 µg) were resolved on a SDS-polyacrylamide gel and subjected to immunoblotting using the anti-hER HC20 antibody, anti-Lamin B Ab-1 antibody (fractionation control), and anti-ß-actin AC-15 antibody (loading control). Representative data from three independent experiments are shown. C, The relative expression of hER66 and hER46 were quantified by densitometry analysis of the three experiments and normalized to ß-actin signals. Values shown correspond to the average ± SEM of the three independent experiments.

hER46 expression remains rather stable during estrogen-induced MCF7 cell cycle

View larger version (33K): [in this window] [in a new window]   FIG. 2. hER46 expression during estradiol-induced MCF7 cell cycle. Forty percent confluent MCF7 cells growing for 72 h in phenol red-free medium supplemented with 2.5% charcoal-treated FCS (T0) were synchronized at the G1/S transition by a combined treatment with E2 (10 nM) and aphidicolin (5 mg/ml) during 48 h. Cells were collected at the indicated time points after release from the aphidicolin blockade (time 0 h). A, After propidium iodide labeling, asynchronous (T0) and synchronized MCF7 cells were analyzed by flow cytometry. B, After subcellular fractionation, protein extracts (20 µg) were resolved on a SDS-polyacrylamide gel and subjected to immunoblotting with the anti-hER HC20 antibody and the anti-ß-actin AC-15 antibody (loading control). The relative expression of hER66 and hER46 were quantified by densitometry analysis of three independent experiments and normalized to ß-actin signals. Values shown correspond to the average ± SEM.

Overexpression of hER46 blocks MCF7 cells in G₀/G₁ phases
The above results likely suggest that hER46 influences MCF7 growth. To confirm this assumption, MCF7 cell subclones (MCF7 pIND, pIND hER66, and pIND hER46) were established using ecdysone-inducible vectors expressing either hER isoforms. After a 48-h treatment with 5 x 10^–5 M ponasterone A, an ecdysone-like molecule, Western blots confirmed an inducible overexpression of the hER46 isoform in growing MCF7 pIND hER46 cells (Fig. 3A). In contrast, modifications of the hER66 expression pattern were not apparent in the pIND hER66 subclone after ponasterone A treatment. This is likely because of the particularly high levels of endogenous hER66 already present in MCF7 cells. Similar results were also observed in MCF7 subclones stably transfected with vectors directing a constitutive expression of hER66 (data not shown). Consequences of ponasterone A-driven expression of either hER isoforms were first assessed on 40% confluent MCF7 cells growing in normal medium (5% FCS; Fig. 3, B and C). Flow cytometry analysis clearly demonstrated that ponasterone A specifically decreased the population of MCF7 pIND hER46 cells in S phase by 65%, compared with untreated cells (Fig. 3C). Furthermore, treatment with ponasterone A specifically induced the accumulation of MCF7 pIND hER46 cells in the G₀/G₁ phase of their cell cycle. These results were confirmed on another series of MCF7 pIND hER66 and hER46 subclones (data not shown).

View larger version (32K): [in this window] [in a new window]   FIG. 3. Overexpression of hER46 blocks MCF7 cell in G0/G1 phase. MCF7 cells were stably transfected with pVgRXR plasmid and pIND, pIND hER66, or pIND hER46 ecdysone-inducible expression vectors. Three clones, MCF7-pIND, MCF7-pIND hER66, and MCF7-pIND hER46 were selected as described in Materials and Methods. A, Western blot analysis probing the expression of both hER66 and -46 forms in MCF7-pIND clones treated or not with an ecdysone-like molecule, ponasterone A (Ponas. A, 5 x 10–5 M), during 48 h. B, MCF7-pIND clones growing in normal medium complemented with 5% FCS were treated or not with 5 x 10–5 M Ponas. A during 48 h, and the cycle phase distribution of cell populations were determined by a dual BrdU/propidium iodide pulse labeling and flow cytometry analysis. C, Graphic representation of the percentage of cells in S phase deduced from B. Values are the average ± SD of four independent experiments. D, Consequences of a Ponas. A treatment on E2- or FCS-induced cell proliferation of MCF7-pIND clones. MCF7-pIND, MCF7-pIND hER66, and MCF7-pIND hER46 were grown for 3 d in phenol red-free medium supplemented with 2.5% charcoal-treated FCS before being treated or not with either 10 nM E2 or 10% serum during 24 h. Ponas. A treatment was or not performed during the last 48 h before harvesting. The percentage of cells in S phase was determined by propidium iodide labeling and FACS analysis.

In contrast to hER66, hER46 does not mediate estrogen-induced cell proliferation
The question of whether hER46 may mediate cell proliferation induced by estrogen was next addressed. To reach this aim, we first had to select a cell line in which stable expression of hER66 provokes E₂ to exhibit mitogenic effects. The establishment of such a system remained critical because estradiol treatment often inhibits rather than stimulates the growth of ER-negative cell lines stably transfected with the ER66 cDNA, in contrast to the situation observed in ER-positive breast carcinomas (35). Among the different cell lines tested, PC12 cells gave the expected response, with E₂ having no impact on PC12 growth (PC12 control) and stimulating proliferation of PC12 cells stably expressing the hER66 cDNA (PC12 hER66). The PC12 cell line was therefore selected as biological system to probe the capability of hER46 to mediate the mitogenic activity of estrogens. Stable transfection of hER46 in PC12 cells did not confer an estradiol-induced cell proliferation, in contrast to the 2-fold increase in thymidine incorporation observed in PC12 hER66 cells (Fig. 4). These results demonstrate that hER46 is unable to mediate mitogenic activity of estrogen, in contrast to hER66.

View larger version (28K): [in this window] [in a new window]   FIG. 4. hER46 does not mediate estrogen-induced cell proliferation in PC12 cells. After transfection with pCR 3.1, pCR hER66, or pCR hER46 expression vectors, three stable PC12 clones, PC12 control, PC12 hER66, and PC12 hER46 were selected. A, Western blot analysis probing the expression of hER66 and hER46 forms in PC12 clones. B, Effects of E2 on [3H]thymidine incorporation into PC12 clones. Cells seeded in 24-well plates (5 x 104 cells/well) with DMEM/F12 containing 7.5% charcoal dextran-treated FCS and 2.5% charcoal dextran-treated horse serum were maintained for 2 d in the presence of the indicated concentration of E2 before assessing [3H]thymidine incorporation on the final day of culture. Values shown correspond to the average ± SD of three experiments.

Overexpression of hER46 inhibits the estrogenic induction of AF-1 permissive target genes in MCF7 cells

View larger version (23K): [in this window] [in a new window]   FIG. 5. Overexpression of hER46 inhibits the estrogenic induction of the c-fos and cyclin D1 gene transcriptional activity in MCF7 cells. MCF7 cells, maintained in phenol red-free medium supplemented with 5% charcoal-treated calf serum, were transiently transfected with c-fos-Luc (A) or cyclin D1-Luc (B) reporter genes (200 ng) together with pCR 3.1 alone or with increasing quantity of pCR hER46 or pCR hER66 (50–200 ng). CMV-ß-Gal (100 ng) was used as internal control. Forty-eight hours after transfection, cells were treated for 12 h with 10 nM E2 or ethanol (EtOH). Luciferase activities were normalized with ß-galactosidase activities and the values standardized to the reporter activity measured in the presence of pCR 3.1 alone without E2. Values correspond to the average ± SEM of at least three separate transfection experiments.

View larger version (30K): [in this window] [in a new window]   FIG. 6. hER46 represses hER66 AF-1 transcriptional activity on ERE-controlled genes in MCF7 cells. Two ERE-controlled reporter genes with no intrinsic preference for AF-1 and AF-2 hER transactivation functions were selected for this study: the C3-Luc (A) and the ERE-TK-Luc (B). MCF7 cells, maintained in phenol red-free medium supplemented with 2.5% charcoal-treated calf serum, were transiently transfected with 200 ng of the reporter genes together with 50 ng of pCR 3.1 expression vectors (empty or encoding either hER) alone or with increasing quantity of pCR hER46 (0–200 ng). One hundred nanograms of CMV-ß-Gal was used as internal control. Cells were treated for 36 h with 10 nM E2, 2 µM 4-OHT, or ethanol (EtOH), as indicated within the panels. Luciferase activities were normalized with ß-galactosidase activities, and results were expressed as a percentage of the reporter activity measured in the presence of the expression vector pCR hER66 alone and E2 (or 4-OHT). Values correspond to the average ± SEM of at least three separate transfection experiments.

The hER46 homodimer has more affinity for an ERE than a hER66 homodimer
The ability of hER46 to behave as an effective AF-1-negative competitor on ERE-controlled genes might result from its aptitude to compete for the binding of hER66 to an ERE. We therefore assessed the ability of hER46 to compete for the binding of hER66 to an ERE in EMSAs. To do so, we produced in vitro rabbit reticulocyte lysate extracts containing constant levels of hER66 proteins in conjunction with increasing amounts of hER46, as verified in Western blots (Fig. 7A). Subsequent EMSAs revealed an ERE/hER66 homodimer complex, a fast migrating ERE/hER46 homodimer complex, and an intermediate ERE/hER66/46 heterodimer complex. Interestingly, when little amounts of hER46 are coproduced with the hER66, it is the heterodimer complex that is preferentially formed; with the inverse also verified (Fig. 7A and data not shown). Importantly, increasing the amounts of hER46 protein destabilized the ERE/hER66 homodimer complex. These results might reflect differences in the respective affinity of the hER isoforms dimers for an ERE. We thus followed the binding of each isoform to DNA with increasing quantities of radiolabeled ERE in EMSAs, and the results were next evaluated by Scatchard analysis (Fig. 7B). These experiments demonstrate that the hER46 homodimer has a twice more potent intrinsic affinity for the ERE than does the hER66 homodimer, with a calculated affinity constant of 0.11 and 0.2 nM, respectively. Unfortunately, the affinity of the hER66/46 heterodimer for the ERE could not be defined by this approach due to the impossibility to produce protein extracts containing only the heterodimer.

View larger version (38K): [in this window] [in a new window]   FIG. 7. hER46 homodimer is more affine for an ERE than hER66 homodimer. A, In vitro transcription/translation in rabbit reticulocyte lysate used plasmid mixes (completed to 1 µg with empty vector pCR 3.1) containing 0.2 µg pCR hER66 and increasing amounts of pCR hER46 (0, 0.005, 0.01, 0.02, 0.04, and 0.8 µg). Then 2.5 µl of in vitro-translated products were subjected to EMSA through incubation with 0.05 ng of 32P-labeled ERE and resolved on a 5% polyacrylamide gel. The positions of the three specific complexes (ERE/hER66 homodimer, ERE/hER66/46 heterodimer, and ERE/hER46 homodimer) are indicated. The relative amounts of these three complexes were then quantified by densitometry. Values correspond to the average of two independent experiments. In parallel, 2.5 µl of in vitro-translated products were resolved on a 10% SDS-polyacrylamide gel and then subjected to immunoblotting with the anti-ER HC20 antibody. B, Plasmid samples (completed to 1 µg with empty vector pCR 3.1) containing either 0.2 µg of pCR hER66 or pCR hER46 were in vitro transcribed and translated in rabbit reticulocyte lysate. Then 2.5 µl of in vitro-translated products were incubated with an increasing amount of 32P-labeled ERE (0.0625, 0.125, 0.25, 0.5, and 1 ng) and resolved on a 5% polyacrylamide gel. The amounts of homodimers were quantified and subjected to Scatchard analysis.

The hER66/hER46 heterodimer is AF-1 permissive
The ability of the hER46 to act as an effective AF-1-negative competitor might also result from its ability to form heterodimers with the hER66. Because these heterodimers contain only one AF-1 region, we next assessed whether they might be inactive in cellular contexts strictly permissive to this transactivation function. However, the binding of both hER homodimers and hER66/46 heterodimer to EREs prevent the specific determination of the transcriptional activity of the hER66/46 on ERE-containing reporters. To circumvent this, we set up a strategy similar to the one previously used by Tremblay et al. (39) when defining the transcriptional properties of the ER/ERß heterodimer. This method takes advantage of the mutation of three residues within the ER DNA binding domain that change its DNA binding specificity to that of a glucocorticoid receptor (Fig. 8A) (40). This hER_GR mutant induces transcription of a GRE-TK-Luc but not of an ERE-TK-Luc reporter gene (Fig. 8B). To measure the specific activity of the hER_GR/hER46 and hER_GR/hER66 heterodimers, we used a reporter gene whose transcription is under the control of two hybrid E/GRE DNA-responsive elements [(E/GRE)₂-SV-Luc]. Importantly, in strict AF-1 (HepG2) or strict AF-2 (HeLa) permissive cell lines, an E₂-induced transcriptional activity of this reporter gene occurred only when hER_GR was coexpressed with either hER66 or hER46 (Fig. 8C). Similar results were obtained in MCF7 cells, with an induction of the reporter gene in the presence of E₂ observed when expressing only hER_GR due to its heterodimerization with endogenous hER. These results indicate that the hER66/46 heterodimer is as potent as a hER66 homodimer for activating transcription in both AF-2- and AF-1-permissive cell contexts. They also suggest that a single AF-1 region is sufficient for a hER66 homodimer to function. The AF-1 dominant-negative action of the hER46 on ERE-driven gene is therefore not a consequence of its ability to form a heterodimer with hER66.

View larger version (30K): [in this window] [in a new window]   FIG. 8. An AF-1 activity is retained within the hER66/hER46 heterodimer. A, Schematic representation of the first zinc finger of the DNA binding domain of hER. The positions of the three amino acids that contribute to DNA binding specificity are indicated. These residues were mutated to substitute the specificity of hER66 binding to ERE for a specific binding to a GRE (hERGR66 mutant). The structure of the (E/GRE)2-SV-Luc reporter gene with its two hybrid E/GRE DNA-responsive elements is also indicated. B, HeLa cells were transfected with the ERE-TK-Luc or GRE-TK-Luc reporter genes (100 ng) in conjunction with pCR 3.1, pCR hER66, pCR hER66GR, or pCR hER46 (50 ng). One hundred nanograms of CMV-ß-Gal was used as internal control. Luciferase activities were normalized with ß-galactosidase activities, and the results were standardized to the reporter activity measured in the presence of pCR 3.1 without E2. Values correspond to the average ± SD of at least three separate transfection experiments. C, AF-2-permissive HeLa cells, AF-1-permissive HepG2, and MCF7 cells were transfected with 200 ng of (E/GRE)2-SV-Luc reporter and pCR 3.1, pCR hER66, pCR hER66GR, or pCR hER46 (50 ng) alone or in combination as indicated on the bottom of the graph. Results are expressed and normalized as in B.

	Discussion

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First, we show that during MCF7 cell growth, hER46 is mainly expressed in the nucleus at levels remaining relatively low, whereas hER66 accumulates in the nucleus and, to a lesser extent, in the cytoplasm, as previously reported (41). When cells reach hyperconfluency and become quiescent, the situation reverses, with a strong accumulation of hER46 within the nucleus concomitant with a decrease in hER66 levels. We have previously shown that the amounts of hER46 present in whole-cell extracts are constant, when comparing confluent and nonconfluent (20% confluence) MCF7 cells (26). This apparent discrepancy with the present data are explained by the fact that the previous analysis used cells that just reached confluence, when hER46 expression is still relatively low. As shown in Fig. 1, an accumulation of hER46 within the nucleus requires the cells to be hyperconfluent. Consequently, when cells have reached confluency, the expression of hER46 is obviously subject to additional controls, whose mechanisms remain to be defined.

Interestingly, this accumulation of hER46 correlates with a stage when cells become refractory to E₂-induced growth. Indeed, several years ago, electrophoretic analysis of in vivo-labeled ER with ³H-tamoxifen aziridine showed that the size of ER protein was dependent on cell confluency: whereas growing MCF7 cells expressed a monomeric binding entity of 62 kDa, hyperconfluent cells presented a 47-kDa binding entity (42). Furthermore, during the different phases of the estrous cycle, both entities coexist in distinct proportions during the diestrous (1/2) and proestrous (1/1). Importantly, only the smaller form was detected during the estrous phase, a phase that is associated with the uterus being refractory to E₂ stimulation (43). Altogether, these data suggest that high expression levels of ER46 correlate with cells being refractory to the mitogenic effects of E₂.

Our experiments using an ecdysone-inducible system clearly show that an increase in hER46 expression in nonconfluent MCF7 cells reduces the percentage of cells in S phase after estrogen or serum induction of cell growth. Other studies have shown that the permissiveness of osteoblast-like SaOS cells to E₂ mitogenic effects, obtained through the exogenous expression of hER66, is altered in a dose-dependent manner by hER46 (27). Therefore, hER46 obviously behaves as a cell growth inhibitor when it is overexpressed in MCF7 cells, probably through controlling the proliferative influence of hER66. To validate these conclusions, we used ER-negative PC12 cell line, in which the stable expression of hER66 but not hER46 allows estrogen to mediate cell proliferation. This further indicates that the hER A/B domains and probably its AF-1 activity are required for the receptor to exhibit a proliferative influence. Corroborating this result, Fujita et al. (44) previously reported that a fully activated AF-1 induces growth of ER-positive breast cancers. In ER^–/– mice generated by an insertional disruption of the ER gene in the first coding exon, critical E₂-induced growth deficiencies were observed in breast and uterus tissues (9). Although totally abolishing the production of the full-length ER, this disruption does not suppress ER46 expression (45). This further emphasizes the importance of AF-1 in ER proliferative activity.

Mediation of estrogen-induced cell proliferation by hER66 results in part from modifications in the expression patterns of genes, e.g. those involved in the control of the cell cycle such as c-fos and cyclin D1. Previous studies clearly demonstrated the importance of AF-1 activity in the estrogenic induction of these genes. Notably, a truncated hER devoid of the A/B domain (HE19, equivalent to hER46) did not transactivate the c-fos and cyclin D1 promoters (21, 36, 37). Extending these data, the present study clearly demonstrates that increasing expression of hER46 in MCF7 cells abolishes the estrogenic induction of both of these promoters in a dose-dependent manner. In parallel, we determined MCF7 cells as providing an environment permissive to both AFs, with nevertheless an increased sensitivity to AF-1. In these cells, AF-2-permissive reporter genes such as pS2-Luc (data not shown) are equally sensitive to both hER isoforms, and increasing the amounts of hER46 does not impact hER66 transcriptional activity. In contrast, hER46 inhibited the transcriptional activity of hER66 on AF-1-sensitive genes in a dose-dependent manner. Consequently, changes within the respective levels of expression of hER isoforms as occurs when cells reach confluence should specifically inhibit hER66-mediated transcription of E₂ target genes sensitive to AF-1 but not AF-2. These data are particularly relevant because the proliferative activity of hER66 seems to be mediated, as previously mentioned, by its AF-1 activity.

Interestingly, hER46 shares several functional similarities with ERß. For instance, both of these ER forms are devoid of the AF-1 present in hER66, although sharing relatively conserved DNA and ligand binding domains (7, 26). Consequently, hER46 and ERß induce the transcription of ERE-driven genes mainly via their AF-2 (26, 46). Recent studies also showed that, as does hER46, ERß counteracts the activity of ER66 in many cellular systems. Indeed, the stable expression of ERß inhibits the E₂-stimulated proliferation of the ER-positive MCF7 or T47D breast cancer cells (47, 48). Furthermore, unlike ER66, ERß represses cyclin D1 gene transcription and blocks ER66-mediated induction when both receptors are present (38). Finally, the expression of ERß decreases in invasive breast cancers tissues, compared with adjacent normal mammary gland (12), suggesting that the ER66 to ERß ratio increases during carcinogenesis. Correspondingly, the highest ER66 to ER46 ratios are observed in growing MCF7 breast cancer cells and the lowest in hyperconflent MCF7 cells being refractory to E₂ mitogenic effect or in primary human cultures from vascular endothelial cells (28, 29) or osteoblasts (27). Although the specific functions of ER46 and ERß in cancer are not known, there is increasing evidence that these ER proteins deficient in AF-1 have inhibitory effects on cellular proliferation.

Several mechanisms might explain the ability of hER46 to efficiently suppress the AF-1 activity of hER66. First, hER46 may compete the binding of hER66 to ERE or other transcription factors (AP-1 and Sp1 proteins) in ERE-independent mechanisms. Indeed, both forms efficiently bind EREs and physically interact with AP-1 and specificity protein 1 (49, 50). We show in this report that, in vitro, increasing amounts of hER46 squelches the binding of hER66 to ERE. As determined by Scatchard analysis, this competition is facilitated by a 2-fold increased affinity of the hER46 for an ERE, compared with the hER66 homodimer. This is in accordance with previous studies ascribing a better affinity of receptors deleted from their N-terminal A/B domains for their hormone-responsive elements (51, 52). For instance, deletion of the A/B domain from the Xenopus ER increases by 2-fold its affinity for an ERE (52).

EMSAs using in vitro-translated proteins also revealed that hER46 heterodimerizes with hER66, generating a protein complex that has only one AF-1 function. Because this would provide a mean for hER46 to inhibit the AF-1 of its partner, we evaluated whether the AF-1 domain of hER66 is still functional when heterodimerized with hER46. To specifically monitor the transactivation properties of the heterodimer, we used a hER66 mutant (hER66_GR) that specifically binds glucocorticoid receptor elements (GREs) (40). Expression of this mutant together with hER46 results in the formation of a hER66_GR/hER46 heterodimer whose specific activity was assayed on a reporter gene placed under the control of a hybrid E/GRE-responsive element. The heterodimer efficiently activated the reporter gene in AF-2-sensitive cells such as HeLa cells but, surprisingly, also in strictly AF-1-permissive HepG2 cells. This means that heterodimerization with hER46 does not impact on the activity of hER66 mediated by its AF-1. Interestingly, within the ER/ERß heterodimer, each AF-1 domain can be activated independently (39). This demonstrates that ER AF-1 retains its transcriptional properties within the context of ER/ERß and hER66/hER46 heterodimers and suggests that only one AF1 domain is sufficient for ER to function.

We conclude from these results that the AF-1 dominant-negative action of hER46 is not due to an inhibition of the AF-1 activity within a hER66/46 heterodimer. Whereas a transcriptional activity of the hER66/46 heterodimer was detected in MCF7 cells using the hER66 _GR mutant, we failed to detect the presence of endogenous heterodimers in these cells by coimmunoprecipitation experiments (data not shown), suggesting that hER46 more readily homodimerizes than heterodimerizes with hER66 in MCF7 cells.

The accumulation of hER46 in the nucleus during MCF-7 cells growth arrest can inhibit the activity of hER66, at least through competition for the binding to a shared ERE. Besides this passive mechanism, an active process can also be envisioned, in which the substitution of hER66 by hER46 on the ERE would direct the specific recruitment of corepressors. Indeed, in contrast to the hER66 that interacts with recruitment of corepressors only when liganded to antiestrogens such as 4-OHT, the hER46 isoform can recruit these cofactors in the absence of any ligand (30, 31). However, this hypothesis would imply that a fraction of the large amounts of hER46 produced when cells reached confluence stays unliganded. This remains to be determined.

When MCF-7 cells reach confluence, some of the intracellular hER46 is detected in the cytosolic fraction. This suggests that the mediation of cell growth arrest by hER46 can also involve the activation or the inhibition of nongenomic pathways. In vascular endothelial cells, a pool of hER46 was found associated with cell membrane in a palmitoylation-dependent manner (28, 29). In these cells, hER46 modulates the actions of estrogens initiated at the level of the cell membrane. As an example, hER46 activates the endothelial nitric oxide synthase pathway more efficiently than hER66 (28, 29). Although we did not succeed in identifying a pool of hER46 associated with MCF7 cells membrane (data not shown), the occurrence of specific nongenomic regulations initiated by hER46 in MCF-7 cells cannot be ruled out.

In conclusion, the generation of hER46 proteins in mammary cells constitutes a key regulatory element in the estrogenic control of cell growth. Actions of hER46 are obviously mediated in part through genomic effects by interfering with the transcriptional activity of hER66. Further studies are now required to identify genes whose transcription is placed under the specific control of either hER isoforms.

	Acknowledgments

 
We gratefully acknowledge Dr. F. Gannon, G. Reid, Dr S. Denger, D. Manu and H. Brand for their helpful comments. We thank P. Chambon for the gift of the expression vector HE82.

	Footnotes

 
This work was supported by fellowships from the "Région Bretagne" and the Association pour la Recherche Contre le Cancer (ARC) (to G.P.) and funds from Rennes Métropole, the Centre National de la Recherche Scientifique, the ARC, the Ligue Contre le Cancer, and the European network program GENOSPORA (QLK6-1999-02108).

First Published Online September 8, 2005

¹ G.P. and C.L.P. contributed equally to this work

Abbreviations: AF, Activation function; BrdU, 5-bromo-2'-deoxyuridine; ER, estrogen receptor; ERE, estrogen-responsive element; FACS, flow cytometry analysis; FCS, fetal calf serum; GRE, glucocorticoid receptor element; hER, human estrogen receptor-; IFA, immunofunctional assay; NP-40, Nonidet P-40; 4-OHT, 4-hydroxytamoxifen.

Received July 12, 2005.

Accepted for publication August 30, 2005.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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