The Human Estrogen Receptor- Isoform hER46 Antagonizes the Proliferative Influence of hER66 in MCF7 Breast Cancer Cells
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《内分泌学杂志》
Equipe d’Endocrinologie Moleculaire de la Reproduction, Unite Mixte de Recherche Centre National de la Recherche Scientifique 6026, 35042 Rennes Cedex, France
Abstract
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
GROWTH AND DIFFERENTIATION of the female reproductive tracts are under the critical influence of estrogens such as 17-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 - (NR3A2) (6, 7, 8). Targeted disruption of ER and ER genes clearly demonstrated that the postnatal development of uterus and mammary glands rely on ER rather than ER (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 ER 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
Plasmids
The reporter plasmids ERE-TK-Luc, hC3-Luc, and pCMV--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 hER66GR were generated by cloning the coding region of hER66 (+228/+2030), hER46 (+727/+2030), and hER66GR (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 -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 -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--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
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 -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).
hER46 expression remains rather stable during estrogen-induced MCF7 cell cycle
The experiments depicted above might suggest the existence of a correlation between the expression pattern of hER46 and specific phases of the cell cycle. To verify this hypothesis, we designed experiments aiming at analyzing the expression of hER66 and hER46 throughout an estrogen-induced cell cycle. To do so, 40% confluent MCF7 cells maintained in steroid-free medium [2.5% charcoal dextran-treated FCS] during 72 h were treated with 10 nM E2 and synchronized in their cell cycle at the G1/S phase transition using a 48-h aphidicolin treatment. Release of the aphidicolin block through washings then allowed the cells to progress throughout their cycle. The efficient completion of the synchronization step was confirmed by flow cytometry analysis, with 70% of the cells stopped in the G1/S phase transition (Fig. 2A). Cells progressed through the S phase 6 h after aphidicolin withdrawal. At 9 h, cells went through the G2/M phases and finally returned in an asynchronous state 12 h later (time point 24 h) with approximately 70% cells in G0/G1 phase (Fig. 2A). Assessing the relative distribution of either hER isoforms within the nuclear and cytoplasmic fractions by Western blots showed that the nuclear amounts of hER46 are stable up to S phase, slightly decrease during the G2/M phases, and return to higher level when cells engage again in G0/G1 phases (Fig. 2B). In contrast, a strong decrease in nuclear and cytoplasmic hER66 signals was observed during the G1 phase after E2 treatment. These expression levels remain repressed through the other phases of the cell cycle (Fig. 2B).
Together these data suggest that high levels of hER46 are not found in quiescent MCF7 cells arrested in the G0/G1 phase but rather within MCF7 cells becoming refractory to growth, a state that is reached when cells are hyperconfluent.
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).
The impact of a ponasterone A-induced expression of hER46 on E2-induced cell proliferation was subsequently analyzed. MCF7 subclones were maintained in medium complemented with 2.5% charcoal-treated FCS during 72 h prior treatment or not with 10 nM E2 or 10% serum for 24 h. Subsequent flow cytometry analysis showed that the specific overexpression of hER46 abolishes the hormonal stimulation of MCF7 growth, with this repressive effect occurring in the absence or presence of E2 (Fig. 3D). Altogether, these experiments demonstrate that an overexpression of the hER46 isoform affects MCF7 growth, mainly leading to a G0/G1 phase arrest.
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.
Overexpression of hER46 inhibits the estrogenic induction of AF-1 permissive target genes in MCF7 cells
hER46 is a potent ligand-inducible transcription factor in promoter and cell contexts sensitive to hER AF-2 but has no transcriptional activity and behaves as a powerful inhibitor of hER66 activity in contexts in which AF-1 predominates over AF-2 (26, 33). The consequences of an increased expression of hER46 on estrogen target gene activity will therefore depend on the relative permissiveness of MCF7 cells and target genes to hER AF-1 and AF-2. The transcriptional properties of hER46 were thus evaluated on reporter constructs placed under the control of different E2-sensitive promoters. Taking into account the divergent roles that hER isoforms have on E2-mediated cell proliferation, we first selected promoters from genes involved in this process, exemplified by c-Fos and cyclin D1. These genes are transcriptionally induced by hER66 in an ERE-independent mechanism requiring a functional AF-1domain (21, 22, 36, 37, 38). In hER-positive MCF7 cells, the transcriptional activity of both promoters is 2.5-fold up-regulated by E2; and, importantly, increasing amounts of hER46 strongly inhibits this estrogenic induction (Fig. 5). In contrast, increasing amounts of pCR hER66 enhances the estrogenic response of c-Fos promoter (Fig. 5A) and negatively impact cyclin D1 promoter activity only at the highest concentration (Fig. 5B). These results indicate that, in MCF7 cells, decreasing the hER66 to -46 ratio by an overexpression of hER46 inhibits the estrogenic induction of c-Fos and cyclin D1 promoters.
To assay the generality of this observation, we subsequently analyzed the impact of increasing concentrations of hER46 on the complement 3 promoter (C3-Luc), which contains an ERE and has no intrinsic preference for AF-1 or AF-2 (33). In the presence of E2, hER46 exhibited a 70% lower transactivation capability than hER66 on this mixed AF-1/AF-2 reporter gene (Fig. 6A). Therefore, MCF7 cells are less sensitive to AF-2 than AF-1. Despite this prevalence of the MCF7 cell context toward AF-1, increasing amounts of pCR hER46 had no effect on C3-Luc activation by hER66 in the presence of E2 (Fig. 6A). This contrasted with the expected inhibition of endogenous hER66 activity occurring in strict AF-1-sensitive cell context. We therefore treated transfected MCF7 cells with 4-OHT, a partial hER agonist whose estrogenic activity exclusively depends on AF-1, i.e. detectable only in cell and promoter contexts sensitive to AF-1 (14). Furthermore, the C3-Luc gene is a well-characterized 4-OHT-responsive reporter system (16). The 4-OHT-induced transcriptional activity of the C3-Luc gene was inhibited with increasing hER46 expression (Fig. 6A). In these conditions, hER46 thus behaves as an inhibitor of hER AF-1 activity, revealing a cell-context mainly sensitive to AF-1. Analysis of the ERE-TK-Luc, the second reporter gene with no intrinsic preference for AF-1 and AF-2, seemed to confirm this assumption. In contrast to the C3-Luc reporter, the direct evaluation of the respective activities of either hER isoforms was biased by the high activity of the ERE-TK-Luc reporter induced by endogenous hER proteins (Fig. 6B). However, increasing amounts of exogenous hER46 inhibited E2-induced hER66 transcriptional activity on this reporter gene, confirming the AF-1 permissiveness of MCF-7 cells.
Altogether, these results demonstrate that MCF7 cells are mainly sensitive to the AF-1 function of hER, however, with a low permissiveness to AF-2. In such context, changes in the hER66 to hER46 ratio should mainly impact the transcriptional activity of AF-1-permissive estrogen target genes.
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.
In conclusion, with a 2-fold higher affinity for the ERE, the hER46 dimer is able to compete the binding of the hER66 homodimer and, by such means, would be able to inhibit the transcriptional activity of AF-1-permissive genes induced by the hER66.
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/ER 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 hERGR 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 hERGR/hER46 and hERGR/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 hERGR 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 hERGR 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.
Discussion
The role of estrogens in the promotion and development of breast cancers was initially established by clinical and epidemiological observations, such as the therapeutical efficiency of ovariectomy and antiestrogen therapy. Moreover, E2 has a potent mitogenic effect on ER-positive breast cancer cell lines such as MCF7 cells (4, 10, 11). However, to date, the molecular mechanisms through which E2 controls the growth of ER-positive breast cancer cells are poorly understood. A first step toward the understanding of these processes was reached through the identification of an isoform of the hER, hER46, which is coexpressed with the full-length hER66 in MCF7 cells (26). Being devoid of the A/B domain containing the AF-1, the hER46 harbors specific functional properties (26). We hypothesized that hER46 may influence the E2-induced growth of MCF7 cells and therefore sought to determine whether a direct correlation exists between the expression of hER46 and cell growth and to define the underlying mechanisms.
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 ER. 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 ER induce the transcription of ERE-driven genes mainly via their AF-2 (26, 46). Recent studies also showed that, as does hER46, ER counteracts the activity of ER66 in many cellular systems. Indeed, the stable expression of ER inhibits the E2-stimulated proliferation of the ER-positive MCF7 or T47D breast cancer cells (47, 48). Furthermore, unlike ER66, ER represses cyclin D1 gene transcription and blocks ER66-mediated induction when both receptors are present (38). Finally, the expression of ER decreases in invasive breast cancers tissues, compared with adjacent normal mammary gland (12), suggesting that the ER66 to ER 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 ER 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 (hER66GR) that specifically binds glucocorticoid receptor elements (GREs) (40). Expression of this mutant together with hER46 results in the formation of a hER66GR/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/ER 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/ER 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 "Region Bretagne" and the Association pour la Recherche Contre le Cancer (ARC) (to G.P.) and funds from Rennes Metropole, 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.
Accepted for publication August 30, 2005.
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Abstract
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
GROWTH AND DIFFERENTIATION of the female reproductive tracts are under the critical influence of estrogens such as 17-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 - (NR3A2) (6, 7, 8). Targeted disruption of ER and ER genes clearly demonstrated that the postnatal development of uterus and mammary glands rely on ER rather than ER (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 ER 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
Plasmids
The reporter plasmids ERE-TK-Luc, hC3-Luc, and pCMV--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 hER66GR were generated by cloning the coding region of hER66 (+228/+2030), hER46 (+727/+2030), and hER66GR (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 -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 -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--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
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 -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).
hER46 expression remains rather stable during estrogen-induced MCF7 cell cycle
The experiments depicted above might suggest the existence of a correlation between the expression pattern of hER46 and specific phases of the cell cycle. To verify this hypothesis, we designed experiments aiming at analyzing the expression of hER66 and hER46 throughout an estrogen-induced cell cycle. To do so, 40% confluent MCF7 cells maintained in steroid-free medium [2.5% charcoal dextran-treated FCS] during 72 h were treated with 10 nM E2 and synchronized in their cell cycle at the G1/S phase transition using a 48-h aphidicolin treatment. Release of the aphidicolin block through washings then allowed the cells to progress throughout their cycle. The efficient completion of the synchronization step was confirmed by flow cytometry analysis, with 70% of the cells stopped in the G1/S phase transition (Fig. 2A). Cells progressed through the S phase 6 h after aphidicolin withdrawal. At 9 h, cells went through the G2/M phases and finally returned in an asynchronous state 12 h later (time point 24 h) with approximately 70% cells in G0/G1 phase (Fig. 2A). Assessing the relative distribution of either hER isoforms within the nuclear and cytoplasmic fractions by Western blots showed that the nuclear amounts of hER46 are stable up to S phase, slightly decrease during the G2/M phases, and return to higher level when cells engage again in G0/G1 phases (Fig. 2B). In contrast, a strong decrease in nuclear and cytoplasmic hER66 signals was observed during the G1 phase after E2 treatment. These expression levels remain repressed through the other phases of the cell cycle (Fig. 2B).
Together these data suggest that high levels of hER46 are not found in quiescent MCF7 cells arrested in the G0/G1 phase but rather within MCF7 cells becoming refractory to growth, a state that is reached when cells are hyperconfluent.
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).
The impact of a ponasterone A-induced expression of hER46 on E2-induced cell proliferation was subsequently analyzed. MCF7 subclones were maintained in medium complemented with 2.5% charcoal-treated FCS during 72 h prior treatment or not with 10 nM E2 or 10% serum for 24 h. Subsequent flow cytometry analysis showed that the specific overexpression of hER46 abolishes the hormonal stimulation of MCF7 growth, with this repressive effect occurring in the absence or presence of E2 (Fig. 3D). Altogether, these experiments demonstrate that an overexpression of the hER46 isoform affects MCF7 growth, mainly leading to a G0/G1 phase arrest.
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.
Overexpression of hER46 inhibits the estrogenic induction of AF-1 permissive target genes in MCF7 cells
hER46 is a potent ligand-inducible transcription factor in promoter and cell contexts sensitive to hER AF-2 but has no transcriptional activity and behaves as a powerful inhibitor of hER66 activity in contexts in which AF-1 predominates over AF-2 (26, 33). The consequences of an increased expression of hER46 on estrogen target gene activity will therefore depend on the relative permissiveness of MCF7 cells and target genes to hER AF-1 and AF-2. The transcriptional properties of hER46 were thus evaluated on reporter constructs placed under the control of different E2-sensitive promoters. Taking into account the divergent roles that hER isoforms have on E2-mediated cell proliferation, we first selected promoters from genes involved in this process, exemplified by c-Fos and cyclin D1. These genes are transcriptionally induced by hER66 in an ERE-independent mechanism requiring a functional AF-1domain (21, 22, 36, 37, 38). In hER-positive MCF7 cells, the transcriptional activity of both promoters is 2.5-fold up-regulated by E2; and, importantly, increasing amounts of hER46 strongly inhibits this estrogenic induction (Fig. 5). In contrast, increasing amounts of pCR hER66 enhances the estrogenic response of c-Fos promoter (Fig. 5A) and negatively impact cyclin D1 promoter activity only at the highest concentration (Fig. 5B). These results indicate that, in MCF7 cells, decreasing the hER66 to -46 ratio by an overexpression of hER46 inhibits the estrogenic induction of c-Fos and cyclin D1 promoters.
To assay the generality of this observation, we subsequently analyzed the impact of increasing concentrations of hER46 on the complement 3 promoter (C3-Luc), which contains an ERE and has no intrinsic preference for AF-1 or AF-2 (33). In the presence of E2, hER46 exhibited a 70% lower transactivation capability than hER66 on this mixed AF-1/AF-2 reporter gene (Fig. 6A). Therefore, MCF7 cells are less sensitive to AF-2 than AF-1. Despite this prevalence of the MCF7 cell context toward AF-1, increasing amounts of pCR hER46 had no effect on C3-Luc activation by hER66 in the presence of E2 (Fig. 6A). This contrasted with the expected inhibition of endogenous hER66 activity occurring in strict AF-1-sensitive cell context. We therefore treated transfected MCF7 cells with 4-OHT, a partial hER agonist whose estrogenic activity exclusively depends on AF-1, i.e. detectable only in cell and promoter contexts sensitive to AF-1 (14). Furthermore, the C3-Luc gene is a well-characterized 4-OHT-responsive reporter system (16). The 4-OHT-induced transcriptional activity of the C3-Luc gene was inhibited with increasing hER46 expression (Fig. 6A). In these conditions, hER46 thus behaves as an inhibitor of hER AF-1 activity, revealing a cell-context mainly sensitive to AF-1. Analysis of the ERE-TK-Luc, the second reporter gene with no intrinsic preference for AF-1 and AF-2, seemed to confirm this assumption. In contrast to the C3-Luc reporter, the direct evaluation of the respective activities of either hER isoforms was biased by the high activity of the ERE-TK-Luc reporter induced by endogenous hER proteins (Fig. 6B). However, increasing amounts of exogenous hER46 inhibited E2-induced hER66 transcriptional activity on this reporter gene, confirming the AF-1 permissiveness of MCF-7 cells.
Altogether, these results demonstrate that MCF7 cells are mainly sensitive to the AF-1 function of hER, however, with a low permissiveness to AF-2. In such context, changes in the hER66 to hER46 ratio should mainly impact the transcriptional activity of AF-1-permissive estrogen target genes.
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.
In conclusion, with a 2-fold higher affinity for the ERE, the hER46 dimer is able to compete the binding of the hER66 homodimer and, by such means, would be able to inhibit the transcriptional activity of AF-1-permissive genes induced by the hER66.
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/ER 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 hERGR 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 hERGR/hER46 and hERGR/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 hERGR 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 hERGR 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.
Discussion
The role of estrogens in the promotion and development of breast cancers was initially established by clinical and epidemiological observations, such as the therapeutical efficiency of ovariectomy and antiestrogen therapy. Moreover, E2 has a potent mitogenic effect on ER-positive breast cancer cell lines such as MCF7 cells (4, 10, 11). However, to date, the molecular mechanisms through which E2 controls the growth of ER-positive breast cancer cells are poorly understood. A first step toward the understanding of these processes was reached through the identification of an isoform of the hER, hER46, which is coexpressed with the full-length hER66 in MCF7 cells (26). Being devoid of the A/B domain containing the AF-1, the hER46 harbors specific functional properties (26). We hypothesized that hER46 may influence the E2-induced growth of MCF7 cells and therefore sought to determine whether a direct correlation exists between the expression of hER46 and cell growth and to define the underlying mechanisms.
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 ER. 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 ER induce the transcription of ERE-driven genes mainly via their AF-2 (26, 46). Recent studies also showed that, as does hER46, ER counteracts the activity of ER66 in many cellular systems. Indeed, the stable expression of ER inhibits the E2-stimulated proliferation of the ER-positive MCF7 or T47D breast cancer cells (47, 48). Furthermore, unlike ER66, ER represses cyclin D1 gene transcription and blocks ER66-mediated induction when both receptors are present (38). Finally, the expression of ER decreases in invasive breast cancers tissues, compared with adjacent normal mammary gland (12), suggesting that the ER66 to ER 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 ER 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 (hER66GR) that specifically binds glucocorticoid receptor elements (GREs) (40). Expression of this mutant together with hER46 results in the formation of a hER66GR/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/ER 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/ER 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 "Region Bretagne" and the Association pour la Recherche Contre le Cancer (ARC) (to G.P.) and funds from Rennes Metropole, 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.
Accepted for publication August 30, 2005.
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