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Cancer Research 67, 5513, June 1, 2007. doi: 10.1158/0008-5472.CAN-07-0967
© 2007 American Association for Cancer Research
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Endocrinology

Differential Regulation of Estrogen Receptor {alpha} Turnover and Transactivation by Mdm2 and Stress-Inducing Agents

Vanessa Duong1,2, Nathalie Boulle1,2, Sylvain Daujat3, Jérôme Chauvet1,2, Sandrine Bonnet1,2, Henry Neel3 and Vincent Cavaillès1,2

1 Institut National de la Santé et de la Recherche Médicale U540; 2 Université Montpellier I; and 3 Institut de Génétique Moléculaire de Montpellier, Montpellier, France

Requests for reprints: Vincent Cavaillès, Institut National de la Sante et de la Recherche Medicale U540, 60 rue de Navacelles, Montpellier, F-34090 France. Phone: 33-4-67-61-24-05; Fax: 33-4-67-61-37-87; E-mail: v.cavailles{at}valdorel.fnclcc.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In mammalian cells, the level of estrogen receptor {alpha} (ER{alpha}) is rapidly decreased upon estrogen treatment, and this regulation involves proteasome degradation. Using different approaches, we showed that the Mdm2 oncogenic ubiquitin-ligase directly interacts with ER{alpha} in a ternary complex with p53 and is involved in the regulation of ER{alpha} turnover (both in the absence or presence of estrogens). Several lines of evidence indicated that this effect of Mdm2 required its ubiquitin-ligase activity and involved the ubiquitin/proteasome pathway. Moreover, in MCF-7 human breast cancer cells, various p53-inducing agents (such as UV irradiation) or treatment with RITA (which inhibits the interaction of p53 with Mdm2) stabilized ER{alpha} and abolished its 17ß-estradiol–dependent turnover. Interestingly, our data indicated that ligand-dependent receptor turnover was not required for efficient transactivation. Altogether, our results indicate that the Mdm2 oncoprotein and stress-inducing agents complexly and differentially regulate ER{alpha} stability and transcriptional activity in human cancer cells. [Cancer Res 2007;67(11):5513–21]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Estrogens are key regulators of cell differentiation and proliferation, and these hormones play important roles in female reproduction physiology and tissue homeostasis. They exert their biological action via specific receptors (ER{alpha} and ERß), which are members of a superfamily of hormone nuclear receptors, acting as gene regulatory transcription factors (1). Upon ligand binding, ERs regulate gene expression through the binding to their cognate estrogen response elements (ERE) or via protein-protein interactions with transcription factors such as activator protein-1 or specificity protein-1. In the presence of hormone, ERs undergo a major conformational change allowing the recruitment of transcriptional cofactor complexes, which in turn engage the basal transcription machinery and/or act to locally modify chromatin structure and subsequently stimulate expression of estrogen-responsive genes.

Liganded nuclear receptors recruit various types of enzymatic activities that participate in gene expression regulation (2). Previous studies have suggested that ubiquitin-conjugating enzymes or ubiquitin-protein ligases, such as UbcH5/UbcH7 (3, 4), RPF1/RSP5 (5), or E6-AP (6), interact with members of the nuclear receptor superfamily and modulate their transactivation functions. Similarly, ATPase subunits of the proteasome complex, such as TRIP1/SUG1 (7) or TBP1 (8), also bind nuclear receptors and modulate their functions.

More than 30 years ago, Jensen et al. have shown that 17ß-estradiol (E2) treatment significantly reduces ER{alpha} levels in the uterus of ovariectomized rats (9). More recently, several studies have shown that binding of E2 to ER{alpha} significantly decreases its stability. This shorter half-life in the presence of hormone seems to implicate the ubiquitin/proteasome pathway because ER{alpha} has been shown to be ubiquitinated (10), and the ligand-dependent down-regulation is blocked by proteasome inhibitors (3, 11, 12). Interestingly, although partial antiestrogens, such as tamoxifen, also increase ER{alpha} accumulation, pure antihormones, such as the ICI 182,780 compound, strongly decrease receptor stability (10, 13). Several reports have suggested that the proteasome may control not only ER{alpha} protein levels but also hormone-dependent transcription (14). This effect is associated with the immobilization of ER{alpha} on the nuclear matrix as determined by fluorescence recovery after photobleaching (15). Although several candidates have been proposed to account for the E2-dependent regulation of ER{alpha} expression, the exact molecular mechanisms still remain unraveled.

The Mdm2 oncogene is overexpressed in a wide variety of human cancers (16), and its role in tumorigenesis is linked to its ability to act as an E3 ubiquitin-ligase (17), which mediates the ubiquitination and proteasome-dependent degradation of several growth regulatory proteins including p53 (18, 19). Interestingly, it has been previously suggested that Mdm2 could directly interact with ER{alpha} (20, 21), and a positive effect of Mdm2 overexpression on ER{alpha} activity has also been reported (20).

In the present study, we show that the Mdm2 oncoprotein is involved in both ligand-dependent and ligand-independent decrease of ER{alpha} stability. Our data indicate that Mdm2 regulates ER{alpha} expression as a ternary complex with p53, and in support of this observation, we show that various stress-inducing agents (which stabilize p53) block E2-dependent regulation of ER{alpha} stability in MCF-7 human breast cancer cells. Finally, this study provides several lines of evidence showing that the E2-dependent turnover of the receptor is not necessary for ERE-mediated transactivation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids and reagents. The ER{alpha} expression vectors (wild type and deletion mutants) were given by P. Chambon [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Strasbourg, France]. The GST-AF2wt vectors (22), the plasmids encoding Gal-GRIP1 and ER-VP16 (23), and the ERE-ßGlob-Luc and 17M5ßGlob-Luc (24) reporter constructs were described elsewhere. The GST-p53 and the pcDNA3 plasmids encoding full-length p53 (25) were obtained from U. Hibner [Institut de Génétique Moléculaire de Montpellier (IGMM), Montpellier, France]. The pXJ-Mdm2 vector was obtained from B. Wasylyk (IGBMC), and the pcMI-Hdm2 contained the Mdm2 cDNA (BamHI/EcoRI fragment) subcloned into the pCMI1 vector (26). The pcMI-Hdm2{Delta}RING was obtained by inserting into the pCMI1 vector a PCR-amplified BamHI/EcoRI fragment corresponding to amino acids 1 to 435. The GST-Mdm2 was constructed by cloning a XhoI/NotI fragment of the human Mdm2 cDNA into the pGEX4T3 vector (Amersham Biosciences). The Gal-Mdm2 was generated by introducing a blunted NcoI/BglII fragment from pXJMdm2 into the SmaI site of the pSG424 plasmid (27). The RITA compound (NSC652287) was obtained from Dr. R.J. Schultz (NIH, Developmental Therapeutics Program, Drug Synthesis and Chemistry Branch, Rockville, MD).

Cell culture. Monolayer cell cultures (MCF-7, HeLa, and BG1) were grown, respectively, in Ham's F-12/Dulbecco's modified Eagle's medium (1:1, F12/DMEM) or DMEM alone supplemented with 10% FCS (Invitrogen) and antibiotics. Before hormonal treatments, cells were stripped of endogenous estrogens as previously described (28). When indicated, cells were irradiated using a Stratalinker UV cross-linker (model 1800) from Stratagene. The MELN cell line derived from MCF-7 cells stably transfected with the ERE-ßGlob-Luc-SVneo plasmid (29). Mouse embryo fibroblasts (MEF) null for both p53 and Mdm2 (MEFdKO; ref. 30) were obtained from G. Lozano (Houston, TX) and cultured in F12/DMEM.

Transient transfection and luciferase assays. Cells were plated in six-well plates (105 per well) 24 h before DNA transfection (4 µg of total DNA) by the calcium phosphate method using CMV-ßGal expression vector as an internal control. Luciferase values from transient transfection were normalized by the ß-galactosidase activities as described (28), and all data were expressed as mean ± SD.

Western blot analysis. Whole-cell extracts were prepared in high-salt lysis buffer (HSB) containing 500 mmol/L NaCl, 50 mmol/L Tris (pH 8), 1% NP40, 1 mmol/L DTT, and proteases inhibitors (Roche Diagnostics). Proteins were quantified using the Bradford assay (Bio-Rad Laboratories), and 30 µg were usually loaded on SDS-PAGE and transferred to polyvinylidene difluoride membrane. Blots were saturated in TBST buffer [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.1% Tween 20 (v/v), 5% nonfat dehydrated milk (w/v)]; incubated with specific primary antibodies for ER{alpha}, p53, or ubiquitin (Tebu), Mdm2 (clone 2A10; gift from J. Piette, IGMM), pS2 (clone p2802; gift from M.C. Rio, IGBMC), or actin (Sigma); and probed with the appropriate secondary antibody (Sigma). Detection was done using the Chemiluminescence Reagent Plus kit (Perkin-Elmer Life Science).

Immunoprecipitation experiments. Transfected MCF-7 or HeLa cells were lysed either in HSB for ubiquitination detection or in NETN buffer for coimmunoprecipitation. Immunoprecipitations were carried out using anti-ER{alpha} or anti-Mdm2 antibody (clone 4B11; gift from J. Piette) and protein G+ agarose beads (Tebu). Beads were washed four times in lysis buffer and boiled in 2x SDS sample buffer.

GST pull-down assays. In vitro translation and glutathione S-transferase (GST) pull-down assays were done as previously described (28). Protein interactions were analyzed by SDS-PAGE followed by quantification using a Phosphorimager (Fujix BAS1000).

Apoptosis assay and quantitative PCR. These assays have been described elsewhere (31).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ER{alpha} is involved in a ternary complex with p53 and Mdm2. To better characterize protein-protein interactions between Mdm2 and ER{alpha}, we first did GST pull-down experiments. As shown in Fig. 1A (left) , we observed a ligand-independent interaction of in vitro translated ER{alpha} with Mdm2 expressed as a fusion protein with GST, whereas no detectable interaction was obtained with GST alone. The binding of Mdm2 to ER{alpha} was mediated by the ligand-binding domain (LBD) of the receptor, as shown by the recruitment of in vitro expressed Mdm2 by the GST-LBD protein, which was also unaffected by E2 (right). Moreover, the use of a GST-LBD mutated in the conserved AF2 activation domain indicated that this interaction did not require an intact AF2 transactivation domain (data not shown). The use of ER{alpha} deletion mutants (shown in Fig. 1B, left) confirmed that both the DBD (HE11 mutant) and the NH2-terminal region (HE19 mutant) were not necessary for the binding to Mdm2 (Fig. 1B, right). By contrast, our data confirmed that the COOH-terminal LBD of ER{alpha} was required for the in vitro interaction because the HE15 mutant poorly associated with GST-Mdm2. Altogether, these results showed a direct ligand- and AF-2–independent interaction of ER{alpha} with Mdm2.


Figure 1
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  		Figure 1. Interaction of ER{alpha} with p53 and Mdm2. A, GST pull-down assays were carried out as described in Materials and Methods using bacterially expressed GST, GST-Mdm2, or GST-LBD proteins to retain 35S-labeled ER{alpha} or Mdm2 in the presence of vehicle (C), E2 (10–6 mol/L), or 4-hydroxytamoxifen (OHT; 10–6 mol/L). B, schematic representation of the ER{alpha} mutants. In vitro translated full-length ER{alpha} or various deletion mutants were analyzed for their interaction with the GST-Mdm2 fusion protein. Inputs, 10% of the material used in the assay (as in A). C, ER{alpha} and Mdm2 expression vectors were transfected into p53/Mdm2+/+ U2OS cells (lanes 1 and 2) or p53/Mdm2–/– MEFs (MEFdKO; lanes 3 and 4). MCF-7 cells were transfected with p53 and Mdm2 expression vectors (lanes 5–7). Whole-cell extracts were subjected to immunoprecipitation as described in Materials and Methods using anti-Mdm2 antibody (lanes 2 and 4), anti-ER{alpha} (lane 6), or with an irrelevant antibody (lane 7). Western blotting using either anti-Mdm2, anti-ER{alpha}, or anti-p53 antibody was done as indicated. Whole-cell extracts (lanes 1, 3, and 5). Inputs, 10% (left) and 6% or 18% (respectively, for ER{alpha} and for p53/Mdm2; right). D, mammalian two-hybrid assay was carried out using E2-treated MCF-7 cells transfected with the 17M5-Luc reporter together with expressing vectors for Gal4, Gal-GRIP1, or Gal-Mdm2 with VP16 or ER-VP16, in the presence or not of p53. Luciferase activity was expressed as % control in presence of Gal4. Columns, mean of three values; bars, SD. *, P < 0.05 (using Student's t test).

 
To confirm that these interactions also occurred in intact cells, we first set up coimmunoprecipitation experiments. As illustrated in Fig. 1C, ER{alpha} was found associated with immunoprecipitated Mdm2, as determined by probing the Western blot with a polyclonal antibody specific for the receptor (lanes 1 and 2). Interestingly, when the same experiment was carried out in MEFdKO embryonic fibroblasts (32) derived from p53–/– and Mdm2–/– mice (lanes 3 and 4), ER{alpha} was not coprecipitated with Mdm2, suggesting that p53 could be required for the interaction between ER{alpha} and Mdm2. Indeed, the formation of a ternary complex among ER{alpha}, p53, and Mdm2 was further shown by coimmunoprecipitation of both p53 and Mdm2 with endogenous ER{alpha} from MCF-7 cells (Fig. 1C, right).

Moreover, the fact that ER{alpha} could be engaged in a ternary complex with p53 and Mdm2 was also confirmed using a modified mammalian two-hybrid system (Fig. 1D). An expression plasmid coding for Mdm2 fused to the Gal4 DBD (Gal-Mdm2) was cotransfected in MCF-7 cells together with a Gal4-responsive reporter plasmid and an expression vector coding for ER{alpha} fused to the VP16 activation domain or for VP16 alone. In our experimental conditions, a slight but significant increase in luciferase activity (>2-fold versus 7-fold with Gal-GRIP used as a positive control) was obtained when ER-VP16 was coexpressed with Gal-Mdm2 (compared with the activity obtained with VP16 alone). Interestingly, when a p53 expression plasmid was cotransfected with Gal-Mdm2 and ER-VP16, we observed a significant increase in luciferase activity (>8-fold), suggesting that p53 indeed stabilize the interaction between ER{alpha} and Mdm2. Altogether, these results suggest that ER{alpha}, p53, and Mdm2 coexist within the same protein complex in intact cells.

p53 and Mdm2 are required for ligand-dependent ER{alpha} turnover. To evaluate the role of Mdm2 and p53 in a ligand-dependent down-regulation of ER{alpha}, we first used the MEFdKO model (32). As shown in Fig. 2A , in this p53/Mdm2–/– background, estrogen treatment did not decrease ER{alpha} accumulation (as observed in parallel in p53/Mdm2+/+ wild-type MEFs) but instead slightly increased receptor levels. To show that the expression of p53 and Mdm2 was required for the E2-dependent inhibition of ER{alpha} accumulation, we transiently transfected the expression vectors for both p53 and Mdm2 together with the expression vector for ER{alpha} in p53/Mdm2–/– MEFs (Fig. 2B). In this condition, we not only abolished the positive effect of E2 on ER{alpha} accumulation that we observed in control p53/Mdm2–/– cells, but we also restored the negative hormonal regulation of ER{alpha} expression.


Figure 2
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  		Figure 2. p53 and Mdm2 regulate E2-dependent expression of ER{alpha}. A, ER{alpha} accumulation was analyzed by Western blot using an anti-ER{alpha} antibody. MEFWT and MEFdKO cells (p53/Mdm2–/–) were transfected with an ER{alpha} expression vector and treated 20 h after transfection with vehicle or E2 (10–8 mol/L) for 20 h. B, Western blot analysis of ER{alpha} accumulation in MEFdKO cells transiently transfected with an ER{alpha} expression vector (150 ng) together with p53 and Mdm2 expression plasmids (250 ng for each vector) or empty vectors alone. Cells were cultured 20 h after transfection in the presence or absence of E2 (10–8 mol/L). *, nonspecific band detected by anti-ER{alpha} antibody. C, HeLa cells were transfected with ER{alpha} alone (1.5 µg) or ER{alpha} together with Mdm2 expression vector (150 or 500 ng, top and 1.5 µg, bottom). Cells used (bottom) were treated with MG132 (4 µmol/L) or DMSO alone for 24 h. Actin, ER{alpha}, and Mdm2 levels were quantified by Western blot 24 h after transfection. D, HeLa cells were transfected either with ER{alpha} (1.5 µg) or ER{alpha} and Mdm2 (1.5 µg each) expression vectors. Cycloheximide (CHX; 50 µmol/L) was added to inhibit ER{alpha} synthesis (time 0), and experiments were stopped at the indicated times. Mdm2 overexpression (at time 0) and ER{alpha} half-life were analyzed by Western blot.

 
Mdm2 regulates ligand-independent expression of ER{alpha}. Because some of the data shown in Fig. 1 suggested that Mdm2 could also bind ER{alpha} in an E2-independent manner, we investigated the effect of Mdm2 overexpression on ER{alpha} accumulation in the absence of ligand. As shown in Fig. 2C (top), when increasing amounts of Mdm2 expression vectors were transiently cotransfected with ER{alpha} in untreated HeLa cells, the accumulation of the receptor was significantly decreased. This effect involved the ubiquitin/proteasome pathway because the Mdm2-dependent down-regulation of ER{alpha} was completely relieved by the proteasome inhibitor MG132 (Fig. 2C, bottom). To emphasize the importance of Mdm2 in ligand-independent turnover of ER{alpha}, we measured the effect of Mdm2 overexpression on the apparent stability of the ER{alpha} protein by chase experiment in HeLa cells using cycloheximide as a protein synthesis inhibitor. As shown in Fig. 2D, we noticed a significant decrease in ER{alpha} apparent stability in the presence of Mdm2. This effect of Mdm2 on ER{alpha} stability was also observed in the presence of E2 (data not shown), thus emphasizing the role of Mdm2 in the E2-dependent and E2-independent posttranslational regulation of ER{alpha} expression.

Mechanisms involved in Mdm2 regulation of ER{alpha} stability. To further decipher the molecular mechanisms involved in ER{alpha} degradation by Mdm2, we first used a mutant of Mdm2 (Mdm2{Delta}RING) deleted in the COOH-terminal part of the protein, which contains the RING domain required for its ubiquitin-ligase activity (33). As shown in Fig. 3A , this mutant still interacted with ER{alpha} in GST pull-down experiment. Interestingly, overexpression of the Mdm2{Delta}RING mutant did not decrease ER{alpha} accumulation compared with the effect of its wild-type counterpart (Fig. 3B), suggesting that the E3 ubiquitin-ligase activity of Mdm2 is directly involved in ER{alpha} degradation. The implication of the ubiquitin/proteasome pathway was further shown in ubiquitination assays. As shown in Fig. 3C, after immunoprecipitation of ER{alpha}, slowly migrating forms of the receptor were detected both using anti-ER{alpha} (bottom) or anti-ubiquitin (top) antibodies, thus confirming that the level of ER{alpha} ubiquitination increased upon Mdm2 overexpression.


Figure 3
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  		Figure 3. Mechanisms of Mdm2-dependent degradation of ER{alpha}. A, a GST pull-down experiment was done to determine the in vitro interaction between 35S-labeled Mdm2 or Mdm2{Delta}RING (deleted of the COOH-terminal region) with the GST-AF2 fusion protein that contains the ligand-binding domain of ER{alpha}. B, HeLa cells were transfected either with the ER{alpha} expression plasmid alone (1.5 µg) or together with Mdm2 or Mdm2{Delta}RING expression vector (1.5 µg). The accumulation of ER{alpha} and Mdm2 proteins (wild type or Mdm2{Delta}RING) was analyzed by Western blot. C, ER{alpha} expression vector (1.5 µg) was transfected into HeLa cells with or without Mdm2 (1.5 µg). The ubiquitination status of ER{alpha} was then determined by immunoprecipitation of ER{alpha} from whole-cell extracts (as described in Materials and Methods) followed by Western blotting using anti-ubiquitin antibody (top) or with an ER{alpha} antibody (bottom). D, HeLa cells were transiently transfected with ER{alpha} expression vector alone (500 ng; lanes 1, 2, 4, and 5) or together with the Mdm2 expression vector (1 µg; lanes 3 and 6). Cells were treated with E2 (10–8 mol/L, lanes 2 and 5) or with vehicle alone (lanes 1, 3, 4, and 6). The expression of ER{alpha} and Mdm2 was analyzed by immunoblotting. The same experiment was done using the wild-type receptor or the HE11 ER{alpha} mutant (deleted of the DBD).

 
Finally, in an attempt to compare the E2-dependent turnover of the receptor with the ligand-independent degradation observed upon Mdm2 overexpression, we used a mutant of ER{alpha} deleted in the central DNA-binding domain involved in the binding of p53 (see Fig. 1B).4 This ER{alpha}-{Delta}DBD mutant (HE11) was no longer degraded upon E2 stimulation (Fig. 3D, lane 5) in support with the role of this domain in recruiting p53 and consistent with the formation of a ternary complex among the receptor, p53, and Mdm2 (Fig. 1). By contrast, the ligand-independent increase in ER{alpha} turnover observed upon Mdm2 overexpression was comparable for the wild-type protein and the DBD-deleted mutant (Fig. 3D, lane 3 for ER{alpha} wild type and lane 6 for HE11). Altogether, these data support the hypothesis that different mechanisms might be involved in the regulation of ER{alpha} turnover upon E2 treatment or Mdm2 overexpression.

Stress-inducing agents block ligand-dependent turnover of ER{alpha}. Previous studies reported that in MCF-7 human breast cancer cells, the E2-dependent decrease of ER{alpha} accumulation was abolished by the MG132 proteasome inhibitor (3, 11, 12). Results shown in Fig. 4A confirmed that MG132 was able to reverse the effect of various ligands, such as E2, estrone (E1), estriol (E3), or diethylstilbestrol, on ER{alpha} levels in MCF-7 cells. Interestingly, proteasome blockade is a cellular stress that increased accumulation of p53 (Fig. 4A, compare right and left) and, consequently, induced apoptosis, as shown by quantification of cytoplasmic nucleosomes (Fig. 4B).


Figure 4
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  		Figure 4. Effect of p53-inducing agents on ER{alpha} signaling. A, MCF-7 cells were treated for 20 h with 10–7 mol/L E1, E2, E3, or diethylstilbestrol (DES) in the absence (control) or presence of MG132 (4 µmol/L). ER{alpha} and p53 protein levels were analyzed by Western blot. B, MCF-7 cells were cultured with MG132 (MG; 4 µmol/L), actinomycin D (ActD; 10 nmol/L), or exposed to UV (150 J/m2) for 20 h, and apoptosis was measured by cytoplasmic nucleosomes quantification. Values were normalized by DNA quantification assay (measurement of DNA with DABA). C, MCF-7 cells were irradiated (UV), treated with MG132 (4 µmol/L), actinomycin D (10 nmol/L), or untreated (control) in the presence of vehicle or E2 (10–8 mol/L). Extracts were prepared and Western blotted with antibodies for ER{alpha} and p53. D, MCF-7 cells were irradiated by UV or not (control) and cultured in the presence of vehicle, E2 (10–8 mol/L), 4-hydroxytamoxifen (10–8 mol/L), or ICI 182,780 (ICI; 10–8 mol/L) for 20 h. ER{alpha} and p53 accumulation were analyzed by Western blot.

 
Based on our results concerning the role of Mdm2 in the regulation of ER{alpha} turnover, we thought that the effect of MG132 could be linked (at least in part) to its ability to dissociate Mdm2 from p53 and/or ER{alpha}. We therefore analyzed the effect of other stress-inducing agents (genotoxic or non-genotoxic) for their ability to regulate E2-dependent turnover. As shown in Fig. 4B, both UV irradiation and inhibition of transcription (actinomycin D treatment) increased significantly programmed cell death in MCF-7 cells. In all conditions, the accumulation of p53 was significantly increased to levels comparable with those obtained after MG132 treatment and, very interestingly, both treatments that increase p53 levels concomitantly suppressed the hormone-dependent down-regulation of ER{alpha} (Fig. 4C).

We then characterized the effect of UV irradiation on the regulation of ER{alpha} accumulation. We first showed that the blockade of E2-dependent turnover after UV exposition was rapid since observed 4 h after irradiation and still detectable 24 h later (data not shown). We then investigated the effect of UV irradiation on the response to antiestrogens, which have also been shown to regulate ER{alpha} expression. Indeed, as previously reported, incubation with the partial antagonist 4-hydroxytamoxifen increased the level of ER{alpha}, whereas pure antiestrogens, such as the ICI 182,780 molecule, negatively regulated the accumulation of the receptor (10). As shown in Fig. 4D, UV irradiation did not affect the stabilization of ER{alpha} upon 4-hydroxytamoxifen treatment. Very interestingly, it did not antagonize the effect of ICI 182,780, whereas it completely reversed the agonist-dependent decrease of ER{alpha} levels. This suggested that degradation of ER{alpha} by pure antiestrogens involved different mechanisms than those required for hormone-dependent degradation.

Concomitant stabilization of ER{alpha} and p53 by stress-inducing agents. To confirm that UV irradiation modulated the stability of the ER{alpha} protein, we did chase experiments with cycloheximide. As shown in Fig. 5A and B , the stability of the receptor in the absence of de novo protein synthesis decreased upon E2 treatment, and UV irradiation completely inhibited the hormone-dependent degradation. As expected, upon UV treatment, p53 seemed very stable, and its accumulation was strongly increased (Fig. 5A). Altogether, these data suggest that stress-inducing agents induce a co-stabilization of p53 and ER{alpha} in MCF-7 breast cancer cells. Our hypothesis was that disruption of the p53/Mdm2/ER{alpha} complex upon cellular stress was at the basis of the loss of E2-dependent turnover of the receptor.


Figure 5
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  		Figure 5. Effect of stress-inducing agents on ER{alpha} stability. A, the steady-state level of ER{alpha} was measured by pulse-chase assay with cycloheximide. MCF-7 cells were irradiated with UV (bottom) or not (top) and treated concomitantly with E2 (10–8 mol/L) for 4 h. Cycloheximide was then added (time 0) during 2, 4, or 6 h. ER{alpha} stability was measured by Western blot. B, quantification of the experiment in (A). The intensity of the bands corresponding to ER{alpha} levels was determined by PCBAS imaging. Values were normalized by quantifying actin expression on the same blot. Results are expressed as % control (time 0). C, kinetics of ER{alpha} and p53 accumulation by Western blot in stably transfected MELN cells treated or not with E2 (10–8 mol/L) in presence or not of RITA (1 µmol/L) during 2, 4, or 8 h.

 
To further emphasize this point, we used the NSC652287 compound, also known as RITA (which stands for reactivation of p53 and induction of tumor cell apoptosis) and recently described as an inhibitor of the p53/Mdm2 interaction (34). As shown in Fig. 5C, treatment of MCF-7 cells with RITA at 1 µmol/L significantly abolished the ligand-dependent decrease in ER{alpha} accumulation. This effect was detectable 4 h after the beginning of the treatment and very interestingly, it correlated again with the increase in p53 accumulation. As a control of UV irradiation and RITA treatment, we checked by quantitative reverse transcription-PCR that no up-regulation of the ER{alpha} mRNA was associated with the blockade of the E2-dependent decrease observed at the protein level (data not shown). Combined, these data therefore suggest that dissociation of the p53/Mdm2/ER{alpha} complex upon cellular stress leads to a co-stabilization of p53 and ER{alpha} proteins.

Dissociation between ligand-dependent ER{alpha} expression and transactivation. Because previous studies proposed that the E2-dependent decrease of ER{alpha} accumulation was required for transcriptional activity of the receptor (14), we analyzed whether these two variables were indeed linked in our hands. We first investigated the ability of transiently transfected ER{alpha} to increase the transcription of a reporter gene in MEFs expressing or not p53/Mdm2. As shown in Fig. 6A , we found that ER{alpha} strongly activated transcription in cells where E2 up-regulated its accumulation (Control). Very interestingly, we found that overexpression of p53/Mdm2 only slightly modify ER{alpha} transactivation although it completely inversed the effect of E2 on its accumulation (see Fig. 2B).


Figure 6
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  		Figure 6. E2-dependent transactivation of ER{alpha} does not require receptor turnover. A, MEFdKO cells were transiently transfected either with the 17Eß-Luc reporter plasmid (300 ng) and ER{alpha} (100 ng) alone or together with p53 and Mdm2 expression vectors (same transfection experiment as for the Western blot analysis in Fig. 2B). Cells were treated with vehicle or E2 (10–8 mol/L), and the luciferase activity was quantified as described in Materials and Methods. Results were expressed relative to control in presence of ER{alpha} alone (% control). Columns, mean of three values; bars, SD. B, MCF-7 cells stably transfected with ERE-ßGlob-Luc (MELN cells) were treated with vehicle (control) or E2 (10–8 mol/L) for 20 h, 4 h after irradiation (UV) or without being irradiated (C). Luciferase activity was measured as described in Materials and Methods. Columns, mean relative activity (% E2 without irradiation) of three values; bars, SD. C, MCF-7 cells were UV irradiated (150 J/m2) or not (control) in the presence (E2, 10–8 mol/L) or in the absence (C) of E2 for 20 h. pS2, p53, and actin protein levels were analyzed by Western blot. D, left, BG1 ovarian cancer cells were treated or not (C) for 20 h with E2 (10–8 mol/L), and ER{alpha} protein levels were analyzed by Western blot. Right, MCF-7 and BG1 cells were transiently transfected with ERE-ßGlob-Luc, treated with vehicle or E2 (10–8 mol/L), 4-hydroxytamoxifen (10–8 mol/L) or ICI 182,780 (10–8 mol/L) for 20 h, and the luciferase activity was quantified and expressed as in (A).

 
We then analyzed the effect of UV irradiation on endogenous ER{alpha} activity. To this aim, we used MELN cells that are MCF-7 cells stably transfected with an E2-regulated luciferase reporter gene, allowing easy monitoring of endogenous ER{alpha} activity (24). MELN cells were treated with E2 4 h after irradiation by UV (i.e., when E2-dependent turnover of ER{alpha} was no longer detectable; see Fig. 4). As shown in Fig. 6B, the transcriptional activity of ER{alpha} was significantly increased both in the absence of ligand or in the presence of E2. In all conditions, the induction by E2 was detectable, confirming that E2-dependent turnover was not a prerequisite for transactivation even on endogenous ER{alpha}. Moreover, the same results were obtained on the endogenous E2-regulated pS2 gene (Fig. 6C), thus confirming the dissociation of stress-inducing agents on the accumulation of ER{alpha} and on its transactivation.

Finally, we screened several ER{alpha}-expressing human cancer cell lines for the negative E2-dependent regulation of ER{alpha} accumulation. As shown in Fig. 6D, we found that BG1 ovarian cancer cells exhibited a strong positive regulation of ER{alpha} levels in response to E2 treatment, contrasting with the negative regulation observed in MCF-7 breast cancer cells (see Figs. 4 and 5). Of interest, when we analyzed the transactivation properties of ER{alpha} in these two cell lines, we found that at least on the reporter that we used in transient transfection, the respective endogenous receptors were able to transactivate to similar extent (Fig. 6D). Altogether, our results strongly suggest that the effect of E2 on ER{alpha} turnover could be dissociated from its effect on transcriptional activation at least on some target promoters.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Modulation of ER{alpha} levels is a critical variable in determining the hormonal response of breast cancer cell proliferation. The control of ER{alpha} expression is under a complex regulation that takes place both at the transcriptional and posttranscriptional levels. Previous data suggested that ER{alpha} was down-regulated in the presence of E2 through a proteasome-dependent mechanism (3, 11, 12). In this study, we have investigated the mechanisms regulating the hormone-induced ER{alpha} turnover, and several lines of evidence indicate that Mdm2, an oncogenic ubiquitin-ligase, plays an important role in the regulation of ER{alpha} accumulation by E2.

First, using coimmunoprecipitation and a modified two-hybrid assay, we show that Mdm2 interacted with ER{alpha} in a ternary complex with p53. The direct in vitro interaction between the receptor and both Mdm2 and p53 was also shown using GST pull-down assays. Our data suggested that Mdm2 binding involved the LBD of ER{alpha} but did not require the ligand-dependent AF2 interface. Interestingly, p53 interacted with a different region of the receptor (i.e., the central region; data not shown), thus supporting the formation of a ternary complex.

The use of p53/Mdm2–/– cells showed that the two proteins were required for the E2-dependent down-regulation of ER{alpha}. Moreover, we showed that the COOH-terminal region of Mdm2, which encompassed the E3 activity, was required for the degradation of ER{alpha} and that overexpression of Mdm2 increased ER{alpha} ubiquitination. Previous studies highlighted the role of the Mdm2 ubiquitin-ligase in the degradation of steroid hormone nuclear receptors. In the case of the glucocorticoid receptor (GR), Sengupta and Wasylyk showed that disruption of the p53/Mdm2 interaction prevented ubiquitination of GR and that the ligand-dependent trimeric complex between GR, p53 and Mdm2 enhanced proteasomal degradation of the receptor (35). Moreover, the E3 ligase activity of Mdm2 was also necessary for the ubiquitination and degradation of the androgen receptor (36).

From the data presented in our study, we propose that p53 and Mdm2 are involved in the ligand-dependent degradation of ER{alpha}. Interestingly, in support of our data, it has been recently shown, by chromatin immunoprecipitation, that Mdm2 was recruited on the ER{alpha}-regulated pS2 promoter upon E2 stimulation (37, 38). Moreover, both Mdm2 and p53 expression levels are increased by E2 in MCF-7 breast cancer cells (39). This positive regulation could be of importance in the E2-dependent down-regulation of the receptor. Obviously, other factors associated with p53 or Mdm2 could be involved in such a degradation complex. A strong candidate is the coactivator AIB1, which has been shown to interact with p53 (40) and to be required for the E2-dependent turnover of ER{alpha} (41). The involvement of AIB1 could explain why the AF2-mutated ER{alpha} is no longer degraded upon hormone stimulation.5 Interestingly, in breast cancer, AIB1 amplification correlates with that of Mdm2 (42), and AIB1 expression is regulated by ubiquitination (43).

Our results dealing with the effect of stress-inducing agents on ER{alpha} expression clearly indicated that the increased turnover of the receptor in the presence of the pure antiestrogen ICI 182,780 involves different mechanisms than those implicated in the E2-dependent degradation. Indeed, the effect of ICI182,780 on ER{alpha} levels were not abolished in UV-irradiated MCF-7 cells (Fig. 4D) and in MEFdKO (data not shown). Similar dissociation between the effects of E2 and ICI 182,780 were previously reported (44), and other pathways involving, for example, the NEDD8 ubiquitin-like protein (45) or CSN5/Jab1 (46) could account for the degradation in the presence of pure antihormones.

Previous data have highlighted the existence of a ligand-independent degradation of ER{alpha} and showed the role of CHIP (COOH terminus of Hsc70-interacting protein) in the ubiquitination of misfolded ER{alpha} (47). More recently, E6-AP has also been shown to be involved in a calmodulin-dependent degradation pathway of ER{alpha} (48). Our data suggest that, when overexpressed, Mdm2 is also involved in the ligand-independent turnover of ER{alpha}. In these conditions, several evidences indicate that p53 could be dispensable for the effect of Mdm2, and this could reflect the fact that, upon overexpression of Mdm2, the binding equilibrium between ER{alpha} and Mdm2 could be strongly displaced towards complex formation, thus avoiding the requirement for p53 to stabilize the interaction. Finally, although we show that Mdm2 increased ER{alpha} ubiquitination, further work will be required to define which particular lysine residues are modified by Mdm2 in the presence or absence of E2.

The present work also highlights the regulation of ER{alpha} expression by cellular stress. We show that several stress-inducing agents that stabilize p53 also increase ER{alpha} levels and block its E2-dependent down-regulation. The stabilization of p53 upon treatment with stress-inducing agents results from its dissociation from Mdm2 due to posttranslational modifications such as phosphorylation (49). It is interesting to note that phosphorylation of nuclear receptors has also been linked to their turnover (36, 50, 51). Concerning ER{alpha}, it has very recently been reported that S118 is an essential determinant of ER{alpha} degradation (38), and a previous work has shown that extracellular signal-regulated kinase 7 enhances the destruction of the receptor in a ligand-independent manner (52). By contrast, another report suggests that in MCF-7 cells, inhibition of mitogen-activated protein kinase results in an increased degradation of ER{alpha} (53). Altogether, these data suggest that phosphorylation is directly or indirectly involved in ER{alpha} turnover. Interestingly, we found that the degradation observed upon Mdm2 overexpression was no longer detected when we tested ER{alpha} mutated on the phosphorylated Ser118 residues (data not shown).

Finally, although it has been suggested that the E2-dependent turnover of ER{alpha} was required for efficient transactivation of the receptor (14), the data presented herein do not support such a conclusion. We first show that in p53/Mdm2–/– cells where E2 treatment led to an increase in ER{alpha} accumulation, a strong hormone-dependent transactivation was observed. Interestingly, the same result was obtained with endogenous ER{alpha} in BG1 ovarian cancer cells. In addition, in MCF-7 cells, the blockade of ER{alpha} E2-dependent degradation upon UV irradiation (or MG132; data not shown) did not abolish the transcriptional response to E2 cells both on a stably transfected ERE-containing reporter gene and on the well-known pS2 target gene. Altogether, the present work dissociates the effect of E2 on ER{alpha} turnover from the effect of hormone on transactivation, thus supporting previous studies done using MG132, which suggested that the two events are not linked (54).

In conclusion, we propose that within a same protein complex, ER{alpha} and p53 are both targeted by the Mdm2 E3 ubiquitin ligase; consequently, the stability of ER{alpha} and p53 is concomitantly increased in response to stress-inducing agents. This study therefore emphasizes the relevance of protein-protein interactions among nuclear receptors, the Mdm2 oncogene, and the p53 tumor suppressor. Several physiologic and pathologic consequences of these interactions have been proposed for the glucocorticoid receptor (55). Our data showing the involvement of the Mdm2 oncoprotein in the regulation of ligand-dependent and ligand-independent ER{alpha} expression are in accordance with a recent study that reported that Mdm2 protein expression is a negative prognostic marker in breast carcinoma whose expression showed a negative correlation with ER{alpha} (56). Such an inverse association might seem surprising because this receptor is believed to mediate proliferative signaling of estrogens. However, our laboratory has shown that in the absence of ligand, ER{alpha} also exerts anti-invasive activity in breast cancer cell lines (57), and this effect could therefore be lowered in tumor cells overexpressing Mdm2. Altogether, the present work highlights the role of Mdm2 on hormone signaling and will be at the basis of future investigations to decipher its importance in various physiopathologic situations.


   Acknowledgments
 
Grant support: Institut National de la Santé et de la Recherche Médicale, University of Montpellier I, Ligue Nationale contre le Cancer, and Association pour la Recherche sur le Cancer.

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

We thank P. Chambon and R.J. Schultz for plasmids and reagents and Jean-Marc Vanacker, Stephan Jalaguier, Patrick Augereau, Jean-Claude Nicolas, and Audrey Castet for critical reading of the manuscript.


   Footnotes
 
Note: V. Duong was a recipient of fellowships from the French Minister of Research and the Association pour la Recherche sur le Cancer.

Current address for S. Daujat: Max Planck Institute for Immunology, 79108 Freiburg, Germany.

4 Our unpublished results. Back

5 V. Duong, unpublished data. Back

Received 7/28/06. Revised 3/13/07. Accepted 3/22/07.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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