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Molecular Endocrinology 17 (2): 287-299
Copyright © 2003 by The Endocrine Society

Receptor-Interacting Protein 140 Binds c-Jun and Inhibits Estradiol-Induced Activator Protein-1 Activity by Reversing Glucocorticoid Receptor-Interacting Protein 1 Effect

Catherine Teyssier1, Karine Belguise1, Florence Galtier, Vincent Cavailles and Dany Chalbos

Institut National de la Santé et de la Recherche Médicale, Endocrinologie Moléculaire et Cellulaire des Cancers (U 540), 34090 Montpellier, France

Address all correspondence and requests for reprints to: Dr. Dany Chalbos, Unité 540 Institut National de la Santé et de la Recherche Médicale (INSERM), Endocrinologie Moléculaire et Cellulaire des Cancers, 60 Rue de Navacelles, 34090 Montpellier, France. E-mail: chalbos{at}u540.montp.inserm.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the presence of estradiol, estrogen receptor-{alpha} (ER{alpha}) increases transcription triggered by activator protein-1 (AP-1). We have previously shown that induction is mediated by the direct interaction between c-Jun and ER{alpha}, which stabilizes a multiprotein complex containing the coactivator GRIP1 (glucocorticoid receptor interacting protein 1). The effect of receptor-interacting protein 140 (RIP140) in this regulation was assessed in the present study. We report that overexpression of RIP140 inhibits estradiol-induced AP-1-dependent transcription in a dose-dependent manner. Inhibition is not affected by trichostatin A, suggesting that histone deacetylase recruitment is not implicated. RIP140, which binds Jun proteins in pull-down assays and in intact cells, as shown by coimmunoprecipitation analysis and a mammalian one-hybrid system, participates in a multiprotein complex containing c-Jun and ER{alpha}. Moreover, the negative effect of RIP140 on AP-1-mediated transcription is relieved by GRIP1 overexpression and, conversely, RIP140 inhibits the stimulatory effect of GRIP1. The two cofactors compete for binding to c-Jun and ER{alpha} both in vitro and in intact cells, and GRIP1 interaction with both ER{alpha} and c-Jun is required for an efficient competition. These overall results suggest that the ratio between RIP140 and GRIP1 could determine, as proposed for hormone response element-mediated responses, the efficacy of estradiol in stimulating transcription of genes under AP-1 control.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGENS PLAY A fundamental role in the development of female sex organs and in the reproductive process in vertebrates. Their action is mediated by specific intracellular estrogen receptors (ER{alpha} and ERß) belonging to the steroid/nuclear receptor (NR) superfamily of hormone-inducible transcription factors. These receptors share structurally related functional domains, including the central and highly conserved DNA-binding domain (DBD) and the moderately conserved C-terminal ligand-binding domain. Two distinct activation functions (AFs), which are promoter and cell type specific, and in most cases synergize with each other, contribute to transcriptional activity. The constitutively active AF-1 domain is present in the N-terminal part of the receptor. The ligand-dependent AF-2 function is located at the C-terminal end of the ligand-binding domain and implicates an amphipathic {alpha}-helical motif (helix 12). Hormone binding to the receptor induces swinging of helix 12 and formation of a new interaction surface for accessory factors which either contact components of the basal transcriptional machinery or modulate chromatin structure to facilitate binding of the transcription initiation complex (reviewed in Refs. 1 and 2).

Proteins that bind to receptors in a ligand- and AF-2-dependent manner and enhance their transactivation potential have been identified in recent years. These coactivators include the cAMP response element-binding protein CBP/p300 and a family of highly related molecules called p160 proteins, comprising steroid receptor coactivator 1 (SRC-1), transcriptional intermediary factor-2 (TIF2)/glucocorticoid receptor interacting protein I (GRIP1), and RAC3 (receptor-associated coactivator 3)/p/CIP (p300/CBP interacting protein)/AIB1 (amplified in breast cancer 1)/ACTR (activator of thyroid and retinoic acid receptors). CBP/p300, SRC-1, and ACTR were recently shown to have an intrinsic histone acetyltransferase activity that could influence the accessibility of transcription factors to chromatin templates (2, 3). The coactivator effect on NRs is less clear for other factors such as receptor-interacting protein 140 (RIP140). Although RIP140 interaction with receptors occurs only in the presence of an agonist and is abolished by mutations that inactivate AF-2, the major effect of RIP140 overexpression in transient transfections of mammal cells is inhibition of ER{alpha} activity (4). RIP140 also acts as a repressor of orphan receptor TR2 (5), in a ligand-independent manner, and of fatty acid (peroxisomal proliferator-activated receptor-{gamma}) (6), glucocorticoid (7), and retinoic acid (retinoic acid receptor/retinoic X receptor) (8) receptors in the presence of cognate agonist. Moreover, it decreases cooperative ER/Pit-1 activation of the pituitary-specific prolactin promoter (9). It has been proposed that RIP140 might regulate NR AF-2 activity by competition for p160 coactivators such as SRC-1 (6, 9). In addition, RIP140 has been shown to possess an intrinsic inhibitory function (5), and direct recruitment of histone deacetylase (HDAC) or carboxyl-terminal binding protein (CtBP) could be responsible for its negative effect (10, 11).

NRs also regulate gene transcription by interfering with other transcription factors (12, 13, 14). We and others, for instance, have shown that estradiol can modulate activator protein 1 (AP-1) activity (15, 16, 17, 18). AP-1 complexes correspond mainly to dimers of proteins encoded by fos and jun gene families, which have been widely implicated in differentiation, cell proliferation, and transformation (19). Jun (c-Jun, JunB, JunD) and Fos (c-Fos, Fra-1, Fra-2, and FosB) proteins share a conserved region containing the basic DBD and the leucine zipper dimerization motif. Jun proteins form either homodimers or heterodimers with proteins of the Fos family and regulate gene transcription through interaction with a specific DNA sequence, the 12-O tetra-decanoyl-phorbol-13 acetate-responsive element, also referred to as the AP-1 site (19, 20, 21). Some NR coactivators such as CBP/p300 (22, 23) and SRC-1 (24), which are considered as molecular integrators, can physically interact with c-Jun and c-Fos and enhance transcriptional activation by AP-1.

In the case of ERs, AP-1-mediated transcription is generally potentiated by hormone, contrary to what is observed with other NRs such as RAR, which always has an inhibitory effect (25). The estradiol-mediated regulation of AP-1 activity requires an integral ER{alpha} AF-2 domain, and it is enhanced by overexpression of the NR coactivator GRIP1 (26, 27). This cross-talk does not involve binding of activated ER{alpha} to DNA and is likely due to protein-protein interactions (15, 18, 24, 28). Previous studies demonstrated that ER{alpha} could bind to various members of the Jun family in vitro. This interaction was also observed in intact cells and appeared important in stabilizing a multiprotein complex containing GRIP1 (26).

The aim of this study was to evaluate the RIP140 effect on ER{alpha}-regulated AP-1 activity. We report that RIP140 physically interacts with Jun proteins in vitro and in intact cells and inhibits estradiol enhancement of AP-1-mediated transcription. We also show, using a modified mammalian two-hybrid system, that RIP140 can participate in the formation of a ternary complex with ER{alpha} and c-Jun. Finally, our results suggest that RIP140, the inhibitory effect of which can be reversed by GRIP1, could compete with the coactivator within this complex.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RIP140 Inhibits Estradiol-Induced AP-1 Activity
The effects of RIP140 on AP-1-dependent transcription regulated by estradiol were evaluated by transient transfection experiments in MCF7 breast cancer cells. Increasing amounts of a RIP140 expression vector (pEFRIP) were transfected together with the (AP-1)4-TK-CAT reporter plasmid (Fig. 1AGo). RIP140 inhibited estradiol activation of AP-1-dependent chloramphenicol acetyltransferase (CAT) activity in a dose-dependent manner, and the hormonal regulation was totally abolished for the highest tested RIP140 concentration. In contrast, transfection of a RIP140 antisense expression vector, in the same experiment, increased the estradiol effect by about 50%. Transfection of both sense and antisense RIP140 had no significant effect on the basal level of AP-1-mediated CAT activity (Fig. 1AGo). As shown in Fig. 1BGo, they also only marginally affected AP-1-dependent transcription induced by c-Jun or c-Fos overexpression. In addition, no significant regulation was detected on the ß-galactosidase control plasmid (not shown) and on the pGL3 vector, which constitutively expresses luciferase under the control of Simian virus 40 promoter and enhancer sequences (Fig. 1CGo).



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  		Figure 1. RIP140 Overexpression Represses Estradiol-Induced AP-1 Activity

A, Steroid-stripped MCF7 cells were transfected with 1 µg of (AP-1)4-TK-CAT together with 0.5, 1, or 1.5 µg of pEFRIP (RIP140) or pEFRIP antisense (ASRIP)/when indicated. Cells were then incubated for 28 h with 10 nM 17ß-estradiol (E2) or vehicle (C). CAT activity was evaluated in whole-cell extracts after normalization for ß-galactosidase activity, as described in Materials and Methods. The results are expressed in % conversion of [14C]chloramphenicol and represent the mean (±SD) calculated from triplicate wells from one experiment representative of three separate assays. B, Steroid-stripped MCF7 cells were transfected with 1 µg (AP-1)4-TK-CAT and 1.5 µg of pEFRIP (RIP140), pEFRIP antisense (ASRIP), or empty vector (C). They were cotransfected with 0.2 µg pCI-c-Jun or 0.1 µg pCI-c-Fos when indicated. Cells were then incubated in F12/DMEM 1% DCC for 28 h. CAT activity was measured and calculated as described in panel A. C, Steroid-stripped MCF7 cells were transfected with 1 µg pGL3 together with 1.5 µg of pEFRIP (RIP140), pEFRIP antisense (ASRIP), or empty vector (C). After 28-h incubation in F12/DMEM 1% DCC, CAT activity was measured and calculated as described in panel A.

 
The Inhibitory Effect Is Not Trichostatin A (TSA) Sensitive
To assess whether or not the repression of estradiol-mediated regulation of AP-1 activity by RIP140 involved histone deacetylases, we tested the effect of TSA, a potent inhibitor of these enzymes, on AP-1-mediated transcription (Fig. 2Go). In MCF7 cells transfected by the (AP-1)4-TK-CAT reporter construct, TSA increased AP-1-mediated CAT activity in control cells, in agreement with published results (29), and in estradiol-treated cells. However, TSA treatment did not relieve the inhibitory effect of RIP140, suggesting that histone deacetylases did not take part in the suppression of estradiol induction.



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  		Figure 2. Effect of TSA on the Inhibition of AP-1 Activity by RIP140

Steroid-stripped MCF7 cells were transfected with 1 µg (AP-1)4-TK-CAT, 0.4 µg ER{alpha} expression vector HEO, and increasing concentrations of pEFRIP. Cells were then incubated for 28 h with 10 nM 17ß-estradiol (E2) or vehicle (C) in the absence or presence of TSA (25 or 100 ng/ml). CAT activity was evaluated as described in Fig. 1AGo. CAT activities represent the mean (±SD) calculated from triplicate wells from one experiment representative of three separate assays.

 
RIP140 Binds to Jun Protein in Vitro and in Intact Cells
Pull-down experiments were then performed to determine whether RIP140 could directly bind to c-Jun, as noted for p160 cofactors (24). As shown in Fig. 3AGo, 35S-methionine-labeled RIP140 was retained by purified hexahistidine c-Jun fusion proteins bound on nickel-chelating resin. The interaction of in vitro translated-RIP140 deletion mutants with c-Jun fusion protein was then analyzed to localize the RIP140 domains required for the interaction. All the tested deletion mutants, except RIP140 (917–1158), bound to c-Jun with diverse efficiencies also always lower than that observed for the entire protein, suggesting that multiple parts of RIP140 contribute to the interaction.



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  		Figure 3. RIP140 Physically Interacts with c-Jun

A, In vitro translated radiolabeled RIP140 and RIP140 deletion mutants were incubated overnight with nickel-chelating resin loaded or not with purified histidine-tagged c-Jun (10 µg), as described in Materials and Methods. After extensive washes, proteins were eluted and subjected to SDS-PAGE and fluorography. Ten percent inputs of the radiolabeled proteins used in the assays are shown (input). Quantification was performed with a Fuji Photo Film Co., Ltd. BAS1000 Bioimaging Analyser. B, In vitro35S-labeled c-Jun and c-Jun deletion mutants were incubated overnight with GST or GST-RIP140 fusion proteins preloaded on glutathione sepharose, as described in Materials and Methods. Inputs represent 10% of the radiolabeled c-Jun mutants used in the assays. C, Coimmunoprecipitation analysis. MCF7 cell extract was subjected to immunoprecipitation with rabbit anti-c-Jun antibody or nonimmune rabbit immunoglobulins (IgG). Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted by using anti-RIP140 and anti-c-Jun antibodies. Input (I) lane contains 10% of total cell extract used in the assay. Black arrows indicate the position of RIP140 and c-Jun and open arrow indicates the position of immunoglobulins.

 
The reciprocal experiment was then performed to confirm this interaction. As shown in Fig. 3BGo, 35S-labeled c-Jun was retained by fusion proteins harboring RIP140 amino acids 27–439 or 683-1158 (~9% input for the two constructs) but not by the RIP140 residues 917-1158 fusion, in agreement with Fig. 3AGo. Multiple bands observed after migration of in vitro translated c-Jun in sodium dodecyl sulfate gels most likely correspond to Ser-63, Ser-73, or both phosphorylated forms of c-Jun, as previously described by Bannister et al. (23). All phosphorylated forms of c-Jun bound to glutathione-S-transferase (GST)-RIP140 fusion proteins to the same extent, indicating that, at least in our in vitro GST pull-down assay, phosphorylation of Ser-63/73 did not modify the interaction with the cofactor. In fact, use of c-Jun deletion mutants demonstrated that only the C-terminal part, which contains both the leucine zipper and the basic DBD, was involved in the interaction.

To confirm that RIP140 interacts with c-Jun in intact cells, endogenous protein complexes associated with c-Jun were immunoprecipitated with anti-c-Jun antibody, and the precipitated proteins were subjected to immunoblotting with anti-RIP140 antibody (Fig. 3CGo). RIP140 was specifically detected in complexes recognized by anti-c-Jun antibody.

Tripartite Complex with ER{alpha}, RIP140, and c-Jun
As shown above, RIP140 could directly bind to Jun proteins, and it is well established that RIP140 interacts physically with activated ER (4, 30). In addition, we previously reported that ER{alpha} can also associate with c-Jun in a protein-protein interaction (26). We therefore attempted to determine whether the three molecules could coexist within the same protein complex.

In a first approach, 35S-in vitro-labeled RIP140, preincubated or not with 35S-in vitro-labeled c-Jun, was incubated with a GST fusion protein containing the ER{alpha} hinge domain (GST-CD) which was previously shown to directly interact with c-Jun (26), but not the receptor AF-2 domain (Fig. 4AGo). RIP140, which, as expected, did not bind to the fusion protein when added alone, was retained by GST-CD in the presence of c-Jun, demonstrating that c-Jun could interact in the same time with ER{alpha} and RIP140.



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  		Figure 4. c-Jun Participates in Protein Complexes Containing ER{alpha} and RIP140

A, In vitro translated radiolabeled RIP140 was preincubated with radiolabeled c-Jun or reticulocyte lysate for 30 min at room temperature. It was then incubated with GST-CD fusion protein, containing ER{alpha} residues 179–312, preloaded on glutathione sepharose beads equilibrated in TKE buffer (10 mM Tris, pH 7.5, 75 mM KCl, 0.5 mM EDTA), overnight at 4 C. After five washes in TKE buffer, proteins were eluted and subjected to SDS-PAGE and fluorography as described in Materials and Methods. Ten percent inputs of radiolabeled proteins used in the assays are shown on the left. B, The GST-ER{alpha} fusion proteins, GST-AF2 (ER{alpha} amino acids 313–595), and GST-DAF2 (amino acids 251–595) were preloaded on glutathione sepharose beads. They were then preincubated for 4 h with or without 10 µg histidine-tagged c-Jun (c-Jun°) or c-Fos (c-Fos°) purified on nickel-chelating resin as described in Materials and Methods. After extensive washes, fusion proteins were incubated overnight with in vitro translated radiolabeled c-Jun (top panel) or RIP140 (middle and bottom panel). Estradiol (E2, 1 µM) was present during incubation when indicated. Proteins were then eluted and subjected to SDS-PAGE and fluorography as described in Materials and Methods. Twenty percent (top and middle panel) or ten percent (bottom panel) input of radiolabeled proteins used in the assays are shown on the left. Quantification was performed with a BAS1000 Bioimaging Analyser (Fuji Photo Film Co., Ltd.). B, In vitro35S-labeled c-Fos was incubated overnight with GST or GST-RIP140 fusion proteins preloaded on glutathione sepharose, as described in Materials and Methods. Input represents 10% of the radiolabeled c-Fos used in the assays. D, Steroid-stripped MCF7 cells were transfected with 1 µg GAL4luc and with vector encoding the DBD of GAL4 fused to RIP140 (residues 848-1158) (GAL4-RIP140, 1 µg). They were cotransfected with vector encoding the activation domain of VP16 fused to ER{alpha} (VP16-ER{alpha}, 1 µg) and pCI-c-Jun (2 µg) when indicated. Nonfused GAL4 DBD and VP16 activation domains were used in control experiments. Cells were incubated with vehicle (C) or 10 nM 17ß-estradiol (E2) for 28 h. Luciferase activity was evaluated in whole-cell extracts as described in Materials and Methods. The results shown are the mean (±SD) of luciferase activities calculated from triplicate wells from one experiment representative of three separate assays.

 
We then tested binding of RIP140 on preformed ER{alpha}/c-Jun complexes. GST-ER fusion proteins, with (GST-DAF2) or without (GST-AF2) the c-Jun interaction domain, were preloaded on glutathione-coupled beads and preincubated with or without excess of purified unlabeled hexahistidine c-Jun protein (Fig. 4BGo). After extensive washes, beads were incubated with 35S-in vitro-labeled c-Jun or RIP140 in the absence or presence of estradiol. Preincubation with c-Jun drastically decreased the consecutive interaction of ER{alpha} with labeled c-Jun, demonstrating that most GST-DAF2 fusion molecules were bound to unlabeled c-Jun in these experimental conditions (top panel). In agreement with previous results (26) showing that in vitro binding of c-Jun on ER{alpha} is estradiol independent, similar results were observed in the presence as in the absence of ligand. In contrast, 35S-labeled RIP140 efficiently interacted with bead-bound GST-DAF2/c-Jun complexes (middle panel) in a ligand-dependent manner, demonstrating that ER{alpha} could bind RIP140 and c-Jun at the same time. Addition of c-Jun, in fact, increased RIP140 binding, especially in the absence of ligand (11% vs. 3% input), but also in the presence of estradiol (47% vs. 26%). This enhancement was not observed when GST-AF2 was used in the assay (Fig. 4BGo). c-Fos, which does not bind to ER{alpha} (26), interacted with RIP140 in pull-down experiments (Fig. 4CGo). Similarly, no difference in RIP140 binding was detected when purified unlabeled hexahistidine c-Fos was used instead of c-Jun (Fig. 4BGo, bottom panel). The increased binding of RIP140 to GST-DAF2 in the presence of c-Jun was therefore likely to reflect the direct interaction of RIP140 with c-Jun and its incorporation into a ternary complex.

The formation of RIP140/ER{alpha}/c-Jun complexes was then confirmed in intact cells using a modified mammalian two-hybrid system. Expression plasmid coding for RIP140 amino acids 848-1158 fused to the GAL4 DBD (GAL4-RIP140) and pCI-c-Jun were cotransfected in MCF7 with a GAL4-responsive promoter, in the absence or presence of an expression vector coding for ER{alpha} fused to the VP16 activation domain (Fig. 4BGo). In our experimental conditions, overexpression of c-Jun increased, by 3-fold, the GAL4-driven luciferase activity measured in the presence of GAL4-RIP140 alone. As expected, increased reporter gene transcription was noted when GAL4-RIP140 and VP16-ER{alpha} were coexpressed in cells stimulated by estradiol, but not in control cells. Moreover, overexpression of c-Jun, together with the two fusion proteins, drastically amplified luciferase activity in the presence of hormone. We therefore conclude that c-Jun could bind ER{alpha}/RIP140 complexes in intact cells.

The Inhibitory Effect of RIP140 Is Partly Reversed by GRIP1 Overexpression
We then investigated whether the inhibitory effect of RIP140 could be reversed by other cofactors which, by tethering to ER{alpha}, mediated the AF-2 transactivation function. To test this hypothesis, GRIP1 and RIP140 effects were assayed on the estradiol regulation of AP-1-dependent transcription. Constant amounts of RIP140 or GRIP1 expression vectors were cotransfected into MCF7 cells with increasing amounts of GRIP1 or RIP140, respectively, along with the (AP-1)4-TK-CAT construct (Fig. 5AGo). Results showed that GRIP1 partly reversed the RIP140-induced inhibition in a dose-dependent manner. In agreement with published results (27), GRIP1 overexpression alone amplified the hormonal effect. This stimulatory effect was inhibited when RIP140 was overexpressed and was nearly abolished upon addition of the highest amount of RIP140 expression vector.



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  		Figure 5. GRIP1 Competes with RIP140 on AP-1 Activity and Within the Multiprotein Complex

A, Steroid-stripped MCF7 cells were transfected with 1 µg (AP-1)4-TK-CAT and either 0.5 µg of pEFRIP plus 0, 0.5, 1, or 1.5 µg of pSG5-GRIP1 or 0.5 µg of pSG5-GRIP1 plus 0, 0.5, 1, or 1.5 µg of pEFRIP. Cells were then incubated for 28 h with 10 nM 17ß-estradiol (E2) or vehicle (C). CAT activity was evaluated as described in Fig. 1AGo. The results represent the mean (±SD) calculated from triplicate wells from one experiment representative of three separate assays. B, In vitro translated radiolabeled GRIP1 was incubated overnight with nickel-chelating resin loaded with purified histidine-tagged c-Jun in the absence or presence of increasing amounts (20 µg and 100 µg) of GST or GST-RIP140 (27–439), as described in Materials and Methods. After extensive washes, proteins were eluted and subjected to SDS-PAGE and fluorography. C, Steroid-stripped HeLa cells were transfected with 1 µg GAL4luc and vector encoding the DBD of GAL4 fused to GRIP1 (GAL4-GRIP1, 0.5 µg). They were cotransfected with vector encoding the activation domain of VP16 fused to ER{alpha} (VP16-ER{alpha}, 0.5 µg), pCI-c-Jun (2 µg), and pEFRIP (3 µg) when indicated. Cells were then incubated with vehicle (C) or 10 nM 17ß-estradiol (E2) for 28 h. Luciferase activity was evaluated as described in Materials and Methods. The results shown are the mean (±SD) of normalized luciferase activities calculated from triplicate wells from one experiment representative of three separate assays.

 
Competition Between GRIP1 and RIP140 for Protein-Protein Interactions
GRIP1 reversion of the inhibitory effect of RIP140 on AP-1 activity could suggest that the two cofactors competed for interaction site(s) within the ER{alpha}-c-Jun protein complex. Recent reports have documented competitive in vitro binding of RIP140 and p160 coactivators to NRs (6, 7). We thus investigated whether such competition could also exist for the interaction with c-Jun. Pull-down competition assays were performed with hexahistidine c-Jun fusion protein bound on nickel-chelating resin and 35S-methionine-labeled GRIP1 in the absence or presence of GST or GST-RIP140 proteins. As shown in Fig. 5BGo, increasing the amount of GST-RIP140, but not that of GST, reduced binding of GRIP1 to c-Jun. Inhibition was observed with GST fusions containing either amino acids 27–439 (Fig. 5BGo) or amino acids 683-1158 (not shown) of RIP140, which both interacted with c-Jun (Fig. 3BGo).

The competition between GRIP1 and RIP140 within ternary complexes containing ER{alpha} and c-Jun was then examined in intact cells. HeLa cells were transfected with a GAL4-responsive construct and an expression plasmid coding for GRIP1 fused to the GAL4 DBD (GAL4-GRIP1). As shown in Fig. 5CGo, cotransfection of a vector encoding either c-Jun or ER{alpha} fused to the activating domain of VP16 (VP16-ER{alpha}) enhanced reporter gene transcription due to GRIP1/c-Jun (24, 26) or GRIP1/ER{alpha} (2, 3, 24, 31) interactions, respectively. Formation of GRIP1/ER{alpha}/c-Jun complexes was reflected by the synergistic effect of VP16-ER{alpha} and c-Jun on GAL4-GRIP1-regulated transcription, in agreement with previous data (26). The addition of RIP140 decreased luciferase activity observed in the presence of GRIP1 together with either c-Jun or VP16-ER{alpha} alone, demonstrating a partial inhibition of GRIP1 binding to c-Jun and ER{alpha}. In addition, it inhibited by about 50% the synergistic effect observed in the presence of the three partners. Comparable results were obtained in the reverse experiment in which the effect of GRIP1 overexpression was measured in the presence of GAL4-RIP140 along with VP16-ER{alpha} and/or c-Jun (Fig. 6Go, B–D).



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  		Figure 6. GRIP1 Binding to Both ER{alpha} and c-Jun Are Necessary for Competition with RIP140

A, Steroid-stripped MCF7 cells were transfected with 1 µg (AP-1)4-TK-CAT and either 1.5 µg of pSG5-GRIP1 wild type, or mutants, or 0.5 µg pEFRIP plus 0, 1, or 1.5 µg of pSG5-GRIP1 wild type or mutants. Cells were then incubated for 28 h with 10 nM 17ß-estradiol (E2) or vehicle (C). CAT activity was evaluated as described in Fig. 1AGo. The results represent the mean (±SD) calculated from triplicate wells from one experiment representative of three separate assays. B, Steroid-stripped HeLa cells were transfected with 0.5 µg GAL4luc and vector encoding the DBD of GAL4 fused to RIP140 residues 848-1158 (GAL4-RIP140, 0.5 µg). They were cotransfected with vector encoding the activation domain of VP16 fused to ER{alpha} (VP16-ER{alpha}, 0.5 µg) together with pSG5-GRIP1 or a pSG5-GRIP1 mutant (3 µg) as indicated. Cells were then incubated with vehicle (C) or 10 nM 17ß-estradiol (E2) for 28 h. Luciferase activity was evaluated as described in Materials and Methods. The results shown are the mean (±SD) of luciferase activity calculated from triplicate wells from one experiment representative of three separate assays. C, Steroid-stripped HeLa cells were transfected with 0.5 µg GAL4luc and 0.5 µg GAL4-RIP140. They were cotransfected with pCI-c-Jun (1 µg) together with pSG5-GRIP1 or a pSG5-GRIP1 mutant (3 µg) as indicated. After 28 h in fresh medium, luciferase activity was evaluated as described in panel A. D, Steroid-stripped HeLa cells were transfected with 0.5 µg GAL4luc and 0.5 µg GAL4-RIP140. They were cotransfected with VP16-ER{alpha} (0.5 µg) and pCI-c-Jun (1 µg) together with pSG5-GRIP1 or a pSG5-GRIP mutant (1.5 or 3 µg) when indicated. Cells were then incubated with vehicle (C) or 10 nM 17ß-estradiol (E2) for 28 h. Luciferase activity was evaluated as described in panel A.

 
GRIP1 Interaction with ER{alpha} and c-Jun Are Both Necessary for an Efficient Competition with RIP140
We previously showed that regulation of AP-1-mediated transcription by estradiol required the ER{alpha} AF-2 domain while AF-1 domain is dispensable (26). The importance of the three LXXLL motifs, clustered in the central region of the p160 coactivator polypeptide chain, for binding to NRs in a ligand- and AF-2dependent manner has been established (26, 32, 33, 34). On the other hand, the C-terminal part of SRC-1 has been demonstrated to directly bind c-Jun in vitro (24), and it is also the case for GRIP1 since amino acids 1121–1462 are necessary and sufficient for an efficient interaction with c-Jun (our unpublished data). To determine the importance of GRIP1/ER{alpha} and GRIP1/c-Jun interactions on RIP140 inhibition of estradiol-induced AP-1 activity, we tested the effect of two GRIP1 mutants, deleted of residues 1121–1462 (mutJun) or mutated in the three NR boxes (mutNR) in the absence or the presence of RIP140 expression vector. As shown in Fig. 6AGo, no significant effect of these two mutants was detected in the absence of RIP140 overexpression, contrary to the results obtained with wild-type GRIP1. In addition, neither of them reversed the inhibitory effect of RIP140 on estradiol activation of AP-1-dependent transcription.

The ability of the GRIP1 mutants to compete with RIP140 for binding to ER{alpha} and c-Jun was then examined in one- and two-hybrid mammalian systems. Mutant mutJun was as efficient as wild-type GRIP1 to displace VP16-ER{alpha} from GAL4-RIP140 whereas no effect was observed with mutNR (Fig. 6BGo). On the contrary, mutNR but not mutJun competed with GAL4-RIP140 for c-Jun binding (Fig. 6CGo). Finally, when both VP16-ER{alpha} and c-Jun were present, the two mutants had no significant effect (mutJun) or only slightly decreased (mutNR) GAL4-driven luciferase activity, suggesting that GRIP1 interaction with both ER{alpha} and c-Jun is necessary to displace RIP140 from the ternary complexes (Fig. 6DGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
RIP140 appears to have a unique function as compared with other proteins that bind to receptors in a ligand- and AF2-dependent manner and that act as coactivators. In fact, a repressive effect of RIP140 on NR-mediated regulation of genes containing hormone-responsive element has been documented by several groups (6, 7, 8, 9). Interestingly, this repression was observed for both positive and negative glucocorticoid-triggered regulations of various promoters (7). We report here the negative effect of RIP140 on estradiol induction of AP-1-mediated transcription.

We demonstrated that, as reported for CBP/p300 (23) and p160 coactivators (24, 26), RIP140 directly bound to c-Jun in vitro and interacted with c-Jun in intact cells in coimmunoprecipitation and mammalian one-hybrid assays. As for the ER{alpha}/RIP140 interaction (30), multiple domains of RIP140 contribute to its interaction with c-Jun. Both N-terminal and C-terminal parts, which contain two or three NR boxes, respectively, among the nine motifs harbored by the entire protein, were sufficient for binding to c-Jun and, despite our efforts, we failed to isolate a RIP140 deletion mutant interacting only with ER{alpha} or c-Jun. This suggests that LXXLL signatures, which are contained in all coactivators and are crucial and sufficient for interactions between NRs and coactivators (2, 3), may be involved in the RIP140/c-Jun interaction. It is worth mentioning that cyclin D1, which acts as a physical bridge between ER{alpha} and SRC-1, also binds the cofactor through LXXLL motifs (35). However, a single LXXLL motif appeared insufficient for the interaction with c-Jun since the RIP140 917-1158 deletion mutant (Fig. 3Go) and a GST fusion protein containing only one LXXLL motif from transcriptional intermediary factor-1{alpha} (Boulahtouf, A., and V. Cavailles, unpublished data) did not interact with c-Jun, whereas they efficiently bound ER{alpha}.

The increase of in vitro RIP140 binding to ER{alpha} when complexed to c-Jun (Fig. 4BGo) and the drastic synergistic effect observed when the third partner was added in the modified two-hybrid system (Figs. 4DGo and 6DGo) strongly suggest a cooperative interaction of the three proteins. In fact, in the absence of estradiol, RIP140 overexpression had no or only minor effects on basal and c-Jun/c-Fos-induced AP-1-mediated transcription. RIP140 thus seemed to be an efficient repressor of the estradiol effect on AP-1 activity but not of c-Jun/c-Fos transcriptional activity (Fig. 1Go). This suggests that the c-Jun/RIP140 interaction alone is not sufficient for the regulation and that RIP140 needs the presence of activated ER{alpha} to exert its repressive effect.

Recent studies have shown that HDACs might contribute to the repression exerted by RIP140. RIP140 directly binds to class I HDACs (11, 36) and to the corepressor CtBP (10), which itself interacts with class I and class II HDACs (37, 38). Trichostatin A, an HDAC inhibitor, partly overcomes the inhibitory effect of RIP140 on retinoic acid receptor/retinoid X receptor-mediated transcription (11) as well as the repressive effect of CtBP in some circumstances (39). In our experiments, TSA actually increased AP-1 transactivation in the absence and presence of estradiol, in agreement with the findings of Lee et al. (29). However, it had no effect on RIP140 action (Fig. 2Go), showing that recruitment of HDACs, directly or through interaction with a corepressor such as CtBP (29, 37, 38, 39), was not required for the inhibition. We cannot, however, totally rule out the potential involvement of CtBP, which has been suggested to act via HDAC-dependent and HDAC-independent mechanisms (39).

The negative effect of RIP140 on estradiol-regulated AP-1 activity was partly reversed by p160 coactivator overexpression. This antagonistic effect of RIP140 could result from its inhibition of the binding of AF-2 interacting coactivators. The in vitro interaction between RIP140 and p160 coactivators to NRs peroxisomal proliferator-activated receptor-{gamma} (6) and glucocorticoid receptor (7) was reported to be partly competitive. RIP140 and GRIP1 also inhibited each other for binding to ER{alpha} both in GST pull-down assays (not shown) and in a two-hybrid mammalian system (Figs. 5CGo and 6BGo). In addition, as reported here, RIP140 could compete with the p160 coactivator for the physical interaction with c-Jun in vitro (Fig. 5BGo) and in intact cells (Figs. 5CGo and 6CGo). Finally, GRIP1 deletion mutants unable to bind either ER{alpha} or c-Jun failed to efficiently compete with RIP140 on estradiol-induced AP-1 activity (Fig. 6AGo) and in the ternary complex formation (Fig. 6DGo). Competition for both ER{alpha} or c-Jun binding are therefore likely important for GRIP1 displacement by RIP140, and changes from GRIP1/ER{alpha}/c-Jun complexes (responsible for estradiol induction of AP-1-mediated transcription) to RIP140/ER{alpha}/c-Jun complexes (unable to trigger the hormonal effect) could be sufficient to explain the inhibitory effect of RIP140 (Fig. 7Go). Detailed analysis of the relative binding affinities of GRIP1 and RIP140 for c-Jun and ER{alpha}, however, will be necessary to assess the importance of the two kinds of interactions in the antagonist effect.



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  		Figure 7. Model of RIP140 Inhibition of Estradiol-Induced AP-1 Transcriptional Activity

In cells expressing a low RIP140 level, ER{alpha} enhances, in the presence of estradiol (E2), AP-1-mediated activity by direct binding to c-Jun protein, and this interaction is stabilized by the coactivator GRIP1, which interacts with both c-Jun and ER{alpha} (28 ). RIP140, which also interacts with ER{alpha} and c-Jun, competes with GRIP1 within the ternary complex. The resulting RIP140/c-Jun/ER{alpha} complex, either by a passive mechanism or by corepressor recruitment, prevents the hormonal effect. To simplify the scheme, ER{alpha} is represented by a monomer whereas a monomer or, more likely, a dimer could be present in the complex.

 
In conclusion, our results indicate that RIP140 physically interacts with c-Jun and inhibits estradiol-induced AP-1-mediated transcription. RIP140, which also binds to ER{alpha}, could be considered as an integrator with remarkable properties. Unlike CBP/p300 and p160 coactivators (23, 24), binding of RIP140 to c-Jun is probably not sufficient to trigger the biological effect, but this interaction could be necessary to GRIP1 displacement and stabilization of inactive multiprotein complexes. The ratio between the two cofactors might therefore determine, as reported for hormone response element-mediated responses (6, 7, 8, 9), the efficacy of estradiol in stimulating transcription of genes under AP-1 control.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
Reporter plasmids (AP-1)4-TK-CAT (16) and GAL4luc (40) have been previously described. ER{alpha} and VP16-ER{alpha} expression vectors were donated by P. Chambon (41) and B. O’Malley (42), respectively. PCI-c-Jun, pCI-c-Fos (14), and GST-ER{alpha} truncation mutants (26) were as described. The expression plasmid for GST-c-Jun fusion protein (pGEX-3X-c-Jun) was provided by T. Kouzarides (23, 30, 41). PST19-c-Jun{Delta}224–334 and pTarget-c-Jun{Delta}1–238 (26), pEFRIP (4), GST-RIP140 27–439, and GST-RIP140 683-1158 (30) have been previously described. For antisense RIP140 construction, a 630-bp fragment from the 5'-end of the RIP140 cDNA [XbaI/HindIII fragment from pBRIP140 (4)] was inserted in the SmaI site of the pCI vector and then transferred as a XbaI/BamHI fragment in the corresponding sites of the pEFBOS plasmid to generate the ASRIP vector, thereby expressing an antisense RNA that encompasses 288 nucleotides of the 5'-untranslated region, the ATG, and 342 nucleotides of RIP140 coding sequence. Expression plasmids for GAL4-RIP140 fusion protein harboring RIP140 residues 848-1158 were donated by M. Parker. Full-length RIP140 cDNA (SpeI/KpnI fragment from pEFRIP) was subcloned into the XbaI/KpnI sites of pCDNA3.1 (Invitrogen, Gronigen, The Netherlands) to generate pCRIP140. Truncated mutants 1–282, 1–480, and 1–735 were produced by inserting a stop codon containing oligonucleotide at the XhoI, EcoRV, or AflII sites, respectively. Mutants 479-1158 and 917-1158 were constructed by inserting, at the EcoRV and Bpu1102I sites, an oligonucleotide containing the RIP140 translation initiation site. Both pSG5-GRIP1 and GAL4-GRIP1 constructs (43) contain the entire GRIP1 cDNA sequence. GRIP1 mutJun mutant, which corresponds to GRIP1 deleted of residues 1121–1462, has been described previously (44). GRIP1 mutNR mutant, mutated in the three NR boxes, was a generous gift from M. Stallcup. PGL3-Control vector (Promega Corp., Charbonnières, France) contains luciferase cDNA under the control of Simian virus 40 promoter and enhancer sequences.

Cell Culture
MCF7 and HeLa cells were maintained in DMEM/Ham’s F-12 (1:1). Media were supplemented with 10% fetal calf serum (FCS) and 50 µg/ml gentamycin. For transient transfection experiments, cells were stripped of endogenous steroids by successive passages in phenol red-free medium containing 10% (2 d), and then 3% (3 d) dextran-coated charcoal (DCC) stripped FCS (DCC-FCS), as previously described (16). They were then plated at about 80% confluence (106–2 x 106 cells per 35-mm diameter well) 24 h before transfection.

Transient Transfection, CAT, and Luciferase Assays
Twenty-four hours after plating, the medium was changed and cells were transfected for 16 h using the calcium phosphate DNA coprecipitation method, as described previously (16). When cells were transfected by an expression vector, the same amount of empty vector was transfected in control cells. One microgram of the ß-galactosidase expression plasmid pCMVß (CLONTECH Laboratories, Inc., Palo Alto, CA) was used for internal control of transfection efficiency. PSPT19 DNA was added up to 5 µg total DNA per well when necessary. Cells were washed twice with phenol red-free medium and treated, as indicated, for 24 h in phenol red-free medium containing 1% DCC for AP-1-mediated transcription assays and 3% DCC for two-hybrid experiments. CAT enzyme assays were performed in whole-cell extracts after normalization for ß-galactosidase activity (16). Acetylated and nonacetylated forms of [14C]chloramphenicol were separated by thin layer chromatography. Quantification was performed with a Fuji Photo Film Co., Ltd. BAS1000 Bioimaging Analyser (Raytest, Paris, France). For luciferase assays, cells were lysed for 15 min in the cell culture lysis reagent from Promega Corp. Luciferase activity was measured using an LKB luminometer (LKB Instruments, Rockville, MD) and normalized for ß-galactosidase activity, as previously described (45).

Coimmunoprecipitation
Cells extracts were prepared as described by Stein et al. (46) and aliquots stored at -80 C. For immunoprecipitation, 700 µg protein were incubated overnight at 4 C, with rabbit polyclonal immunoglobulins (IgG) directed against c-Jun (N; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rabbit nonimmune IgG (6 µg) in HNTG buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.1% Triton X-100; and 10% glycerol) in the presence of protease inhibitors (Complete, Roche Molecular Biochemicals, Meylan, France). Prewashed protein G beads (Amersham Pharmacia Biotech, Orsay, France) were then added and the incubation continued for 1 h at 4 C. Immunoprecipitates were recovered by centrifugation, washed four times in HNTG buffer, and resolved by SDS-PAGE. Proteins were analyzed by Western blotting using rabbit polyclonal anti-RIP140 (H-300, Santa Cruz Biotechnology, Inc., dilution 1:200) and anti-c-Jun (dilution 1:200) followed by horseradish peroxidase-conjugated goat antirabbit IgG (Sigma-Aldrich Corp., Saint Quentin Fallavier, France; dilution 1:4000). Signals were visualized by chemiluminescence (Renaissance, NEN Life Science Products Life Science, Le Blanc Mesnil, France).

Purification of Hexahistidine and GST Fusion Proteins and Pull-Down Assays
The hexahistidine c-Jun and c-Fos fusion proteins were purified, from Escherichia coli transformed with pDS56-c-Jun and pDS56-c-Fos, respectively (47), by nickel affinity chromatography in the presence of 6 M urea. They were renatured by extensive dialysis, as previously described (26). Protein renaturation was verified by gel retardation assay using a consensus collagenase12-O tetra-decanoyl-phorbol-13 acetate-responsive element (28). Production and purification of GST fusion proteins were done as previously described (26). GST-RIP140-(27–439) and GST-RIP140-(683–1158) hybrid proteins used for competition assays were eluted from glutathione-coupled beads (Amersham Pharmacia Biotech) in 20 mM glutathione; 100 mM Tris, pH 8; 120 mM NaCl in the presence of protease inhibitors (Complete, Roche Molecular Biochemicals Mannheim). After concentration on an Ultrafree centrifugal filter (Millipore Corp., Saint Quentin en Yvelines, France), the protein extract was extensively dialyzed against NETN buffer (0.5% Nonidet P-40; 1 mM EDTA; 20 mM Tris, pH 8; 100 mM NaCl). Histidine fusion protein and GST fusion proteins, preloaded on Ni-NTA nickel-chelating resin (QIAGEN, Courtabeuf, France) or glutathione-coupled beads, respectively, were incubated overnight at 4 C with 35S-labeled proteins generated by the TNT in vitro transcription-coupled translation system from Promega Corp.. After three washes with NETN containing protease inhibitors, samples were boiled in 2x sodium dodecyl sulfate sample buffer and analyzed by SDS-PAGE. Signals were amplified by fluorography (Amplify, Amersham Pharmacia Biotech) and gels were exposed at -80 C. 35S-Labeled proteins were quantified with a Fuji Photo Film Co., Ltd. BAS1000 Bioimaging Analyzer (Raytest, Paris, France).


   ACKNOWLEDGMENTS
 
We are grateful to M. Parker, M. Stallcup, T. Kouzarides, M. Karin, A. Castet, and P. Chambon for providing plasmids. We thank J. Y. Cance for figure illustration.


   FOOTNOTES
 
This work was supported by INSERM, the Association pour la Recherche sur le Cancer (Grants 1411 and 5444), the French Ministère de la Recherche et de l’Enseignement Supérieur (fellowship to C.T. and K.B.), and la Ligue Nationale Contre le Cancer (fellowship to C.T.).

1 C.T. and K.B. contributed equally to this study. Back

Abbreviations: AF, Activation function; AP-1, activator protein 1; CAT, chloramphenicol acetyltransferase; CBP, cAMP response element binding protein; CtBP, carboxy-terminal binding protein; DBD, DNA-binding domain; DCC, dextran-coated charcoal; ER, estrogen receptor; FCS, fetal calf serum; GRIP1, glucocorticoid receptor interacting protein 1; GST, glutathione-S-transferase; GST-CD, GST fusion protein containing ER{alpha} hinge domain; HDAC, histone deacetylase; mutNR, GRIP1 mutant mutated in the three NR boxes; mutJun, GRIP1 mutant deleted of residues 1121–1462; NR, nuclear receptor; RIP140, receptor-interacting protein 140; SRC-1, steroid receptor coactivator 1; TSA, trichostatin A.

Received for publication September 13, 2002. Accepted for publication November 12, 2002.


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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα
Coregulators:   RIP140  |  GRIP1
Ligands:   17β-Estradiol



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