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Molecular Endocrinology 20 (5): 1035-1047
Copyright © 2006 by The Endocrine Society

Receptor-Interacting Protein 140 Differentially Regulates Estrogen Receptor-Related Receptor Transactivation Depending on Target Genes

Audrey Castet, Adrien Herledan, Sandrine Bonnet, Stéphan Jalaguier, Jean-Marc Vanacker and Vincent Cavaillès

Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 540 (A.C., A.H., S.B., S.J., V.C.), Endocrinologie Moléculaire et Cellulaire des Cancers and Université de Montpellier I, 34090 Montpellier, France; and Equipe Mixte INSERM 0229 (J.-M.V.), Centre Régional de Lutte contre le Cancer Val d’Aurelle, 34298 Montpellier, France

Address all correspondence and requests for reprints to: Vincent Cavailles, Institut National de la Santé et de la Recherche Médicale Unité 540, Endocrinologie Moléculaire et Cellulaire des Cancers and Université de Montpellier I, 60 rue de Navacelles, 34090 Montpellier, France. E-mail: v.cavailles{at}montp.inserm.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have investigated the effects of receptor-interacting protein 140 (RIP140) on transcriptional regulation by estrogen receptor-related receptors (ERRs). We first show that RIP140 inhibits transactivation by ERR{alpha}, ß, and {gamma} on natural or artificial reporter genes containing different types of response elements. This repression correlates with a strong in vitro binding between several regions of RIP140 and the three ERR isoforms. Surprisingly, although RIP140 inhibits transactivation of the thyroid hormone receptor-{alpha} gene by ERRß, it significantly increases its regulation by ERR{alpha} and ERR{gamma}. Mutagenesis and transient transfections in SL2 cells indicate that thyroid hormone receptor-{alpha} promoter expression involved Sp1 sites. In support of this observation, we demonstrate that RIP140 also positively regulates ERRs transactivation of other known Sp1 targets such as the p21 gene. This effect requires the two proximal Sp1 binding sites of the promoter and is partially dependent on the activation function 2 domain of ERRs. Finally, we provide evidences for a role of histone deacetylases in the regulation of p21 promoter by RIP140. Altogether, these data indicate that RIP140 differentially regulates ERR activity depending on the target sequence on the promoters.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE NUCLEAR RECEPTOR (NR) superfamily comprises transcription factors that control various developmental and physiological pathways (1). They share a common modular structure containing a centrally located conserved DNA binding domain (DBD), a carboxyl-terminal ligand binding domain (LBD) with a hormone-dependent domain [activation function (AF) 2], and a variable amino-terminal region encompassing a constitutive activation function 1 (AF 1). Depending of the subtype, NR can bind to specific DNA response elements within the regulatory regions of target genes either as monomers, homodimers, or heterodimers with other members of the family such as the retinoic X receptors (for a review, see Ref. 2).

The NR family not only includes ligand-regulated receptors, but also orphan receptors, for which no natural ligand has been identified to date. Among this group, estrogen receptor (ER)-related receptors (ERRs) {alpha} and ß were the first orphan NRs to be identified on the basis of their sequence similarity to ER{alpha} (3). A third ERR (ERR{gamma}) has been isolated more recently (4). The three receptors are very similar as they display 90% sequence identity within the DBD and more than 60% within the LBD. Both ERR{alpha} and ERR{gamma} are highly expressed in muscle, heart, and adipose tissues, as well as in the central nervous system (for a review, see Ref. 5) and noticeably, ERR{alpha} is also highly expressed in bone (6). ERR{alpha} has recently been shown to be implicated in energy storage, mitochondrial biogenesis, and oxidative phosphorylation (7, 8, 9). ERRß is required for placentation as evidenced in knockout animals (10). Finally, recent publications show that ERR{alpha} may represent a biomarker of poor prognosis in breast and ovarian cancers whereas ERR{gamma} seems to be associated with a favorable clinical course in these cancers (11, 12).

In terms of structure, ERRs are very close to the ERs because sequence alignment reveals a 60% identity in the DBD regions and a moderate similarity (<35%) of the LBDs, which is consistent with the incapacity of ERRs to bind estradiol (for a review, see Ref. 5). Nevertheless, it has been demonstrated that ERRs can interfere with estrogen signaling (reviewed in Ref. 13). Indeed, these receptors recognize the same DNA binding elements, namely the steroidogenic factor 1 response element (SFRE thus also called ERRE for ERR-response element) or the classical estrogen response element (ERE) (14). ERRs are expressed in tissues in which estradiol has important physiological functions and share common target genes with ERs such as osteopontin (14), lactoferrin (15), or pS2 (16).

NR activity is controlled by a large set of proteins that modulate chromatin structure and the recruitment of the basal transcription machinery (2). Among them, several cofactors regulate the activities of both ERs and ERRs, such as the steroid receptor coactivators (17), peroxisome proliferator-activated receptor-{gamma} coactivator-1 (18, 19), or small heterodimer partner (20, 21), suggesting another level of cross-talk between ERs and ERRs. One of the first proteins to be identified as a hormone-recruited cofactor was receptor-interacting protein 140 (RIP140) (22). The human RIP140 gene, located on chromosome 21, encodes a protein of 1158 residues, which interacts with a large number of nuclear receptors (23). RIP140 is widely expressed and is essential for female fertility and energy homeostasis as evidenced in knockout mice (24, 25). Despite its recruitment by agonist-liganded receptors, RIP140 exhibits a strong transcriptional repressive activity, which involves histone deacetylase (HDACs) (26), CtBPs (27), and additional inhibitory partners yet to be characterized (28, 29).

In this study, we have analyzed the role of RIP140 in the regulation of gene transcription by ERRs. We first measured the repressive effect of RIP140 on ERR transactivation through ERE or ERRE and compared the in vitro interaction of RIP140 with the three isoforms. We then demonstrated that ERRs were also able to activate transcription from various promoters through Sp1 response elements. The Sp1-mediated transactivation of ERR{alpha} was further increased by RIP140 through a mechanism which involves histone deacetylases. Altogether, our data indicate that the regulation of ERR signaling by RIP140 is complex and depends on the promoter context.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ERE-Independent Inhibition of pS2 Gene Transcription by RIP140
The repressive activity of RIP140 on estrogen signaling has been demonstrated in transient transfection experiments using, in most cases, artificial reporter plasmids bearing consensus response elements. As shown in Fig. 1AGo, a strong inhibition of ER{alpha} activity was also observed on the natural pS2 promoter (pS2-Luc construct) upon RIP140 overexpression. The transcriptional regulation of pS2 gene by estrogens is dependent on both an ERE and an ERRE site (16). In our hands, mutation of the ERE sequence produced a strong decrease of both the basal and 17ß-estradiol (E2)-induced levels of the pS2-Luc reporter (Fig. 1BGo). However, RIP140 still exerted a significant repressive effect on the mutated pS2-Luc construct, which could reflect its ability to inhibit ERRE-mediated transactivation of either ERs or ERRs.


Figure 1
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  		Fig. 1. Effect of RIP140 on Transcriptional Regulation of the pS2 Promoter

HeLa human cancer cells were transiently transfected, with the pS2-Luc reporter construct (1 µg), either wild-type (pS2-Luc) or mutated on the ERE sequence (pS2-Luc{Delta}ERE), together with expression vectors for ER{alpha} (0.5 µg) and RIP140 (dark bars) or the corresponding empty vector alone (white bars) (2.5 µg). Cells were then treated by 17ß-estradiol 10–8 M (E) or vehicle alone (C) for 24 h, and luciferase activity was quantified as indicated in Materials and Methods. Results are expressed as relative luciferase activity and are the mean (±SD) of three independent experiments.

 
RIP140 Decreases ERE and ERRE-Mediated Transactivation of ERRs
As shown in Fig. 2AGo, upon transient transfection of ERR{alpha}, ß, and {gamma} expression vectors, we observed a significant increase of pS2 transcription. This regulation required a functional AF2 domain within the ERRs because both ERR{alpha} and ERRß mutants deleted of the C-terminal region, which contains the conserved AF2 activation domain, were no longer able to transactivate (data not shown). As previously reported for several members of the nuclear receptor superfamily, ERR transactivation of the pS2 promoter was strongly repressed by RIP140.


Figure 2
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  		Fig. 2. Transcriptional Repression of ERR Activity by RIP140

HeLa cells were transiently transfected with the following reporter constructs: pS2-Luc (1 µg) in panel A, ERE-ßGlob-Luc or 17M5bGlob-Luc (0.5 µg) in panel B, and ERRE-SV40-Luc or SV40-Luc also called pGL2-promoter (0.5 µg) in panel C. Transfections were performed in six-well dishes for panel A and in 12-well dishes for panels B and C. The expression vectors encoding wild-type ERR{alpha}, ERRß, or ERR{gamma} (0.5 µg in panels A and B; 0.25 µg in panel C) were cotransfected in the presence of RIP140 expression plasmid or empty vector (2 µg in panel A and 1 µg in panels B and C). After 48 h, luciferase activity was quantified as indicated in Materials and Methods. Results are expressed as relative luciferase activity (% of control) and are the mean (±SD) of values from two independent experiments.

 
To confirm that the negative effect of RIP140 on pS2 transcription could be due to the inhibition of ERR activity acting through both an ERE and ERRE, we analyzed whether RIP140 was able to negatively regulate ERR{alpha} transcriptional activity on the corresponding isolated response elements. As shown in Fig. 2Go, B and C, the luciferase activity from ERE- or ERRE-containing reporter plasmids was increased by all three ERRs transiently expressed into HeLa cells whereas no significant effect was detected on control reporter construct. As observed in the case of the pS2 promoter, RIP140 efficiently decreased ERR transactivation on both ERE- or ERRE-containing reporter plasmids. Similar effects were obtained in MCF-7 human breast cancer cells (data not shown), thus confirming that RIP140 negatively controls ERR transactivation on different response elements corresponding either to a palindromic sequence or to an extended half-site.

In Vitro Interaction of RIP140 with ERRs
To analyze the interaction of RIP140 with ERRs, we performed glutathione-S-transferase (GST) pull-down assays. As shown in Fig. 3AGo, we observed a significant binding of radiolabeled ERR{alpha}, ß, and {gamma} with chimeric GST-RIP140 proteins encompassing the amino-terminal region (residues 27–429), the central region (residues 429–582), or the carboxyl-terminal moiety of the molecule (residues 683-1158). Results shown in Fig. 3BGo also indicate that, when bacterially expressed as a GST-fused protein, the LBD of ERR{alpha} or ERR{gamma} efficiently interacted with the full-length RIP140 molecule or with either the amino-terminal (residues 1–480) or the carboxyl-terminal (residues 479-1158) domains. Interestingly, in all cases, however, the interaction of ERR{alpha} appeared lower than that of ERRß and ERR{gamma} (Fig. 3AGo). Moreover, as shown in Fig. 3BGo, binding of RIP140 to GST-ERR{alpha} was weaker than that obtained with GST-ERR{gamma} (i.e. comparable, respectively, to the interaction of RIP140 with unliganded or liganded GST-ER{alpha}). Altogether, these experiments demonstrated that several regions of RIP140 specifically interacted with the three ERRs.


Figure 3
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  		Fig. 3. In Vitro Interaction of RIP140 with ERRs

A, GST pull-down assays were performed using bacterially expressed GST or GST-RIP proteins and 35S-labeled wild-type ERR{alpha}, ERRß, or ERR{gamma}. B, GST-ERR{alpha}, GST-ERR {gamma}, and GST-ER{alpha} proteins (containing only the LBDs of the corresponding receptors) were used to pull down radiolabeled RIP140 either full length or deleted of its N- or C-terminal moiety. The interaction between GST-ER{alpha} and RIP140 was measured in the presence of 10–6 M E2 or ethanol (panel C). Inputs represent 10% of the material used in the assays.

 
Regulation of the Thyroid Hormone Receptor-{alpha} (TR{alpha}) Promoter by ERRs and RIP140
We then investigated whether RIP140 could also modulate the ERR-dependent activation of other endogenous ERR{alpha}-target genes. As shown in Fig. 4Go, A and B, transient transfection experiments in HeLa cells demonstrated that transcription from the TR{alpha} promoter (30) was significantly increased by ERR{alpha} (2.7- to 4.3-fold increase) and ERRß (6.2- to 7.1-fold increase) whereas under the same conditions ERR{gamma} increased TR{alpha} promoter expression less efficiently (1.3- to 3-fold increase).


Figure 4
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  		Fig. 4. Regulation of the TR{alpha} Promoter by ERR, RIP140, and Sp1

A, Transient transfections were performed in HeLa cells using the TR{alpha}-Luc reporter plasmid (0.5 µg), together with ERRs or empty expression vectors (1 µg), in the presence of RIP140 or empty expression vectors (1.8 µg). Luciferase activity was quantified as indicated in Materials and Methods, and results are expressed as relative activity (% of control) and are the mean (±SD) of three values. B, ERR{alpha}-, ERRß-, and ERR{gamma}-expressing vectors (0.5 µg) were tested in HeLa cells on the TR{alpha}-Luc reporter construct bearing either a wild-type (WT) or an ERRE-mutated sequence (0.25 µg). Relative luciferase activities are the mean (±SD) of three values. C, SL2 cells were transiently transfected as indicated in Materials and Methods, with the TR{alpha}-Luc reporter plasmid (0.25 µg) together with increasing amounts of Sp1 expression vector. Luciferase activity represents the mean (±SD) of three values.

 
Although RIP140 slightly repressed the activity of ERRß, we observed an unexpected significant 2.5-fold increase of ERR{alpha}- and ERR{gamma}-driven transactivation. This suggests that the regulation of the TR{alpha} promoter could involve complex mechanisms different from those observed on ERE or ERRE-containing reporters. We therefore compared the activity of the different ERRs on the wild-type and ERRE-mutated promoter constructs (Fig. 4BGo). Under our conditions (i.e. using a 2-fold ratio between ERR plasmids vs. reporters), mutation of the ERRE did not significantly affect the response to the three ERRs, suggesting that the ERR-driven regulation might involve additional sites.

Candidate response elements to mediate such an activity could be Sp1-binding sites. Indeed, different papers have reported that Sp1 factors are able to support transactivation by various nuclear receptors, in the case of ER{alpha} in particular (31). In addition, several Sp1 sites are present within the TR{alpha} promoter sequence used in this study (32). To verify the functionality of these sites, we investigated whether the TR{alpha} promoter was Sp1 responsive. As shown in Fig. 4CGo, transient transfection of the TR{alpha} reporter construct in Drosophila SL2 cells (which are devoid of Sp1 transcription factor) demonstrated that Sp1 indeed transactivates the TR{alpha} promoter. Altogether these data indicated that TR{alpha} is an Sp1-target gene the ERR{alpha}- and ERR{gamma}-dependent regulation of which is positively modulated by RIP140.

ERRs and RIP140 Activate Gene Expression through Sp1 Sites
To confirm and extend the effect of ERRs and RIP140 on Sp1-mediated transactivation, we used the natural Sp1-responsive promoter region of the p21 gene. Upon transient cotransfection, the p21 promoter construct (pWP101) was activated by ERR{alpha} and ERRß, but weakly by ERR{gamma} (Fig. 5AGo). Overexpression of RIP140 further increased ERR{alpha} and ERR{gamma} transactivation capacities. Interestingly, transactivation driven by ERRß was rather decreased by RIP140 supplementation. This pattern of regulation of p21 promoter by ERRs and RIP140 is strikingly similar to that observed for TR{alpha} promoter (Fig. 4AGo) and was also obtained using the SRY promoter, which is also targeted by the Sp1 transcription factor (33) (data not shown). Furthermore, increasing the amount of transfected ERR{alpha} in the presence of a fixed amount of RIP140 resulted in an enhancement of promoter activity, suggesting that the positive effect of RIP140 was dependent on ERR expression (Fig. 5BGo). Similarly, as shown in Fig. 5CGo, we also observed that the positive effect of RIP140 was dependent on the concentration of expression vector transiently transfected. It has been previously reported on the osteopontin promoter (34) that the relative induction of luciferase activity by ERRs was highly dependent on the ratio between the quantities of receptor expression vector vs. those of reporter plasmid used in the transfection. This held true for the regulation of the pWP101 reporter and explained why, in Fig. 5BGo, we failed to detect any regulation by ERR{alpha} in the absence of RIP140 overexpression. This also accounted for the variations observed in the relative regulation of pWP101 by ERR{alpha} alone or in the presence of RIP140 (see Fig. 5Go, A–C).


Figure 5
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  		Fig. 5. Positive Regulation of the p21 Promoter by ERRs and RIP140

A, HeLa cells were transiently transfected in 12-well dishes with the pWP101 expression plasmid (0.25 µg) together with the plasmids encoding the various ERRs (0.5 µg) in the presence or not of RIP140 (black bars) (0.9 µg). Luciferase activities were quantified as indicated in Materials and Methods. B, Transient transfections were performed in HeLa cells (12-well dishes) using the pWP101 reporter plasmid (0.5 µg) together with increasing concentrations of ERR{alpha} (from 10–250 ng) or empty expression vectors, in the presence or not of RIP140 (0.9 µg). Luciferase activity was quantified as indicated in Materials and Methods, and results are expressed as relative activity (% of control without ERR{alpha}) and are the mean (±SD) of three values. C, HeLa cells were transiently transfected with the pWP101 reporter vector (0.5 µg) together with increasing amounts of RIP140 expression plasmid, in the presence or not of ERR{alpha} (0.5 µg). Results are expressed as relative activity (% of control without RIP140) and are the mean of three values. D, MELN cells (50 ) were stably transfected or not with the expression vector of ERRß. p21 mRNA levels were then quantified by quantitative RT-PCR as indicated in Materials and Methods, and normalized to the RS9 levels. The data shown are the mean (±SD) of three values and are representative of several independent experiments.

 
Results presented above suggested that the p21 gene could be an ERR target gene. To verify this hypothesis, we generated MCF-7 breast cancer cells stably transfected with the ERRß expression vector, which was, in our hands, the most potent regulator of TR{alpha} and p21 promoter activities (see Figs. 4AGo and 5AGo). Interestingly, these stable transfectants exhibited a 2.5-fold increase in p21 mRNA accumulation, thus confirming that the regulation by ERRs could also take place on the endogenous p21 gene (Fig. 5DGo).

We then characterized the regulation of the p21 promoter by ERR{alpha} using constructs with point mutations in Sp1-responsive sites (schematized on Fig. 6AGo). As shown in Fig. 6BGo, mutation of the Sp1(3) site only slightly affected ERR-driven transactivation, whereas mutations in the two proximal Sp1(5/6) sites, which are important for the basal activity of the p21 promoter (35), resulted in a dramatic decrease in ERR{alpha} response. Interestingly, the mutation of the Sp1(5/6) sites also abolished the activation by RIP140 whereas the response was maintained on the Sp1(3) mutant (Fig. 6CGo). The involvement of Sp1 factor in this regulation is supported by its ability to interact in vitro with the three ERRs, although the interaction appeared slightly less intense than with ER{alpha} (Fig. 6DGo). Consistently, we also found that a minimal reporter construct containing three Sp1 binding sites in front of a minimal promoter (Sp1-Luc construct) positively responded to ERR{alpha} and RIP140 (Fig. 6EGo) in a manner similar to that observed for TR{alpha} and p21 promoters.


Figure 6
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  		Fig. 6. Activation by ERRs and RIP140 through Sp1-Binding Sites

A, Schematic representation of the different reporter constructs used. The p21 reporter promoter with the Sp1 binding sites that have been mutated is shown together with the Sp1-Luc artificial reporter. B, Transient transfection of HeLa cells (12-well dishes) with the pWP101 reporter constructs (0.4 µg), either wild-type or mutated for different Sp1 binding sites, together with the plasmids encoding ERR{alpha} or empty vector alone (1.6 µg). Results are expressed as relative activity (% of control without ERR{alpha}) and are the mean of three values. C, HeLa cells in 12-well dishes were transiently transfected with the pWP101 indicated reporter plasmids (0.4 µg) together with ERR{alpha} (0.8 µg) in the presence or not of RIP140 (0.9 µg). Luciferase activities (mean ± SD of three values) are expressed as % of control without RIP140. D, GST pull-down assays were performed using bacterially expressed GST or GST-Sp1 proteins and 35S-labeled wild-type ERR{alpha}, ERRß, ERR{gamma}, or ER{alpha}, according to conditions described elsewhere (46 ). E, Transient transfection in HeLa cells using the artificial Sp1-Luc reporter construct, together with the ERR{alpha} expression plasmids in the presence or not of RIP140. Data are expressed as in panel B.

 
Role of the AF2 Function of ERRs
To define whether the AF2 domain of ERR{alpha} was required for its transactivation through Sp1 response elements and for the positive effect of RIP140 overexpression, we used two complementary approaches. We first analyzed the activity of an ERR{alpha} mutant deleted of the AF2 domain. This mutation resulted in a loss of activity on both the pWP101 (Fig. 7AGo) and the pS2-Luc (Fig. 7BGo), although the loss of transactivation on pWP101 was incomplete (Fig. 7AGo and data not shown obtained with a higher ratio of ERR{alpha} expression vector vs. reporter).


Figure 7
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  		Fig. 7. Role of the AF2 function of ERRs in the Response to RIP140

HeLa cells were transiently transfected as indicated in Materials and Methods with either the pS2-Luc (B and D) or the pWP101 (A and C) reporter plasmids, together with the expression vector for ERR{alpha} (wild-type or AF2-deleted) in the presence or not of RIP140 (black bars). In panels A and C, cells were transfected in 12-well dishes using 0.6 µg of reporter plasmid together with expression vectors for ERR{alpha} (0.3 µg) and RIP140 (0.9 µg). In panel B, cells were transfected in six-well dishes using 1 µg of the pS2-Luc plasmid together with expression vectors for ERR{alpha} (0.5 µg) and RIP140 (2 µg). In panel D, cells were transfected in 12-well dishes using the pS2-Luc plasmid (0.4 µg) and the vectors encoding ERR{alpha} (0.8 µg) or RIP140 (0.85 µg). Luciferase activity was measured (when indicated after an 18-h stimulation by XCT790 at 2 µM or vehicle alone), and results are the mean (±SD) of three values.

 
The second approach was to use the recently described XCT790 inverse agonist, which selectively inhibits the constitutive activity of ERR{alpha} (36). When transiently transfected HeLa cells were incubated for 18 h with 2 µM XCT790, we observed a loss of ERR{alpha} activity on both the pWP101 and the pS2-Luc reporter (Fig. 7Go, C and D, respectively). As for ERR{alpha}mutAF2, the inhibitory effect of XCT790 appeared more limited on the p21 promoter than on the pS2 one (Fig. 7CGo and data not shown).

Interestingly, both the positive and negative regulations of ERR{alpha} by RIP140 (on the pWP101 and pS2-Luc reporters, respectively) were still detected in the absence of a functional AF2 (use of the AF2-deleted mutant or in the presence of XCT790). This might be due to the activity of endogenous ERRß or ERR{gamma} (that are not sensitive to XCT790) or suggest that other domains of ERR{alpha} might be implicated in the recruitment of RIP140 on target promoters, as supported by its strong in vitro interaction with ERR{alpha}mutAF2 (data not shown). Alternatively, this could also highlight the fact that the positive effect of RIP140 on Sp1-mediated responses did not involve a direct interaction between RIP140 and the receptor.

Histone Deacetylases Are Involved in the Positive Regulation by RIP140
It has been known for a long time that HDAC1, tethered to DNA by Sp1, is an important regulator of p21 gene expression (37) and is responsible for the huge increase of p21 expression in response to HDAC inhibitors (35). Because we (29) and others (26) previously showed that RIP140 strongly interacts with class I and II HDACs, we therefore tested whether these enzymes may play a role in the positive regulation by RIP140.

We first investigated the role of HDAC1 in the regulation of Sp1-mediated ERR transactivation by RIP140. As shown in Fig. 8AGo, overexpression of HDAC1 resulted in the complete loss of RIP140 effect on ERR{alpha} transactivation of the pWP101 construct, thus indicating that HDACs are directly or indirectly involved in the positive regulation by RIP140. A mechanism that could explain our observations relies on the fact that overexpressed RIP140 could titrate away HDAC1 from Sp1 and therefore lead to a derepression of Sp1 activity.


Figure 8
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  		Fig. 8. Effect of HDACs on the Regulation by RIP140

A, The pWP101 reporter plasmid (0.5 µg) was transfected into HeLa cells (12-well dishes) together with the ERR{alpha} (0.25 µg) and RIP140 (0.375 µg) encoding plasmids in the presence or absence of the expression vector for HDAC1 (0.375 µg). Luciferase activity was then obtained as indicated in Materials and Methods. B, HeLa cells were transiently transfected as indicated in Materials and Methods with the pWP101 reporter plasmid (0.5 µg), together with the expression vector for ERR{alpha} (0.25 µg) in the presence of not of RIP140 (black bars) (0.9 µg). Luciferase activity was measured after a 24-h stimulation by TSA (500 ng/ml) or vehicle alone, and results are the mean (±SD) of three values.

 
To confirm the involvement of HDACs, we analyzed the effect of trichostatin A (TSA), a potent inhibitor of HDAC activity, on the regulation of ERR{alpha} transcriptional activity by RIP140 on the p21 reporter gene (Fig. 8BGo). As expected, we observed that TSA strongly increased basal transcriptional activity (35). Interestingly, the significant induction of ERR{alpha} transactivation by RIP140 was no longer observed in the presence of TSA. This is in accordance with our hypothesis because, when HDAC-mediated repression of Sp1 is relieved by TSA, sequestration of HDACs by RIP140 could no longer produce an increase of Sp1 activity. Altogether, these results therefore suggest that HDAC titration by RIP140 could explain its positive effect on ERR-mediated transactivation through Sp1 sites.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
RIP140 is an atypical regulator of nuclear receptor activity that could be considered as an anticoactivator because it interacts with transcriptionally active receptors but negatively controls their transactivation. In this study, we have characterized the role of RIP140 in transcriptional signaling by the ERR orphan receptors.

Our results first indicate that RIP140 inhibits ERR{alpha}, ß, and {gamma} activity on ERE- or ERRE-containing reporter constructs. This was demonstrated on artificial reporter plasmids and on natural promoters harboring these binding sites such as pS2, (Figs. 1Go and 2Go), ERR{alpha}, or osteopontin (data not shown). Our data thus confirm and extend to ERR{alpha} and ERRß a recent study that reported the negative regulation of ERR{gamma} by RIP140 (38). However, the mechanism of transcriptional repression of ERR activity has still to be demonstrated. Our recent data indicate that four inhibitory domains are present within the RIP140 molecule (29). The two carboxyl-terminal domains appeared very important for the global repression exerted by RIP140 (29) and could therefore be involved in the negative control of ERE- and ERRE-dependent activities of ERRs.

As expected from results obtained with other nuclear receptors, we show that different regions of RIP140 interact in vitro with the three ERRs. The RIP140 protein exhibits nine LxxLL motifs that are involved in binding to nuclear receptors. Our unpublished observations indicate that these motifs exhibit more than 10-fold variation in their affinity for ER{alpha}, and further work will be needed to determine the involvement of the various LxxLL motifs in the interaction with the three ERRs. Interestingly, data presented in Fig. 3Go suggest that the affinity of RIP140 for ERR{alpha} could be lower than for the other ERRs. Previous data suggested that ERR{alpha} is a phosphorylated protein (39), and it has been proposed that this posttranslational modification could be necessary for its full activation (11). To verify this hypothesis, we have tested the ability of RIP140 to interact in vitro with ERR{alpha} and ERRß expressed in mammalian cells (data not shown). We found that the interaction of the two receptors was much more comparable in this condition than when expressed in vitro or in bacteria, thus reinforcing the idea that the interaction of ERR{alpha} with RIP140 could be regulated by factors present in mammalian cells.

The second point of the present study deals with the demonstration that ERRs positively regulate transcription through Sp1 binding sites. This was shown both on transiently transfected promoters (from the TR{alpha}, SRY, and p21 genes) and also on endogenous p21 mRNA accumulation. The involvement of Sp1 binding sites was demonstrated by site-directed mutagenesis and by the use of a reporter containing isolated Sp1 response elements. One hypothesis to explain this regulation is that ERRs increase Sp1 expression as recently observed in COS-7 cells (40). However, we failed to detect a significant increase of Sp1 protein accumulation by Western blot analysis (data not shown). In contrast, our data support a direct interaction of the three ERRs with Sp1 (Fig. 6CGo). Such a regulation of Sp1 activity through direct recruitment by the Sp1 transcription factor has been proposed for other nuclear receptors (for a review, see Ref. 31). In the case of ER{alpha}, the recruitment on the p21 promoter has been recently illustrated by chromatin immunoprecipitation (41). Moreover, it appears that the AF1 domain of ER{alpha} was required for activation at an Sp1 element whereas the AF2 domain was dispensable (42, 43). Concerning ERRs, we found that activation at Sp1 sites did not require the A/B domain (data not shown). This is consistent with the observation that the A/B domain of ERR{gamma} was dispensable for 4-hydroxytamoxifen-dependent transactivation at activator protein 1 sites (44). By contrast, we found that, in the case of ERR{alpha}, Sp1-dependent transactivation required the AF2 domain because it was significantly reduced by AF2 deletion or by the use of the XCT790 inverse agonist (Fig. 7Go).

Finally, the main observation of the present work is that overexpression of the transcriptional repressor RIP140 strongly increased ERR{alpha}- and ERR{gamma}-mediated transactivation via Sp1-response sites (both on isolated sites and on natural promoters). Interestingly, this effect appears quite specific because, under the same conditions, RIP140 decreased, rather than increased, ERRß transactivation. This could be related to differences in ERR isoform sequences and structures that could modify their ability to interact with other factors, in particular with Sp1 or RIP140. Using overexpression of HDAC1 or treatment with a specific inhibitor of the enzymatic activity, we demonstrate that the positive regulation exerted by RIP140 involves HDAC either directly or indirectly. It has been known for a long time that HDACs repress Sp1 activity based, for instance, on the effect of trichostatin A (for a review, see Ref. 45). Interestingly, strong activation of the thymidine kinase promoter by E2F1 (37) or of the p21 promoter by p53 (46) involved the dissociation of HDAC1 from the C terminus of Sp1 as a consequence of the formation of Sp1-E2F1 or Sp1-p53 complexes, respectively. At present, we do not favor such a mechanism (i.e. involving the formation of a Sp1-RIP140 complex), and instead we hypothesize that RIP140, which strongly recruits HDACs, could derepress the p21 promoter by titrating away these enzymes from Sp1. Importantly, our unpublished observations indicate that RIP140 also increases Sp1-dependent transactivation by ER{alpha}. A very recent study has reported that the pure antiestrogen ICI182780 induces p21 gene transcription by releasing HDAC1 and ER{alpha} from Sp1 sites (41). This points out that two different negative regulators of ERE-mediated transactivation, a ligand (ICI182780) and a repressor (RIP140), both increase Sp1-mediated transcription of the p21 gene.

From a physiological point of view, RIP140 therefore appears to be an important inhibitor of the mitogenic effects of liganded ER{alpha} through repression of its transactivation on ERE (22) or AP1 (47) sites. Concerning the E2 regulation of transcription through ER{alpha}/Sp1 interaction (48), target genes are involved in either positive (E2F1, c-fos) or negative (retinoic acid receptor-{alpha}, p21) control of cell proliferation. Depending on the cell and promoter context, on the relative expression of the different actors (receptors and cofactors), or on various physiological and pathological conditions, it is tempting to speculate that the positive regulation of Sp1-mediated responses by RIP140 might only occur on genes that negatively control cell proliferation. This would obviously reinforce the role of RIP140 as a key factor in breast cancer cell proliferation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
pS2-Luc and pS2{Delta}ERE-Luc (16) were kindly given by V. Giguère (McGill University, Montreal, Canada). TR{alpha}-Luc, TR{alpha}mut, ERRE-Luc, and ERRE-SV40-Luc were described elsewhere together with the expression vectors for ERRs (wild type or mutated) (30). Plasmids containing p21WAF1/CIP1 promoter driving the luciferase reporter gene (pWP101wild-type and mutated constructs) (35) were kindly provided by Y. Sowa (Kyoto, Japan). ERE-ßGlob-Luc and 17M5ßGlob-Luc were described elsewhere (49). pGL2-promoter vector was purchased from Promega Corp. (Madison, WI). pSG5-ER{alpha} vector was a gift of P. Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France). pEFRIP140 vector and plasmids encoding GST-RIP and full-length pCRIP140, N deleted or C deleted, were previously described (29). GST-Sp1 vector and Sp1- and HDAC1- expressing plasmids together with Sp1-Luc reporter (37) were obtained from C. Seiser (Vienna BioCenter, Vienna, Austria). PpacSp1 was obtained from R. Tjian (Howard Hughes Medical Institute-University of California, Berkeley, CA). The pSG5-puro was a gift from H. Gronemeyer (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France) (50).

Cell Culture
HeLa and MCF-7 human cancer cells were derived from stocks routinely maintained in the laboratory and grown in DMEM or in Ham’s F-12/ DMEM (1:1) (respectively) supplemented with 10% fetal calf serum (Invitrogen, Cergy-Pontoise, France) and antibiotics. Drosophila SL2 cells were grown at 25 C without CO2 in SF900II medium (Life Technologies, Gaithersburg, MD) supplemented with penicillin and streptomycin sulfate. Stable transfection in MCF-7 cells was carried out using the calcium phosphate technique on 7 x 106 cells plated in a 90-mm Petri dish. pSG-ERRß plasmid (20 µg) was cotransfected with pSG-puro vector (2 µg), and selection of stably transfected cells was initiated 2 d later using puromycin (0.125 µg/ml).

Transient Transfection
For transient transfection experiments, HeLa cells were plated in six-well dishes at about 80% confluence (106 cells per 35-mm diameter well). Plasmids [luciferase reporters, expression vectors for ERR, RIP140, HDAC1, and CMV-ßGal (as an internal control)] were transfected using the calcium phosphate method. Drosophila SL2 cells (5 x 106) were plated in 24-well plates, and transfection was carried out using Lipofectamin 2000 (Invitrogen, Cergy-Pontoise, France). Each well was transfected with 0.25 µg of reporter plasmid, 0.625µg of CMV-ßGal, and with pPac-Sp1 (0–125 ng) supplemented, when necessary, with empty vector to equalize the total DNA transfected. Cells were harvested 48 h after transfection and assayed for luciferase activity as described below. Cell extract preparation was carried out as recommended by Promega Corp. Cells were lysed at 4 C for 10 min in 0.4 ml of lysis buffer (25 mM Tris, pH 7.8; 2 mM EDTA; 10% glycerol; 1% Triton X-100). Luciferase activity was measured on 100-µl supernatant aliquots during 1 sec after injection of 100 µl of luciferase detection solution using a luminometer (Labsystem, Les Ulis, France). Transfection data were normalized by the ß-galactosidase activities determined as described elsewhere (51) and expressed as relative luciferase activities. Trichostatin A and puromycin were from Sigma (Saint-Quentin, France). XCT790 was from Exelixis (San Diego, CA) (36).

GST Pull-Down Assay
In vitro translation and GST pull-down assays were performed as previously described (52). Briefly, 35S-labeled proteins were cell-free synthesized using the TNT lysate system (Promega) and incubated with purified GST fusion proteins overnight at 4 C in NETN buffer containing 0.5% Nonidet P-40, 1 mM EDTA, 20 mM Tris (pH 8), 100 mM NaCl, 10 mM DTT, and protease inhibitors (Roche Diagnostics, Meylan, France). Protein interactions were analyzed by SDS-PAGE followed by quantification using a phosphor imager (Fujix BAS1000). Gels were stained with Coomassie brilliant blue to visualize the GST fusion proteins present in each track.

Quantitative RT-PCR
Classically, 2 µg of total RNA were reverse transcribed as previously described (53). Real-time PCR quantification was performed using a SYBR Green approach (Light Cycler; Roche Diagnostics). PCR was carried out in a final volume of 10 µl using 0.5µl of each primer (10 µM), 2 µl of the supplied enzyme mix, 4.5 µl of H2O, and finally 2.5µl of the template diluted at 1:20. After a 10-min preincubation at 95 C, runs corresponded to 45 cycles of 15 sec at 95 C (denaturation), 7 sec at 64 C (annealing), and 5 sec at 72 C (elongation). Primers for p21 were previously described (53). PCR products were subjected to melting curves analysis using the light cycler system to exclude the amplification of nonspecific products. The p21 mRNA levels were corrected for RS9 mRNA levels (reference gene) and normalized to a calibrator sample.


   ACKNOWLEDGMENTS
 
We thank Drs. Sowa, Giguère, Seiser, Tjian, and Boulanger for the gift of plasmids and Dr. Willy (Exelixis, Inc.) for supplying the ERR{alpha} inverse agonist. We are also grateful to F. Rabenoelina for quantitative RT-PCR and to Dr. Balaguer and to E. Sarrot, for MELN and SL2 Drosophila cells, respectively. This work is dedicated to the memory of Dr. Françoise Vignon.


   FOOTNOTES
 
This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the University of Montpellier I, the Association pour la Recherche sur le Cancer (Grant 3494), the Ligue Régionale contre le Cancer (Grant RAB05002FFA, and comité départemental Drôme), and the Fondation Lejeune. A.C. is the recipient of an INSERM fellowship.

All authors have nothing to declare.

First Published Online January 26, 2006

Abbreviations: AF, Activating function; DBD, DNA binding domain; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; ERR, estrogen receptor-related receptor; ERRE, ERR-response element; GST, glutathione-S-transferase; HDAC, histone deacetylase; LBD, ligand binding domain; NR, nuclear receptor; RIP140, receptor interacting protein; TR{alpha}, thyroid hormone receptor-{alpha}; TSA, trichostatin A.

Received for publication June 9, 2005. Accepted for publication January 17, 2006.


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NURSA Molecule Pages Link:

Nuclear Receptors:   TRα  |  ERα  |  ERRα  |  ERRβ  |  ERRγ
Coregulators:   RIP140  |  HDAC1
Ligands:   17β-Estradiol



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