RIP140 IS A REPRESSOR OF THE ANDROGEN RECEPTOR ACTIVITY
Abbreviated title: AR repression by RIP140
1Sophie CARASCOSSA, Jérôme GOBINET, Virginie GEORGET, Annick LUCAS, Eric
2BADIA, Audrey CASTET, Roger WHITE, Jean-Claude NICOLAS, Vincent CAVAILLÈS
and Stéphan JALAGUIER*
INSERM, U540, Montpellier, F-34090, France
1 present address : Institut Biologie Intégrative, 7, quai Saint-Bernard 75252 PARIS cedex 05, France
2 Institute of Reproductive and Developmental Biology, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom
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*To whom correspondence and reprint requests should be addressed
INSERM, U540, 60 rue de Navacelles F-34090, Montpellier, France
tel (33)467043706, fax (33)467540598
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S. C. received grants from “Association pour la Recherche sur les Tumeurs de la Prostate” and ”Ligue Nationale contre le Cancer”, J. G. and V. G. from “Association pour la Recherche contre le Cancer” and A. C. was a recipient of “Poste d’accueil INSERM”. This work was
funded by the Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine de Montpellier, “Association pour la Recherche contre le Cancer” (grant N? 3494), “Ligue
Nationale contre le Cancer” (grant N? RAB05002FFA) and “Fondation Jérôme Lejeune”.
Keywords : AR, RIP140, transcription repression, HDAC, LNCaP.
The androgen receptor (AR) is a ligand-activated transcription factor which controls growth and survival of prostate cancer cells. In the present study, we investigated the regulation of AR activity by the receptor interacting protein RIP140. We first showed that RIP140 could be co-immunoprecipitated with the receptor when co-expressed in 293T cells. This interaction appeared physiologically relevant since ChIP assays revealed that under R1881 treatment, RIP140 could be recruited to the PSA encoding gene in LNCaP cells. In vitro GST pull-down
assays evidenced that the carboxy-terminal domain of AR could interact with different regions of RIP140. By means of fluorescent proteins we demonstrated that ligand-activated AR was not only able to translocate to the nucleus but also to relocate RIP140 from very structured nuclear foci to a diffuse pattern. Overexpression of RIP140 strongly repressed AR-dependent transactivation by preferentially targeting the ligand binding domain-dependent activity. Moreover, disruption of RIP140 expression induced AR overactivation thus revealing RIP140 as a strong AR repressor. We analysed its mechanism of transrepression and first demonstrated that different regions of RIP140 could mediate AR-dependent repression. We then showed that the carboxy-terminal end of RIP140 could reverse transcriptional intermediary factor TIF2-dependent overactivation of AR. The use of mutants of RIP140 allowed us to suggest that CtBP played no role in RIP140-dependent inhibition of AR activity whereas HDACs partly regulated that transrepression. Finally, we provided evidence for a stimulation of RIP140 mRNA expression in LNCaP cells under androgen treatment, further emphasizing the role of RIP140 in androgen signalling.
Defects in androgen signalling result in a large panel of clinical phenotypes ranging from perturbation in male sexual development to prostate cancer etiology (1, 2). Because androgen stimulation regulates prostate epithelial cell growth, treatment for advanced prostate cancer, the major malignancy in men in Western countries, can be achieved by eradication of androgen action through androgen withdrawal using chemical or surgical castration (3). However the disease invariably progresses to an androgen-independent state. Therefore, elucidation of mechanisms that regulate androgen actions is of critical importance.
The effects of androgens are mediated by the androgen receptor (AR), a transcription factor member of the nuclear receptor superfamily. Unliganded AR is a cytoplasmic protein associated in an inactive state with heat shock proteins (4). Under hormone binding, the receptor undergoes conformational changes which induce its translocation from the cytoplasm to the nucleus. In order to regulate transcription of target genes, AR binds to specific DNA sequences called androgen response elements (ARE) (1). The receptor harbors three main functional domains: the amino-terminal domain where the primary ligand-independent transactivation domain, AF1 (amino acids 142-337) supports the major transactivation function of the receptor (5), the central DNA-binding domain and the carboxy-terminal domain also called ligand-binding domain (LBD) (1). The AR LBD is highly conserved among the steroid receptor family of proteins and contains the weak transcriptional activation domain AF2 (6).
AR-mediated transactivation requires the concerted action of AF1 and AF2 (6). To date a great number of AR cofactors have been described to mediate androgens action (7). Gene activation by the AR is thought to require the general initiation factors that form preinitiation complexes on common core promoter element (8), and different general and specific coactivators that either modulate chromatin structure (9) or serve as direct adaptors between
the receptor and general initiation factors (10). The interest in AR corepression rapidly developed in the recent years and subsequently the number of AR corepressors drastically increased (see (11) for a review). The mechanism of action for many corepressors remains to be discovered. However, recruitment of histone deacetylases (HDACs) is a common way to repress AR activity. In that category, are found different proteins (12-14) including the short heterodimer partner (SHP) (15), which can be all recruited by the agonist-activated AR. RIP140 (receptor interacting protein of 140 kDa) is a protein of 1158 amino acids which is recruited by a large number of agonist-activated receptors, including ER;, TR, RAR and
RXR (16), AR (17), VDR (18), PPAR;/LXR; (19) or GR (20). It was also shown to interact
with other nuclear receptors such as SF1 or DAX-1 (21) or other transcription factors including the aryl hydrocarbon receptor (22), 14-3-3 (23) or c-jun (24). Its mechanism of action not only involves a competition with coactivators such as those belonging to the p160 family (25) but it also implies active repression. We and others recently evidenced four repressive domains in the molecule involving complex mechanisms relying on multiple partners, including HDACs and C-terminal binding proteins (CtBPs) (26, 27). Surprisingly, although widely depicted as a corepressor, a study by Ikonen et al. described
RIP140 as a strong coactivator for AR (17). In order to decipher RIP140 mechanism of action we investigated further its role in the androgen signalling pathway. In the present paper, we first characterized the interaction between RIP140 and AR and provided evidence for a nuclear relocalization of RIP140 upon activation of the receptor. We showed that RIP140 is a strong AR repressor, and to shed light on the mechanism of RIP140-dependent inhibition, we investigated the role of CtBPs and HDAC as well as a competition with a p160 coactivator. Finally, we demonstrated that RIP140 mRNA expression in LNCaP cells was significantly increased by a treatment with R1881, further emphasizing the role of RIP140 in AR activity.
RIP140 interacts with AR
In order to determine whether AR could interact with the coregulator RIP140, we first performed immunoprecipitations between full-length proteins (Figure 1A). 293T cells were either non transfected (lanes 1 and 2) or transfected with pCMV-AR alone (lanes 3 and 4), pCMV-AR and pEF-c-mycRIP140 (lanes 5 and 6) or pEF-c-mycRIP140 alone (lanes 7 and 8),
-8 M R1881. As shown in the Figure, when AR is expressed alone the use and treated with 10
of an anti-AR antibody immunoprecipitated the receptor (lane 4, upper panel). When AR and c-mycRIP140 were co-expressed, the same antibody not only pulled-down AR (lane 6, upper panel) but also c-mycRIP140 (lane 6, lower panel). We noted the band corresponding to c-mycRIP140 was slightly retarded as compared to the input which could be due to differences in salt concentrations. It has to be noticed that, as a control for the immunoprecipitation, when pEF-c-mycRIP140 was transfected alone (lanes 7 and 8), the use of an anti-AR antibody could not pull-down c-mycRIP140, thus strengthening the specificity of the interaction.
To investigate further the interaction and determine which domains of the proteins were involved, we performed in vitro GST pull-downs. In this series of experiments, three
fragments of RIP140 spanning the whole protein were expressed as GST fusion proteins and either full-length or truncated domains of AR were in vitro translated. As indicated in Figure
1B, upper panel, in the presence of R1881, full-length AR interacted with the three regions of RIP140. However, the binding appeared stronger with GST-RIP140(27-439). As observed in Figure 1B, middle panel, only a very faint band corresponding to the binding between GST-RIP(27-439) and AR(1-501) could be detected whereas none was observed with either the central or the carboxy-terminal part of RIP140. In the lower panel was analysed the interaction with the carboxy-terminal part of the receptor in the presence of R1881. As observed, both GST-RIP(27-439) and GST-RIP(683-1118) appeared to have a strong affinity
for AR(618-919) whereas GST-RIP(428-739) displayed a lower but still significant binding. Only a faint band was observed when GST was incubated with either full-length AR or AR(618-919) whereas none appeared with AR(1-501). It must be stated that the experiments with either full-length AR or AR(618-919) were also done in the absence of R1881 and gave the same degree of interaction (data not shown). Coomassie staining of the gels indicated that the amount of GST fusion proteins was kept constant in all experiments (data not shown). To give further credit to the interaction we wondered whether RIP140 could be recruited to an androgen-dependent gene. To this end we performed chromatin immunoprecipitation (ChIP)
-8 M assay with an anti-RIP140 antibody on LNCaP cells previously treated or not with 10R1881. Since a recent work (28) evidenced that transcription factors could differentially recruit the promoter and the enhancer of the PSA gene, these different regions of the gene were then amplified (Figure 1C). As observed on the figure either a 1-hour or 6-hour treatment with the AR agonist induced a clear amplification of both the PSA promoter and the enhancer as quantified by quantitative PCR demonstrating that an AR-responsive gene could be a target of RIP140.
We conclude from these experiments that RIP140 interacts with AR both in vitro and in intact
cells. Furthermore the interaction is mediated on one hand by several regions covering the entire cofactor and on another hand by the ligand binding domain of AR.
AR relocalizes RIP140
Subcellular localization of transcription factors is tightly regulated. Therefore we questioned whether overexpression of one partner could affect the localization of the other. We first transfected COS7 cells with pYFP-RIP140 (see Figure 2A). As observed in the left panel, whatever the treatment of the cells YFP-RIP140 always formed foci in the nucleus, a structure already described (26). In Figure 2A, right panel, the cells were cotransfected with vectors
expressing CFP-AR and YFP-RIP140. When the cells were incubated with ethanol, AR was localized to the cytoplasmic compartment, whereas RIP140 was nuclear and formed regular foci (upper panel). When treated with the agonist R1881, AR was entirely translocated to the nucleus (Figure 2A, middle panel). Remarkably, in the same cell, RIP140 presented a more evenly spread nuclear localization with only rare foci. Interestingly, when the cells were treated with the complete antagonist bicalutamide, AR was translocated to the nucleus as previously described (29) but there, RIP140 formed the same foci as observed in the presence of ethanol. Interestingly, when merged the two signals did not show a colocalization of the two proteins. From these observations we can conclude that translocation of the activated AR relocalized RIP140. Moreover the relocalization was specific to the activated receptor since the bicalutamide-liganded AR was not able to trigger it.
Then, we wondered whether AR amino- or carboxy-terminal domains would induce differential localizations of RIP140. Therefore, we cotransfected COS7 cells with YFP-RIP140 together with either CFP-AR(1-501) (Figure 2B, left panel) or CFP-AR(507-919) (Figure 2B, right panel). As observed in the left panel, whatever the treatment, CFP-AR(1-501) stayed in the cytoplasm whereas YFP-RIP140 was organized in nuclear foci. In Figure 2B, right panel, it is interesting to observe that CFP-AR(507-919) in the absence of agonist ligand was nuclear but evenly spread, whereas YFP-RIP140 still formed foci. Remarkably, under R1881 treatment CFP-AR(507-919) was organized in large foci. In the same conditions, YFP-RIP140 was organized in structures of the same size as CFP-AR(507-919). As observed on the overlay picture, the two proteins were perfectly colocalized. These data strengthen the evidence of a ligand-dependent intracellular interaction between RIP140 and AR mediated by the carboxy-terminal domain of the receptor.
RIP140 inhibits AR-dependent transactivation
We then investigated the role of RIP140 on AR-dependent transactivation. In a first series of experiments, CV1 cells were transfected with pCMV-AR and increasing amounts of pcRIP140 (Figure 3A). As shown in the figure, RIP140 dose-dependently inhibited AR-mediated transactivation with a maximal repression obtained with 2 ？g of transfected
We then asked whether an extinction of RIP140 could affect AR transactivation. To this end we used mouse embryo fibroblasts lacking the RIP140 gene (termed RIPKO-1) (30) as well as the wild type counterpart cells. As observed in Figure 3B, whatever the dose of vector transfected, AR was repeatedly overactivated in RIPKO-1 cells treated with R1881 as compared to the wild-type. It has to be noticed that no difference in AR expression could be observed in either cell type (data not shown). This experiment allowed us to propose that endogenous RIP140 exert a significant repressive effect on AR-dependent transactivation further emphasizing results described above.
RIP140 possesses nine nuclear receptor boxes (31) and results from Figure 1 showed that several regions of RIP140 were able to mediate its interaction with AR. Therefore we investigated in CV1 cells the repressive potential of RIP140 constructs spanning different domains of the cofactor on AR-dependent transcription. As shown in Figure 3C, all the constructs tested displayed a high degree of repression. However, the two fragments encompassing the amino-terminal part of RIP140, i.e. RIP140(1-282) and RIP140(1-480) did
not exhibit a repressive potential as strong as the wild type. By sharp contrast, the carboxy-terminal fragment of RIP140 and more precisely RIP140(917-1158) exhibited an even stronger repression than full-length RIP140. We concluded that different domains of RIP140 can mediate AR-dependent repression.
It has already been evidenced that RIP140 can compete away coactivators to bind nuclear receptors (25). Moreover, results presented above evidenced the carboxy-terminal domain of RIP140 as a strong inhibitor. Therefore we investigated whether RIP140(917-1158) could compete with an AR coactivator, TIF2, for repression of the receptor. As evidenced in Figure 3D, when CV1 cells were cotransfected with pSG5-TIF2, AR-dependent transcription was augmented. Remarkably, cotransfections with increasing amounts of pcRIP140(917-1158) not only reversed AR overactivation, but also completely down-regulated AR activity. Noticeably the same experiment was done with full-length RIP140 and the same results were obtained (data not shown). These data allowed us to propose that the carboxy-terminal part of RIP140 can act as a strong competitor for p160-mediated activation of AR.
AR exhibiting two transactivation domains, lying in the amino- and the carboxy-terminal parts of the receptor, we asked whether results from protein-protein interactions would be corroborated by transactivation assays. We first studied the effect of RIP140 on the constitutively active AR(1-660). CV1 cells were transfected with a constant dose of pCMV-AR(1-660) and increasing amounts of pcRIP140. As evidenced in Figure 4A, whatever the quantity of pcRIP140 transfected, AR(1-660)-dependent activity could not be modulated, indicating that the main activation domain of AR, when isolated, was not a target for RIP140. We then asked whether RIP140 could inhibit AR LBD-dependent transactivation. We thus used the deletion mutant AR(507-919) that lacks the amino-terminal domain and displays no transcriptional activity per se. In order to restore its transcriptional ability in a strictly ligand dependent manner, CHO cells were cotransfected with an AR coactivator, TIF2 (Figure 4B). In those conditions, as shown in the Figure, a dose as low as 50 ng pcRIP140 was sufficient to completely reverse TIF2-induced activity of AR(507-919).