9-cis-Retinoic Acid Inhibits Androgen Receptor Activity through Activation of Retinoid X Receptor

Kuang-Hsiang Chuang1, Yi-Fen Lee1, Wen-Jye Lin, Chin-Yi Chu, Saleh Altuwaijri, Yu-Jui Yvonne Wan and Chawnshang Chang

George Whipple Laboratory for Cancer Research, Department of Pathology (K.-H.C., Y.-F.L., W.-J.L., C.-Y.C., S.A., C.C.), University of Rochester Medical Center, Rochester, New York 14642; and Department of Pharmacology, Toxicology and Therapeutics (Y.-J.Y.W.), The University of Kansas Medical Center, Kansas City, Kansas 66160-7417

Address all correspondence and requests for reprints to: Chang Chawnshang, George Whipple Laboratory for Cancer Research, Department of Pathology, University of Rochester Medical Center, Rochester, New York 14642. E-mail: chang{at}urmc.rochester.edu; web site: www.urmc.rochester.edu/changARlab.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although the retinoic X receptor (RXR) forms heterodimers with many members of the estrogen receptor subfamily, the interaction between RXR and the members of the glucocorticoid receptor subfamily remains unclear. Here we show that the RXR can form a heterodimer with the androgen receptor (AR) under in vitro and in vivo conditions. Functional analyses further demonstrated that the AR, in the presence or absence of androgen, can function as a repressor to suppress RXR target genes, thereby preventing the RXR binding to the RXR DNA response element. In contrast, RXR can function as a repressor to suppress AR target genes in the presence of 9-cis-retinoic acid, but unliganded RXR can function as a weak coactivator to moderately enhance AR transactivation. Together, these results not only reveal a unique interaction between members of the two nuclear receptor subfamilies, but also represent the first evidence showing a nuclear receptor (RXR) may function as either a repressor or a coactivator based on the ligand binding status.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE NUCLEAR RECEPTOR (NR) superfamily is a large group of more than 150 related transcription factors, which control cellular homeostasis, growth, differentiation, and development (1). This superfamily can be divided into two subfamilies based on the DNA response elements they recognize (2). One subfamily includes the estrogen receptor (ER), which recognizes the 5'-(A/G)GTCA-3' DNA segment, and the other subfamily includes the glucocorticoid receptor recognizing the 5'-(A/G)GACA-3' DNA segment. Members of the NR superfamily are characterized by a highly conserved DNA-binding domain (DBD), which contains two zinc finger modules for sequence-specific DNA binding (3). The formation of receptor homodimers and/or heterodimers also requires an extensive carboxyl (C)-terminal dimerization interface that resides within the ligand-binding domain (LBD) (4, 5). Moreover, NRs possess transactivation functions (AFs), which can confer activation potential to heterologous DBDs. Transactivation is mediated by both constitutive and inducible AFs (AF-1 and AF-2, respectively), with the ligand-inducible AFs located within the LBD (6).

The androgen receptor (AR), when activated by androgen, translocates from the cytoplasm to the nucleus and binds to the androgen response element (ARE) as a homodimer in the regulatory regions of AR target genes (7, 8, 9). In conjunction with coregulator proteins such as SRC-1 (steroid receptor coactivator-1), SRC-2, and SRC-3 (10, 11, 12), the liganded ARs form transcriptional activation complexes within target gene promoters to facilitate gene expression. Therefore, the AR may function as a pivotal molecular switch that controls the expression of appropriate proteins in a highly coordinated manner from embryogenesis to adulthood. It is possible that AR coregulator proteins play important roles in regulating AR transactivation in tissue-specific and stage-specific manners. Recently, several AR-associated coregulators (ARAs), such as ARA24, ARA54, ARA55, ARA67, ARA70, ARA160, ARA267, breast cancer-1, cAMP response element binding protein (CREB)-binding protein/p300, human Rad9, retinoblastoma protein, Smad3, Testicular orphan nuclear receptor 4, and transcription factor IIH (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26), were identified. The study of AR coregulators involved in modulating AR-mediated gene regulation should expand our understanding of how coregulators influence gene expression.

The retinoic X receptor (RXR) is the NR for the vitamin A metabolite, 9-cis-retinoic acid (9cRA) (27, 28), and is involved in many biological processes, including cell growth, differentiation, metabolism, morphogenesis, and homeostasis during embryonic development and postnatal life (29, 30). Once bound with 9cRA, RXRs can bind to cognate DNA regulatory elements and activate transcription as homodimers (31). In the absence of ligand, RXRs form stable tetramers or serve as obligatory partners with a large number of NRs (32, 33). Therefore, the intracellular state of RXR is a dynamic equilibrium between tetramers, heterodimers, or homodimers, the populations of which are dependent on the vitamin A moiety and homeostasis.

To date, the cross-talk between the androgen signaling pathway and retinoid signaling pathway remains unclear. Here we identified the RXR as a novel coregulator for the AR by showing the interaction between the AR and RXR and their mutual regulation. The cross-talk between the AR and RXR not only expands the functions of both receptors but also contributes to the understanding of the complex gene network regulated by the NR superfamily.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
9cRA Represses AR Target Genes Both in Vitro and in Vivo
Like the RXR, the AR acts as a transcription factor to activate many androgen target genes. To explore the cross-talk between the retinoid signaling pathway and the androgen signaling pathway, we investigated the potential regulatory effects of the RXR on AR-mediated transactivation. In PC-3 cells, the AR activated mouse mammary tumor virus (MMTV)-ARE-Luc activity in the presence of dihydrotestosterone (DHT) (Fig. 1AGo, lanes 6 and 9), which could then be further induced by the addition of the unliganded RXR{alpha} (lanes 18, 19, and 20 vs. lane 9). In contrast, without AR, RXR{alpha} by itself has no effect on the MMTV-ARE-Luc activity in the absence or presence of RXR ligands (lanes 2, 3, 4, and 5). However, in the presence of 9cRA, RXR{alpha} significantly suppressed DHT-induced AR transactivation in a dose-dependent manner (lanes 11, 12, and 13 vs. lane 9) but did not influence the AR expression (Fig. 1BGo). Furthermore, addition of higher concentrations of 9cRA has stronger suppressive effect than a lower concentration (10–6, 10–7, and 10–8 M 9cRA vs. 10–9 and 10–10 M 9cRA). An RXR-selective ligand, LG101305, was also used to examine the effect of activated RXR{alpha} on AR-mediated transcriptional activity. In the presence of LG101305, DHT-induced AR transactivation was also repressed by the RXR{alpha} in a dose-dependent manner (lanes 15, 16, and 17 vs. lane 9), although LG101305 was shown less potent than 9cRA (lanes 15, 16, and 17 vs. lanes 11, 12, and 13). Similar suppression effects also occurred when we replaced the MMTV-ARE-Luc reporter with the p(ARE)4-Luc reporter (Fig. 1CGo) or with the prostate-specific antigen (PSA)-Luc reporter (Fig. 1DGo), another AR target gene that is widely used as a marker for prostate cancer progression. To rule out the potential artificial effects linked to transfected reporter assays, the expression of endogenous PSA in LNCaP cells was measured by Northern and Western blot analyses. As shown in Fig. 2AGo, the expression of the PSA mRNA was dramatically induced after 24 h of DHT treatment. Addition of 1 µM 9cRA can clearly repress the expression of endogenous PSA mRNA in the presence of 10 nM DHT. The level of intracellular PSA protein detected by Western blotting was also reduced after the treatment of 1 µM or 1 nM RXR ligands (Fig. 2BGo). In addition, we also observed that in LNCaP cells transfected with PSA-Luc reporter, endogenous RXR{alpha} repressed DHT-induced AR transactivation in the presence of 9cRA or LG101305 (Fig. 2CGo). This in vivo 9cRA-mediated suppressive effect strongly supports the reporter assay data in Fig. 1Go and demonstrates that the RXR may function as a repressor in the presence of 9cRA to suppress AR target gene expression. In contrast, unliganded RXR can function as a weak coactivator to slightly enhance AR transactivation.




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Fig. 1. 9cRA/RXR Repression of AR-Mediated Transactivation

A, PC-3 cells were cotransfected with 300 ng MMTV-ARE-Luc and 100 ng pSG5-AR (lanes 6–20) with increasing amounts of pCMX-RXR{alpha} (+, 100 ng; ++, 300 ng; +++, 600 ng) as indicated. The total plasmid amount was adjusted with pCMX or pSG5 parent vector to 1 µg for each 35-mm transfection using SuperFect. phRL-tk-Luc (30 ng) was cotransfected as the control for normalization. B, PC-3 cells were cotransfected with 1 µg pSG5-AR, 3 µg MMTV-ARE-Luc, 0.1 µg phRL-tk-Luc, and increasing amounts of pCMX-RXR{alpha} (+, 1 µg; ++, 3 µg; +++, 6 µg) using SuperFect for each 100-mm transfection for 24 h, and then treated with 10 nM DHT and/or 1 µM 9cRA for 16–18 h. Total protein was extracted, and Western blot was performed to examine the expression of RXR and AR. C, PC-3 cells were cotransfected with 300 ng p(ARE)4-Luc and 100 ng pSG5-AR (lanes 6–20) with increasing amounts of pCMX-RXR{alpha} (+, 100 ng; ++, 300 ng; +++, 600 ng) as indicated. D, PC-3 cells were cotransfected with 300 ng PSA-Luc and 100 ng pSG5-AR (lanes 6–20) with increasing amounts of pCMX-RXR{alpha} (+, 100 ng; ++, 300 ng; +++, 600 ng) as indicated. After 24 h transfection, cells were treated with DHT, 9cRA, and/or LG101305 as indicated. After 16–18 h incubation, cells were harvested for the Dual-Luciferase reporter assay. Each Luc activity is presented relative to the transactivation observed in the absence of DHT and is the mean ± SD of four experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (Figure continues on next page.)

 


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Fig. 2. 9cRA Repression of PSA Gene Expression in LNCaP Cells

A, Total RNA (25 µg) from LNCaP cells, which were treated with 10 nM DHT and/or 1 µM 9cRA for 24 h, was loaded onto a formaldehyde RNA gel, transferred onto a nylon membrane, and then hybridized with a 32P-end-labeled PSA gene fragment from exon 1. ß-Actin was used as an internal control. B, Total protein (80 µg) from LNCaP cells, which were treated with DHT and/or 9cRA as indicated for 16–18 h, was loaded onto a 10% polyacrylamide gel, transferred onto a polyvinylidine difluoride membrane, and then incubated with a monoclonal anti-PSA antibody (DAKO Corp., Carpinteria, CA) or an anti-AR antibody. C, LNCaP cells were transfected with 1 µg PSA-Luc for each 35-mm transfection using SuperFect. phRL-tk-Luc (50 ng) was cotransfected as the control for normalization. After 24 h transfection, cells were treated with DHT, 9cRA, and/or LG101305 as indicated. After 16–18 h incubation, cells were harvested for the Dual-Luciferase reporter assay. Each Luc activity is presented relative to the transactivation observed in the absence of DHT and is the mean ± SD of four experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Ab, Antibody.

 
The AR Represses RXR{alpha}-Mediated Transactivation
The effect of the AR on RXR{alpha}-mediated transcriptional activity was also examined by transactivation reporter assay. The full-length AR and RXR{alpha} in eukaryotic expression vectors (pSG5-AR and pCMX-RXR{alpha}) were cotransfected with a Luc reporter containing a RXR-response element (pCRBP II-Luc) into PC-3 cells. As shown in Fig. 3AGo, in the presence of 9cRA, the Luc activity induced by pCMX-RXR{alpha} could be repressed in a dose-dependent manner by cotransfection of pSG5-AR in the absence of DHT (lanes 6, 7, and 8 vs. lane 5), although addition of 10 nM DHT moderately reduced such repression (lanes 10, 11, 12 vs. 6, 7, 8). However, as shown in Fig. 3BGo, the increasing AR did not affect the protein expression level of RXR{alpha}. Using another strategy of cotransfection of an AR short interfering RNA (siRNA) plasmid into PC-3 cells, we found that AR siRNA, but not BS/U6, vector rescues the suppression effect of 9cRA-induced RXR{alpha} transactivation by the AR (Fig. 3CGo, lanes 4, 5, and 6 vs. lane 3), in a dose-dependent manner.



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Fig. 3. The Effects of the AR on RXR{alpha}-Mediated Transcriptional Activity

A, PC-3 cells were cotransfected with 100 ng pCMX-RXR{alpha} (lanes 4–12), 300 ng pCRBP II-Luc, 30 ng phRL-tk-Luc, and increasing amounts of pSG5-AR expression vector (+, 100 ng; ++, 300 ng; +++, 600 ng) in the presence (+) or absence (–) of 10 nM DHT and 1 µM 9cRA. The total plasmid amount was adjusted with pCMX or pSG5 parent vector to 1 µg for each 35-mm transfection using SuperFect. B, PC-3 cells were cotransfected with 1 µg pCMX-RXR{alpha}, 3 µg pCRBP II-Luc, 0.1 µg phRL-tk-Luc, and increasing amounts of pSG5-AR expression vector (+, 1 µg; ++, 3 µg; +++, 6 µg) using SuperFect for each 100-mm transfection for 24 h, and treated with 10 nM DHT and/or 1 µM 9cRA for 16–18 h. Total protein was extracted, and Western blot was performed to examine the expression of RXR and AR. C, PC-3 cells were cotransfected with 60 ng pCRBP II-Luc and 15 ng pCMX-RXR{alpha} in the presence or absence of 90 ng pSG5-AR and increasing amounts of AR siRNA or BS/U6 empty vector (+, 90 ng; ++, 450 ng; +++, 900 ng). pRL-TK (15 ng) was cotransfected as the control for normalization. The total plasmid amount was adjusted with pCMX, pSG5, or BS/U6 parent vector to 1 µg for each 35-mm transfection using SuperFect. After 24 h of transfection, cells were treated with or without 1 µM 9cRA. After 16–18 h incubation, cells were harvested for the reporter assay. Each Luc activity is presented relative to the transactivation observed in the absence of 9cRA and is the mean ± SD of four experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
RXR{alpha} Binding to the RXR-Response Element (RXRE) Is Inhibited by the AR
The EMSA using 32P-end-labeled RXRE-direct repeat 1 (DR1) probe (25) was applied to further dissect the mechanism of how the AR represses RXR{alpha}-mediated transactivation. As shown in Fig. 4Go, AR could not bind to DR1 (lanes 2 and 3) and the specific RXR-DR1 band was decreased with the addition of increasing amounts of the AR (lanes 5, 6, and 7 vs. lane 4). This result suggests that the AR might be able to repress RXR{alpha}-mediated transactivation by sequestering RXR{alpha} away from its target DNA.



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Fig. 4. Inhibition of RXR{alpha} DNA Binding by the AR

In vitro translated RXR{alpha} protein (1 µl) was incubated with increasing amounts of in vitro translated AR (1 µl, 2 µl, and 4 µl) in EMSA reaction buffer (10 mM HEPES/pH 7.9, 2% (vol/vol) glycerol, 100 mM KCl, 1 mM EDTA, 5 mM MgCl2, and 1 mM dithiothreitol) for 15 min. 32P-end-labeled DR1 was added into the protein mixture and incubated for 15 min before loading. The reaction mixtures were then applied to a 5% native polyacrylamide gel. The radioactive gel was analyzed by autoradiography.

 
AR-RXR Interaction in the Glutathione-S-Transferase (GST) Pull-Down Assays
The potential direct interaction between the RXR and the AR was then examined by the GST pull-down assay. The GST pull-down assay using GST-RXR{alpha} fusion protein demonstrated that RXR{alpha} could physically interact with the AR (Fig. 5AGo). To map the regions in the AR that interact with the RXR{alpha}, various AR deletion mutants were tested in the GST pull-down assay. As shown in Fig. 5AGo, GST-RXR{alpha} can interact with the retinoic acid receptor-ß and an in vitro-translated 35S-labeled AR deletion construct, the LBD of AR (AR-LBD), but not the N-terminal domain of AR (AR-N), the DBD of AR (AR-DBD), or the ER{alpha}. These results indicate that the RXR{alpha} can specifically interact with the AR-LBD. We also identified the regions in the RXR{alpha} that can interact with the AR. The N-terminal domain and the C-terminal domain of RXR{alpha} fused with GST (GST-RXR{alpha}-N+DBD and GST-RXR{alpha}-LBD, respectively) were used in the GST pull-down assay. As shown in Fig. 5BGo, in vitro-translated 35S-labeled AR can interact with both GST-RXR{alpha}-N+DBD and GST-RXR{alpha}-LBD. The minimal interaction regions in the N-terminal domain and the C-terminal domain of RXR{alpha} were defined by testing more RXR{alpha} fragments in the GST pull-down assay. As shown in Fig. 5CGo, the AR-LBD interacted with GST-RXR{alpha}-N and GST-RXR{alpha}H4–6 but not GST-RXR{alpha}-DBD, GST-RXR{alpha}H1–3, GST-RXR{alpha}H7–9, and GST-RXR{alpha}H10–12, suggesting that the N-terminal domain and helix 4/5/6 region of the RXR could bind to the LBD of the AR. The region in the AR-LBD domain required for interaction with RXR{alpha} was defined further by construction and testing of additional AR deletion derivatives in the GST pull-down assay. With serial deletion of AR-LBD starting from the C terminus, we created several truncated AR mutants. As shown in Fig. 6AGo, GST-RXR{alpha} had stronger interaction with both full-length AR and AR{Delta}H8–12 than with AR{Delta}H6–12, AR{Delta}H4–12, AR{Delta}H3–12, AR{Delta}H1–12, and AR-N. Interaction with RXR{alpha} is not affected by deletion of helix 8 to helix 12 of AR but is affected by deletion of helix 6 to helix 12 of AR. These results indicate that helix 6/7 region of AR may play an important role in the AR-RXR{alpha} interaction. Based on the crystal structure of the human AR LBD (amino acids 663–919) in complex with the ligand metribolone (R1881) (35), helices 6 and 7 are shown to be exposed on the surface of AR-LBD by using 3D-Mol Viewer analysis (InforMax, Bethesda, MD) (Fig. 6BGo). The GST pull-down data combined with the structural analysis result imply that the helix 6/7 region of AR may serve as the interaction interface for the AR binding to the RXR{alpha}.



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Fig. 5. Physical Interaction between the AR and the RXR{alpha} in Vitro

A, AR interaction with the RXR{alpha} in the GST pull-down assay. The GST-RXR{alpha} fusion protein and GST control protein were purified as instructed by the manufacturer (Pharmacia Biotech, Piscataway, NJ). Five microliters of in vitro translated [35S]methionine-labeled AR, ER{alpha}, and retinoic acid receptor-ß, and different AR deletion mutants, AR-N, AR-DBD, and AR-LBD, were incubated with the GST-RXR{alpha} or GST bound to glutathione-Sepharose beads in the pull-down assay. B, Localization of the interaction domain within RXR{alpha}. The AR was in vitro translated and incubated with GST, GST-RXR{alpha}, and two deletion mutants, GST-RXR{alpha}-N+DBD and GST-RXR{alpha}-LBD. C, The in vitro-translated AR-LBD was incubated with GST, GST-RXR{alpha}, GST-RXR{alpha}-N, GST-RXR{alpha}-DBD, GST-RXR{alpha}H1–3, GST-RXR{alpha}H4–6, GST-RXR{alpha}H7–9, and GST-RXR{alpha}H10–12 in the pull-down assay. Comparable abundance of each fusion protein was used in the GST pull-down assay. The pull-down complex was loaded onto a 10% or 15% polyacrylamide gel and visualized by autoradiography.

 


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Fig. 6. Mapping of the Interaction Region within the LBD of AR

A, Deletion derivatives of AR were tested in GST pull-down assays. The GST-RXR{alpha} fusion protein was purified. In vitro translated [35S]methionine-labeled AR (5 µl), and different AR deletion mutants, AR{Delta}H8–12, AR{Delta}H6–12, AR{Delta}H4–12, AR{Delta}H3–12, AR{Delta}H1–12, and AR-N were incubated with the GST-RXR{alpha} bound to glutathione-Sepharose beads in the pull-down assay. The input represents 5% of the amount of each labeled protein used in the pull-down assay. The pull-down complex was loaded onto a 10% polyacrylamide gel and visualized by autoradiography. B, Helices 6 and 7 in the AR LBD. Helices 6 and 7 of AR are blue; the other regions of AR are yellow; and the metribolone (R1881) molecule is a blue ball-and-stick representation.

 
Coimmunoprecipitation of the RXR-AR Complex
We then used a coimmunoprecipitation assay to demonstrate that the AR could interact with the RXR in vivo. As shown in Fig. 7Go, A and B, the AR was detected in the anti-RXR{alpha} or anti-RXRß antibody-precipitated complex from COS-1 cells cotransfected with the AR and the RXR{alpha} or the RXRß, but not in the complex from cells transfected only with the AR, the RXR{alpha}, or the RXRß. To further test whether the AR-RXR complex exists under physiological conditions, we used coimmunoprecipitation to detect an endogenous AR-RXR complex in cell lysates. As shown in Fig. 7CGo, the AR was detected in the anti-RXR{alpha} antibody-precipitated complex from LNCaP and PC-3(AR)2, but not PC-3 whole-cell extracts. Together, data from GST pull-down (Fig. 5Go, A and B, and Fig. 6AGo), and coimmunoprecipitation assays (Fig. 7Go) all demonstrated that the RXR interacts with the AR in vitro and in vivo.



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Fig. 7. In Vivo Interaction between the AR and the RXR

Coimmunoprecipitation of overexpressed flag-AR and RXR. A, COS-1 cells were cotransfected with 10 µg pCMX-RXR{alpha} and/or 10 µg pIRES-flag-AR. B, COS-1 cells were cotransfected with 10 µg pCDNA3-RXRß and/or 10 µg pIRES-flag-AR. The total plasmid amount was adjusted with pCMX, pIRES-flag, or pCDNA3 parent vector to 20 µg for each 100-mm transfection by the calcium phosphate precipitation method. Coimmunoprecipitation of the endogenous AR and RXR{alpha}. C, LNCaP, PC-3(AR)2, and PC-3 cells were pretreated with or without DHT and/or 9cRA. Cell extracts were prepared and immunoprecipitations were performed using an anti-RXR{alpha} antibody, anti-RXRß antibody, or normal rabbit IgG, followed by immunoblotting using an antibody to the flag tag or to the AR. IP, Immunoprecipitation.

 
RXR{alpha} Recruitment in the AREs in the Promoter of the PSA Gene Increases in Response to DHT Treatment but Decreases in the Presence of 9cRA and LG101305
Three AREs have been identified in the PSA gene. ARE I and ARE II reside in the –630 bp promoter region, and ARE III is in the enhancer region located –4 kb upstream of the PSA transcription initiation site (36). To examine whether the RXR can form a protein complex with the AR, which binds to an ARE in the promoter region of PSA gene, chromatin immunoprecipitation assays were performed to detect the recruitment of RXR{alpha} to the ARE I and ARE III regions after treatment with DHT. LNCaP cells were cultured in RPMI 1640 with 10% charcoal-treated fetal bovine serum (FBS) for 4 d followed by treatment with 10 nM DHT, 1 µM 9cRA, and/or 1 µM LG101305 for 3 h. Soluble chromatin was prepared after formaldehyde treatment of the cell cultures. Specific RXR{alpha} antibodies were used to immunoprecipitate protein-bound chromatin fragments. The chromatin DNAs were analyzed by quantitative PCR using two specific pairs of primers spanning the ARE I region (–39 to –250 bp) and the ARE III region (–4170 to –3978 bp) in the promoter of PSA gene. As shown in Fig. 8AGo, RXR{alpha} recruitment to ARE I was detected in LNCaP cells after treatment with DHT, but not 9cRA or LG101305, suggesting that the unliganded RXR{alpha} binds to the DHT-AR-ARE complex, which leads to the modest induction of AR transactivation (See Fig. 1AGo, lanes 18, 19, 20 vs. lane 9). However, in the presence of 9cRA or LG101305, RXR{alpha} recruitment to ARE I was significantly decreased in LNCaP cells with DHT treatment, which may result in the suppression of AR transactivation (see Fig. 1AGo, lanes 11, 12, 13 vs. lane 9, and lanes 15, 16, 17 vs. lane 9). Also, with DHT treatment, AR recruitment to ARE I was reduced in LNCaP cells in the presence of 9cRA or LG101305. No recruitment was detected to the promoter of the ß-actin gene (data not shown). Similar results were also observed with the ARE III region in the promoter of PSA gene (Fig. 8BGo). As the coimmunoprecipitation results showing that the interactions between the AR and the RXR{alpha} were not influenced by DHT and/or 9cRA treatment, these chromatin immunoprecipitation assay data indicate that the activated RXR bound to 9cRA or LG101305 may have little effect on the interaction between AR and RXR{alpha}, yet may play a major role in reducing the interaction between the AR and the ARE, leading to the repression of AR transactivation.



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Fig. 8. Chromatin Immunoprecipitation Assays of RXR Binding on ARE Regions of PSA Gene Promoter

LNCaP cells were treated with 10 nM DHT, 1 µM 9cRA, and/or 1 µM LG101305 for 3 h. Sonicated chromatin fragments were prepared from formaldehyde-treated cell cultures. Polyclonal RXR{alpha} antibodies were used to immunoprecipitate chromatin fragments. A normal rabbit IgG was used as a negative control (*) to immunoprecipitate chromatin fragments, whereas anti-AR antibodies were used as a positive control (**). A, These DNA fragments were then amplified by PCR using a specific pair of primers spanning the ARE I region in the PSA gene promoter. B, DNA fragments were amplified by PCR from immunoprecipitates above using a specific pair of primers spanning the ARE III region in the PSA gene promoter.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have demonstrated a direct protein-protein interaction between the RXR{alpha} and AR and their mutual regulation of transcriptional activity. Figure 9AGo shows a model for AR suppressed RXR-mediated transactivation, with the 9cRA-inducible RXRE segment representing the entire RXR target gene promoter. In the presence of 9cRA, the RXR can form a homodimer and induce RXR-target gene transactivation through RXR homodimer binding to the RXRE. After AR binding to the RXR, the AR/RXR complex may dissociate from the RXRE, resulting in the repression of RXR target gene expression. However, because unliganded-AR, which is mainly located in the cytoplasm, has a stronger suppressive effect than liganded AR, it is also possible that the unliganded AR may bind to newly synthesized RXR in the cytoplasm and interfere with its nuclear translocation. On the other hand, Fig. 9BGo shows a model for the RXR modulation of AR-mediated transactivation. Without 9cRA binding, the RXR may stabilize the DHT-AR-ARE complex and enhance AR transactivation. In the presence of 9cRA, however, the AR-RXR complex may dissociate from the ARE, resulting in the suppression of AR transactivation.



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Fig. 9. Schematic Models of Functional Interaction between AR and RXR

A, Model of RXR-target gene inactivation by the AR. B, Model of AR-target gene inactivation by 9cRA/RXR.

 
It is also possible that mutual suppression between the AR and the RXR is due to competitive squelching of common limiting transcriptional factors. However, this is unlikely because our findings show that DHT-AR has a less suppressive effect on 9cRA-induced RXR transactivation than unliganded-AR. In contrast, only liganded-RXR represses AR-mediated transactivation.

The potential impact of this model may be significant. First, in contrast to the classic roles of RXR as a transcriptional coactivator to enhance the transactivation of its partner receptors, such as the vitamin D receptor, thyroid hormone receptor, and peroxisome proliferator activated receptor (37, 38, 39, 40), we show here that RXR can suppress the transactivation of the AR, a member of another NR subfamily.

Second, because the presence or the absence of 9cRA binding can influence whether RXR functions as a repressor or weak coactivator for AR transactivation of reporter gene expression, our studies suggest that both ligand and receptor play important roles in the diverse functions of RXR. Similarly, the demonstration that DHT, a potent ligand for the AR, can reduce AR suppression of RXR transactivation also emphasizes that both ligand (DHT) and receptor (AR) may be necessary for proper AR function.

Third, because most evidence suggested that the AR functions as a transcription factor to activate its target genes via binding to AREs, data shown here demonstrate that the AR could also function as a repressor via protein-protein interaction to influence the expression of non-AR target genes, which represents a new dominant function for AR.

Fourth, both RXR and AR are widely expressed in various human tissues (4, 41, 42, 43, 44), and cellular levels of RXR are generally substantially higher than AR levels. Early reports also established an inhibitory role of 9cRA in prostate carcinogenesis (45). The detailed mechanism by which 9cRA can suppress prostate tumor growth, however, remains unclear. Here we provide possible explanations by demonstrating that 9cRA may go through RXR-AR interaction to suppress PSA expression. Whether this newly discovered pathway can be used as a target to develop new drug(s) for the battle against prostate cancer remains to be elucidated. Nevertheless, our conclusion that the AR and RXR modulate regulation of each other’s target genes is in agreement with a recent animal study showing that androgen negatively regulates RXR-mediated gene expression (46).

In summary, our demonstration of the AR-RXR interaction may represent a unique mechanism among NRs, and the transcriptional regulation between two receptors may help us to better understand the cross-talk between androgen and retinoid signaling pathways.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals and Plasmids
DHT and 9cRA were obtained from Sigma Chemical Co. (St. Louis, MO). LG101305 was kindly provided by Ligand Pharmaceuticals, Inc. (San Diego, CA) pSG5-AR, MMTV-ARE-Luc, PSA-Luc, and pCMX-RXR{alpha} were used in our previous reports (25, 34). pIRES-flag-AR was constructed by inserting flag-tagged AR cDNA into pIRESneo vector (CLONTECH, Palo Alto, CA). pCDNA3-RXRß was from Renata Polakowska. p(ARE)4-Luc was kindly provided by Michael L. Lu (Harvard University, Boston, MA) (47). The AR siRNA expression vector that produces a siRNA targeting AR in mammalian cells was constructed by digesting and inserting a double-stranded polynucleotide 5'-GTCGGGCCCTATCCCAGTCCCACTTG-CTCGAGCAAGTGGGACTGGGATAGGGCTTTTTGAATT-CGC-3' into the ApaI-EcoRI site of a DNA-based BS/U6 vector (Ambion, Inc., Austin, TX). pCRBPII-Luc was a gift from Vimla Band (Tufts University, Boston, MA).

Construction of Deletion Derivatives of AR and RXR{alpha}
To construct pCDNA3-flag AR (1–919), AR{Delta}H8–12 (1–800), AR{Delta}H6–12 (1–771), AR{Delta}H4–12 (1–729), AR{Delta}H3–12 (1–696), AR{Delta}H1–12 (1–666), and AR-N (1–556), PCR amplication was used to generate the indicated fragment from the pSG5-AR with specific oligonucleotides that introduced in-frame BamHI and XbaI sites. PCR fragments were subcloned into pCDNA3-flag vector, a flag-tag insertion derivative of pCDNA3 vector (Invitrogen, San Diego, CA). To construct GST-RXR{alpha}-N (114–202), GST-RXR{alpha}-DBD (114–202), GST-RXR{alpha}H1–3 (203–265), GST-RXR{alpha}H4–6 (266–338), GST-RXR{alpha}H7–9 (339–409), and GST-RXR{alpha}H10–12 (410–462), PCR amplication was used to generate indicated fragments, which were then subcloned into pGEX-KG vector, from the pCMX-RXR{alpha}.

Glutathione-S-transferase (GST) Pull-Down Assay
GST-RXR{alpha}, GST-RXR{alpha}-N+DBD, GST-RXR{alpha}-LBD, GST-RXR{alpha}-N, GST-RXR{alpha}-DBD, GST-RXR{alpha}H1–3, GST-RXR{alpha}H4–6, GST-RXR{alpha}H7–9, and GST-RXR{alpha}H10–12 fusion proteins and GST control protein were obtained by transforming expressing plasmids into BL21 (DE3) pLysS strain-competent cells followed with 1 mM isopropyl-D-thiogalactoside induction. GST fusion proteins then were purified by glutathione-Sepharose 4B as instructed by the manufacturer (Amersham Biosciences Corp., Piscataway, NJ). In vitro translated [35S]methionine-labeled proteins (5 µl) generated with the TNT-coupled reticulocyte lysate system (Promega Corp., Madison, WI) were mixed with the glutathione-Sepharose bound GST proteins at 4 C for 3 h to perform the pull-down assay as described previously (48). The bound proteins were separated on a 10% or 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel and visualized by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Cell Culture and Transfections
COS-1 cells and human prostate cancer PC-3 cells were maintained in DMEM containing penicillin (25 U/ml), streptomycin (25 µg/ml), and 10% FBS. Human prostate cancer LNCaP cells were maintained in RPMI 1640 with 10% charcoal-treated FBS. Briefly, 2 x 105 cells were plated on 35-mm dishes, 4 x 105 cells were plated on 60-mm dishes, and 106 cells were plated on 100-mm dishes 24 h before transfection. The cells were transfected using SuperFect (QIAGEN, Chatsworth, CA) or a modified calcium phosphate precipitation method (37), and the total plasmid amount was adjusted with parent vectors. After 24 h of transfection, cells were treated with DHT, 9cRA, and/or LG101305 as indicated. After 16–18 h of incubation, cells were harvested. At least three independent experiments were carried out in each case. To normalize the transfection efficiency, the phRL-tk-Luc (Promega) was cotransfected for the Dual-Luciferase reporter assay (Promega). The PC-3(AR)2 cells are AR stably-expressed PC-3 cells, a gift from T. J. Brown (University of Toronto, Toronto, Ontario, Canada).

EMSA
The EMSA was performed as described previously (37). Briefly, the reaction was performed by incubating the 32P-end-labeled DR1 probe with in vitro-translated RXR{alpha} with or without an increasing amount of the AR (1, 2, or 4 µl). The EMSA incubation buffer used included 10 mM HEPES/pH 7.9, 2% (vol/vol) glycerol, 100 mM KCl, 1 mM EDTA, 5 mM MgCl2, and 1 mM dithiothreitol. DNA-protein complexes were resolved on a 5% native polyacrylamide gel. The radioactive gel was analyzed by PhosphorImager scanning (Molecular Dynamics, Inc.).

Coimmunoprecipitation
Cells plated on 100-mm dishes were incubated in DMEM or RPMI plus 10% charcoal-treated FBS for 16 h and then treated with or without 10 nM DHT and/or 1 µM 9cRA for an additional 16 h. After washing with ice-cold 1x PBS, cells were solubilized in 1 ml RIPA buffer [10 mM NaHPO4/pH 7.0, 150 mM NaCl, 2 mM EDTA, 0.5% (wt/vol) Nonidet P-40, 0.1% (wt/vol) SDS, 1% (wt/vol) sodium deoxycholate, and 1 mM phenylmethylsulfonylfluoride]. Insoluble material was removed by centrifugation (16,000 x g; 10 min at 4 C). Polyclonal anti-RXR{alpha} or anti-RXRß antibodies (10 µl) (200 µg/ml; Santa Cruz Biotechnology, Inc.) were added to the cell lysates and incubated for 2 h at 4 C. Immunoprecipitates were collected with protein A/G-Sepharose beads (Santa Cruz Biotechnology, Inc.), washed four times in RIPA buffer, and then analyzed by Western blotting with mouse anti-flag (1:1000) or rabbit anti-AR (1:3000) (NH-27) antibodies.

Western Blotting
Cell lysates or immunoprecipitated proteins were separated by SDS-PAGE through 10% gels, electroblotted onto polyvinylidine difluoride membrane, and Western blotted with the indicated antibodies. Blots were blocked at 4 C with Tris-buffered saline with 0.5% Tween 20 (TBST) plus 5% dry nonfat milk and antibodies were diluted in this buffer as suggested by the manufacturers. Blots were incubated in primary antibodies for 2 h at room temperature or overnight at 4 C, washed three times in TBST, and then incubated with the appropriate alkaline phosphatase-conjugated goat antimouse or antirabbit IgG secondary antibodies (Bio-Rad Laboratories, Inc., Hercules, CA) diluted in TBST plus 5% milk for 1 h at room temperature. ß-Actin was detected by a monoclonal anti-ß-actin antibody (Santa Cruz Biotechnology) as an internal control for equivalent protein loading. Immunoblots were visualized using an alkaline phosphatase conjugation kit (Bio-Rad) or an ECL Western blotting detection kit (Amersham Pharmacia Biotech).

Northern Blotting Analysis
At the indicated time points, total cellular RNA was isolated from the cells using TRIzol reagent (Life Technologies, Gaithersburg, MD). Total RNA (25 µg) was size fractionated in a 1% agarose-2% formaldehyde gel and blotted onto a nylon membrane. Hybridization was performed in QuikHyb solution (Stratagene, La Jolla, CA) with a probe from exon 1 of the PSA gene labeled with [{alpha}-32P]dCTP. A ß-actin cDNA probe (Ambion) was used as a control for equivalent RNA loading. Hybridization intensity was quantitated by PhosphorImager scanning (Molecular Dynamics).

Chromatin Immunoprecipitation
LNCaP cells were cultured in RPMI 1640 with 10% charcoal-treated FBS for 4 d, treated with appropriate ligands for 3 h, washed with PBS, and cross-linked with 1% formaldehyde at 37 C for 10 min. Then the cross-linking was stopped by adding glycine to a final concentration of 125 mM for 5 min at room temperature. Cells were rinsed twice with ice-cold PBS, collected into PBS, and centrifuged. The pellets were then resuspended in cell lysis buffer [5 mM piperazine-N,N'-bis(2-ethanesulfonic acid-KOH/pH 8.0, 85 mM KCl, and 0.5% Nonidet P-40] with 1x protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) and incubated for 10 min on ice. Nuclei were pelleted by centrifugation and then resuspended in nuclear lysis buffer (50 mM Tris-HCl/pH 8.0, 10 mM EDTA, and 1% SDS) for 10 min on ice. Nuclear lysates were then sonicated three times by using a Branson Sonifier 250 (Branson Ultrasonics Corp., Danbury, CT) with a microtip in 20-sec bursts followed by 1 min of cooling on ice for a total sonication time of 3 min per sample. Debris was cleared by centrifugation for 10 min at 4 C, and supernatants were collected, diluted 5-fold in dilution buffer (1.0% Triton X-100, 1 mM EDTA, 150 mM NaCl, and 15 mM Tris-HCl/pH 8.0), followed by immunoclearing with 80 µl of a salmon sperm DNA-protein A-Sepharose slurry for 30 min at 4 C with agitation. After a brief centrifugation, 20% of the total supernatant was saved as total input control. Of the 80% remaining supernatant, half was incubated with and half was incubated without 5 µg of polyclonal anti-RXR{alpha} antibody (Santa Cruz Biotechnology) overnight at 4 C with rotation. After immunoprecipitation, 60 µl of the salmon sperm DNA-protein A-Sepharose slurry were added, and the incubation was continued for an additional 1 h at 4 C with rotation. Sepharose beads were washed sequentially for 5 min each in low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl/pH 8.0, and 150 mM NaCl), high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl/ pH 8.0, and 500 mM NaCl), and LiCl wash buffer (0.25 M LiCl; 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl/pH 8.0). Beads were then washed two times with TE buffer (10 mM Tris/pH 8.0, 1 mM EDTA) and extracted two times with 250 µl of elution buffer (1% SDS, 0.1 M NaHCO3). Two hundred fifty microliters of 10 µg/ml ribonuclease and 5 M NaCl to a final concentration of 0.3 M were added into pooled eluates and then heated at 65 C for 5 h to reverse the formaldehyde cross-linking. DNA fragments were purified with a DNA purification kit (QIAquick Spin Kit; QIAGEN). For quantitative PCR, 5 µl of 50 µl DNA extraction were used in 25 cycles of amplification. Two pairs of primer sequences were as follows: PSA ARE I primer A: 5'-TCTGCCTTTGTCCCCTAGAT-3'; PSA ARE I primer B: 5'-AACCTTCATTCCCCAGGACT-3'; PSA ARE III primer A: 5'-CCTCCCAGGTTCAAGTGATT-3'; and PSA ARE III primer B: 5'-GCCTGTAATCCCAGCACTTT-3'.

Statistics
Data are presented as the mean ± SD. Comparisons between groups were done using a two-tailed Student’s t test by GraphPad InStat software (GraphPad Software, Inc., San Diego, CA). P values < 0.05 are considered significant.


    ACKNOWLEDGMENTS
 
We thank Ligand Pharmaceuticals for providing LG101305. We also thank T. J. Brown, Renata Polakowska, Vimla Band, and Michael L. Lu for their valuable cells and plasmids. We are grateful to Karen Wolf and Erik R. Sampson for manuscript preparation, and we thank Patricia M. Hinkle and Patricia J. Simpson-Haidaris for critical review.


    FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK 60948 and a George Whipple Professorship Endorsement.

First Published Online January 13, 2005

1 K.-H.C. and Y.-F.L. contributed equally to this paper. Back

Abbreviations: AF, Transactivation function; AR, androgen receptor; ARA, AR-associated coregulator; ARE, androgen response element; CAT, chloramphenicol acetyltransferase; 9cRA, 9-cis-retinoic acid; CRBP II, cellular retinol-binding protein II; DBD, DNA-binding domain; DHT, dihydrotestosterone; ER, estrogen receptor; FBS, fetal bovine serum; GST, glutathione-S-transferase; LBD, ligand-binding domain; Luc, luciferase; MMTV, mouse mammary tumor virus; NR, nuclear receptor; PSA, prostate-specific antigen; RXR, retinoid X receptor; RXRE, RXR-response element; SDS, sodium dodecyl sulfate; siRNA, short interfering RNA; SRC, steroid receptor coactivator; TBST, Tris-buffered saline-Tween 20.

Received for publication April 29, 2004. Accepted for publication January 4, 2005.


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