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The Androgen Receptor Recruits Nuclear Receptor CoRepressor (N-CoR) in the Presence of Mifepristone via Its N and C Termini Revealing a Novel Molecular Mechanism for Androgen Receptor Antagonists

2004; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês

10.1074/jbc.m408972200

ISSN

1083-351X

Autores

Myles C. Hodgson, Inna Astapova, Shinta Cheng, Larissa J. Lee, Manon C. Verhoeven, Eunis Choi, Steven P. Balk, Anthony N. Hollenberg,

Tópico(s)

Hormonal and reproductive studies

Resumo

The androgen receptor (AR) activates target gene expression in the presence of agonist ligands via the recruitment of transcriptional coactivators, but recent work shows that overexpression of the nuclear corepressors NCoR and SMRT attenuates this agonist-mediated AR activation. Here we demonstrate using NCoR siRNA and chromatin immunoprecipitation that endogenous NCoR is recruited to and represses the dihydrotestosterone (DHT)-liganded AR. Furthermore this study shows that NCoR and coactivators compete for AR in the presence of DHT. AR antagonists such as bicalutamide that are currently in use for prostate cancer treatment can also mediate NCoR recruitment, but mifepristone (RU486) at nanomolar concentrations is unique in its ability to markedly enhance the AR-NCoR interaction. The RU486-liganded AR interacted with a C-terminal fragment of NCoR, and this interaction was mediated by the two most C-terminal nuclear receptor interacting domains (RIDs) present in NCoR. Significantly, in addition to the AR ligand binding domain, the AR N terminus was also required for this interaction. Mutagenesis studies demonstrate that the N-terminal surface of the AR-mediating NCoR recruitment was distinct from tau5 and from the FXXLF motif that mediates agonist-induced N-C-terminal interaction. Taken together these data demonstrate that NCoR is a physiological regulator of the AR and reveal a new mechanism for AR antagonism that may be exploited for the development of more potent AR antagonists. The androgen receptor (AR) activates target gene expression in the presence of agonist ligands via the recruitment of transcriptional coactivators, but recent work shows that overexpression of the nuclear corepressors NCoR and SMRT attenuates this agonist-mediated AR activation. Here we demonstrate using NCoR siRNA and chromatin immunoprecipitation that endogenous NCoR is recruited to and represses the dihydrotestosterone (DHT)-liganded AR. Furthermore this study shows that NCoR and coactivators compete for AR in the presence of DHT. AR antagonists such as bicalutamide that are currently in use for prostate cancer treatment can also mediate NCoR recruitment, but mifepristone (RU486) at nanomolar concentrations is unique in its ability to markedly enhance the AR-NCoR interaction. The RU486-liganded AR interacted with a C-terminal fragment of NCoR, and this interaction was mediated by the two most C-terminal nuclear receptor interacting domains (RIDs) present in NCoR. Significantly, in addition to the AR ligand binding domain, the AR N terminus was also required for this interaction. Mutagenesis studies demonstrate that the N-terminal surface of the AR-mediating NCoR recruitment was distinct from tau5 and from the FXXLF motif that mediates agonist-induced N-C-terminal interaction. Taken together these data demonstrate that NCoR is a physiological regulator of the AR and reveal a new mechanism for AR antagonism that may be exploited for the development of more potent AR antagonists. The androgen receptor (AR), 1The abbreviations used are: AR, androgen receptor; DHT, dihydrotestosterone; PSA, prostate-specific antigen; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; LBD, ligand binding domain; RLU, relative light units; RID, receptor interacting domains; HA, hemagglutinin; DBD, DNA binding domain; CMV, cytomegalovirus; NCoR, nuclear corepressor; siRNA, small interfering RNAs. 1The abbreviations used are: AR, androgen receptor; DHT, dihydrotestosterone; PSA, prostate-specific antigen; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; LBD, ligand binding domain; RLU, relative light units; RID, receptor interacting domains; HA, hemagglutinin; DBD, DNA binding domain; CMV, cytomegalovirus; NCoR, nuclear corepressor; siRNA, small interfering RNAs. a member of the steroid/nuclear receptor superfamily, plays a critical role in normal male development, including the development of the prostate gland. In addition, AR action plays a fundamental role in the development and progression of prostate cancer (1Catalona W.J. N. Engl. J. Med. 1994; 331: 996-1004Crossref PubMed Scopus (266) Google Scholar, 2Brinkmann A.O. Blok L.J. de Ruiter P.E. Doesburg P. Steketee K. Berrevoets C.A. Trapman J. J. Steroid Biochem. Mol. Biol. 1999; 69: 307-313Crossref PubMed Scopus (256) Google Scholar, 3Gelmann E.P. J. Clin. Oncol. 2002; 20: 3001-3015Crossref PubMed Scopus (733) Google Scholar). Prostate cancers are initially androgen responsive such that targeted therapies aimed at lowering circulating androgen levels are the treatment of choice for metastatic disease. In most cases, however, the disease becomes progressive and unresponsive to androgen ablation therapies. This progressive stage of the disease, referred to as hormone refractory or androgen-independent prostate cancer, is generally heralded by the re-expression of androgen-regulated genes such as prostate-specific antigen (PSA). AR gene amplification or mutations may contribute to this re-expression of androgen-regulated genes, but it occurs mainly through undefined molecular mechanisms that allow for AR signaling in the absence of ligand or at reduced systemic androgen levels (4Culig Z. Hobisch A. Cronauer M.V. Cato A.C. Hittmair A. Radmayr C. Eberle J. Bartsch G. Klocker H. Mol. Endocrinol. 1993; 7: 1541-1550Crossref PubMed Scopus (397) Google Scholar, 5Visakorpi T. Hyytinen E. Koivisto P. Tanner M. Keinanen R. Palmberg C. Palotie A. Tammela T. Isola J. Kallioniemi O.P. Nat. Genet. 1995; 9: 401-406Crossref PubMed Scopus (1229) Google Scholar, 6Taplin M.E. Bubley G.J. Shuster T.D. Frantz M.E. Spooner A.E. Ogata G.K. Keer H.N. Balk S.P. N. Engl. J. Med. 1995; 332: 1393-1398Crossref PubMed Scopus (1030) Google Scholar, 7Feldman B.J. Feldman D. Nat. Rev. Cancer. 2001; 1: 34-45Crossref PubMed Scopus (1945) Google Scholar, 8Balk S.P. 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In contrast, in the presence of a hormone antagonist, steroid receptors fail to recruit coactivators. Moreover, certain antagonists preferentially stimulate the recruitment of nuclear receptor corepressors to target promoters, which in turn recruit a multiprotein complex that leads to histone deacetylation and transcriptional repression (11Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.A. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2917) Google Scholar, 12Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2224) Google Scholar, 13Jackson T.A. Richer J.K. Bain D.L. Takimoto G.S. Tung L. Horwitz K.B. Mol. Endocrinol. 1997; 11: 693-705Crossref PubMed Scopus (383) Google Scholar, 14Smith C.L. Nawaz Z. O'Malley B.W. Mol. Endocrinol. 1997; 11: 657-666Crossref PubMed Scopus (556) Google Scholar).The AR is structurally similar to other steroid receptors in that it is recruited to target elements as a homodimer and contains a high affinity, steroid-specific ligand binding domain. However the AR differs in the molecular mechanisms by which it recruits both coactivators and corepressors. Unlike other family members, the ligand-binding domain of the AR preferentially recruits its own AF-1 domain via an N-terminal FXXLF motif, which interacts strongly with the hydrophobic cleft of the LBD created by ligand binding (15Wong C.I. Zhou Z.X. Sar M. Wilson E.M. J. Biol. Chem. 1993; 268: 19004-19012Abstract Full Text PDF PubMed Google Scholar, 16Doesburg P. Kuil C.W. Berrevoets C.A. Steketee K. Faber P.W. Mulder E. Brinkmann A.O. Trapman J. Biochemistry. 1997; 36: 1052-1064Crossref PubMed Scopus (181) Google Scholar, 17Ikonen T. Palvimo J.J. Janne O.A. J. Biol. 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Endocrinol. 2003; 213: 79-85Crossref PubMed Scopus (43) Google Scholar, 28Berrevoets C.A. Umar A. Trapman J. Brinkmann A.O. Biochem. J. 2004; 379: 731-738Crossref PubMed Scopus (48) Google Scholar) have shown that the agonist-liganded AR can recruit the corepressors NCoR and SMRT, suggesting that agonist-dependent activation of the AR may be dependent upon the relative levels of these corepressors versus coactivators.Besides the physiological agonists testosterone and DHT, the AR can also interact with many other steroidal or nonsteroidal drugs that function as relatively pure antagonists (such as hydroxyflutamide and bicalutamide) or as partial agonists (29Berrevoets C.A. Umar A. Brinkmann A.O. Mol. Cell. Endocrinol. 2002; 198: 97-103Crossref PubMed Scopus (58) Google Scholar). Bicalutamide, which is widely used for prostate cancer treatment, can stimulate the AR to bind DNA, but fails to recruit coactivators and can mediate the recruitment of NCoR to the androgen-regulated PSA gene, indicating that corepressor recruitment may contribute to antagonist activity (30Masiello D. Cheng S. Bubley G.J. Lu M.L. Balk S.P. J. Biol. Chem. 2002; 277: 26321-26326Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 31Shang Y. Myers M. Brown M. Mol. Cell. 2002; 9: 601-610Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 32Chen C.D. Welsbie D.S. Tran C. Baek S.H. Chen R. Vessella R. Rosenfeld M.G. Sawyers C.L. Nat. Med. 2004; 10: 33-39Crossref PubMed Scopus (1938) Google Scholar). Nonetheless, bicalutamide has limited efficacy in the advanced androgen-independent stage of prostate cancer, and other AR antagonists are similarly ineffective at this stage of the disease (33Joyce R. Fenton M.A. Rode P. Constantine M. Gaynes L. Kolvenbag G. DeWolf W. Balk S. Taplin M.E. Bubley G.J. J. Urol. 1998; 159: 149-153Crossref PubMed Scopus (185) Google Scholar). As enhancement of corepressor recruitment to the AR may be an effective approach for blocking AR signaling in prostate cancer, this study further examines corepressor recruitment by AR agonists and antagonists.Loss of function experiments were carried out initially using NCoR siRNA, in conjunction with chromatin immunoprecipitation (ChIP). These confirmed that endogenous NCoR could negatively regulate the activity of the DHT-liganded AR, and indicated that AR activity may be regulated by the relative levels of NCoR and coactivators. Although multiple other ligands could mediate AR-NCoR interaction, the AR partial agonist RU486 (mifepristone) functioned uniquely, at nanomolar concentrations, as a strong enhancer of this interaction. Significantly, while NCoR RIDs and the AR LBD contributed to AR-NCoR binding, this was markedly enhanced by a further interaction with the AR N terminus via a site that was independent of the N-terminal FXXLF motif. These results demonstrate that NCoR is a biological regulator of AR action, and identify a new role for the AR N terminus in the AR-NCoR interaction. Moreover, the marked enhancement of the AR-NCoR interaction by RU486, but not by AR antagonists currently in clinical use, indicates that this interaction is a target for the development of new potent AR antagonists.MATERIALS AND METHODSPlasmids and Reagents—Expression vectors for AR (pSVARo) ERα (pcDNA-ERα), NCoR (PKCR2-NCoR), and SRC1 (pSG5-SRC1) have been described previously (24Cheng S. Brzostek S. Lee S.R. Hollenberg A.N. Balk S.P. Mol. Endocrinol. 2002; 16: 1492-1501Crossref PubMed Scopus (94) Google Scholar, 34Cohen R.N. Brzostek S. Kim B. Chorev M. Wondisford F.E. Hollenberg A.N. Mol. Endocrinol. 2001; 15: 1049-1061Crossref PubMed Scopus (102) Google Scholar, 35Makowski A. Brzostek S. Cohen R.N. Hollenberg A.N. Mol. Endocrinol. 2003; 17: 273-286Crossref PubMed Scopus (63) Google Scholar). The NCoRc vector (pKCR2-NCoRc) was referred to previously as NCoRI and encodes the C-terminal amino acids 1574–2454 of NCoR (numbering is based on murine NCoR). VP16-NCoRc encodes the three NCoR RIDs from NCoRc fused to the VP16 transactivation domain in the AASVVP16 vector (amino acids 1806–2454). Additional fusion protein constructs with VP16 were constructed in the pACT vector, while fusions to the Gal4 DNA binding domain were in the pBind vector (Promega, Madison, WI). The VP16 fusions with AR include VP16-AR (encoding the full-length AR), VP16-ARDBD-LBD (encoding amino acids 501–919), VP16-ARNTD (amino acids 1–500), VP16-ARLBD (amino acids 661–919), and have been described (24Cheng S. Brzostek S. Lee S.R. Hollenberg A.N. Balk S.P. Mol. Endocrinol. 2002; 16: 1492-1501Crossref PubMed Scopus (94) Google Scholar). Additional NCoR fragments fused to VP16 were generated by ligating the indicated PCR-amplified regions (numbering is based on the murine NCoR sequence) into pACT with a C-terminal HA tag. GST-NCoR (N2N1) contains amino acids 2063–2300 of hNCoR fused to GST in PGEX4T1 and has been described previously (36Cohen R.N. Wondisford F.E. Hollenberg A.N. Mol. Endocrinol. 1998; 12: 1567-1581Crossref PubMed Scopus (63) Google Scholar, 37Cohen R.N. Putney A. Wondisford F.E. Hollenberg A.N. Mol. Endocrinol. 2000; 14: 900-914Crossref PubMed Scopus (90) Google Scholar) The AR mutant F23A was kindly provided by E. Wilson. The mutants LF26,27AA and ARdel23–27 were generated from pSVARo using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The Gal4-NCoRc CoRNR box mutants were described previously (34Cohen R.N. Brzostek S. Kim B. Chorev M. Wondisford F.E. Hollenberg A.N. Mol. Endocrinol. 2001; 15: 1049-1061Crossref PubMed Scopus (102) Google Scholar, 35Makowski A. Brzostek S. Cohen R.N. Hollenberg A.N. Mol. Endocrinol. 2003; 17: 273-286Crossref PubMed Scopus (63) Google Scholar). The reporter constructs ARE4-luciferase, containing four tandem copies of a synthetic ARE, and the estrogen response element (ERE2)-luciferase containing two EREs have been described (24Cheng S. Brzostek S. Lee S.R. Hollenberg A.N. Balk S.P. Mol. Endocrinol. 2002; 16: 1492-1501Crossref PubMed Scopus (94) Google Scholar). pG5-luciferase, regulated by five tandem Gal4 binding sites, and pRL-CMV, a CMV promoter-regulated Renilla control, were from Promega. Dihydrotestosterone (DHT), estradiol (E2), and RU486 were from Sigma and were used as 1:1000 stock solutions in ethanol.Cell Culture and Transfection—CV1 and 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). Cells in 48-well tissue culture plates in DMEM containing 10% charcoal dextran-stripped fetal bovine serum (CDS-FBS, Hyclone) were cotransfected using Lipofectamine 2000 (Invitrogen). Cells were transfected with 50 ng of reporter vector, expression vector, and coregulators (NCoR, NCoRc, SRC1) except where indicated, and with 1.25 ng of pRL-CMV Renilla vector for normalization. After 24 h the medium was replaced with fresh DMEM/10% CDS-FBS medium containing hormones or drugs at the indicated final concentrations. Following a further 24 h, firefly, and Renilla luciferase activities were assayed with the dual-luciferase assay system from Promega as per the supplier's instructions. All samples were in triplicate or quadruplicate and firefly luciferase activities were normalized for cotransfected Renilla activity. To assess expression of transfected proteins, lysates from replicate wells were prepared in 1% SDS, run on 8 or 10% SDS-PAGE, and immunoblotted with anti-HA (Covance) or anti-AR antibodies (Santa Cruz Biotechnology) (1:1000 dilutions incubated overnight at 4 °C), followed by horseradish peroxidase-conjugated anti-mouse or rabbit Ig secondaries (Promega) and ECL (Amersham Biosciences).RNA Interference—Vectors expressing hairpin small interfering RNAs (siRNA) under the control of the mouse U6 promoter were constructed by inserting pairs of annealed DNA oligonucleotides into pBS/U6 plasmid (a gift of Y. Shi) at ApaI-EcoRI sites (38Sui G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google Scholar). The target sequence for NcoR was: 5′-GGGCTTATGGAGGACCCTATGA-3′. To assess effects on AR transactivation, this siRNA plasmid (pU6-NCoR) or a control laminin siRNA plasmid (pU6-laminin) were cotransfected with the AR expression vector and the indicated reporters. To assess protein expression, cells in 6-well plates were transfected with 0.1–1.6 μg of vector per well. At 48 h after transfection the cells were collected, and nuclear extracts were isolated (39Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3908) Google Scholar). Nuclear proteins were separated by electrophoresis in 10% SDS-polyacrylamide gels and transferred overnight to nitrocellulose membranes. Blots were probed with a 1:500 dilution of an affinity-purified anti-NCoR antiserum in Tris-buffered saline containing 5% nonfat milk and 0.05% Tween 20, followed by horseradish peroxidase-conjugated anti-rabbit antibody (Amersham Biosciences) at 1:1000 dilution (24Cheng S. Brzostek S. Lee S.R. Hollenberg A.N. Balk S.P. Mol. Endocrinol. 2002; 16: 1492-1501Crossref PubMed Scopus (94) Google Scholar). The blots were visualized using ECL Plus Western blotting detection system.Chromatin Immunoprecipitation—LNCaP prostate cancer cells grown to ∼80% confluence in 10-cm plates were switched to steroid hormone-depleted medium (RPMI 1640/10% CS-FCS) for 48 h and then exposed to 10 nm DHT for varying times. Plates were then rinsed with PBS and fixed for 10 min at room temperature with 1% formaldehyde in PBS. After rinsing twice with ice-cold PBS, cross-linking was terminated by scraping cells into 1 ml of 100 mm Tris, pH 9.4 and 10 mm dithiothreitol and incubating at 30 °C for 15 min. Cell pellets were then washed twice with PBS and resuspended in 0.3 ml of lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris, pH 8.1, and protease inhibitors). Chromatin was sheared to 300–1000 bp with three sequential 10 s pulses at 70% power with a microtip ultrasonic cell dismembrator (Fisher). Cell debris was pelleted at 10,000 × g, and the supernatant was precleared with 10 μg of sheared salmon sperm DNA, 20 μl of non-immune sera, and 20 μl of a 50% slurry of protein A-Sepharose. Immunoprecipitation was with 500 ng of AR N-terminal rabbit polyclonal antibody (PG-21, Upstate Biotechnology), affinity-purified NCoR C-terminal anti-peptide antibody (24Cheng S. Brzostek S. Lee S.R. Hollenberg A.N. Balk S.P. Mol. Endocrinol. 2002; 16: 1492-1501Crossref PubMed Scopus (94) Google Scholar) or a nonspecific control antibody, 2 μg of sheared salmon sperm DNA, and 20 μl of a 50% slurry of protein A-Sepharose. Precipitates were washed three times with 300 mm NaCl, 50 mm Tris, pH 8.0, 2.7 mm KCl, 0.05% Tween-20 and 1% deoxycholate. Three additional washes with 10 mm Tris, 1 mm EDTA were performed and then beads were eluted three times with 35 μl of 1% SDS and 100 mm NaHCO3 at 37 °C for 10 min each. Eluates were pooled and incubated at 65 °C overnight to reverse cross-links. Products were then purified with QiaQuick PCR purification spin resin (Qiagen, Valencia, CA) and 10% of the eluate was subjected to 50 cycles of PCR amplification with steps of 94 °C, 55 °C, and 72 °C for 1 min each. The primers for the p21 cyclin-dependent kinase promoter were 5′-AAGCTCCCTCCTGGACCCAGA-3′ and 5′-CAAGCCTCAGAGCATCAG-3′; and for the PSA promoter 5′-GAGAGCTAGCACTTGCTGTT-3′ and 5′-AGTTCTAGTTTCTGGTCTCA-3′. PCR products were analyzed by gel electrophoresis and ethidium bromide staining in 4% agarose gels.GST Pull-down Assays—GST-NCoR (N2N1) and GST alone were produced as described previously and purified using glutathione-agarose beads (36Cohen R.N. Wondisford F.E. Hollenberg A.N. Mol. Endocrinol. 1998; 12: 1567-1581Crossref PubMed Scopus (63) Google Scholar, 37Cohen R.N. Putney A. Wondisford F.E. Hollenberg A.N. Mol. Endocrinol. 2000; 14: 900-914Crossref PubMed Scopus (90) Google Scholar) and there integrity was visualized on SDS-PAGE. To derive the indicated AR moiety, CV1 cells in 60-mm plates were transfected with either full-length AR, AR N-DBD, or D-LBD as described above. After 24 h, medium was replaced with fresh medium with or without 10 nm DHT or RU486. Cells were washed twice with ice-cold PBS and extracts prepared by scraping cells into 0.75 ml of lysis buffer (PBS, 5% glycerol, 0.05% Triton X-100 and protease inhibitors). Lysates were incubated on ice for 5 min and sonicated with two sequential 5-s pulses at 30% power with a microtip ultrasonic dismembrator (Fisher) and centrifuged at 4 °C to pellet insoluble material. Cell extracts were precleared with GST agarose beads for 2 h at 4 °C on a rotating wheel. Cleared lysates were incubated for 16 h with or without 10 nm DHT or RU486 at 4 °C on a rotating wheel in the presence of equal amounts of GST or GST-NCoR (N2N1) agarose beads. The beads were washed once with ice-cold lysis buffer and three times with ice-cold PBS and bound proteins were eluted by boiling in Laemmli sample buffer (Promega), run on 12% SDS-PAGE and blotted onto nitrocellulose membranes. To visualize AR proteins, Western blots were probed with either a polyclonal antiserum recognizing the first 20 or last 19 residues of hAR (Santa Cruz Biotechnology). Five percent of total cell lysates was loaded as input.RESULTSNCoR Down-regulation Enhances AR Transcriptional Activity—Previous studies have shown that transfected NCoR could repress the transcriptional activity of the DHT-liganded AR (24Cheng S. Brzostek S. Lee S.R. Hollenberg A.N. Balk S.P. Mol. Endocrinol. 2002; 16: 1492-1501Crossref PubMed Scopus (94) Google Scholar). RNAi was used to directly address whether endogenous NCoR functions as a negative regulator of AR transcriptional activity. CV1 cells were transfected with an AR expression vector and AR-regulated luciferase reporter gene, with or without plasmids encoding an NCoR or control siRNA expression vector (pU6-NCoR or pU6-laminin, respectively). As shown in Fig. 1A, AR transcriptional activity was augmented by the NCoR siRNA, but not the control siRNA plasmid. Consistent with this result, immunoblotting showed that NCoR protein expression was markedly down-regulated by the NCoR siRNA (Fig. 1B).We showed previously that repression of AR transcriptional activity by transfected NCoR was dependent upon an interaction between NCoR receptor interacting domains and the AR LBD (24Cheng S. Brzostek S. Lee S.R. Hollenberg A.N. Balk S.P. Mol. Endocrinol. 2002; 16: 1492-1501Crossref PubMed Scopus (94) Google Scholar). Therefore, another control for nonspecific siRNA effects was to examine whether enhancement of AR activity by the NCoR siRNA was dependent on the AR LBD. In contrast to full-length AR, an AR expression vector with the LBD-deleted (AR N-DBD, encoding the N terminus and DBD) was constitutively active in the absence of DHT and was not stimulated by the NCoR siRNA (Fig. 1C).As a further control, we examined the estradiol-liganded ERα. Whereas NCoR can interact with ERα when it is liganded by certain partial agonists, the estradiol-liganded ERα is not represssed by NCoR transfection. As shown in Fig. 2, the NCoR siRNA did not enhance the activity of the estradiol-liganded ERα, while AR activity was again markedly enhanced in the same experiment. Taken together, these data supported the conclusion that endogenous NCoR functions to suppress the transcriptional activity of the agonist-liganded AR.Fig. 2NCoR levels do not influence ER-mediated action. CV1 cells were transfected with either ERα and an ERE-Luc reporter or the AR and the ARE4-Luc reporter. All transfections also included the pRL-CMV control as well as well as the indicated amounts of pU6-NCoR. Cells were treated with the indicated ligands for 24 h, and luciferase versus Renilla luciferase activities were determined from triplicate samples (expressed as RLU ± S.E.).View Large Image Figure ViewerDownload Hi-res image Download (PPT)NCoR Is Recruited to Androgen-regulated Genes by the Endogenous DHT-liganded AR in Prostate Cancer Cells—Although we have been able to markedly down-regulate NCoR expression by siRNA and enhance AR activity in CV1 and 293T cells (data not shown) efforts to substantially down-regulate NCoR protein in a prostate cancer cell line expressing endogenous AR have not yet been successful. Therefore, chromatin immunoprecipitation was used as an alternative approach to test the hypothesis that NCoR associates with the DHT-liganded endogenous AR and is recruited to AR-regulated genes. Androgen responsive LNCaP prostate cancer cells were grown in steroid hormone-depleted medium for 2 days, and were then pulsed with DHT. Chromatin was cross-linked with formaldehyde at varying times after the DHT pulse, and sheared chromatin was immunoprecipitated with anti-AR, anti-NCoR, or control antibodies. PCR was then used to assess AR and NCoR recruitment to AREs in the p21 cyclin-dependent kinase inhibitor promoter and PSA regulatory regions. As shown in Fig. 3A, DHT treatment led to the recruitment of AR to the p21 gene. Significantly, NCoR was not associated with the p21 ARE in the absence of DHT, consistent with the lack of AR binding, but became associated after DHT treatment. Interestingly, the binding of both AR and NCoR appeared to be transient based on this method, as has been reported previously for AR and ERα (31Shang Y. Myers M. Brown M. Mol. Cell. 2002; 9: 601-610Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar).Fig. 3NCoR is recruited to both the p21 and PSA promoters in the presence of DHT.A, LNCaP cells were treated with DHT for the indicated number of minutes, fixed, sheared, and immunoprecipitated with the indicated antibodies (rabbit anti-PDGF receptor antibody was used as a negative control). After reversing the cross-links the solubilized chromatin was subjected to PCR with primers spanning the p21 ARE or control primers for the human cd1 gene. The resulting PCR products were resolved on ethidium bromide-stained agarose gels. Also shown is PCR from input chromatin used with the AR, NCoR, and control antiserum. B, an identical paradigm was used on precipitated DNA complexes treated as above and PCR-amplified with primers in the PSA promoter flanking the ARE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DHT similarly induced the association of both AR and NCoR with the PSA promoter (Fig. 3B), as well as the ARE in the PSA enhancer (data not shown). In this experiment the AR and NCoR association was not detected until 30–45 min, and it persisted for at least 2 h. More rapid and transient association of both AR and NCoR have been observed in other experiments (data not shown), and the basis for the variable recruitment kinetics are not yet clear. Nonetheless, a consistent correlation has been observed between AR and NCoR recruitment in response to DHT. It should be noted

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