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Originally published In Press as doi:10.1074/jbc.M106399200 on November 19, 2001

J. Biol. Chem., Vol. 277, Issue 3, 2207-2215, January 18, 2002
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Cyclin D1: Mechanism and Consequence of Androgen Receptor Co-repressor Activity*

Christin E. PetreDagger §, Yelena B. WetherillDagger , Mark Danielsen||, and Karen E. KnudsenDagger **

From the Dagger  Department of Cell Biology, the University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521 and the || Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D. C. 20007

Received for publication, July 9, 2001, and in revised form, October 11, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Androgen receptor regulation is pivotal for prostate growth and development. Activation of the receptor is dictated by association with androgen (ligand) and through interaction with co-activators and co-repressors. We have shown previously that cyclin D1 functions as a co-repressor to inhibit ligand-dependent androgen receptor activation. We demonstrate that cyclin D1 directly binds the N terminus of the androgen receptor and that this interaction is independent of ligand. Furthermore, we show that the interaction occurs in the nucleus and does not require the LXXLL motif of cyclin D1. Although two distinct transactivation domains exist in the N terminus (AF-1 and AF-5), the data shown support the hypothesis that cyclin D1 targets the AF-1 transactivation function. The constitutively active AF-5 domain was refractory to cyclin D1 inhibition. By contrast, cyclin D1 completely abolished androgen receptor activity, even in the presence of potent androgen receptor co-activators. This action of cyclin D1 at least partially required de-acetylase activity. Finally, we show that transient, ectopic expression of cyclin D1 results in reduced cell cycle progression in androgen-dependent LNCaP cells independent of CDK4 association. Collectively, our data support a model wherein cyclin D1 has a mitogenic (CDK4-dependent) function and an anti-mitogenic function (dependent on regulation of the AF-1 domain) that can collectively control the rate of androgen-dependent cellular proliferation. These findings provide insight into the non-cell cycle functions of cyclin D1 and provide the impetus to study its pleiotropic effects in androgen-dependent cells, especially prostatic adenocarcinomas.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The androgen receptor (AR)1 is a 110-kDa ligand-dependent transcription factor whose mis-regulation is implicated in the formation and progression of prostatic adenocarcinoma (1-3). Development, growth, and survival of prostatic epithelia are dependent on serum androgen, which exerts its biological effect through the AR (4-6). It has been suggested that aberrant or inappropriate activation of the AR facilitates the formation of prostate hyperplasias or neoplasias. Given the importance of the AR in prostate cancer progression, it is critical to determine its precise mode of regulation.

The AR is expressed at high levels in prostatic epithelial cells and is activated in this cell type by dihydrotestosterone (DHT), a high affinity ligand for the AR (1, 7). Prior to DHT binding, the AR exists diffusely throughout the cytoplasm and nucleus of the cell and is held inactive through the interaction of specific heat shock proteins (8, 9). Upon ligand binding, heat shock proteins are displaced. The receptor then dimerizes, translocates to the nucleus, and modulates transcription from discrete DNA sequences, termed androgen-responsive elements (10-12). From androgen-responsive elements, the receptor is capable of mediating transactivation and potential transcriptional repression (13). Although activation of the AR is clearly required for prostate proliferation, its critical transcription targets have yet to be identified. The best known target of the AR is prostate-specific antigen (PSA), whose expression levels are monitored clinically to diagnose aberrant prostate growth (14-16).

Like all nuclear receptors, the AR is loosely divided into three functional domains as follows: a C-terminal ligand binding domain, a DNA binding domain, and a variable N-terminal region (17, 18). Although in other nuclear receptors the C-terminal ligand binding domain contains a potent transactivation function, the C-terminal transactivation domain of the AR (AF-2) is relatively weak (18-20). The AR is unique in that two additional transactivation functions exist in the N terminus, termed AF-1 and AF-5 (21, 22). Like AF-2, the AF-1 transactivation function is dependent on ligand binding to the receptor (2). By contrast, AF-5 transactivation is constitutively active (21, 22). In the absence of ligand, it is hypothesized that the C terminus folds in such a way as to inhibit N-terminal transactivation domains (2, 19, 23-25). Deletion of the C terminus permits ligand-independent AF-5 activation, supporting this hypothesis (21). In the full-length receptor it is suggested that ligand binding fosters a conformational change that promotes interaction of the N-terminal and C-terminal transactivation functions (25). Apart from these regulatory mechanisms, emerging evidence makes clear that meaningful activation of transcription also requires the recruitment of transcriptional co-modifiers (13, 26, 27).

The AR is known to interact with a series of co-activators, and a smaller subset of co-repressors (13, 26, 27). Interaction with these regulatory proteins shows some specificity with regard to transactivation functions. For example, TIF-1 and GRIP2 are known to act on the C-terminal AF-2 domain, whereas SRC1 also interacts with and activates the N-terminal transactivation functions (19, 20, 28, 29). The net result of co-activator recruitment is to stimulate transcription of target genes, at least in part through the modification of histones. Several known co-activators (e.g. P/CAF, p300, and SRC-1) contain inherent histone acetylation activity (HAT) (30-32). This enzymatic activity is critical because histone acetylation is thought to relax chromatin, thereby facilitating DNA unwinding required for gene transcription (33, 34). Co-activators that lack HAT activity (e.g. ARA70) are speculated to function by recruiting HATs to the promoter complex (35).

Counter-balancing the effect of these co-activators are the co-repressors. As might be expected, these proteins are thought to either harbor intrinsic histone deacetylase (HDAC) activity or to recruit HDACs to the receptor complex (33, 34). The AR is speculated to interact with general HDAC recruiting proteins, such as NcoR and SMRT (36, 37). The mechanisms governing these interactions have yet to be fully explored.

Intriguingly, we have previously identified cyclin D1 as a co-modifier of AR activity (38). Cyclin D1 has an important role in cell cycle control as a required component of the CDK4 kinase complex (39). Cyclin D1 binds CDK4 directly and initiates CDK4-mediated phosphorylation of the retinoblastoma tumor suppressor protein, RB (40, 41). Because inactivation of RB is required for cell cycle progression, cyclin D1 is requisite for cellular proliferation (42). Outside of this role in the cell cycle, cyclin D1 is known to bind estrogen receptor (ER)-alpha and activate its transactivation function in the absence of ligand (43, 44). This function of cyclin D1 requires its LXXLL motif and is thought to involve the recruitment of SRC-1 (45). In contrast, we have demonstrated previously (38) an additional role for cyclin D1. We showed that cyclin D1 interacts with the AR in vivo and inhibits its transactivation potential, without affecting AR expression. This inhibition is independent of CDK4 and thus also independent of the role of cyclin D1 in RB phosphorylation.

We demonstrate that cyclin D1 binds with high affinity to the N terminus of the AR and inhibits the AF-1 transactivation function. We show that the inhibitory action of cyclin D1 is dominant to both HAT and non-HAT co-activators and that the complete action of cyclin D1 requires regulation of acetylation. Interestingly, ectopic expression of cyclin D1 or a mutant incapable of binding to CDK4 reduced the proliferative index of an androgen-dependent prostatic cell line (LNCaP), indicating that cyclin D1-induced AR inhibition acts to limit cellular proliferation in this cell type. These data put forth the hypothesis that cyclin D1 has dual roles in androgen-dependent cell types as follows: one that is CDK4-dependent and initiates cell cycle progression and another that is independent of CDK4 and counter-balances its mitogenic activity in androgen-dependent cells. Thus, our results classify cyclin D1 as a potent AR co-repressor and provide the impetus to study deregulation of the cyclin D1/AR interaction in prostate carcinoma.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Treatment-- CV1, C33A, and LNCaP cell lines were obtained from ATCC and maintained in a 5% CO2 incubator. CV1 and C33A cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA), 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin (Mediatech, Herndon, VA). For CV1 and C33A reporter assays, 10% charcoal dextran-treated FBS (HyClone Laboratories, Logan, UT) and phenol red-free Dulbecco's modified Eagle's medium were utilized. LNCaP cells were maintained in Iscove's modified Eagle's medium supplemented with 5% heat-inactivated FBS, 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin. The HDAC inhibitor trichostatin A was obtained from Sigma.

Transfection and Transcriptional Reporter Assays-- CV1 or C33A cells were transfected in the absence of androgen with the indicated plasmid constructs using the BES/calcium phosphate protocol (46). LNCaP cells were transfected with the indicated plasmids using FuGENE6 transfection reagent (Roche Molecular Biochemicals), in accordance with the manufacturer's recommended protocol. Post-transfection, CV1 and C33A cells were allowed to recover for a period of 5-6 h and then supplemented with 0.1 nM dihydrotestosterone (DHT; Sigma), 1 nM R1881, or 0.1% ethanol vehicle (ETOH) for 18 h. Following stimulation, cells were harvested, and luciferase activity was quantified using the Promega luciferase assay kit (Promega, Madison, WI). beta -Galactosidase activity was used as an internal control for transfection efficiency and measured by classic colorimetric assay or Galacto-Star protocols (Tropix, Bedford, MA).

Plasmids-- The pSG5AR wild type androgen receptor expression plasmid and pSG5-ARA70 were kindly provided by Dr. Chawnshang Chang (University of Rochester, Rochester, NY) (35). The pAR5 constitutively active androgen receptor construct was the gift of Dr. Albert O. Brinkmann (Erasmus University Rotterdam, Rotterdam, The Netherlands) (47). The pSG5-AR-T877A biologically relevant androgen receptor mutation construct was kindly provided by Dr. David Feldman (Stanford University School of Medicine, Stanford, CA). The CMV-beta -galactosidase construct was the gift of Dr. Jean Wang (University of California at San Diego, La Jolla, CA). The PSA61LUC reporter was kindly provided by Dr. Kitty Cleutjens (Erasmus Universiteit (48)) and contains 6.1 kb of the human PSA promoter. The pRc/CMV-cyclin D1 and pRc/CMV-cyclin D1-LALA constructs were gifts of Dr. R. Bernards (The Netherlands Cancer Institute, Amsterdam, The Netherlands) (45). The pFlex-D1-T286A nuclear cyclin D1 expression plasmid was the gift Dr. Alan Diehl (University of Nebraska Medical Center, Omaha, NE) (49). The pCR3.1 hSRC-1A construct was the gift of Dr. B. W. O'Malley (Baylor College of Medicine, Houston, TX) (50). Plasmid encoding H2B-GFP was a gift of Dr. G. Wahl (Salk Institute, La Jolla, CA). pCMVp300 was the gift of Dr. T. Kouzarides (Wellcome/CRC Institute). CMV-P/CAF was the generous gift of Dr. S. Y. R. Dent (M. D. Anderson Cancer Center, Houston, TX). The pGEMARDelta C, wtAR-pGEM, pAR1-501, pAR1-661, pAR506-918, and pAR623-918 have been described previously (51). The CMV-CD44 construct was the generous gift of Dr. L. Sherman (University of Cincinnati, Cincinnati, OH). pGEX 3× cyclin D1-GST was provided by Dr. M. Roussel (St. Jude Children's Hospital, Memphis, TN). The cyclin D1-KE construct was provided by Dr. R. Weinberg (Whitehead Institute for Biomedical Research, Cambridge, MA). The green fluorescent protein (GFP) encoding plasmid, Green lantern 2, was supplied from Invitrogen.

Immunoprecipitation and Immunoblots-- Cells transfected as described above were pelleted and lysed in a NETN (20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), and 0.5% Nonidet P-40) solution containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml 1,10-phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM sodium fluoride, 1 mM sodium vanadate, and 60 mM beta -glycerophosphate. Lysates were subjected to brief sonication and clarified by centrifugation. For immunoblots, equal protein was loaded and subjected to SDS-PAGE. For co-immunoprecipitations, equal amounts of protein were immunoprecipitated with antibodies directed against cyclin D1 (NeoMarkers, Fremont, CA), AR (Santa Cruz Biotechnology, Santa Cruz, CA), E1A (Santa Cruz Biotechnology), or MDM2 (Santa Cruz Biotechnology) antibodies. Precipitates were recovered through incubation with protein A-Sepharose beads (Amersham Biosciences). Input, bound, and flow-through fractions were obtained from each reaction and subjected to SDS-PAGE. Proteins were transferred to Immobilon (Millipore Corp., Bedford, MA) and immunoblotted for the indicated proteins. Antisera against GFP were purchased from Roche Molecular Biochemicals. Horseradish peroxidase-conjugated protein A (Bio-Rad) and enhanced chemiluminescence enhancer (PerkinElmer Life Sciences) were used to visualize proteins.

Binding Assays-- 35S-Labeled wild type and mutant androgen receptor proteins were generated using the TnT-coupled Reticulocyte Lysate system for in vitro transcription and translation (Promega), in accordance with the manufacturer's recommended protocol. NEG-772 Easytag Express Protein Labeling Mix was utilized for 35S-protein labeling (PerkinElmer Life Sciences). Reactions were performed in a total volume of 50 µl. GST-cyclin D1 was transformed into BL21 bacteria as specified by Novagen (Madison, WI). Cyclin D1 expression was induced through 1 mM isopropyl-1-thio-beta -D-galactopyranoside addition for a period of 3-5 h. Bacteria were harvested by centrifugation and resuspended in 4 ml of NET-N plus 0.003% Sarkosyl, phenylmethylsulfonyl fluoride, and protease inhibitors (NET-N + SPP). The resuspended pellets were sonicated and then supplemented with 1% Triton X-100. Lysed pellets were subjected to clarification. A portion of the remaining lysate was run on a SDS-PAGE gel and Coomassie-stained to verify expression (data not shown). From the total lysate 3 ml were removed and added to 50 µl of glutathione-agarose beads, which were incubated for 3 h at 4 °C. Beads were then washed 6 times with 1 ml of NET-N+SPP. In vitro translated proteins were incubated with the beads for 1.5 h at 4 °C with rotation. Beads were subsequently washed 5 times with 1 ml of NET-N + SPP. Input (4% of reaction), flow-through (3% of initial unbound fraction), and bound (10% of bound proteins prior to washing) were subjected to 10% SDS-PAGE. Following electrophoresis, the gel was incubated in Fluoro-Hance (Research Products International Corp., Mount Prospect, IL), as specified by the manufacturer. Proteins were detected via autoradiography.

Immunofluorescence-- LNCaP cells were seeded in 6-well dishes on poly-L-lysine-coated glass coverslips. On the following day, cells were transfected using FuGENE6 Transfection Reagent (Roche Molecular Biochemicals) as described in the manufacturer's protocol. 4 µg of wild type pRc/CMV-cyclin D1, cyclin D1-KE, or pcDNA3 (Invitrogen Carlsbad, CA) constructs were transfected overnight along with 1 µg of H2B-GFP. Following transfection, the media were replaced, and cells were permitted to express the constructs for 18 h. Subsequently, bromodeoxyuridine (BrdUrd, Amersham Biosciences) was added for a 16-h labeling period. Coverslips were fixed in 3.7% formaldehyde at room temperature for 15 min and then washed in phosphate-buffered saline. BrdUrd incorporation was determined by indirect immunofluorescence as previously described (52). Experiments were performed in duplicate and at least 200 transfected cells were tallied per coverslip.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cyclin D1 Regulation of the Androgen Receptor Is Independent of the LXXLL Motif-- We have shown previously that cyclin D1 does not affect AR expression but binds to the AR and inhibits its ligand-dependent transactivation (38). However, the mechanism underlying this inhibitory action of cyclin D1 was not determined. Because cyclin D1 modulates ER-alpha activity through its LXXLL motif (43-45), we hypothesized that the ability of cyclin D1 to inhibit AR activity may also rely on this interaction site. To test this hypothesis, we employed a characterized mutant of cyclin D1 (cyclin D1-LALA) that is defective in the LXXLL motif and lacks the ability to modulate ER function (45). CV1 cells, which express no endogenous AR (data not shown), were co-transfected in the absence of steroid hormones with plasmids encoding the human AR (pSG5-AR), PSA61LUC (a reporter for the AR containing a 6.1-kb fragment of the endogenous prostate-specific antigen promoter), and either wild type cyclin D1 or cyclin D1-LALA. CMV-beta -gal was included in all transfections for reporter assay, as a control for transfection efficiency. In parallel experiments, androgen receptor expression was monitored via immunoblot and compared with co-transfected GFP. Subsequent to transfection, cells were treated with either 1 nM R1881 (Fig. 1A), 0.1 nM DHT (Fig. 1B), or 0.1% ethanol vehicle. Treatment was continued for 16 h at which time cells were harvested, lysed, and monitored for either luciferase and beta -galactosidase activity or for AR and GFP expression. Relative luciferase activity is shown. In accordance with previous observations (38), R1881 up-regulated AR activity ~5-fold during the stimulation period (Fig. 1A), whereas the physiological concentration of DHT stimulated AR activity ~10-12-fold above vehicle treatment (Fig. 1B, upper panel). Transfection of multiple constructs did not affect AR expression (Fig. 1B, lower panel), but in both instances, wild type cyclin D1 and cyclin D1-LALA reduced its activity in the presence of ligand to basal levels (Fig. 1, A and B). These data indicate that the LXXLL motif of cyclin D1 is dispensable for inhibition of ligand-dependent AR transactivation potential.


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  		Fig. 1.   The LXXLL motif is dispensable for AR inhibition. A, CV1 cells were cultured in charcoal dextran-treated serum that contains growth factors but is devoid of steroids. Cells were subsequently co-transfected with constructs encoding beta -galactosidase (CMV-beta gal, 1.0 µg), wild type AR (pSG5-AR, 1.5 µg), 6.1 kb of the human PSA promoter linked to a luciferase reporter (PSA61LUC; 2 µg), and either wild type, mutant cyclin D1 (cyclinD1-LALA), or empty vector (pCDNA3; 4.5 µg). Following transfection, cells were stimulated with either R1881 (solid bars) or 0.1% ethanol vehicle (ETOH, striped bars) as indicated. After 16 h, cells were harvested, and luciferase activity was measured. These values were then normalized against beta -galactosidase activity. Experiments were performed at least in triplicate, and vehicle-treated AR transfection values were set to 1. Bars represent mean induction, and error bars indicate S.D. B, experiments were performed as in A, but 0.1 nM DHT was utilized to stimulate ligand-dependent AR activation (upper panel). Lysates from parallel experiments wherein 1.0 µg of the H2B-GFP plasmid was transfected in place of CMV-beta gal were subject to SDS-PAGE and immunoblotting (lower panel). C, CV1 cells were transfected with pSG5AR and cyclin D1-LALA at a 1:1 ratio. 48 h post-transfection, cells were lysed, and clarified extracts were used for immunoprecipitations (IP) with anti-AR, anti-cyclin D1, anti-MDM2, or anti-E1A antibodies. Protein antibody complexes were recovered through binding to protein A-Sepharose beads and washed extensively with NETN. The bound fraction was then boiled and protein content resolved on a 10% SDS-PAGE gel. Resolved proteins were transferred to an Immobilon membrane and probed with antibodies to recognize either the cyclin D1 (left panel) or the AR (right panel).

These findings predict that the cyclin D1-LALA mutant would retain binding to the AR. To verify this hypothesis, CV1 cells were transfected at equal ratios with expression plasmid for the wild type AR and cyclin D1-LALA. Transfected cells were lysed, and protein complexes were recovered using antisera generated against cyclin D1, AR, or negative controls (E1A or MDM2). Immunoprecipitated complexes were subjected to SDS-PAGE and immunoblotted for either the AR or cyclin D1. As shown in Fig. 1C, complexes immunoprecipitated using anti-cyclin D1 contained both cyclin D1-LALA and the AR (lanes 4 and 7, respectively). Conversely, complexes immunoprecipitated using anti-AR antisera contained both the AR and cyclin D1-LALA (lanes 2 and 8). Lysates immunoprecipitated using antisera against E1A or MDM2 failed to recover either cyclin D1 or the AR (lanes 3 and 6). Thus, cyclin D1-LALA retains its ability to bind AR. In addition, these data indicate that the mechanism by which cyclin D1 modulates AR activity is distinct from its ability to regulate the estrogen receptor.

Nuclear Cyclin D1 Inhibits Androgen Receptor Activity-- Cyclin D1 localization is known to transition from the nucleus to the cytoplasm throughout the cell cycle and in response to mitogen stimulation (53, 54). In a similar fashion, AR localization between nucleus and cytoplasm changes due to an external proliferative signal, androgen (ligand) (10, 11). Because it is possible that cyclin D1 inhibits AR activity through cytoplasmic sequestration, we employed an exclusively nuclear cyclin D1 mutant protein, cyclin D1-T286A (49). This well characterized mutant form of cyclin D1 cannot be phosphorylated by GSK-3beta or associate with CRM1 and therefore is not exported from the nucleus. By using this construct we assessed the ability of nuclear cyclin D1 to diminish AR transactivation through reporter assay. The nuclear cyclin D1 construct was transfected into CV1 cells along with wild type AR and reporter constructs as in Fig. 1. Transfected cells were subsequently stimulated with physiological levels of DHT (0.1 nM) for a period of 16 h. As seen in Fig. 2 (upper panel), cyclin D1-T286A retained AR inhibitory activity, reducing ligand-dependent AR transactivation to basal levels, comparable with that seen with wild type cyclin D1. Transfection of cyclin D1 constructs did not affect androgen receptor expression as shown by immunoblot (Fig. 2, lower panel). These results demonstrate that cyclin D1 has the ability to repress AR transactivation without preventing AR expression levels, nuclear translocation, or previous cytoplasmic complex formation.


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  		Fig. 2.   Nuclear cyclin D1 inhibits androgen receptor transactivation. CV1 cells were co-transfected with reporters and wild type or nuclear cyclin D1 T286A as in Fig. 1A, with the ratios indicated. AR activity in the absence of ligand was set to 1. Experiments were performed at least in triplicate (upper panel). Lysates from parallel experiments wherein 1.0 µg of the H2B-GFP plasmid was transfected in place of CMV-beta gal were subject to SDS-PAGE and immunoblotting (lower panel).

Cyclin D1 Interacts with the N-terminal Transactivation Functions-- Upon activation and DNA binding, the AR has the ability to modulate transcription of target genes. The specific form of transcriptional modification depends on the recruitment of co-activators or co-repressors (13, 27). To determine the mechanism by which cyclin D1 functions as a co-repressor for ligand-dependent AR transactivation, we first mapped the site of cyclin D1 interaction. Initially, interaction sites were determined using GST pull-down experiments (Fig. 3). Immobilized GST-cyclin D1 was recovered on glutathione-agarose. A series of previously characterized AR deletion constructs (51) were utilized to generate [35S]methionine-labeled proteins via in vitro transcription/translation. Plasmid encoding the cell surface protein CD44 was also subjected to in vitro transcription/translation as a negative control for the binding assay. In vitro translated proteins were incubated with immobilized GST-cyclin D1, washed, and collected. Input, bound, and flow-through fractions were resolved by SDS-PAGE, and proteins were detected by autoradiography. As shown in Fig. 3, in vitro translated CD44 was not retained by GST-cyclin D1-agarose (compare lanes 1-3), whereas wild type AR remained bound to the column (lanes 4-6). This finding is important, as we had demonstrated previously (38) cyclin D1 binding to AR only in vivo, via co-immunoprecipitation experiments. The in vitro binding studies shown here demonstrate that cyclin D1 binding to the AR does not require ligand and occurs in the absence of steroid receptor accessory factors. By using N- and C-terminal truncations of the androgen receptor, it was evident that N-terminal AR proteins (amino acids 1-661 and 1-502) bound strongly to GST-cyclin D1-agarose (compare lanes 7-9 and 10-12, respectively). By contrast, C-terminal fragments of the AR (amino acids 506-918 and 623-918) failed to bind cyclin D1 (compare lanes 13-18). These data demonstrate that a cyclin D1-binding site resides within amino acids 1-502 of the AR.


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  		Fig. 3.   Cyclin D1 binds the N terminus of the androgen receptor. GST-conjugated wild type cyclin D1 was expressed in E. coli and purified. Purification was verified by SDS-PAGE (data not shown). GST-cyclin D1 was bound to glutathione-agarose beads, washed, and subsequently incubated with [35S]methionine-labeled full-length or truncated AR proteins or CD44 control (as shown) at 4 °C with rotation for 1.5 h. GST-cyclin D1 beads were subsequently washed 5 times in NETN supplemented with 0.003% Sarkosyl. Column input, flow-through, and bound were subjected to SDS-PAGE and detected via autoradiography.

AF-5 Activity Is Refractory to Cyclin D1-mediated Inhibition-- The N terminus of the AR contains two independent transactivation domains, AF-1 and AF-5 (21, 22). The AF-1 function is ligand-dependent and requires the presence of the C terminus to invoke transactivation potential (2). In contrast, the AF-5 transactivation function is ligand-independent and does not require the presence of the C terminus. Thus, deletion of the C terminus results in a constitutively active receptor, dependent solely on the AF-5 activity (21). This mutant receptor, AR5, has been characterized previously to exhibit constitutive and yet diminished transactivational potential. Indeed, in our system, AR5 demonstrated a ligand-independent activity that was ~3-4-fold lower than the ligand-induced wild type receptor (data not shown). Because our findings implicated a binding region for cyclin D1 within the AR5 construct (amino acids 1-627), we tested the ability of cyclin D1 to abrogate AR5 transactivation. Strikingly, whereas AR5 expression levels remained constant, co-expression of cyclin D1 failed to inhibit the AF-5 transactivation function (Fig. 4A, upper panel). This finding was also recapitulated in reporter assays performed in C33A cells (data not shown). As with the wild type AR, expression levels of AR5 were unchanged by cyclin D1 co-expression (Fig. 4A, bottom panel).


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  		Fig. 4.   Cyclin D1 binds AR5 but fails to inhibit the constitutively active androgen receptor. A, CV1 cells were co-transfected with reporters as in Fig. 1A with expression plasmid for AR5 (a known constitutively active form of the receptor) in the presence or absence of cyclin D1, with the plasmid ratios indicated. Cells were treated with 0.1 nM DHT or ethanol vehicle, lysed, and relative luciferase activity measured. Constitutive AR5 activity in the presence of vehicle alone was set to 100% (upper panel). Reporter assays were performed at least in duplicate. Lysates from parallel experiments in C33A wherein 1.0 µg of the H2B-GFP plasmid was transfected in place of CMV-beta gal were subject to SDS-PAGE and immunoblotting (lower panel). B, CV1 cells cultured in the presence of steroid were transfected with pAR5 and/or cyclin D1 at a 1:1 ratio. As in Fig. 1B, cells were lysed, and protein was extracted and immunoprecipitated with anti-AR, anti-cyclin D1, and anti-E1A antibodies. Complexes were recovered on protein A-Sepharose beads, washed, and then subjected to SDS-PAGE. Resolved proteins were transferred to Immobilon and immunoblotted for cyclin D1.

To determine the ability of cyclin D1 to bind AR5, in vivo co-immunoprecipitation experiments were performed. Briefly, CV1 cells were co-transfected with expression plasmids for cyclin D1 and AR5. Post-transfection, cells were harvested, lysed, and subjected to co-immunoprecipitation using antisera generated against the AR. As can be seen in Fig. 4B, AR5 efficiently co-precipitates cyclin D1 (lane 4), in contrast to control antisera (lane 3). Thus, cyclin D1 binds and yet fails to inhibit constitutive AF-5 transactivation. Taken together, these data suggest that cyclin D1 acts through the AF-1 function to repress ligand-dependent transactivation.

Cyclin D1 Is Dominant to Known AR Co-activators-- Because our data implicated the AF-1 transactivation function as the site of cyclin D1 co-repressor activity, we began by examining the effect of cyclin D1 on general co-activators known to activate N-terminal AR transactivation domains (SRC-1) or harbor intrinsic HAT activity (P/CAF, p300) (19, 20, 28, 29). (Fig. 5, A-C). For these experiments, CV1 cells were transfected with plasmid encoding the wild type AR, PSA61LUC reporter, and specific N-terminal co-activators in the presence or absence of cyclin D1. Although each of these molecules has been shown to super-activate ligand-dependent AR transactivation, some of these initial characterizations were performed with relatively high ratios of AR to co-activator (up to 1:8 ratio). Because cyclin D1 demonstrates efficient AR inhibition at 1:3 ratios with the AR (or less (38)), each co-activator was tested for receptor activation at a 1:3 ratio. As shown in Fig. 5, A-C, DHT stimulated AR-dependent PSA61LUC activity ~12-fold. Co-expression of SRC-1, p300, and P/CAF at a 1:3 ratio enhances DHT-stimulated AR transactivation between 17- and 27-fold over cells treated with ethanol vehicle alone. These results clearly demonstrate that enhanced activation occurs even at relatively low receptor to co-activator ratios. Strikingly, equal expression of cyclin D1 (1:1 ratio with co-activator) reduced activation of the PSA reporter to basal (non-ligand dependent) levels. Immunoblots demonstrate that AR expression was not affected by the transfection of multiple constructs, as compared with the GFP control (Fig. 5E, compare lanes 1-7). These data demonstrate the dominance of cyclin D1 as a co-repressor against co-activators with intrinsic HAT activity.


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  		Fig. 5.   Cyclin D1 is dominant to androgen receptor co-activator function. CV1 cells were transfected as in Fig. 1A in the presence or absence of co-activator (A, SRC1; B, P/CAF; C, p300; and D, ARA70) and/or cyclin D1, at the relative plasmid ratios shown. Cells were stimulated, harvested, and monitored for beta -galactosidase activity as in Fig. 1A. AR activity in the presence of ethanol vehicle (ETOH) was set to 1 (upper panel). Data bars represent mean relative luciferase activity, and error bars indicate S.D. E, lysates from parallel experiments wherein 1.0 µg of the H2B-GFP plasmid was transfected in place of CMV-beta gal were subject to SDS-PAGE and immunoblotting.

In addition, we tested the ability of cyclin D1 to inhibit ARA70, a co-activator specific to the AR that binds the C terminus and lacks HAT activity (35). Although not a strong co-activator in all systems (55), ARA70 enhanced AR activity on the PSA promoter to ~32-fold above vehicle-treated cells, as compared with 12-fold activation by DHT alone (Fig. 5D). Co-expression of cyclin D1 once again demonstrated its potency in inhibiting AR transactivation potential, reducing activity to basal levels when transfected at equal ratios with ARA70. Although ARA70 binds the C terminus of the AR, it is known that interaction of this region with the N terminus is important for full activity (19, 25, 56). It is perhaps this action that is blocked by cyclin D1 binding. Taken together, these data demonstrate cyclin D1 co-repressor activity is dominant to both HAT and HAT recruiting co-activators.

Cyclin D1 Inhibition Is Partially Dependent on Histone Deacetylase Activity-- Because cyclin D1 binds the N terminus of AR and inhibits transactivation even in the presence of co-activators, we hypothesized that cyclin D1 action may be mediated through changes in chromatin acetylation. In general, co-activators harbor inherent histone acetylase activity (e.g. SRC1, p300, and P/CAF) or recruit histone acetylases to target sites (13, 27, 33). Similarly, many described co-repressors harbor HDAC activity (57); no such function has been ascribed to cyclin D1. To test the hypothesis that cyclin D1 action is dependent upon chromatin state, we examined the effect of TSA, a specific inhibitor of HDACs (58), to abrogate cyclin D1 function. It has been shown previously that TSA induces AR activity in the presence of ligand (59). For these experiments, cells were transfected as usual with AR and PSA61LUC in the presence or absence of cyclin D1. After transfection, cells were treated with either ethanol or DHT in the presence or absence of TSA. First, a dose-response curve was performed to determine the level of TSA required to maximally activate AR activity in our system, because TSA can have nonspecific effects on transcription. In our system maximal induction was achieved at a dose of 50 nM TSA (data not shown). Therefore, this concentration was chosen to study the ability of TSA to reverse cyclin D1 repression. As shown in Fig. 6, 50 nM TSA partially reversed the ability of cyclin D1 to inhibit AR transactivation (12-fold reduction in cyclin D1 repression). These data demonstrate that cyclin D1 is partially dependent on deacetylation to inhibit AR activity.


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  		Fig. 6.   Cyclin D1 action is partially abrogated through HDAC inhibition. CV1 cells were transfected as in Fig. 1A, with the relative plasmid ratios as indicated. Following recovery, cells were treated with 50 nM TSA and either 0.1 nM DHT or ethanol. Cells were incubated 16 h under these conditions and then harvested and monitored for beta -galactosidase and luciferase activity. AR activity in the absence of ligand was set to 1. Experiments were performed in triplicate.

Cyclin D1 Inhibits Androgen-dependent Proliferation-- Together, the data shown demonstrate the potency of cyclin D1 as a co-repressor of the androgen receptor. Androgen receptor activity is required for proliferation of prostate cells and of early prostatic adenocarcinomas. This dependence is utilized clinically to treat prostate cancer, wherein AR antagonists (e.g. bicalutamide) are commonly used as therapeutic agents (60). Given the strength of AR inhibition we observed, we tested the possibility that transient overexpression of cyclin D1 in AR-dependent prostatic carcinoma cells would impact proliferation. For these studies, androgen-dependent LNCaP cells were utilized, which arrest in G1 in response to androgen withdrawal (52). These cells express a tumor-derived endogenous AR (AR-T877A) that is androgen-responsive and whose activation is required for cell cycle progression (61-63). Initially, reporter assays were carried out, which demonstrated that AR-T877A is inhibited by cyclin D1 in a manner comparable with wild type AR, shown in Fig. 7A (upper panel). This observation was not due a decrease in AR-T877A expression, as levels remained constant even in the presence of cyclin D1 constructs (Fig. 7A, lower panel). To determine the consequence of this inhibition, LNCaP cells were then transiently transfected with expression plasmids encoding cyclin D1 constructs, and tested for cell cycle progression via BrdUrd incorporation. Histone H2B-coupled GFP was co-transfected with each of the cyclin D1 constructs such that transfection positive cells could easily be identified. As shown in Fig. 7B, cells transfected with wild type cyclin D1 demonstrated a reduced BrdUrd incorporation as compared with cells transfected with parental vector. Similar results were observed with the cyclin D1-KE allele, which cannot bind CDK4 (44) but still inhibited AR activity. These data provide evidence that cyclin D1 harbors a distinct anti-mitogenic function in androgen-dependent prostatic adenocarcinoma cells, via antagonizing AR-mediated mitogenic signaling.


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  		Fig. 7.   Ectopic cyclin D1 abrogates cell cycle progression in androgen-dependent prostatic adenocarcinoma cells. A, CV1 cells were transfected with the PSA61LUC reporter, T877A mutant AR, and/or cyclin D1 as in Fig. 1A, with the relative plasmid ratios shown. Cells were stimulated, harvested, and monitored for both luciferase and beta -galactosidase activity as in Fig. 1A. Experiments were performed in triplicate, and averages are shown (upper panel). Lysates from parallel experiments wherein 1.0 µg of the H2B-GFP plasmid was transfected in place of CMV-beta gal were subject to SDS-PAGE and immunoblotting (lower panel). B, LNCaP cells containing the endogenous T877A mutant androgen receptor were transfected with either parental vector, wild type cyclin D1, or the non-CDK binding allele of cyclin D1 (cyclin D1-KE) and histone H2B-coupled GFP as described under "Experimental Procedures." Cells were pulsed with BrdUrd for a period of 16 h, and incorporation was monitored via indirect immunofluorescence. Transfected (GFP-positive) were scored for percent BrdUrd incorporation. Experiments were performed at least in duplicate, and averages are shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report, we show that cyclin D1 is a critical negative regulator of the AR. We demonstrate that cyclin D1 functions in the nucleus, independent of its LXXLL motif, to bind the N terminus of the AR and inhibit transactivation. This repressive function of cyclin D1 is likely manifested through an inhibitory effect on the AF-1 domain. Cyclin D1-mediated repression is dominant to both HAT and HAT recruiting co-activators and is dependent on deacetylase activity. The inhibition of AR activity by cyclin D1 has profound anti-mitogenic effects on androgen-dependent prostatic carcinoma cells, underscoring the importance of relative cyclin D1 levels on the proliferative status of androgen-dependent cell types.

Nuclear Cyclin D1 Inhibits AR Activity through Interaction with the N Terminus-- It is well documented that cyclin D1 harbors functions independent of its role in the cell cycle. Specifically, cyclin D1 is known to bind the ERalpha and induce ligand-dependent transactivation (43, 44). This function of cyclin D1 requires a leucine-rich "LXXLL"-like motif, which serves as a bridging factor between the receptor and steroid receptor co-activators (45). Mutation of the leucine-rich region in cyclin D1 (cyclin D1-LALA, mutation of leucines 254 and 255 to alanine) results in a protein that binds the estrogen receptor but fails to recruit co-activators. As a result, this protein acts as a dominant negative for estrogen receptor activation. In contrast to the estrogen receptor, cyclin D1 strongly inhibits ligand-dependent AR activation (38). However, the mechanism underlying this inhibition was unknown. We postulated that this same motif of cyclin D1 was likely required for AR regulation, perhaps serving to compete for co-activator binding or inhibit co-activator activity. Surprisingly, this motif is dispensable for both AR binding and for inhibition of AR activity (Fig. 1). These data indicate that cyclin D1 modifies AR activity through mechanisms distinct from ER-alpha regulation.

Cyclin D1 and the AR both cycle between the nucleus and cytoplasm in response to cellular cues. Specifically, cyclin D1 is exported from the nucleus in response to anti-mitogenic signals, and nuclear export is initiated via GSK-3beta -mediated phosphorylation and increased association with CRM1 (49, 54). Because the AR translocates to the nucleus after ligand binding (10, 11), it was possible that cyclin D1 inhibited AR activity through cytoplasmic sequestration. To test this possibility we utilized cyclin D1-T286A, a constitutively nuclear allele that cannot be phosphorylated by GSK-3beta (49). This mutant retained full AR repressor activity (Fig. 2). Moreover, overexpression of cyclin D1 did not prevent ligand-dependent AR nuclear translocation, as judged by indirect immunofluorescence (data not shown). These observations demonstrate that nuclear cyclin D1 is capable of AR inhibition.

Mapping studies were used to identify the site of cyclin D1 action. We identified the AR N terminus as a principal region of cyclin D1 binding, between amino acids 1 and 502 (Figs. 3 and 4). We observed minimal cyclin D1 interaction with AR fragments encoding amino acids 506-918 and 623-918, as compared with N-terminal truncations (1-661 and 1-502) (Fig. 3). However, while this manuscript was in preparation, another report emerged demonstrating some binding of cyclin D1 to amino acids 633-668 (reported to be ~2-fold above background), which lies within the hinge region of the AR (64). Therefore, whereas a binding site for cyclin D1 may exist within the AR hinge region, a predominant site of interaction lies within the N-terminal transactivation domain. Our data indicate that cyclin D1 interacts with the N-terminal transactivation functions of the AR, AF-1, and AF-5. This finding is of importance, because critical co-activator proteins are known to bind this region and modulate AR function.

Cyclin D1 Inhibition Is Dominant to Co-activators and Involves Deacetylase Activity-- Several co-activators have been shown to act through direct interaction and enhance ligand-dependent AR activity. These co-activators function to bridge the receptors to the pre-initiation complex and facilitate transcription. A subset of these harbor intrinsic HAT activity, thought to loosen nucleosome structure and allow access of additional transcription factors to DNA. However, HAT activity does not always predict co-activator function, as the recently identified HBO1 protein harbors HAT activity but serves as a co-repressor for the AR through an N-terminal interaction (65). We examined the ability of known HAT-containing AR co-activators (SRC-1, P/CAF, and p300) to relieve cyclin D1-mediated repression. Each co-activator enhanced ligand-dependent AR transactivation, as expected. However, cyclin D1 proved dominant to all three activities and reduced AR transactivation potential to basal levels (Fig. 5). It has been suggested recently that cyclin D1 may compete with P/CAF for AR binding and that excess co-activator expression may abrogate the repressor function of cyclin D1, using the murine mammary tumor virus promoter as a readout and increased concentrations of ligand (10-7 M DHT) (64). In our experiments, it is noteworthy that the dominance of cyclin D1 over co-activator function did not require excessive expression; in each experiment cyclin D1 was co-transfected at 1:1 ratios with the co-activator in question. Moreover, experiments were performed using a physiological target of the AR (the PSA promoter) and biologically relevant levels of ligand (10-10 M DHT). These data show that cyclin D1 is dominant to the effect of known AR co-activators.

Interestingly, the activity of a C-terminal binding co-activator, ARA70, was also abrogated by cyclin D1 action. Whereas ARA70 is controversial in its ability to stimulate AR transactivation (55), co-activators do show variant activation capacity dependent on cell type and promoter analyzed (66). Clearly, ARA70 acts as an AR co-activator for PSA transactivation under the conditions utilized, and co-expression of cyclin D1 at a 1:1 ratio abolished AR activity (Fig. 5D). Although somewhat surprising, it is speculated that interaction between the N terminus and C terminus of the AR is required for maximal transactivation potential (25). Therefore, recruitment of a co-activator to the C terminus may not overcome the action of a co-repressor on the N terminus. These results demonstrate the potency of cyclin D1 as repressor of the AR.

Although co-repressors are thought to recruit HDAC activity (e.g. SMRT or NCoR) (67), cyclin D1 is not known to harbor this activity. However, the HDAC inhibitor TSA partially reversed the inhibitory action of cyclin D1 on the AR (Fig. 6). These data indicate that deacetylation may be involved in the co-repressor function and suggest that cyclin D1 may recruit an HDAC molecule to the AR complex. Alternatively, it is possible that cyclin D1 action is dependent on de-acetylase activity that is independent of histones. For example, it has been shown recently that the AR can be acetylated by p300 and p300/cAMP response element-binding protein and that mutation of lysines 632 and 633 (sites of p300-driven acetylation) results in decreased transactivation potential (68). Whereas it is possible that cyclin D1 may recruit a deacetylase that acts directly on the AR to reduce transcriptional activation, it is equally conceivable that the required deacetylase acts on an intermediary protein. Future investigations will distinguish among these possibilities.

Cyclin D1 Serves Dual Roles in Androgen-dependent Prostatic Adenocarcinoma Cells-- The data shown demonstrate the potency of cyclin D1 as a co-repressor of the androgen receptor. In early prostatic adenocarcinoma cells, AR activity is required for proliferation (69, 70). We have shown that androgen induces cyclin D1 expression in prostatic adenocarcinoma cells as part of its mitogenic signal (52). Although androgen also induces CDK2/cyclin E activity (71), activation of cyclin D1 expression and cyclin D1/CDK4 kinase activity is required for cell cycle progression (39). Thus, a paradox exists for androgen-dependent cells, wherein cyclin D1 harbors a mitogenic, androgen-responsive function (induction of CDK4 activity) (52) and an anti-mitogenic function (repression of AR activity) that occurs independent of CDK4 (38). These observations suggest a model wherein androgen stimulation induces cyclin D1 expression, CDK4 is activated, and cell cycle progression ensues. Indeed, after clonal selection, LNCaP cells that modestly overexpress cyclin D1 exhibit a slight growth advantage (72). These procedures would likely select against any anti-mitogenic function of cyclin D1. It is well documented that the main cell cycle target of cyclin D-CDK4 complexes is the retinoblastoma tumor suppressor protein, RB (42). After CDK4 mediated RB phosphorylation in early G1, cyclin D1 expression persists (39). Our data showing that ectopic expression of cyclin D1 or CDK4-refractory cyclin D1 actually inhibits cell cycle progression (Fig. 7) supports the model that after RB inactivation, androgen-induced cyclin D1 expression serves to negatively regulate AR activity and thereby limit the rate of future mitogenic activation and cell cycle progression.

The balance of these opposing actions may contribute to the equivocal results observed in human prostate cancers upon examination of cyclin D1 expression. Whereas some studies report relatively high frequency of cyclin D1 overexpression (up to 30%) in prostatic adenocarcinomas (73, 74), others report that this is a rare event (75) or that cyclin D1 overexpression does not correlate with tumor grade or progression (76). A study of 213 patients also demonstrated no prognostic value for cyclin D1 expression alone in prostate tumors (77).

In summary, we demonstrate that cyclin D1 is a critical negative regulator of AR transactivation. These effects involve a mechanism unrelated to the LXXLL motif and are dominant to the known AR co-activators. This function of cyclin D1 abrogates AR signaling and androgen-dependent mitogenesis in prostatic adenocarcinoma cells. These data demonstrate that cyclin D1 contains both mitogenic (CDK4 dependent) and anti-mitogenic (dependent on regulation of AF-1) interactions in androgen-dependent LNCaP cells. Given the importance of AR regulation in the progression and treatment of prostatic adenocarcinomas, these data provide the impetus to delineate further the regulation of cyclin D1 activity in this tumor type.

    ACKNOWLEDGEMENTS

We thank Drs. A. Brinkmann, R. Bernards, C. Chang, K. Cleutjens, A. Diehl, D. Feldman, T. Kouzarides, B. O'Malley, S. Y. R. Dent, M. Roussel, L. Sherman, J. Y. J. Wang, and R. Weinberg for the generous supply of reagents; Drs. W. Cavenee, K. Arden, S. Khan, and R. Hennigan for critical reading of the manuscript; and Dr. E. Knudsen for technical assistance and critical ongoing discussions.

    FOOTNOTES

* This work was supported in part by grants from the National Institutes of Health and American Cancer Society (to K. E. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by the University of Cincinnati Distinguished Graduate Assistantship award.

Supported by NCI Training Grant ES07250-13 from the National Institutes of Health.

** To whom correspondence should be addressed. Tel.: 513-558-7371; Fax: 513-558-4454; E-mail: Karen.Knudsen@uc.edu.

Published, JBC Papers in Press, November 19, 2001, DOI 10.1074/jbc.M106399200

    ABBREVIATIONS

The abbreviations used are: AR, androgen receptor; DHT, dihydrotestosterone; PSA, prostate-specific antigen; HAT, histone acetylation; HDAC, histone deacetylase; FBS, fetal bovine serum; GFP, green fluorescent protein; TSA, trichostatin A; BrdUrd, bromodeoxyuridine; RB, retinoblastoma; ER, estrogen receptor; BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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