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J. Biol. Chem., Vol. 277, Issue 2, 1240-1248, January 11, 2002

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The Androgen Receptor Represses Transforming Growth Factor- Signaling through Interaction with Smad3^*

Jerry E. Chipuk^§¶, Susan C. Cornelius^§, Nicole J. Pultz^§, Joan S. Jorgensen, Michael J. Bonham^**, Seong-Jin Kim, and David Danielpour^§

From the ^§ Ireland Cancer Center Research Laboratories,  Department of Pharmacology, Case Western Reserve University/University Hospitals of Cleveland, Cleveland, Ohio 44106 and  Laboratory of Cell Regulation and Carcinogenesis, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, September 13, 2001, and in revised form, November 13, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the prostate, androgens negatively regulate the expression of transforming growth factor- (TGF-) ligands and receptors and Smad activation through unknown mechanisms. We show that androgens (dihydrotestosterone and R1881) down-regulate TGF-1-induced expression of TGF-1, c-Fos, and Egr-1 in the human prostate adenocarcinoma cell line, LNCaP. Moreover, 5-dihydrotestosterone (DHT) inhibits TGF-1 activation of three TGF-1-responsive promoter constructs, 3TP-luciferase, AP-1-luciferase, and SBE4_BV-luciferase, in LNCaP cells either with or without enforced expression of TGF- receptors (TRI and TRII). Similarly, DHT inhibits the activation of Smad-binding element (SBE)4_BV-luciferase by either constitutively activated TRI (T204D) or constitutively activated Smad3 (S3*). Activation of SBE4_BV-luciferase by S3* in the NRP-154 prostatic cell line, which is androgen receptor (AR)-negative but highly responsive to TGF-1, is blocked by co-transfection with either full-length AR or AR missing the DNA binding domain. Immunoprecipitation and GST pull-down assays show that AR directly associates with Smad3 but not Smad2 or Smad4. Electrophoretic mobility shift assays indicate that the AR ligand binding domain directly inhibits the association of Smad3 to the Smad-binding element. In conclusion, our data demonstrate for the first time that ligand-bound AR inhibits TGF- transcriptional responses through selectively repressing the binding of Smad3 to SBE.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Normal prostatic epithelium depends on androgens for growth, development, secretory function, and survival (1-4). Most remarkably, androgen ablation induces massive apoptosis of prostatic epithelium (2, 5-8). Loss of androgen dependence occurs invariably during prostate carcinogenesis, accounting for poor long term success of androgen ablation therapy (9). Recent studies (10) show that acquisition of androgen autonomy occurs despite retention or elevated expression of the androgen receptor (AR)¹ in the majority of prostate tumors. AR, a 110-kDa zinc finger transcription factor belonging to the nuclear receptor superfamily, is activated by phosphorylation (11) and dimerization upon ligand binding. This promotes nuclear localization and binding of AR to androgen-responsive elements in the promoters of androgen-regulated genes. AR-mediated transcription is regulated by many AR-interacting proteins such as ARA₇₀ (AR-associated proteins) (12) and ARA₁₆₀ (13), along with cAMP-response element-binding protein (14), AP-1 (9, 15), and Ets (16). The growing list of recently discovered AR transcriptional co-regulators supports the notion that complex networks of signals tightly regulate transcription by androgens. Understanding how these signals promote growth and maintain cell viability will certainly impact on the therapeutic strategies for the prevention and cure of prostate cancer.

TGF-, a potent regulator of cell growth, differentiation, apoptosis, and carcinogenesis in the prostate (17-20), is under androgenic control. TGF- signals through a cooperative interaction with two cell surface serine/threonine kinase receptors, TRI and TRII (21-25). TGF- first associates with constitutively active dimeric TRII, which then recruits and activates TRI kinase by transphosphorylation at a juxtamembrane glycine-serine repeat (21, 26). With the help of Smad anchor for receptor activation (27), phosphorylated TRI is able to activate Smads 2 and 3 by phosphorylating their carboxyl-terminal serine-serine-Xaa-serine motifs (28). Active Smads 2 and 3 can form heteromeric complexes with co-Smad4, and either directly or through interactions with transcription factors and co-regulators bind to Smad-binding elements (SBEs) in TGF--regulated genes (29-31). Further activation of Smads 2 and 3 is blocked by Smad7, whose expression is induced upon TGF- stimulation (32).

Androgens negatively regulate TGF-1 ligand (17, 33) and receptor expression (34, 35), along with Smad expression and activation (36) in the prostate. Recent reports show AR associates with Smad3 and that this association may either enhance (37) or inhibit (38) AR-mediated transcription. Here we report the first in vitro demonstration that DHT inhibits TGF- signaling in prostatic epithelial cells through interaction of AR with Smad3. Our results support that the binding of ligand-bound AR to activated Smad3 inhibits TGF- transcriptional responses by blocking the association of Smad3 with SBE.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Passaging-- LNCaP cells (ATCC) were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) in polylysine-coated flasks and passaged every 3-4 days at subconfluence. Cells were trypsinized with 5 ml of trypsin-EDTA for 10 min at 37 °C, and cell clumps were triturated with 5 ml of DMEM/F-12 + 10% FBS. NRP-154 were cultured as described (39).

Quantitation of TGF-1-3-- A suspension of 250,000 cells/ml in DMEM/F-12, 10% dextran charcoal-stripped FBS was dispensed in polylysine-coated 6-well dishes. Following 48 h, wells were washed twice with DMEM/F-12 supplemented with 100 µg/ml bovine serum albumin fraction V, 5.0 µg/ml human transferrin, 20 ng/ml mouse epidermal growth factor, and 10 ng/ml sodium selenite and replaced with 2 ml of the above medium. After 2-3 h at 37 °C, the medium was replaced as before, and various test factors were added. Following 48-72 h of treatment, conditioned media from these cultures were harvested to assay for TGF-1-3 by sandwich enzyme-linked immunosorbent assay (40-42). Samples were prepared as follows. Conditioned medium was treated with protease inhibitors (2 µg/ml aprotinin, 2 µg/ml leupeptin, and 0.2 mg/ml phenylmethylsulfonyl fluoride) and clarified at 13,000 × g for 10 min. 55 µl of a 100% solution of trichloroacetic acid was added per ml of conditioned medium, vortexed, set on ice for 30 min, and microcentrifuged at 14,000 × g for 10 min. The pellets were washed with 1 ml of cold ether/ethanol (1:1, v/v), lyophilized for 10 min, and dissolved in 125 µl of 4 mM HCl, 150 mM NaCl, and 0.5 mg/ml bovine serum albumin. Daily TGF-1 secretion rates were normalized to DNA content of producer cells. DNA was measured by fluorescence with 3323 Hoeschst (43).

Northern Blot Analysis-- Total RNA was purified using RNeasy^TM columns (Qiagen) as modified by Bonham and Danielpour (44). 10 µg of total RNA was separated by electrophoresis through a 1% agarose, 0.66 M formaldehyde gel containing 0.72 µg/ml ethidium bromide. Equal loading and even transfer were assessed by visualization of the 18 S and 28 S rRNAs. Gels were treated with 60 mM NaOH for 20 min, neutralized with 50 mM Tris-HCl (pH 7.4) and 10 mM NaCl for 20 min, and then blotted onto Nytran (0.45-µm) for 16-24 h with 10× SSPE. After membranes were cross-linked with UV light, they were pre-hybridized for 2 h, hybridized overnight at 65 °C, and washed at the same temperature under modified Church's hybridization conditions (45). The cDNA probes were labeled with [³²P]dCTP by a random priming reaction. Hybridization was done with 2-3 × 10⁶ cpm/ml.

Generation of Active Smads 2 and 3-- The carboxyl-terminal Ser-Ser-X-Ser phosphorylation motif was changed to Asp-Asp-X-Asp by site-directed mutagenesis (QuikChange Site-directed Mutagenesis Kit, Stratagene). PCR mutagenesis was performed by using the following primers: 5'-GATCCCCTTCAGTGCGTTGCGATGACATGGACTAATCTAGACCCGGGTGGCATC-3' and its complement (S2*); 5'-CCAAGCATCCGCTGTGACGATGTGGATTAGA GAGACATCAAGTGCTCTAGAGG-3' and its complement (S3*). 30 ng of pCMV2-FLAG-Smad was used as the PCR template.

Production of GST-Androgen Receptor and GST-Smad Fusions-- The amino-terminal region (a.a. 1-556) and ligand binding domain (a.a. 666-919) were directionally cloned into pGEX-6P-1 (5' BamHI and 3' XhoI sites) to generate 5' GST fusions. Each domain was PCR-amplified using the following primers that contained 5' BamHI and 3' XhoI adapter sequences: amino-terminal region, 5'-CGGATCCGAAGTGCAGTTAGGGCTGGGAAGGGTCT-3' and 5'-ATCTCGAGCTAGGGTGGAAAGTAATAGTCAATGG-3'; and ligand binding domain, 5'-CGGATCCCACATTGAAGGCTATGAATGTCAGCCC-3' and 5'-CGGATCCCACATTGAAGGCTATGAATGTCAGCCC-3'. Full-length GST-Smad3 and -4 were synthesized by PCR and subcloned into the EcoRI-XhoI sites of pGEX-4T-1 (Amersham Biosciences) as described (46).

GST fusions were purified as follows: Luria Burtani/Miller broth in 500-ml batches containing 100 µg/ml of ampicillin were inoculated with 1-ml overnight cultures of BL21 (Stratagene) cells transformed with pGEX-Smads or pGEX-AR constructs. Following 1.5 h of incubation at 37 °C, cultures were brought to room temperature and induced overnight with 50 µg/ml isopropyl-1-thio--D-galactopyranoside. Bacterial pellets were lysed by sonication using a microprobe (50 strokes, 50% duty cycle, setting 3) in 8 ml of cold TNE (10 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA) containing Complete Mini EDTA-free Protease Inhibitor Mixture (Roche Molecular Biochemicals), 0.2 mg/ml phenylmethylsulfonyl fluoride, and 2 mM dithiothreitol. Lysates were treated with 1% Nonidet P-40 for 5 min on ice, clarified at 20,000 × g for 15 min at 4 °C, and mixed overnight at 4 °C with 0.4 ml of glutathione-Sepharose. Resin-containing fusion proteins were washed twice with 10 ml of TNE containing 1% Nonidet P-40 and 0.5 mM dithiothreitol and three times with TNE containing 0.5 mM dithiothreitol. Fusion proteins were either used directly on resin or eluted at 4 °C by a 45-min incubation with 10 mM glutathione, 50 mM NaCl, and 50 mM Tris (pH 8.7) and then dialyzed extensively in EMSA buffer without glycerol. GST fusion proteins were quantified by Coomassie Blue staining of Tris glycine gels, using bovine serum albumin standards.

Transfection of NRP-154 Cells-- Cells (6.0 × 10⁴) were plated overnight in 12-well dishes and transfected using a standard calcium phosphate co-precipitate method for 3 h in GM2 (39) made with dextran charcoal-stripped FBS. The calcium phosphate/DNA co-precipitate was washed away, and the cells were glycerol-shocked (15% glycerol in 1× HEPES-buffered saline) for 90 s. Cells were washed twice with phosphate-buffered saline, allowed to recover overnight in GM3 (47) (made with dextran charcoal-stripped calf serum) ± 10 nM DHT (Sigma) (or vehicle, 70% ethanol), and then treated with 10 ng/ml TGF-1 (R & D Systems) or vehicle (4 mM HCl, 1 mg/ml bovine serum albumin). Luciferase was measured 24 h later using Promega's Dual Luciferase Assay Kit and a Dynex ML3000 Microtiter Plate Luminometer.

Transfection of LNCaP Cells-- Cells (2.5 × 10⁵) were plated overnight in 6-well polylysine-coated plates with 2 ml of media (DMEM/F-12 with 15 mM HEPES, 1% dextran charcoal-stripped FBS). Cells were transfected with LipofectAMINE Plus reagent according to the manufacturer's protocol (Invitrogen) for 3 h before replacing with above medium (+20 ng/ml epidermal growth factor, except for SBE4_BV-luciferase assays) and ±10 nM DHT. 10 ng/ml TGF-1 was added the next day, and the cells were harvested 24-48 h later. Luciferase was measured using Promega's Dual Luciferase Assay Kit and a ML3000 Microtiter Plate Luminometer.

Immunoprecipitation-- NRP-154 cells (8.0 × 10⁵) were plated overnight in 100-mm² dishes with 5 ml of GM2. Cells were transiently transfected with 5 µg each pCMV2-FLAG-S3* and pCMV5-AR using a standard calcium phosphate co-precipitate method. 10 nM DHT (or vehicle) was added after the transfection and again 4 h prior to harvest. Cells were lysed at 4 °C with 400 µl of cold radioimmunoprecipitation (RIPA) buffer (containing Complete Mini-EDTA-free Protease Inhibitor Mixture and 0.2 mg/ml phenylmethylsulfonyl fluoride) using a 25-gauge needle and then clarified at 10,000 rpm for 10 min (4 °C). Lysates were precleared using 0.5 µg of mouse IgG and 20 µl of protein A/G Plus-agarose (Santa Cruz Biotechnology) for 1 h, immunoprecipitated with either 2 µg of mouse anti-FLAG M2 (Sigma) or mouse anti-AR (Lab Vision) overnight in the presence of 10 nM DHT (or vehicle), and treated with 20 µl of protein A/G Plus-agarose for 2 h at 4 °C. The resin was washed four times with RIPA buffer and eluted with 50 µl of 1× SDS-PAGE loading buffer. Samples were separated by electrophoresis using 4-12% NuPAGE BisTris gel (Invitrogen) with 1× MOPS buffer (Invitrogen) and transferred to nitrocellulose (Invitrogen). Western blot detection was done as described (47). Antibodies for Western blot detection were from Santa Cruz Biotechnology (human androgen receptor C-19, (1:4000)) and Sigma (FLAG M2 (1:500)).

In Vitro Transcription/Translation and GST Pull Down-- Full-length AR cDNA (a.a. 1-919) was cloned into pcDNA3 (Invitrogen) and in vitro transcribed/translated using a T7 TnT® Coupled Reticulocyte Lysate System (Stratagene). 5 µl of the ³⁵S-labeled TnT product was added to equal volumes of glutathione-Sepharose-GST-Smad fusions in buffer A (20 mM Tris (pH 7.8), 180 mM KCl, 0.5 mM EDTA, 5 mM MgCl₂, 0.5 mM ZnCl₂, 10% glycerol, 0.1% Nonidet P-40, 0.05% milk, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and rotated overnight at 4 °C. The glutathione-Sepharose beads were then centrifuged (5 min at 3000 rpm) and washed 3 times with 500 µl of cold buffer A. GST-Smad·AR complexes were eluted with 50 µl of 1× SDS-PAGE loading dye and heated to 70 °C for 10 min prior to resolving the complexes in a 4-12% NuPAGE BisTris gel with 1× MES buffer (Invitrogen). The proteins were then transferred to nitrocellulose and detected by a Molecular Dynamics PhosphorImager.

Electrophoretic Mobility Shift Assay-- SBE oligonucleotides (Santa Cruz Biotechnology) were labeled with [-³²P]ATP using T4 polynucleotide kinase (Promega) and ethanol-precipitated. 50,000 cpm of labeled oligonucleotides were mixed with GST fusion proteins in binding buffer (10 mM Tris (pH 7.8), 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 2 mM ZnCl₂, 50 ng of polydeoxyinosinic-deoxycytidylic acid), ±10 nM DHT, for 20 min at 25 °C. Complexes were resolved in a 6% DNA retardation gel (Invitrogen) using 0.5× TBE buffer (Invitrogen); the gel was dried at 80 °C for 1 h and exposed to a phosphorscreen. The sequences of the double-stranded oligonucleotides were 5'-TCGAGAGCCAGACAAAAAGCCAGACATTTAGCCAGACAC-3' and its complement.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Androgens Block the TGF-1-induced Autoinduction of TGF-1 Ligand and c-Fos and Egr-1 Expression in LNCaP Cells-- Androgens negatively regulate expression of TGF- ligands in prostatic cells in both animals (17) and rat cell culture (48). We determined whether androgens could also down-regulate TGF-1 autoinduction (49) in the androgen-responsive human prostatic adenocarcinoma cell line, LNCaP, under serum-free conditions, using isoform-specific sandwich enzyme-linked immunoadsorption assays for TGF-s. From a list of common hormones and growth factors added to LNCaP cells, only TGF-s substantially elevated protein levels of TGF-1 (data not shown; referred to as "TGF autoinduction"). A physiological concentration of DHT (10 nM), the active metabolite of testosterone, inhibited the induced expression of TGF-1 protein (Fig. 1A) and mRNA (Fig. 1B). The stable androgen analogue, R1881, also blocked TGF-2-induced expression of TGF-1 (Fig. 1, B-D). We used R1881 to study the kinetics of TGF-1 mRNA loss following an initial (72 h) induction by TGF-1 (Fig. 1D). Changes in the expression of two transcription factors (c-Fos and Egr-1), shown to be induced by TGF- and involved in TGF- autoinduction (50-53), were also determined (Fig. 1D). The induced expression of TGF-1 ligand, c-Fos, and Egr-1 mRNAs was inhibited by R1881 in a time-dependent manner. Of note, LNCaP cells treated with TGF-1 will maintain increased mRNA levels of TGF-1 ligand, c-Fos, and Egr-1 between 2 and 5 days of treatment (data not shown).

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Androgens block the TGF-1-induced autoinduction of TGF-1 ligand and the expression of c-Fos and Egr-1 in LNCaP cells. A, TGF-1 protein expressed in serum-free medium of LNCaP cells following 48 h of treatment with ±10 ng/ml TGF-2 and ±10 nM DHT was measured by a TGF-1 sandwich enzyme-linked immunosorbent assay. Data are expressed as secretion rates and normalized to DNA content of producer cells at the time of collection. B and C, Northern blot analysis of TGF-1 mRNA from LNCaP cells in response to TGF-1 or TGF-2 and ±DHT or R1881 for 48 h. Controls 1 and 2 are the presence or absence of 20 ng/ml EGF, respectively. D, effect of R1881 on temporal changes of TGF-1, c-Fos, and Egr-1 mRNAs induced by TGF-1 from LNCaP cells determined by Northern blot analysis and quantified by a PhosphorImager. LNCaP cells were pre-treated with 10 ng/ml TGF-1 for 72 h. 10 nM R1881 was added at various time points thereafter, and all cells were harvested at the same time 48 h later. E, LNCaP cells were pretreated with 10 ng/ml TGF-1 for 72 h; cells were then harvested at time 0 (0 h) or after 24 h following treatment with either vehicle or various combinations of 10 nM R1881, 1 µg/ml actinomycin D (ActD), 1 µg/ml cycloheximide (CHX). Total RNA was subjected to Northern blot analysis (left panel), and the bands were quantified by a PhosphorImager (right panel). A representative blot of at least three independent experiments is shown.

The decrease in TGF-1 expression by R1881 occurred through transcriptional repression and did not require de novo protein synthesis, as demonstrated by loss of TGF-1 mRNA expression following 24 h of treatment with R1881 in the presence of actinomycin D or cycloheximide (Fig. 1E, right panel). Quantification of the Northern blot in Fig. 1E is also presented (Fig. 1E, left panel). Actinomycin D caused the same loss of TGF-1 mRNA as did R1881, and co-treatment of R1881 and actinomycin D did not enhance loss by R1881 alone. In the presence of cycloheximide, R1881 caused a smaller loss of TGF-1 mRNA expression, perhaps due to a decrease in AR expression. Co-treatment of actinomycin D and cycloheximide had the same effect as actinomycin D alone. Together, these data suggest that AR functions through directly repressing transcription by TGF-.

DHT Inhibits the Transcriptional Activation of Several Response Elements Induced by TGF-1-- We tested the above hypothesis by assaying the effect of DHT on TGF-1-induced transcriptional activation of various response elements. In LNCaP cells, the TGF-1-induced 3TP-luciferase (54) activity was blocked by co-treatment with DHT (Fig. 2A). Previous work supports that LNCaP cells express low levels of either TRI or TRII (55, 56), potentially accounting for their relatively weak response to TGF-1. Therefore, we co-transfected these cells with either TRI or TRII along with 3TP-luciferase. Co-transfection of TRII resulted in >50-fold enhanced activation of luciferase by TGF-1 (Fig. 2B). In contrast, TRI did not enhance TGF-1-induced 3TP-luciferase activity (data not shown), suggesting that TRII but not TRI was limiting in our LNCaP lineage. Overexpression of TRII did not blunt the ability of DHT to inhibit TGF-1-induced 3TP-luciferase (Fig. 2C). Additionally, the inhibition of TGF-1-induced 3TP-luciferase activity was dependent on DHT concentration (Fig. 2D). These data demonstrate that the levels of endogenous AR in LNCaP can fully repress TGF-1-induced 3TP-luciferase activity, even in the presence of overexpressed TRII, when ligand-bound.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   DHT inhibits the transcriptional activation of several TGF-1 response elements. LNCaP cells were transiently co-transfected with 0.5 µg of the specified luciferase reporter and 12.5 ng of pCMV-Renilla and expression vector (except A) prior to treatment with 10 ng/ml TGF-1 and/or 10 nM DHT (except D) for the indicated times. A, inhibition of TGF-1-induced 3TP-luciferase activity by DHT in LNCaP cells following 48 h of treatment with DHT and TGF-1. B, effect of TRII (1.0 µg of pCMV5-TRII or pCMV5 control) co-transfected in LNCaP cells on the activation of 3TP-luciferase by 48 h of TGF-1 treatment. C, inhibition of 3TP-luciferase activity by TGF-1 was not reversed by transfection of 1.0 µg of pCMV5-TRII (or pCMV5 control). D, dose-dependent effect of DHT on the inhibition of TGF-1induced 3TP-luciferase in LNCaP cells co-transfected with 0.5 µg of pCMV5-TRII. E, effect of DHT on TGF-1-induced 3TP-luciferase activity in NRP-154 cells co-transfected with 1.0 µg of pCMV5-AR (or pCMV5 control). F, activation of AP-1-luciferase by TGF-1 in LNCaP cells overexpressing TRII (1.0 µg pCMV5-TRII) is inhibited by DHT. G, activation of SBE4BV-luciferase by TGF-1 is inhibited by DHT in LNCaP cells co-transfected with 1.0 µg of pCMV5-TRII. H, LNCaP cells were transiently co-transfected with 1.0 µg of SBE4BV-luciferase and 1.0 µg of pCMV5-T204D (or pCMV5 control) and treated with 10 nM DHT (or vehicle) for 24 h before harvesting. Data shown are averages (±S.D.) of triplicate independent measurements of luciferase/Renilla readings relative to untreated controls.

We tested the above results and the requirement for AR in another prostatic cell line, NRP-154, which is exquisitely sensitive to TGF-1 but has undetectable levels of AR (39). Transfection of full-length AR (57) enabled DHT to similarly inhibit TGF-1-induced 3TP-luciferase activity in NRP-154 cells (Fig. 2E). 3TP-luciferase has a complex promoter, consisting of 3× TRE elements upstream of a plasminogen activator inhibitor-1 promoter fragment. We co-transfected LNCaP cells with basic promoter constructs, AP-1- or SBE4_BV-luciferase (58), to define better the elements responsible for transcriptional inhibition by DHT. TGF-1 activated both AP-1-luciferase and SBE4_BV-luciferase, and 10 nM DHT substantially inhibited these activities, comparable with 3TP-luciferase (Fig. 2, F and G). Consistent with these data, activation of SBE4_BV-luciferase by a constitutively activated TRI, T204D (59), was also suppressed by DHT (Fig. 2H).

DHT Inhibition of TGF-1-induced SBE4_BV-Luciferase Activity Occurs through Smad3-- The above data suggested Smads were a potential direct target of the observed DHT inhibition on TGF-1 responses. To begin examining this possibility, we transfected LNCaP and NRP-154 cells with constitutively active Smad 2 or 3 (S2* and S3*, respectively) developed by site-directed mutagenesis of their carboxyl-terminal serines to aspartic acids in the SSXS phosphorylation motifs. Transfection of S3* in both LNCaP (Fig. 3A, left panel) and NRP-154 cells (Fig. 3A, right panel) caused pronounced SBE4_BV-luciferase induction, whereas S2* did not induce SBE4_BV-luciferase activity in either cell line. The induction of SBE4_BV-luciferase by TGF-1 in LNCaP cells was blocked by co-transfection of DN-Smad4 or Smad7 (Fig. 3, B and C, respectively), implicating the dependence of Smads on this reporter. Although co-transfection of LNCaP cells with wild-type AR (DHT) had little inhibitory effect on S3*-induced SBE4_BV-luciferase activity, treatment of AR-transfected LNCaP cells with DHT completely blocked transcription by S3* (Fig. 3D). Due to the substantial S3*-induced SBE4_BV-luciferase activity in LNCaP, additional AR was required to fully repress this response (however, endogenous AR could fully inhibit TGF-1-induced SBE4_BV-luciferase activity, Fig. 2G). Similarly, in NRP-154 cells, DHT inhibited S3*-induced SBE4_BV-luciferase activity only in AR co-transfected cells (Fig. 3E), demonstrating the dependence of both androgens and AR on this effect. Finally, to determine whether Smad3 was the major target of DHT-induced inhibition of TGF-1 signaling, we transfected LNCaP cells with wild-type Smad3 in an attempt to reverse the inhibition. As shown in Fig. 3F, overexpression of wild-type Smad3 fully reversed the inhibition of TGF-1-induced SBE4_BV-luciferase activity by AR and DHT.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   DHT inhibition of TGF-1-induced SBE4BV-luciferase activity occurs through Smad3. A, LNCaP (left panel) or NRP-154 (right panel) cells were transiently co-transfected with 0.5 µg of SBE4BV-luciferase and 150 ng of pCMV2-S2* or pCMV2-S3* (or pCMV2 control). LNCaP cells were co-transfected with 0.5 µg of SBE4BV-luciferase, 30 ng of pCMV2-FLAG-S3*, and 1.0 µg of pcDNA3.1-DN-Smad4 (B) or pcDNA3.1-Smad7 (C) (or pcDNA3.1 control). LNCaP (D) or NRP-154 (E) cells were transiently co-transfected with 0.5 µg of SBE4BV-luciferase, 30 ng of pCMV2-FLAG-S3* (or pCMV2 control), and 0.5 µg of pCMV5-AR (or pCMV5 control). F, LNCaP cells were transiently co-transfected with 0.2 µg of SBE4BV-luciferase, 0.25 µg of pCMV5-AR, 0.25 µg of pCMV2-Smad3 (or pCMV2), and 0.3 µg of pCMV5-TRII before 24 h co-treatment with TGF-1 (or vehicle) and DHT. Cells were treated with 10 nM DHT (or vehicle) for 24 h before harvesting. 12.5 ng of pCMV-Renilla/well was also co-transfected for all luciferase assays. Data shown are averages (± S.D.) of triplicate independent measurements of luciferase/Renilla readings relative to untreated controls.

The Androgen Receptor Associates with Smad3 Independent of Ligand-- Recent evidence from other laboratories (37, 38) suggests that AR associates with Smad3 in an androgen-independent manner. We confirmed these results in our system, along with determining if S3* was still able to bind AR due to mutation of its carboxyl terminus, by both GST pull-down assays and co-immunoprecipitation, respectively. NRP-154 cells were co-transfected with FLAG-S3* and AR and treated with DHT (or vehicle) before immunoprecipitation 24 h later. Fig. 4A demonstrates that immunoprecipitating against either FLAG or AR can capture S3*·AR complexes independent of DHT addition. Full-length (a.a. 1-919) in vitro transcribed/translated ³⁵S-labeled AR associated specifically with GST-Smad3 and not with GST-Smad2 or -4 and weakly with GST-Smad1 (Fig. 4B). Thus, our results show that AR associates directly with Smad3 (and S3*) in the absence of DHT (Fig. 4, A and B) but not with any other Smads (Smads 2 or 4) involved in TGF-1 signal transduction.

View larger version (44K): [in this window] [in a new window]   Fig. 4.   AR associates with Smad3 independent of ligand. A, NRP-154 cells were co-transfected with 5 µg each of pCMV5-AR and pCMV2-FLAG-S3*. 10 nM DHT (or vehicle) was added to cells after transfection, and cells were harvested 24 h later. Cell lysates were precleared using non-immunized mouse IgG and immunoprecipitated (IP) with either anti-FLAG or anti-AR antibodies in the presence of 10 nM DHT (or vehicle) overnight at 4 °C. Samples were subjected to SDS-PAGE and immunodetected using anti-FLAG and anti-AR antibodies. Bands were detected using ECL. B, equal volumes of GST-Smad fusions bound to glutathione-Sepharose were mixed with full-length (a.a. 1-919) in vitro transcribed/translated 35S-labeled AR in buffer A overnight at 4 °C. The complexes were washed three times in buffer A before SDS-PAGE and transfer to nitrocellulose. Lanes 1-4 indicate the respective GST-Smad, and GST control indicates GST alone with AR. Bands were detected using a PhosphorImager. A representative blot of three independent experiments is shown.

The Androgen Receptor DNA Binding Domain Is Not Required to Inhibit Smad3 Activity-- To define better the inhibition of S3*-induced SBE4_BV-luciferase activity by AR, we tested various domains of AR for inhibitory function. NRP-154 cells were co-transfected with SBE4_BV-luciferase, S3*, and various AR expression constructs (60, 61) as follows: wild-type AR, 538-614 (no DNA binding domain (DBD)), DBD, and ABC (no hinge/no ligand binding domain (LBD)) (Fig. 5A). Maximum inhibition of AR on S3*-induced SBE4_BV-luciferase activity was observed by expression of wild-type AR or 538-614 in the presence of ligand (Fig. 5B). In contrast to full-length AR that had no inhibitory effect without DHT, 538-614 repressed the majority of S3* activity without ligand. Expression of DBD or ABC had little to no significant inhibitory effect on S3* activity; and as expected, DHT did not change the effectiveness of these (DBD or ABC) regions on inhibiting S3* activity (Fig. 5B). These data suggest that DHT promotes the inhibitory effect of AR on Smad3 in a DBD-independent manner that requires the LBD or some other region in the AR carboxyl terminus.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   AR DBD is not required to inhibit Smad3 activity. A, mammalian expression vectors used for different domains of AR. B, NRP-154 cells were co-transfected with 0.5 µg of SBE4BV-luciferase, 12.5 ng of pCMV-Renilla, 30 ng of pCMV2-FLAG-S3* (or pCMV2 control), and 0.5 µg of different AR domains or full-length AR (or pCMV5 control). 10 nM DHT (or vehicle) was added to cells immediately after transfection and 8 h before harvest. Each point represents the average of at least triplicate determinations ± S.D.

The Ligand Binding Domain of the AR Inhibits Smad3 Binding to the Smad-binding Element-- Inhibition of Smad3 signaling by DHT could occur through several mechanisms. Upon TGF-1 treatment, Smad3 translocates to the nucleus where it induces target gene expression; similarly, AR is localized to the nucleus in the presence of DHT. Therefore, we examined the effect of AR on the association of Smad3 to SBE. Electrophoretic mobility shift assays were performed with ³²P-labeled SBE oligonucleotides and purified GST-Smad3 in the presence of different domains of AR (Fig. 6A; amino-terminal GST-AR, GST-AR DBD, or GST-AR LBD). GST-Smad3 was able to bind to the SBE in the presence of the amino-terminal GST-AR and GST-AR DBD; however, GST-AR LBD inhibited this association (Fig. 6B). This result is consistent with the data presented in Fig. 5B, which suggests the carboxyl terminus (i.e. LBD) of AR confers inhibition of S3* activity. We next determined the effect of DHT on the association of GST-Smad3 binding to SBE in the presence of GST-AR LBD (200, 400, and 800 ng). The addition of 10 nM DHT enhanced GST-AR LBD-induced inhibition of GST-Smad3 binding to SBE (Fig. 6C). This inhibition was specific to GST-Smad3 because no loss of the GST-Smad4·SBE complex (Fig. 6D) in the presence of GST-AR LDB (800 ng) + DHT was observed. Also, DHT alone did not abrogate GST-Smad3 binding to the SBE. Together, these results suggest that DHT binding to AR enhances or promotes inhibition of Smad3 association to SBE, thereby leading to inhibition of Smad3-mediated transcription.

View larger version (101K): [in this window] [in a new window]   Fig. 6.   LBD of AR inhibits Smad3 binding to SBE. A, diagram of different AR domain GST-fusions. B, an EMSA was performed using a 32P-labeled SBE probe (50,000 cpm) with 400 ng of purified GST-Smad3 in the presence of various GST-AR domains (800 ng of amino terminus (N-term) of DBD or LBD). Complexes were resolved using a 6% DNA retardation gel and 0.5× TBE running buffer. The gel was dried and visualized using a PhosphorImager. C, EMSA (as described in B) of the AR LBD (200, 400, and 800 ng) induced inhibition of GST-Smad3 binding to the SBE with and without 10 nM DHT. D, GST-Smad4 binding to SBE in the presence of 800 ng AR LBD + DHT is shown as control. A representative gel of four independent experiments is shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our data show that androgens can block TGF- responses in prostate epithelial cells through an association of AR with Smad3, which inhibits the binding of Smad3 to SBEs in TGF--responsive promoters. This mechanism of cross-talk provides both rapid and direct means by which androgens can inhibit TGF- signaling.

Our data demonstrate that DHT is not necessary for the in vitro association of full-length AR to Smad3 (Fig. 4, A and B). However, the exact localization of ligand-free AR remains controversial and depends on cell type (60-63). It is likely that the DHT-independent association of AR to Smad3 may not occur in intact NRP-154 cells due to cytosolic localization of ligand-free AR and nuclear localization of S3*. If so, the association of AR to Smad3 without DHT would have occurred after cell lysis. In view of the DHT requirement for the loss of TGF-1-induced transcription by AR, we believe this association in NRP-154 cells occurs only by nuclear co-localization of ligand-bound AR and active Smad3. Data presented in Fig. 6 suggest DHT may also promote a modification in the AR·Smad3 complex to inhibit further the association to SBE.

There is a definite requirement for Smads on all TGF-1-responsive reporter constructs used in this study. We have investigated this requirement by observing the effects of DN-Smad4 and Smad7 on TGF-1-induced 3TP-luciferase and AP-1-luciferase. DN-Smad4 or Smad7 inhibit ~70-80% of TGF-1-induced activity on both of these reporters (data not shown). As shown in Fig. 3, B and C, DN-Smad4 or Smad7 also inhibits TGF-1-induced SBE4_BV-luciferase by ~80%.

DHT is required for full-length AR to inhibit S3*-induced SBE4_BV-luciferase (Fig. 5B). 538-614 AR, which lacks the DBD, is able to inhibit ~70% of S3* activity independent of ligand and ~90% (similar to wild type) of S3* activity with ligand (Fig. 5B); therefore, the DBD may prevent DHT-independent inhibition of S3*. The mechanism by which 538-614 without DHT inhibits S3* activity is unclear; however, data show ARDBD (no DBD) is more efficiently translocated to the nucleus compared with an AR nuclear localization mutant (64). This suggests that the nuclear localization signal of AR is bi- or tripartite and may be enhanced in the absence of DBD. Thus, we hypothesize a greater percentage of 538-614 may be localized to the nucleus in the absence of DHT, as compared with wild-type AR. The means by which AR DBD alone reduces S3* activity is not known, because the 538-614 AR data suggest this region is not necessary for complete inhibition. Furthermore, the EMSA results substantiate that the DBD is not essential for AR-mediated loss of S3* activity (Fig. 6A). AR LBD is able to inhibit GST-Smad3 binding to SBE without DHT; nevertheless, addition of ligand does enhance this effect by ~4-fold, perhaps through an AR LBD conformational change.

The above result parallels data from previous reports that demonstrated LBD alone can bind to Smad3 (37). Interestingly, one report (37) showed the AR DBD or AR LBD could bind to Smad3, although no functional consequence of these interactions was presented. The other study revealed the AR amino-terminal region (a.a. 1-563) associates with the MH2 domain of Smad3 to repress androgen receptor-mediated transcription of murine mammary tumor virus-luciferase (38), but the significance of this interaction (amino-terminal AR·Smad3) to TGF- signaling was not examined. We are not able to explain the discrepancy that exists among several groups attempting to characterize the association of AR with Smad3. However, we believe our data are solid due to our EMSA assay observing a function of the AR·Smad3 association and not solely the physical interaction. It is important to note that although AR can inhibit Smad3, we cannot yet rule out that this inhibition is exclusive to Smad3 and does not also involve Smads 2 or 4 through indirect means.

The normal prostate requires an intact androgen signaling pathway for growth and function, whereas prostate carcinomas often escape from this dependence through changes likely to involve AR. AR receptor mutants that are unable to activate androgen-responsive genes or change the sensitivity of the receptor to circulating androgens (or other steroids) may suppress androgen dependence in the prostate (65-67). Also, mutations within the AR carboxyl terminus which decrease steroid affinity may produce a ligand-insensitive and yet transcriptionally active AR (68, 69). An abundance of AR polymorphisms without functional significance have been characterized, especially within the amino-terminal region, which are linked to increased incidence of prostate cancer (70-72). The expression of several ARAs have been implicated in altering AR activity in the prostate (12, 13, 73-75), which may dysregulate AR·Smad3 interaction and function. Moreover, it is apparent that prostate tumors, both localized and metastatic, maintain or increase AR expression and sensitivity following androgen ablation therapy (10, 76-79). The data presented here suggest a novel mechanism by which such aberrations in AR can directly antagonize TGF- effects within the prostate and promote the development and progression of cancer.

Restoration of TGF- receptor levels by overexpression of wild-type TRII in LNCaP cells was reported to promote TGF- responsiveness and suppress tumor growth through reduced cell proliferation and the induction of apoptosis (80). Consistent with a tumor-suppressive role of TGF- in the prostate, we have shown that DN-TRII promotes malignant transformation of two non-tumorigenic prostate epithelial cell lines (19).² Our data showing that TGF- is a potent inducer of apoptosis in the above cell lines further support TGF- may suppress prostate tumor growth through the induction of apoptosis (47, 81). With this in mind, the current work proposes that constitutive or enhanced activation of AR, through means discussed earlier, may cause loss of androgen dependence (e.g. rescue from undergoing apoptosis) partly via loss of TGF- signaling through inactivation of Smad3, because Smads were shown to be critical to the induction of apoptosis by TGF- (82). This loss would allow for prostatic epithelial cells to escape growth inhibition and apoptosis by TGF-, contributing to carcinogenesis of the prostate.

In conclusion, our data demonstrate for the first time that the association of AR with Smad3 can inhibit the ability of Smad3 to bind SBE and activate transcription. Amplification of AR, or variances within the AR (or its signaling pathway) that promote deregulated and enhanced Smad3 binding, may counteract tumor suppression by TGF-. Moreover, these findings strengthen our hypothesis that androgens promote viability of prostatic epithelial cells, in part, by preventing TGF--induced apoptosis. In view of the numerous binding partners for Smads and AR, understanding how such co-regulators affect the inhibition of Smad3 signaling is critical to our understanding of normal homeostatic mechanisms and carcinogenesis of the prostate.

	ACKNOWLEDGEMENTS

We thank Drs. Mark de Caestecker (pcDNA3.1-DN-Smad4 number 4), Olli Janne (GST-AR DBD), Joan Massagué (3TP-luciferase), Kohei Miyazono (pcDNA3.1-Smad7), Jorma Palvimo (GST-AR DBD), Anita Roberts (pCMV2-FLAG-Smad2, pCMV2-FLAG-Smad3, and pcDNA3.1-DN-Smad4 and critical review of this manuscript), David Russell (pCMV5), Dennis Templeton (discussions and protocol on GST fusion purification), Bert Vogelstein (SBE4_BV-luciferase), Elizabeth Wilson (pCMV5-AR, AR mutants: ABC, 538-614 and 554-644 (DBD)), and Jeffrey Wrana (pCMV5-ALK5 and pCMV5-T204D) for their generous gifts of time and reagents.

	FOOTNOTES

^* This work was supported in part by Cancer Center Development Grant P30CA43703, Ohio Cancer Research Associates grant, and NCI Grant 1R01-CA3069-01 from the National Institutes of Health.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 an NCI Research Oncology Training grant predoctoral award to the Cancer Center from the National Institutes of Health.

^** Current address: University of Washington, Dept. of Molecular Biotechnology, Rm. D4-395, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave., Seattle, WA 98109.

To whom correspondence should be addressed: Ireland Cancer Center Research Laboratories, Samuel Gerber Bldg., Ste. 200, Lab. 3, 11001 Cedar Rd., Cleveland, OH 44106. Tel.: 216-844-6959; E-mail: dxd49@po.cwru.edu.

Published, JBC Papers in Press, November 13, 2001, DOI 10.1074/jbc.M108855200

² D. Danielpour, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: AR, androgen receptor; DHT, 5-dihydrotestosterone; DBD, DNA binding domain; DMEM/F-12, Dulbecco's modified Eagle's medium/Ham's F-12; DN, dominant negative; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; GST, glutathione S-transferase; LBD, ligand binding domain; S2*, active Smad 2; S3*, active Smad 3; SBE, Smad-binding element; TRI, TGF-1 type I receptor; TRII, TGF-1 type II receptor; and TGF-1, transforming growth factor-1; a.a., amino acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid; LBD, ligand binding domain; ARA, AR-associated.

	REFERENCES

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