Differential Modulation of Androgen Receptor-mediated

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 46, Issue of November 15, pp. 43749 –43756, 2002 Printed in U.S.A.

Differential Modulation of Androgen Receptor-mediated Transactivation by Smad3 and Tumor Suppressor Smad4* Received for publication, June 6, 2002, and in revised form, September 3, 2002 Published, JBC Papers in Press, September 10, 2002, DOI 10.1074/jbc.M205603200

Hong-Yo Kang‡, Ko-En Huang‡§, Shiuh Young Chang‡, Wen-Lung Ma‡, Wen-Jye Lin¶, and Chawnshang Chang¶储 From the ‡Center for Menopause and Reproductive Medicine Research, Chang Gung University/Memorial Hospital, Kaohsiung, Taiwan 833, the ¶Departments of Pathology, Urology, and Radiation Oncology, George Whipple Laboratory for Cancer Research, and The Cancer Center, University of Rochester, Rochester, New York 14642

Smad proteins have been demonstrated to be key components in the transforming growth factor ␤ signaling cascade. Here we demonstrate that Smad4, together with Smad3, can interact with the androgen receptor (AR) in the DNA-binding and ligand-binding domains, which may result in the modulation of 5␣-dihydrotestosterone-induced AR transactivation. Interestingly, in the prostate PC3 and LNCaP cells, addition of Smad3 can enhance AR transactivation, and co-transfection of Smad3 and Smad4 can then repress AR transactivation in various androgen response element-promoter reporter assays as well as Northern blot and reverse transcription-PCR quantitation assays with prostate-specific antigen mRNA expression. In contrast, in the SW480䡠C7 cells, lacking endogenous functional Smad4, the influence of Smad3 on AR transactivation is dependent on the various androgen response element-promoters. The influence of Smad3/Smad4 on the AR transactivation may involve the acetylation since the treatment of trichostatin A or sodium butyrate can reverse Smad3/ Smad4-repressed AR transactivation and Smad3/Smad4 complex can also decrease the acetylation level of AR. Together, these results suggest that the interactions between AR, Smad3, and Smad4 may result in the differential regulation of the AR transactivation, which further strengthens their roles in the prostate cancer progression.

Smads are a class of proteins that function as central effectors of the transforming growth factor ␤ (TGF-␤)1 superfamily (1, 2). Smads are directly phosphorylated and activated by type I TGF-␤ family receptors (3, 4). TGF-␤ and activin receptors * This work was supported by National Science Council Grant NSC91-2320-B-182-040, NMRPD1073, Chang Gung Memorial Hospital Grant CMRP1020 (to H.-Y. K.), by National Science Council Grants NMRPD9056, NMRPG9145, CMRP845 (to K.-E. H.), and by National Institutes of Health Grant DK60905 (to C. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence may be addressed. Tel.: 585-273-4500; Fax: 585-756-4133; E-mail: [email protected]. 储 To whom correspondence may be addressed. E-mail: chang@urmc. rochester.edu. 1 The abbreviations used are: TGF, transforming growth factor; AR, androgen receptor; wtAR, wild type androgen receptor; ARA, androgen receptor-associated protein; ARE, androgen response element; DHT, 5␣-dihydrotestosterone; DBD, DNA-binding domain; LBD, ligand-binding domain; PSA, prostate-specific antigen; MMTV, mouse mammary tumor virus; CAT, chloramphenicol acetyltransferase; GST, glutathione-S-transferase; TSA, trichostatin A; SRC, steroid hormone receptor coactivator; HDAC, histone deacetylase; TnT, troponin T; TAT, tyrosine aminotransferase; MH2, Mad homology 2. This paper is available on line at http://www.jbc.org

phosphorylate Smad2 and Smad3 (3, 5), whereas bone morphogenetic protein receptors phosphorylate Smads 1, 5, and 8 (6 – 8). Upon phosphorylation, these Smads will associate with Smad4 and move into the nucleus where they assemble transcriptional complexes that activate specific sets of genes. Thus, Smad4 is a shared key component of these various signaling pathways. A distinct structural feature that distinguishes Smad4 from other Smads is the lack of the SSXS motif at the tail of the Mad homology 2 (MH2) domain terminal that can be phosphorylated by the cognate receptor kinases (3, 4, 9, 10). Smad4 was originally identified as a candidate tumor suppressor gene in chromosome 18q21 that was somatically deleted/ mutated/inactivated in many pancreatic or colorectal tumors (11–13). Knock-out Smad4 studies indicated that the Smad4null mouse has early embryonic lethality (11, 12). The introduction of the Smad4 gene into Smad4-null cells also suggested that the wild type Smad4 could decrease the cell growth rate, cause a cell cycle arrest, and induce apoptosis (13). Although Smad4 has been reported to be infrequently mutated or deleted in breast (14), ovarian (14), and prostate cancers (15), a significant increase of Smad4 was observed in the normal ventral prostate, as well as in the prostate tumors after castration. Moreover, a previous report has shown that the staining for Smad4 was present in areas with a large number of apoptotic cells in the prostate after castration (16). The androgen receptor (AR), a member of the steroid receptor superfamily, functions as an androgen-dependent transcriptional factor (17). After binding to ligand, the activated AR is able to recognize palindromic DNA sequences, called androgen response elements (AREs), and form a complex with ARassociated proteins to induce the expression of AR target genes. Several AR coregulators (ARAs), such as ARA24, ARA54, ARA55, ARA70, ARA160, and ARA267, and Rb, BRCA1, and TIFIIH have been isolated and characterized (18 –25). Results from these studies suggest that coregulators not only can enhance AR transactivation, but may also be able to increase the agonist activity of antiandrogens and 17-␤ estradiol (26) in prostate cancer cells. Previously, we discovered that Smad3 could function as a positive coregulator for AR in prostate cancer DU145 cells (27). We further showed that endogenous prostate-specific antigen (PSA) mRNA in LNCaP cells can be induced by 5␣-dihydrotestosterone (DHT) and the Smad3 further enhanced the PSA expression levels. In addition, several studies also showed that Smad3 can interact with other nuclear receptors, such as estrogen receptor and vitamin D receptor, and enhance these receptors-mediated transactivation (28, 29). As Smad4 proteins have been found to functionally interact with Smad3 (3), AP-1 (30), and p300/CBP (31), it is reasonable that Smad4 may also play an important role in the modulation of the androgen-

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Smad3/Smad4 Represses AR-mediated Transactivation

mediated signal pathway. Here, we demonstrate that both Smad4 and Smad3 can interact with AR in the DNA-binding domain (DBD) and the ligand-binding domain (LBD). Moreover, Smad4 can decrease the AR-Smad3 interaction and repress the Smad3-enhanced AR transactivation. The fact that these interesting inhibitory functions of Smad3/Smad4 on AR transactivation can be reversed by the histone deacetylase (HDAC) inhibitors raises the possibility that Smad3/Smad4 may cooperate with HDAC complex to modulate AR acetylation. This linkage not only strengthens the roles of Smad3/ Smad4 in the prostate cancer cells, it may also further indicate the importance of the acetylation activity in the modulation of AR function in prostate cancer. EXPERIMENTAL PROCEDURES

Chemicals and Plasmids—DHT and hydroxyflutamide were from Schering. pSG5-wild type AR (wtAR) and pCMV-AR were used in our previous report (19). Expression plasmids for glutathione S-transferase (GST)-Smad3 and Smad4 and full-length cDNAs of human Smad3 and Smad4 were kindly provided by Rik Derynck (3). Smad4 (G508S), Smad4 (D539H), Smad4 ⌬M1, ⌬M2, ⌬M,3 and ⌬M4 were provided by Dr. Mark P. de Caestecker (32, 33). Cell Culture and Transfections—Human prostate cancer DU145 cells and PC3 cells were maintained in Dulbecco’s Minimum Essential Medium containing penicillin (25 units/ml), streptomycin (25 ␮g/ml), and 5% fetal calf serum. Transfections were performed using the calcium phosphate precipitation method, and cells were harvested after 24 h for the chloramphenicol acetyltransferase (CAT) assay, as described previously (19). The CAT activity was visualized and quantitated by STORM 840 (Molecular Dynamics). At least three independent experiments were carried out in each case. The SW480䡠C7 cells and PC3-AR2 cells were the gifts from Dr. Eric J. Stanbridge and Dr. T. J. Brown. Western Blot Analysis—Cells were lysed in lysis buffer (50 mM TrisHCl, pH 7.4, 150 mmol/l NaCl, 1 mmol/l EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin). An aliquot of each sample was used for determination of protein content using the Bradford protein assay reagent. Samples with equivalent amounts of protein were subjected to SDS-PAGE on a 7.5% acrylamide gel as described previously (19). Blots were developed using the ECL Western blot analysis system from Amersham Biosciences. GST Pull-down Assay—Fusion proteins of GST-Smad3, GST-AR, and GST protein alone were obtained by transforming expressing plasmids into BL21 (DE3) pLysS strain competent cells followed with 1 mM isopropyl-1-thio-␤-d-galactopyranoside induction. GST-fusion proteins then were purified by glutathione-SepharoseTM 4B (Amersham Biosciences). The wtAR and deletion mutant AR proteins labeled with 35S were generated in vitro using the TNT-coupled reticulocyte lysate system (Promega). For the in vitro interaction, the glutathione-Sepharosebound GST-proteins were mixed with 5 ␮l of 35S-labeled TNT proteins in the presence or absence of 1 ␮M DHT at 4 °C for 3 h. The bound proteins were separated on an 8% SDS-polyacrylamide gel and visualized by using autoradiography. Co-immunoprecipitation of AR and Smads—PC3 Cells were cotransfected with AR and FLAG-Smad4 and FLAG-Smad3 for 16 h and then treated with vehicle or 10 nM DHT for another 16 h. LNCaP and PC3-AR2 cells were treated with vehicle or 10 nM DHT for 16 h. The cells were lysed and incubated with monoclonal anti-FLAG antibody (Sigma), polyclonal Smad4 and Smad3 antibodies (Santa Cruz), or control IgG at 4 °C for 2 h depending on the experimental design, followed by addition of protein A/G beads (Santa Cruz) for 1 h at 4 °C. The bound proteins were separated on an 8% SDS-polyacrylamide gel and blotted with polyclonal AR antibody (NH27), Smad4, and Smad3 antibodies or anti-FLAG antibody. The bands were detected using an alkaline phosphatase detection kit (Bio-Rad). Principle of Real-Time PCR—Fluorescence signal from each real time quantitative PCR reaction (PerkinElmer Life Sciences) is collected as normalized values plotted versus the cycle number. Reactions are characterized by comparing threshold cycle (CT) values. The CT is a value defined the fractional cycle number at which the sample fluorescence signal passes a fixed threshold above base. Quantitative values are obtained from the CT number at which the increase in signal associated with an exponential growth of PCR product starts to be detected (using PerkinElmer Life Sciences systems analysis software) according to the manufacturer’s manual. The precise amount of total RNA added to each reaction (based on absorbance) and its quality (i.e.

lack of extensive degradation) are both difficult to assess. Therefore, we also quantified transcripts of the gene ␤-actin as the endogenous RNA control, and each sample was normalized on the basis of its ␤-actin content. The relative target gene expression level was also normalized to the calibrator. The final results, expressed as N-fold differences in target gene expression relative to the ␤-actin gene and the calibrator termed “N target” were determined as follows: N target ⫽ 2⫺(⌬CTsample⫺⌬CTcalibrator), where ⌬CT values of the sample and calibrator are determined by subtracting the average CT value of the target gene from the average CT value of the ␤-actin gene. Oligonucleotide Primers Design—The target cDNA sequence was evaluated using the Primer Express software (PerkinElmer Life Sciences) (see Table I in Fig. 1). The forward and reverse primers were designed to lie in adjacent exons to prevent amplification genomic DNA that may be contained in samples. RNA Extraction—Total RNA was extracted from LNCaP cell lines by using the TRIZOL (Invitrogen). The quality of the RNA samples was determined by electrophoresis through agarose gels and staining with ethidium bromide. The 18 S and 28 S RNA bands were visualized under UV light. PCR Amplification—All of the PCR reactions were performed using an ABI Prism 7700 Sequence Detection System (Invitrogen). PCR was performed using the SYBR威 Green PCR Core Reagents kit (PerkinElmer Life Sciences). The amplification reactions were performed in 25 ml of final volume containing 10⫻ SYBR buffer, 25 mM of MgCl2, 12.5 mM of dNTP, 0.625 units of AmpliTaq Gold and 0.25 units of uracil N-glycosylase. Final AR, PSA, and ␤-actin forward, reverse concentrations were 2.5 ␮M. To reduce variability between replicates, PCR premixes, which contained all reagents except for total RNA, were prepared and aliquoted into 1.5-ml microfuge tubes. The thermal cycling conditions comprised an initial denaturation step at 95 °C for 10 min and 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Specific PCR amplification products were detected by the fluorescent doublestranded DNA-binding dye SYBR Green core reagent kit (PerkinElmer Life Sciences). Experiments were performed with duplicates for each data point. RESULTS

Repression of Smad3-enhanced AR Transactivation by Smad4 in Different Prostate Cancer Cells—To study the potential correlation between AR and Smads in prostate cancer cells, we first chose prostate cancer PC3-AR2 cells, which were stably transfected with wtAR, to examine the effect of Smads on androgen-mediated mouse mammary tumor virus (MMTV) promoter activity. Activation of MMTV-CAT activity was achieved by treating with 10⫺8 M DHT (Fig. 1A, lane 1 versus 2), and this androgen-activated transactivation was further enhanced by the addition of Smad3, but not Smad4 (Fig. 1A, lanes 3 and 4 versus 5 and 6). Interestingly, Smad3-enhanced AR transactivation was significantly repressed by adding Smad4 (Fig. 1A, lane 3 versus 7). Similar results were obtained with LNCaP cells that expressed mutated but functional AR (Fig. 1A). Next, we examined the effect of Smads on AR transactivation by increasing the dose of Smad3, Smad4, and steroid hormone receptor coactivator-1 (SRC-1) in PC3-AR2 cells. As shown in Fig. 1B, in the presence of Smad3, the addition of Smad4 can result in the repression of Smad3-enhanced AR transactivation in a dose-dependent manner (Fig. 1B, lane 3 versus 4, 5, and 6). On the other hand, in the presence of Smad4, AR transactivation was slightly repressed (Fig. 1B, lane 2 versus 10), and adding Smad3 could then further repress AR transactivation (Fig. 1B, lane 10 versus 11, 12, and 13). In contrast, addition of SRC-1 can then increase AR transactivation in the presence of Smad3 or Smad4 (lane 3 versus 7, 8, 9 and lane 10 versus 14, 15, and 16). Together, these results from reporter assays suggested that the amount of Smad4 available might have a major influence on the Smad3 effect on the AR transactivation. Repression of Endogenous AR Target Gene PSA mRNA Expression by Smad3/Smad4 —To further confirm Smad3/Smad4 repression effects on AR transactivation and reduce potential artifact effects related to reporter assays, we then applied


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FIG. 1. The ligand-induced transactivation of AR is enhanced by Smad3 but repressed by Smad3/Smad4. A, CAT assays were performed with extracts from PC3-AR2 cells transfected with the indicated amount of Smad3 or Smad4 expression vector (␮g) in the presence (⫹) or absence (⫺) of 10⫺8 M DHT. LNCaP cells were transfected with Smad3 or Smad4 expression vector instead of PC3-AR2 cells in experiments otherwise identical with those in A. B, PC3-AR2 cells stably transfected with AR were overexpressed with the indicated amounts of Smad3, Smad4, or SRC-1. 3 ␮g of MMTV-CAT was used as a reporter plasmid in all experiments. All values represent the averages ⫾ S.D. of four independent experiments. C, LNCaP cells were transfected with Smad3, Smad4, and parent vector as indicated for 16 h, followed by DHT treatment for another 16 h. PSA expression level was determined by Northern blotting. The probe was obtained from exon 1 of the PSA gene and labeled with [␣-32P]dCTP. A ␤-actin probe was used as a control for equivalent mRNA loading. (Table I in Fig. 1) The RNA samples from A were reverse transcribed to cDNAs, and all of the samples were subjected to real-time PCR using an ABI Prism 7700 Sequence Detection System (PerkinElmer Life Sciences). D, specific PCR amplification products were detected by the fluorescent double-stranded DNA-binding dye SYBR Green core reagent kit. The PSA mRNA relative to ␤-actin is calculated using the equation 2⫺ ⌬⌬CT. Experiments were performed with triplicates for each data point.

Northern blot to assay Smad3/Smad4 repression effect on AR endogenous target gene PSA mRNA expression. In LNCaP cells, 10 nM DHT increases PSA mRNA expression (Fig. 1C, lane 1 versus 2). Addition of Smad3 further enhances DHT-

induced PSA mRNA expression (Fig. 1C, lane 2 versus 3), whereas addition of Smad4 alone has slight repression of PSA mRNA expression (Fig. 1C, lane 2 versus 4). Addition of both Smad3 and Smad4 reverses the Smad3-enhanced PSA mRNA

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FIG. 3. In vitro interaction between Smads and AR. A, a series of S-labeled full-length AR and different AR deletion mutants were incubated with GST-Smad3, GST-Smad4, and GST alone in the presence (⫹) or absence (⫺) of 10 nM DHT to test for interaction domains of AR for Smad3 and Smad4. Complex bound to GST columns was subjected to SDS-PAGE and autoradiography. B, GST-AR-LBD fusion proteins and GST control were incubated with in vitro transcribed/translated 35S-labeled Smad3 or Smad4 in the presence (⫹) or absence (⫺) of 10 nM DHT to test for interaction in the GST pull-down assay. C, the indicated amounts of purified Smad4 proteins were added to fixed volume of in vitro transcribed/translated 35S-labeled full-length AR and GST-Smad3 (2 ␮g) to test for interaction. Bands were detected using a phosphorimaging device. A representative blot of three independent experiments is shown. 35

FIG. 2. In vivo interaction between Smads and AR. A, co-immunoprecipitation of AR and Smad3. A, PC3-AR2 cells that overexpressed FLAG-Smad3, FLAG-Smad4, and AR were treated with or without DHT. Cell extracts were prepared, and immunoprecipitations were performed using anti-FLAG antibody followed by immunoblotting using antibody to AR. B, proteins were precipitated from PC3, PC3-AR2, and LNCaP cells, which were treated with or without DHT (10⫺8 M) for 16 h using a AR-specific antibody or control IgG antibody. Precipitated proteins were analyzed by Western blotting with mouse monoclonal antibodies specific for AR and Smad4. For comparison, a portion of the cell lysates was probed with the same Smad4 antibody.

expression (Fig. 1C, lane 5). Similar results were also obtained with the real-time quantitative reverse transcription-PCR assay to measure PSA mRNA expression (Tables I and II in Fig. 1 and Fig. 1D). Together, Fig. 1, C and D all confirmed the above ARE-reporter assays showing that addition of Smad3 alone can enhance AR transactivation and that addition of both Smad3 and Smad4 can then repress AR transactivation. Interaction between AR, Smad3, and Smad4 in Vitro and in Vivo—Next, we examined the possibility of interaction between AR, Smad3, and Smad4. N-terminal FLAG-tagged, full-length Smad3 and Smad4 were expressed in PC3-AR2 cells and treated with DHT (or vehicle). Cell extracts were prepared for immunoprecipitations by using anti-FLAG or anti-AR antibodies. As shown in Fig. 2A, in the presence of FLAG-Smads, AR was able to be co-immunoprecipitated with Smad3 as we reported previously (27) and Smad4 was detected in the AR complex both in the presence and absence of DHT. Moreover, Fig. 2B demonstrated that immunoprecipitating against AR can also capture Smads/AR complexes. An in vivo co-immunoprecipitation assay was applied to demonstrate that the endogenous Smad4 immunocomplex exists in the absence or presence of DHT in PC3-AR2 cells but not in AR-negative PC3 cells. A similar result was also obtained when we replaced PC3-AR2 with LNCaP cells. To dissect which individual domain of AR can interact with Smad4, we used GST-Smad4 fusion proteins incubated with various AR deletion mutants in pull-down experiments. As

shown in Fig. 3A, the full-length wtAR could interact with Smad4 both in the presence and absence of 10 nM DHT. While AR-DBD/LBD peptides could interact with Smad4, we also found that both AR-DBD and AR-LBD peptides interact with Smad4 but not N-terminal AR peptide. Similar results were obtained with GST-AR, which can also bind to Smad3 or Smad4 (Fig. 3B). These results suggest that both DBD and LBD domains of AR may contain binding sites for Smad4 or Smad3 interactions. In addition, when we used GST-Smad3 to incubate with AR and added different amounts of Smad4 to this protein complex, the full-length wtAR could interact with Smad3. Interestingly, addition of Smad4 can decrease this AR-Smad3 interaction in a dose-dependent manner (Fig. 3C). Previous reports showed that Smad3 can interact with Smad4 (1, 35, 36), and our results further demonstrated that both Smad3 and Smad4 can interact with AR in the DBD and LBD. It is possible that Smad4 may either directly compete with Smad3 for the same AR binding sites or that Smad4 may simply have a higher binding affinity than Smad3 to bind to AR. We reasoned that the identification of the domains in Smad3 and Smad4 required for their association with AR transcriptional activity would provide us with tools to dissect the roles of Smad3 and Smad4 in Smads-mediated repression of AR transactivation. Therefore, we tested which region(s) within the Smad4 are important for repression of Smad3-enhanced AR transactivation. As shown in Fig. 4A, addition of Smad3 increased the AR transactivation (lane 2 versus 3), co-transfection of Smad4 and Smad3 can then repress the Smad3-enhanced AR transactivation (lane 3 versus 7) in PC3 cells. In contrast, replacing Smad4 with a Smad4 mutant, with a Cterminal deletion, can only show partial repression effect (lane 7 versus 8). On the other hand, a C-terminal deletion of Smad3


FIG. 4. Effects of Smad3 and Smad4 mutants on AR-mediated transcriptional activity. A, PC3 cells were co-transfected with 3 ␮g of Smad3, Smad4, Smad3⌬C, or Smad4⌬C mutant expression vectors with 1 ␮g of pSG5-AR and 3 ␮g of MMTV-CAT, in the presence (⫹) or absence (⫺) of 10⫺8 M DHT. B, Smad3 was co-expressed with different Smad4 deletion mutants as indicated in PC3 cells. Each CAT activity is presented relative to the transactivation observed in the absence of DHT, and an error bar represents the mean ⫾ S.D. of four independent experiments.

resulted in the loss of the Smad3-enhanced effect on the AR transactivation (lane 3 versus 4). As previous reports indicated that the MH2 region of the C-terminal Smad proteins is important for homo-oligomerization and hetero-oligomerization (11), it is possible that the C-terminal region is also important for Smad proteins to interact with AR and exert its function on the modulation of AR transactivation. To further dissect the repression domains of Smad4, a series of deletion mutants of Smad4 (Fig. 4B), were tested in the Smad3-enhanced AR transactivation. Among all mutants, ⌬M4, with a deletion of amino acids 274 –322, and Smad4G508S have the greatest effect to reverse the Smad4 repression of Smad3-enhanced AR transactivation, suggesting that the region amino acids 274 –322 and amino acid 508 within the C-terminal of Smad4 may play essential roles to repress Smad3-enhanced AR transactivation. Transcriptional Repression by Smad3/Smad4 Is Associated with Decreasing AR Acetylation—Previously, p300/CBP was shown to be able to acetylate non-histone proteins including AR and the addition of trichostatin A (TSA), a specific inhibitor of histone deacetylase activity, was shown to induce the andro-

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gen-mediated AR transactivation (34 –36). Other reports also suggested that some Smads could interact directly with several HDAC1-associated proteins, such as 5⬘TG3⬘ interacting factor (TGIF), c-ski, and SnoN-(37– 40), which might play roles in the Smads-mediated transcriptional repression. Whether repression of AR by Smad3/Smad4 might involve the deacetylation of AR, however, remains unknown. We first tested if TSA had any influence on the Smad3/Smad4-mediated AR transactivation. As shown in Fig. 5A, addition of TSA can enhance AR transactivation in a dose-dependent manner (lane 2 versus lane 5, 8, and 11). More importantly, addition of TSA can also reverse the Smad3/Smad4-repressed AR transactivation in a dose-dependent manner in PC3-AR2 cells (lane 3 versus 6, 9, and 12). A similar effect was also observed when cells were treated with NaB, another specific inhibitor of histone deacetylase (lane 13–15). To further determine whether Smad3 and Smad4 could influence the acetylation of AR, immunoprecipitation was performed using an AR-specific antibody on cell extracts transfected with different Smad expression vectors. The immunoprecipitate was subjected to Western blotting with an antiacetyl-lysine antibody, AR-specific antibody, or anti-FLAG antibody. As shown in Fig. 5B, acetyl-lysine-immunoreactive bands were detected in the AR antibody IP complex, which have the identical mobility to the AR bands, whereas acetyllysine-immunoreactivity was decreased when cells were cotransfected with Smad3 and Smad4. Together, these results suggest that Smad3/Smad4 may modulate the endogenous deacetylase activity that results in the decrease of AR acetylation, consequently repressing AR transactivation. The Effects of Smads on AR Transactivation Are Promoter Context- and Cell Type-dependent—Using CV-1 cells, Hayes et al. reported that Smad3 could also repress the transcriptional activity of AR (41), which is in contrast with our earlier report showing that Smad3 can enhance AR transactivation in DU145, LNCaP, and SW480䡠C7 cells (27). However, like most of the studies on AR or Smads-mediated transcription, these were carried out in individual cells using different promoters. The homo- or hetero-oligomerization between Smad3 and Smad4 to interact with the different ARE-promoters linked to the reporters could also influence the AR transactivation. Since the Smad3/Smad4-repressed AR transactivation was observed in cells with MMTV promoter, we further compared the effects of individual Smads on AR transcription activity in MMTV and PSA, two of the AR target natural promoters, and two synthetic promoters, tyrosine aminotransferase (TAT)2 and 5X-ARE, which contains two and five copies of a synthetic ARE, respectively. In PC3(M) cells that express a relatively lower amount of Smad4, we found that adding Smad3 alone could enhance all these reporters linked with the four different AREs, and adding Smad3/Smad4 together can then reverse these enhancement effects (Fig. 6A). These results are consistent with the data shown in Fig. 1. In contrast, when we replace PC3(M) cells with SW480䡠C7 cells, a cell line lacking endogenous functional Smad4 (42), we found that adding Smad3 alone can only enhance MMTV-ARE reporter activity (Fig. 6B, lane 2 versus 3) as we reported previously (27). For the other 3 ARE reporters (PSA-ARE, 5X-ARE, and TAT2-ARE), Smad3 alone has some slight repression (Fig. 6B, lane 7 versus 8, lane 12 versus 13, and lane 17 versus 18). Again adding Smad4 and Smad3 together can then repress AR transactivation in all these four different ARE reporters (Fig. 6B, lanes 2 versus 5, lanes 7 versus 10, lanes 12 versus 15, and lanes 17 versus 20). These results, together with previous reports (27, 41), suggest that the modulation of AR functions by Smad3/Smad4 could be dependent on the context of AREs and the availability of Smad3/Smad4 in the cells. Meanwhile, we measured the de-

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FIG. 5. The association of Smad3 and Smad4 with AR deacylation. A, PC3-AR2 cells co-transfected with the MMTV-LUC reporter and the expression vector for the Smad3/Smad4 were treated with DHT in the presence of increasing doses of TSA. All values represent the mean ⫾ S.D. of three independent experiments. B, immunoprecipitation assays were performed with extracts from cells transfected with Smad3, Smad4, or Smad3/Smad4 expression vector in the presence (⫹) or absence (⫺) of 10⫺8 M DHT. Equal amounts of whole cell lysates were subjected to co-immunoprecipitation with an anti-AR antibody. The co-immunoprecipitates were analyzed on Western blot with a specific anti-acetyl lysine antibody. Anti-AR or anti-FLAG was used in immunoblot analysis of the immunoprecipitated complexes to confirm that uniform amounts of AR and Smads were immunoprecipitated.

tectable amount of endogenous Smad4 and compared it to amounts of Smad4 in transfected cells in Fig. 6, and the results showed that PC3(M) cells contain lower amounts of endogenous Smad4 than exogenous Smad4 in transfected PC3(M) cells. It is possible that a lower level of Smad4 in PC3(M) cells could be one of the factors to contribute to the Smad3 enhanced effect, while higher amounts of Smad4 may result in repression, although we are unable to exclude that the different cofactors and the context of the ARE promoters may also contribute to the enhanced effect of Smad3 on AR-mediated transcription in different cell types and origins. DISCUSSION

In the present studies, we have shown that both Smad4 and Smad3 can interact with AR in the DBD and the LBD. Moreover, Smad4 can decrease the AR-Smad3 interaction and repress the Smad3-enhanced AR transactivation; however, detailed mechanisms of how Smad3 and/or Smad4 can modulate AR transactivation, remains unclear. Fig. 7 illustrates a simple model for the modulation on AR mediated-transactivation by Smad3/Smad4. First, when the ratio of Smad3/Smad4 is relatively higher, Smad3 may function as a positive coregulator to enhance AR transactivation in human prostate cancer cells. As

both Smad3 and Smad4 can interact with AR-DBD and ARLBD, it is likely that higher amounts of Smad4 may be able to directly compete with Smad3 to bind to AR. Alternatively, Smad3 may have a higher binding affinity to Smad4 than to AR. The consequence of such multiple effects among Smad3, Smad4, and AR may result in weaker interactions and transactivations between Smads and AR. Second, recruitment of HDAC complex to promoters has emerged as a general mechanism of transcription repression of target genes, and it may be mediated either through direct interactions with regulatory proteins such as transcription factor or through interaction with corepressors containing an HDAC-interacting domain. One possible mechanism to explain how Smad3/Smad4 can modulate AR transactivation is that after interaction with AR, the complex of Smad3/Smad4 may be able to recruit some transcriptional repressors associated with the HDAC complex, which may then result in the repression of AR transactivation via decreasing the acetylation level of AR. HDAC1 is found in a large transcriptional regulatory complex that includes AR, Smad3, Sin3A (34), and N-CoR (36, 39), and the p300 coactivator may compete with some HDAC1-associated proteins, such as c-ski or TGIF, for the binding to the MH2


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FIG. 7. A simplified model for the roles of Smad3 and Smad4 in AR-mediated target genes transactivation.

FIG. 6. Androgen-response element is important for TGF-␤/ Smad3-enhanced AR transactivation. A, PC3(M) and B, SW480䡠C7 cells were transiently co-transfected with either MMTV-CAT, PSACAT, 5X-ARE-CAT, or (TAT)2-CAT (3 ␮g), in the presence of AR, Smad3, Smad4, or Smad3/Smad4 as indicated. Anti-Smad3 and antiSmad4 antibodies were used in Western blot analysis of the cell extracts to compare the amounts of endogenous Smad3/Smad4 and exogenous Smad4 in both PC3(M) and SW480䡠C7. Each CAT activity is presented relative to the transactivation observed in the absence of DHT. All values represent the mean ⫾ S.D. of three independent experiments.

domain of Smad3 (43, 44). Moreover, because TGIF has been demonstrated to interact with AR and Smad proteins (36, 43), it will be of interest to test whether HDAC1 was recruited into

the Smad3/Smad4 complex on the AR-mediated PSA promoter and HDAC1 or whether TGIF has any influence on the Smad3/ Smad4-mediated repression of AR transactivation in the prostate cancer cells. Furthermore, our data showed that a C-terminal deletion of Smad3 resulted in the loss of the Smad3-enhanced effect on the AR transactivation, and a Smad4 mutant, with a C-terminal deletion, can only show partial repression effects. Together, these results indicate that the MH2 domain within the Cterminal of Smad4 may play an important role that interacts with AR and exerts its function on the modulation of AR transactivation. These data are consistent with previous reports that the MH2 domain of Smad proteins was shown to be involved in many biological functions through interaction with other regulatory proteins including glucocorticoid receptor, suggesting an important role for MH2 domain in regulating nuclear hormone receptors (28, 45). As Smad structure-function section of the MH2 region of the C-terminal Smad proteins is important for homo-oligomerization and hetero-oligomerization between Smad4 and R-Smads (11), it is possible that Smad3 may either homodimerize with Smad3 or heterodimerize with Smad4 via their MH2 domains in the presence of AR-coregulators complex. The consequence of such homo- or hetero-oligomerizations can then result in either the enhancement or repression of AR transactivation. Previous experiments demonstrated that both PCAF and p300 acetylated the AR on three lysine residues, Lys-630, Lys632, and Lys-633 within the KLKK motif (34). While acetylation of these amino acids has been demonstrated to enhance the AR transcriptional activity, mutations on these residues have resulted in the loss of the AR-mediated transactivation on target gene expression. The findings that addition of TSA can reverse the Smad3/Smad4-repressed AR transactivation and the acetylation of AR is decreased in the presence of Smad3 and Smad4 provides evidence that Smad3/Smad4 may be involved in the regulation of AR-mediated gene expression by modulating potential changes to the acetylation status of the AR. The ability of Smad3 in combination with Smad4 to repress the transcriptional activity of AR did not depend on the promoter or cell types but the ability of Smad3 alone to regulate the AR transactivation, indeed depended on the cell context and the promoter sequence. Since our results demonstrated that cooperation between Smad3 and Smad4 is essential to show the optimal repression on AR-mediated transcription, it will be of interest to know whether Hayes’s report (41) showed that the repression of AR-mediated transcription in CV-1 cells by Smad3 may reach to the greatest activity when co-transfected with Smad4. Considering the relative expression levels of Smad4 to Smad3 may determine the extent of the potentiation of AR function, together with other unknown AR coregu-

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lators it is possible that the effect of Smad3/Smad4 on AR transactivation may depend on the combination of the SmadsAR-coregulators complex that exists in the cells. Therefore, the balance between coactivator and corepressor, such as Smad3 and Smad4, with which AR complexes interact in the nucleus, is likely to play important roles in androgen action of gene regulation. Expression of different Smads has been studied in several human cancer cells and tissues including malignant prostate. In the ventral prostate, a significant increase of phosphorylated Smad2 was observed after castration. In prostatic tumor cells, an increased expression of Smad2 and phosphorylated Smad2 was observed after treatment of estrogen (16). The levels of Smad3 and, in particular, Smad4 were enhanced in the normal ventral prostate, as well as in the tumors after castration (16). However, direct evidence to link different Smads expression with androgen is currently lacking. Further studies to determine how androgen affects the expression pattern of different Smads and the cellular ratio between Smad3: Smad4:AR can influence Smads modulation of AR transactivation may help us to better understand the potential roles of Smad3 and Smad4 in the AR-mediated prostate cancer progression.

15. 16. 17. 18. 19. 20. 21. 22. 23.

24. 25. 26. 27. 28. 29.

30. 31.

Acknowledgments—We thank Drs. Rik Derynck, Mark P. de Caestecker, Eric J. Stanbridge, Xiao-Fan Wang, and T. J. Brown for their valuable plasmids and cells. We also thank Karen Wolf for manuscript preparation.

33.

REFERENCES

34.

1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12.

13. 14.

Derynck, R., Zhang, Y., and Feng, X. H. (1998) Cell 95, 737–740 Massague´ , J. (1998) Annu. Rev. Biochem. 67, 753–791 Zhang, Y., Feng, X., We, R., and Derynck, R. (1996) Nature 383, 168 –172 Macias-Silva, M., Abdollah, S., Hoodless, P. A., Pirone, R., Attisano, L., and Wrana, J. L. (1996) Cell 87, 1215–1224 Nakao, A., Imamura, T., Souchelnytskyi, S., Kawabata, M., Ishisaki, A., Oeda, E., Tamaki, K., Hanai, J., Heldin, C. H., Miyazono, K., and ten Dijke, P. (1997) EMBO J. 16, 5353–5362 Liu, F., Hata, A., Baker, J. C., Doody, J., Carcamo, J., Harland, R. M., and Massague, J. (1996) Nature 381, 620 – 623 Graff, J. M., Bansal, A., and Melton, D. A. (1996) Cell 85, 479 – 487 Hoodless, P. A., Haerry, T., Abdollah, S., Stapleton, M., O’Connor, M. B., Attisano, L., and Wrana, J. L. (1996) Cell 85, 489 –500 Kretzschmar, M., Liu, F., Hata, A., Doody, J., and Massague, J. (1997) Genes Dev. 11, 984 –995 Liu, X., Sun, Y., Constantinescu, S. N., Karam, E., Weinberg, R. A., and Lodish, H. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10669 –10674 Takaku, K., Oshima, M., Miyoshi, H., Matsui, M., Seldin, M. F., and Taketo, M. M. (1998) Cell 92, 645– 656 Sirard, C., de la Pompa, J. L., Elia, A., Itie, A., Mirtsos, C., Cheung, A., Hahn, S., Wakeham, A., Schwartz, L., Kern, S. E., Rossant, J., and Mak, T. W. (1998) Genes Dev. 12, 107–119 Dai, J. L., Bansal, R. K., and Kern, S. E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1427–1432 Schutte, M., Hruban, R. H., Hedrick, L., Cho, K. R., Nadasdy, G. M.,

32.

35. 36. 37. 38.

39. 40.

41. 42.

43. 44. 45.

Weinstein, C. L., Bova, G. S., Isaacs, W. B., Cairns, P., Nawroz, H., Sidransky, D., Casero, R. A., Jr., Meltzer, P. S., Hahn, S. A., and Kern, S. E. (1996) Cancer Res. 56, 2527–2530 MacGrogan, D., Pegram, M., Slamon, D., and Bookstein, R. (1997) Oncogene 15, 1111–1114 Brodin, G., ten Dijke, P., Funa, K., Heldin, C. H., and Landstrom, M. (1999) Cancer Res. 59, 2731–2738 Chang, C. S., Kokontis, J., and Liao, S. T. (1988) Science 240, 324 –326 Hsiao, P. W., and Chang, C. (1999) J. Biol. Chem. 274, 22373–22379 Kang, H. Y., Yeh, S., Fujimoto, N., and Chang, C. (1999) J. Biol. Chem. 274, 8570 – 8576 Fujimoto, N., Yeh, S., Kang, H. Y., Inui, S., Chang, H. C., Mizokami, A., and Chang, C. (1999) J. Biol. Chem. 274, 8316 – 8321 Yeh, S., and Chang, C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5517–5521 Hsiao, P. W., Lin, D. L., Nakao, R., and Chang, C. (1999) J. Biol. Chem. 274, 20229 –20234 Yeh, S., Miyamoto, H., Nishimura, K., Kang, H., Ludlow, J., Hsiao, P., Wang, C., Su, C., and Chang, C. (1998) Biochem. Biophys. Res. Commun. 248, 361–367 Yeh, S., Hu, Y. C., Rahman, M., Lin, H. K., Hsu, C. L., Ting, H. J., Kang, H. Y., and Chang, C. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11256 –11261 Lee, D. K., Duan, H. O., and Chang, C. (2000) J. Biol. Chem. 275, 9308 –9313 Miyamoto, H., Yeh, S., Wilding, G., and Chang, C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7379 –7384 Kang, H. Y., Lin, H. K., Hu, Y. C., Yeh, S., Huang, K. E., and Chang, C. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3018 –3023 Matsuda, T., Yamamoto, T., Muraguchi, A., and Saatcioglu, F. (2001) J. Biol. Chem. 276, 42908 – 42914 Subramaniam, N., Leong, G. M., Cock, T. A., Flanagan, J. L., Fong, C., Eisman, J. A., and Kouzmenko, A. P. (2001) J. Biol. Chem. 276, 15741–15746 Zhang, Y., Feng, X. H., and Derynck, R. (1998) Nature 394, 909 –913 Feng, X. H., Zhang, Y., Wu, R. Y., and Derynck, R. (1998) Genes Dev. 12, 2153–2163 de Caestecker, M. P., Yahata, T., Wang, D., Parks, W. T., Huang, S., Hill, C. S., Shioda, T., Roberts, A. B., and Lechleider, R. J. (2000) J. Biol. Chem. 275, 2115–2122 de Caestecker, M. P., Hemmati, P., Larisch-Bloch, S., Ajmera, R., Roberts, A. B., and Lechleider, R. J. (1997) J. Biol. Chem. 272, 13690 –13696 Fu, M., Wang, C., Reutens, A. T., Wang, J., Angeletti, R. H., Siconolfi-Baez, L., Ogryzko, V., Avantaggiati, M. L., and Pestell, R. G. (2000) J. Biol. Chem. 275, 20853–20860 List, H. J., Smith, C. L., Rodriguez, O., Danielsen, M., and Riegel, A. T. (1999) Exp. Cell Res. 252, 471– 478 Sharma, M., and Sun, Z. (2001) Mol. Endocrinol. 15, 1918 –1928 Wotton, D., and Massague, J. (2001) Curr. Top. Microbiol. Immunol. 254, 145–164 Leong, G. M., Subramaniam, N., Figueroa, J., Flanagan, J. L., Hayman, M. J., Eisman, J. A., and Kouzmenko, A. P. (2001) J. Biol. Chem. 276, 18243–18248 Liberati, N. T., Moniwa, M., Borton, A. J., Davie, J. R., and Wang, X. F. (2001) J. Biol. Chem. 276, 22595–22603 Xu, W., Angelis, K., Danielpour, D., Haddad, M. M., Bischof, O., Campisi, J., Stavnezer, E., and Medrano, E. E. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5924 –5929 Hayes, S. A., Zarnegar, M., Sharma, M., Yang, F., Peehl, D. M., ten Dijke, P., and Sun, Z. (2001) Cancer Res. 61, 2112–2118 Goyette, M. C., Cho, K., Fasching, C. L., Levy, D. B., Kinzler, K. W., Paraskeva, C., Vogelstein, B., and Stanbridge, E. J. (1992) Mol. Cell. Biol. 12, 1387–1395 Wotton, D., Lo, R. S., Lee, S., and Massague, J. (1999) Cell 97, 29 –39 Wotton, D., Knoepfler, P. S., Laherty, C. D., Eisenman, R. N., and Massague, J. (2001) Cell Growth Differ. 12, 457– 463 Song, C. Z., Tian, X., and Gelehrter, T. D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11776 –11781