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Molecular Biology and Genetics |
Departments of Surgery and Genetics [S. A. H., M. Z., M. S., F. Y., Z. S.] and Urology [D. M. P.], Liem Sioe Liong Molecular Biology Laboratory, Stanford University School of Medicine, Stanford, California 94303, and Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands [P. t. D.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The signals mediated by transforming growth factor ß (TGF-ß) play fundamental roles in the determination of cell fate and growth control (8 , 9) . Of importance to note for our study here, TGF-ß is a potent growth inhibitor of prostatic epithelial cells (10 , 11) . In prostate cancer, the TGF-ß pathway is often inactive, with the loss of normal RNA transcripts and proteins of TGF-ß receptors (11, 12, 13) . Restoration of the TGF-ß pathway in prostate cancer cells that are originally refractory to TGF-ß can suppress in vitro tumorigenic growth by inhibiting cell proliferation (14 , 15) .
TGF-ß signals are transduced through a group of intracellular signal transducers, the Smad protein family (8 , 16) . The Smad proteins can be divided into three distinct subgroups, the receptor-regulated Smads (R-Smads; 1, 2, 3, 5, and 8), the common mediator Smads (Co-Smads; 4), and the inhibitory Smads (I-Smads; 6 and 7), that antagonize the signaling function of the R- and Co-Smads. Upon stimulation by TGF-ß or related growth factors, receptor-activated Smads become phosphorylated by the kinase activity of activated TGF-ß receptors, oligomerize with the common Smads, and move into the nucleus. These activated Smad protein complexes exert their transcriptional function by binding to DNA and forming active complexes with other transcription factors. Smads have been found to complex with transcriptional activators and repressors, such as AP-1 (17) , CBP/P300 (18 , 19) , and TGIF (20) , to regulate a variety of cellular processes. The activities of nuclear hormone receptors are mediated, at least in part, through the Smad proteins. For example, the vitamin D receptor (VDR) interacts with Smad3 to enhance ligand-dependent transcription (21) , and the glucocorticoid receptor (GR) inhibits TGF-ß-responsive promoters through a physical interaction with Smad3 (22) .
In general, Smad proteins consist of three domains including an NH2-terminal MH1 domain, a COOH-terminal MH2 domain, and an intervening linker between these two domains (8 , 9 , 16) . The MH1 domain is very similar in R-Smads and Smad4, but only weakly conserved in I-Smads (23) . Although the functions of MH1 are not clear, it has been shown that the MH1 domain has intrinsic affinity to the MH2 domain and inhibits the MH2 functions. The MH2 domain has been identified as involved in protein-protein interaction (8) . However, in the case of I-Smads, the MH2 domain was shown to be sufficient for their inhibitory effect (24) .
Androgen promotes the growth and proliferation of prostatic epithelial cells and TGF-ß negatively regulates this process, suggesting that the cross-talk between TGF-ß and androgen-signaling pathways may play significant roles in regulating prostate cancer growth and progression. Expression of Smad proteins has been observed in the prostate tissues (25) . However, the mechanism by which TGF-ß/Smads inhibit proliferation and induce apoptosis in prostate cancer cells is not clear. Moreover, it is also unclear whether the cellular effects of TGF-ß in prostate are mediated through AR. We therefore investigated the molecular mechanism by which TGF-ß inhibits transcriptional activation mediated by AR. We demonstrated that Smad3 specifically represses transcriptional activation mediated by AR and identified the region responsible as the MH2 domain. Protein-protein interaction between AR and Smad3 was demonstrated both in vitro and in vivo. These results provide new information for understanding how TGF-ß regulates the androgen-signaling pathway.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The AR expression vector pSV-ARo was provided by Dr. Albert Brinkmann (Erasmus University, Rotterdam, The Netherlands). pMMTV-pA3-luc was provided by Dr. Richard Pestell (Albert Einstein College of Medicine, New York, NY). The reporter plasmids, pPSA7kb-luc and pPSA530bp-luc, with the luciferase gene under the control of promoter fragments of the human prostate-specific antigen were obtained from Trapman (30) and Belldegrun (31) . pSV-ß-gal, an SV40-driven ß-galactosidase reporter plasmid (Promega, Madison, WI) was used in this study as an internal control. The pSG5-ARA70 plasmid, containing the full-length ARA70 cDNA, and the reporter plasmid pARE-luc were kind gifts from Dr. Chang (32) . pCMV-VDR and pVDRE-luc plasmids were provided by Dr. David Feldman (Stanford University, Stanford, CA). A human estrogen receptor (ER) expression construct (pcDNA3-ER) and a luciferase reporter plasmid with three estrogen-responsive elements were kindly provided by Dr. Myles Brown (Dana-Farber Cancer Institute, Boston, MA). A human thyroid hormone receptor ß (TRß) expression vector driven by the SV40 promoter and a luciferase reporter controlled by two TREs were kindly provided by Dr. Anthony Hollenberg (Beth Israel Deaconess Medical Center, Boston, MA).
Cell Cultures and Transient Transfections.
Both a monkey kidney cell line, CV-1, and a human prostate cell line, PC-3, were maintained in DMEM supplemented with 5% fetal bovine serum (FBS) (HyClone, Denver, CO). CV-1 cells have low levels of endogenous steroid hormone receptor activity and are capable of supporting TGF-ß signaling (33)
. Transient transfections were carried out by using a LipofectAMINE transfection kit (Life Technologies, Inc., Gaithersburg, MD). Approximately 3 x 104 cells were seeded into a 24-well plate 16 h before transfection. About 300 ng of total plasmid DNA and 1.2 µl of LipofectAMINE/well were used in the transfection. The total amount of plasmid per well was equalized in all transfections by addition of the pcDNA3 empty vector. Approximately 16 h after transfection, the cells were washed and fed medium containing 5% charcoal-stripped FBS (steroid hormone free; HyClone) in the presence or absence of steroid hormones. Cells were incubated for another 24 h, and luciferase activity was measured as relative light units (RLU) in a Monolight 3010 luminometer (PharMingen, San Diego, CA) according to the manufacturer’s protocol. The RLU from individual transfections were normalized by ß-galactosidase activity in the same samples. Individual transfection experiments were done in triplicate and the results are reported as mean RLU/ß-galactosidase (±SD) from representative experiments.
GST-Pull-Down Assay.
Expression and purification of GST fusion proteins were performed as described previously (34)
. The full-length and truncated Smad3 proteins were generated and labeled in vitro by the TnT-coupled reticulocyte lysate system (Promega). Equal amounts of GST-AR fusion proteins coupled to glutathione-Sepharose beads were incubated with radiolabeled Smad3 proteins at 4°C for 2 h in a modified binding buffer [20 mM Tris-HCl (pH 7.8), 180 mM KCl, 0.5 mM EDTA, 5 mM MgCl2, 50 µM ZnCl2, 10% glycerol, 0.1% NP-40, 0.05% dry nonfat milk, 1 mM DTT, and 0.5 mM PMSF]. Beads were carefully washed three times with 500 µl of binding buffer and then analyzed by SDS-PAGE followed by autoradiography.
Immunoprecipitation and Western Blotting.
The human AR expression vector pARo, alone or with a Flag-tagged pcDNA3-Smad3 expression plasmid, was transfected into CV-1 cells. Transfected cells were cultured in the presence of 10 nM R1881 for 48 h and then harvested in a buffer containing 0.5% NP-40, 150 mM NaCl, 2 mM MgCl2, 50 mM HEPES-KOH (pH 7.4), 1 mM EDTA, 5% glycerol, 1 mM DTT, 0.5 mM PMSF, and 25 mM NaF. Lysates were clarified by incubation on ice and centrifugation for 5 min; 400 µl of clarified lysate from each sample were precleared for 20 min with 10 µ l of protein A-Sepharose beads bound to 1 µg of antimouse IgG (Pharmacia, Arlington Heights, IL). Precleared lysates were incubated with preequilibrated protein A-Sepharose beads with either mouse normal IgG or Flag monoclonal antibody (Sigma, St. Louis, MO) at 4°C for 3 h. The beads were washed three times in 500 µl of lysis buffer and were eluted by boiling in SDS-PAGE sample buffer. Following SDS-PAGE, proteins were transferred to nitrocellulose (BA85; Schleicher & Schuell, Keene, NH) and blocked overnight at 4°C in TBST (50 mM Tris-HCl, 150 mM NaCl, and 0.08% Tween 20) with 5% low-fat milk. The first antibody was a polyclonal antibody against the NH2 terminus of AR and was used at a 1:200 dilution (Santa Cruz Biotechnology, Santa Cruz, CA). The secondary antibody was antimouse IgG conjugated to horseradish peroxidase and was used at a 1:2000 dilution (Bio-Rad). Detection was performed with ECL reagents according to manufacturer’s protocol using ECL Hyperfilm (Amersham).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Ligand-dependent AR activation was repressed nearly 6-fold by cotransfection of the Smad3 expression construct. Expression of other Smad family members, including the R-Smad2 and the Co-Smad4 as well as the I-Smad6 and I-Smad7, showed no effect (Fig. 1)
. The positive control, the known AR coactivator ARA70, brought about an additional 2-fold increase in DHT-dependent transcription (32)
. Thus, Smad3 specifically repressed transcription from an AR-dependent promoter.
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. Smad3 also repressed a minimal PSA promoter, containing only the proximal 530 bp of the PSA promoter (Fig. 2B)
. The 530 bp contains the two androgen-responsive regions and little else. To ensure that the Smad3-mediated inhibition did not reflect toxic or other nonspecific effects of the cotransfected plasmids, luciferase expression in all experiments was normalized using ß-galactosidase production from a cotransfected plasmid. We examined the intracellular steady-state levels of AR protein in the transfectants and found them to be similar, indicating that the Smad3-mediated repression was not due to lowered levels of expression of AR from the cotransfected plasmid (data not shown). The results strongly suggest that Smad3 inhibited AR-regulated transcription from multiple, naturally occurring androgen-responsive promoters.
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. Addition of TGF-ß1 reduced the level of luciferase expression to almost background levels (Fig. 3A)
. TGF-ß1 by itself had no effect on transcription from the MMTV promoter (Fig. 3A)
. The results suggest that the repression mediated by Smad3 is a downstream effect of TGF-ß signaling.
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, either TGF-ß or Smad3 alone did not significantly alter PSA promoter activity. However, in the presence of TGF-ß, Smad3 was able to reduce AR induction to almost baseline levels (Fig. 3B)
. Smad3 proteins were below detection limit in the whole-cell lysates prepared from both CV-1 and PC-3 cells as determined by Western blotting with a specific Smad3 antibody (data not shown). Again, this demonstrates that Smad3 can repress androgen signaling in a prostate-derived cell type, and this repression requires activation of Smad3 by TGF-ß.
As a receptor-activated Smad protein, Smad3 activity is modulated by other Smad proteins including the Co-Smad4 and I-Smads. Previous studies have shown that Smad4 can form a heterodimer with Smad3 and be translocated into the nucleus to activate the transcriptional response (37, 38, 39)
. The I-Smads are believed to function by competing with the R-Smads for phosphorylation by the TGF-ß receptors (24
, 40)
. Although neither the Co-Smad4 nor I-Smads alone affected AR-dependent transcription (Fig. 1)
, we needed to test whether they could modulate the ability of Smad3 to repress AR function. As seen in Fig. 3C
, while expression of Smad3 alone reduced AR-dependent transcription approximately 8-fold, coexpression of Smad4 and Smad3 resulted in greater repression than expression of Smad3 alone. When Smad7 was coexpressed with Smad3, Smad7 alleviated the repression by Smad3 of the MMTV promoter (Fig. 3D)
. This effect was specific to Smad7, as expression of Smad6 showed no significant impact (Fig. 3D)
. Taken together, these data are consistent with our observation that no endogenous Smad3 protein was detected in CV-1 cells and suggest that Smad3-mediated repression shows the features expected of regulation by TGF-ß signal transduction, and that Smad4 (positive) and Smad7 (negative) are regulatory factors in this process.
The specificity of Smad3 repression was further investigated with other nuclear receptors, including the VDR, ER, and TR. Smad3 augmented VDR-mediated transcriptional activation (Fig. 4)
, consistent with previous studies (21)
. There was no significant effect of Smad3 on ER-mediated transcription. In contrast, Smad3 slightly repressed TR-mediated transcription (Fig. 4)
. Taken together, our results suggest that Smad3 may be involved in multiple regulatory processes mediated by nuclear receptors.
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, the Smad3-MH2 domain was specifically retained with GST-AR-TAD, which consisted of the first 563 aa of AR. The MH1 domain showed no binding with any of the AR domains. This not only provided in vitro evidence of an interaction, but also served to map the interaction between the MH2 domain of Smad3 and the transactivation domain of AR.
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. Binding was greatly reduced when aa 330–563 were deleted. AR-TAD containing only the first 240 aa showed virtually no binding of Smad3. The AR-TAD 1–330 molecule also was severely compromised in the transcriptional transactivation function (Fig. 5C)
. These data demonstrate that the transactivation domain of AR overlaps with the Smad3-binding region. This correlation may suggest a potential mechanism for repression of AR function by Smad3.
To verify the association between AR and Smad3, we analyzed the protein-protein interaction by coimmunoprecipitation analysis (Fig. 5D)
. Initially, both Flag-tagged MH1 and MH2 domains of Smad3 were used as bait to coprecipitate the AR. A specific protein complex was detected when Smad3-MH2 and AR were mixed, but not with a Smad3-MH1 and AR mixture (Fig. 5D)
. Thus, the coimmunoprecipitation data support the in vitro binding assay, suggesting that the MH2 domain of Smad3 interacts with the TAD of AR.
To determine whether the interaction between Smad3 and AR occurs in vivo, mammalian two-hybrid analysis was carried out. The AR constructs consisted of aa 1–563 (GAL4DBD-AR-TAD) and aa 564–919 (GAL4DBD-AR-LBD) fused to the DBD of GAL4, while full-length and truncated Smad3 and Smad2 were fused to the VP16 activation domain (Fig. 6A)
. An induction of the GAL4/Luc reporter was observed after cotransfection of the GAL4DBD-AR-TAD and pcDNA3 empty vector, compared to when the GAL4DBD-AR-LBD construct was used (Fig. 6B)
, consistent with previous evidence of an activation domain within aa 1–563 of AR. Transcription was enhanced when either the full-length or the MH2 domain of Smad3 was added, but not when the MH1 domain of Smad3 or full-length Smad2 was used. Furthermore, in agreement with the results of the in vitro binding assays and coimmunoprecipitation, the MH2 domain of Smad3 was shown to have an even stronger interaction with AR than full-length Smad3 in this experiment. On the basis of the data from multiple lines of experimentation, we conclude that Smad3 specifically interacts with AR and that this interaction is mediated by the MH2 domain of Smad3 and the TAD domain of AR.
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. Full repression of AR-induced activation was also achieved by transfection with the MH2 domain alone. In contrast, the MH1 domain had little effect on transcription. Thus, the MH2 domain of Smad3 is sufficient for repression of AR activation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Loss of the TGF-ß-signaling pathway has been frequently observed in prostate cancer cells (42 , 43) . The evidence provided in this study has shown Smad3 repressing AR-mediated transcription on both MMTV and PSA promoters. PSA has been broadly used as a clinical marker to monitor the progression of prostate cancer. Notably, repression was seen with an intact natural promoter from the PSA gene as well as with a mini-promoter containing little more than the androgen response elements. In addition, this Smad3-mediated repression was enhanced by Smad4 but diminished by Smad7. Taken together, these findings indicated a direct role of Smad3 to repress the AR-mediated transcription and suggest a potential link between TGF-ß/Smads signaling and suppression of human prostate cancer progression.
The Smad3-induced repression of AR-mediated transcription is quite novel and involves a mechanism that is distinct from previous reports on the cross-talk between Smads and nuclear hormone receptors. Smad3 was found to enhance VDR-mediated, ligand-dependent transactivation through interaction of the MH1 domain of Smad3 and the LBD of VDR. This complex then recruits the steroid receptor coactivator (21) . In contrast, the GR has been shown to repress Smad3-mediated transcription, illustrating that regulation can also occur in the opposite direction (22) . Our finding that Smad3 represses AR-mediated transcription thus represents a new molecular mechanism for TGF-ß/Smad regulation of nuclear receptors.
The MH2 domain of Smad3 was found to mediate its binding to AR and to be responsible for repressing AR transactivation. These data are consistent with previous studies of Smad structure-function where the MH2 domain was shown to be involved in many biological processes through interaction with other regulatory proteins (8 , 20) . The MH2 domain mediates both homomeric and receptor-induced heteromeric interactions between Smad4 and R-Smads (37 , 39) . The MH2 of Smad3 also interacts with GR, suggesting an important role for the MH2 domain in regulating nuclear receptors (22) . The biological activity of the MH2 domain may be modulated by interaction with the MH1 domain, when the protein is not phosphorylated. Upon receptor-mediated phosphorylation, this interaction may be altered and each domain may display the DNA and protein interactions required for the proper activity of the transcriptional complex (44) . Our results demonstrating that deletion of the MH1 domain was required to detect AR-Smad3 interaction by coimmunoprecipitation are consistent with this observation (8) .
The mechanism for repression of AR by Smad3 is currently unclear. Since the MH2 domain contains a functional activation domain (45) , it is somewhat surprising that the effect of Smad3 is one of repression. Targeting the MH2 of Smad3 to a heterologous DBD (GAL4DBD) creates a transcriptional activator, suggesting that there is no intrinsic repression domain (46) . A previous report has shown that Smad2 can form a complex with a transcriptional repressor, TGIF, and then recruits the histone deacetylase to form a repressor complex (20) . Since Smad3 but not Smad2 mediates repression of AR-dependent promoters, it is unlikely that TGIF is involved in the process. Another repressor, Ski, has been reported recently to interact with Smad2, Smad3, and Smad4 proteins (47, 48, 49) . It will be interesting to investigate whether Ski is involved in the Smad3-mediated repression.
Smad3 contains a DBD, MH1, and can specifically bind to a palindromic element with Smad4 (37 , 38 , 50) . However, since the MH1 domain is dispensable for the repression, it seems unlikely that the repression by Smad3 is attributable to competition with AR for DNA binding. Since the TAD of AR has been mapped to interact with Smad3 protein, one possible mechanism for repression by Smad3 could be attributable to disrupting the conformation of AR and recruiting other coactivators in the complex. Supporting this hypothesis are data showing that upon ligand binding an intrinsic interaction of AR between the NH2- and COOH-terminal fragments occurs to form an active transcriptional complex (51) .
In conclusion, this study demonstrates for the first time that Smad3 selectively represses AR-mediated transcription. The protein-protein interaction between AR and Smad3 may present a novel mechanism for cross-talk between the TGF-ß and androgen-signaling pathways. Given that prostate cancer cells are generally androgen dependent but have defects in TGF-ß receptor expression, these data provide important new information into the molecular consequences of the loss of TGF-ß signaling. Further study of the regulation by TGF-ß and Smad3 of androgen-induced transcription will provide fresh insights into the biology of prostate cancer and may lead to the development of new treatments.
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FOOTNOTES |
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1 This work was supported by National Institutes of Health Grant CA70297 (to Z. S.), American Cancer Society Grant RPG98213 (to Z. S.), and Dutch Cancer Society Grant NKI 2000-2217 (to P. D.). 
2 To whom requests for reprints should be addressed, at Departments of Surgery and Genetics, Stanford University, 300 Pasteur Drive, R135, Stanford, CA 94305-5328. E-mail: zsun{at}stanford.edu
3 The abbreviations used are: AR, androgen receptor; TGF-ß, transforming growth factor ß; R-Smad, receptor-regulated Smad; Co-Smad, common mediator Smad; I-Smad, inhibitory Smad; VDR, vitamin D receptor; GR, glucocorticoid receptor; aa, amino acid; GST, glutathione S-transferase; ER, estrogen receptor; TR, thyroid receptor; FBS, fetal bovine serum; RLU, relative light unit; TGIF, 5'-TG3' interacting factor; DHT, dihydrotestosterone; PSA, prostate-specific antigen; TAD, transactivation domain; DBD, DNA-binding domain; LBD, ligand-binding domain; MMTV, murine mammary tumor virus.
Received 10/ 6/00. Accepted 12/27/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T. J. Spady, R. Shayya, V. G. Thackray, L. Ehrensberger, J. S. Bailey, and P. L. Mellon Androgen Regulates Follicle-Stimulating Hormone {beta} Gene Expression in an Activin-Dependent Manner in Immortalized Gonadotropes Mol. Endocrinol., April 1, 2004; 18(4): 925 - 940. [Abstract] [Full Text] [PDF]
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 M. Rahman, H. Miyamoto, and C. Chang Androgen Receptor Coregulators in Prostate Cancer: Mechanisms and Clinical Implications Clin. Cancer Res., April 1, 2004; 10(7): 2208 - 2219. [Full Text] [PDF]
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 K. Aurrekoetxea-Hernandez and E. Buetti Transforming Growth Factor {beta} Enhances the Glucocorticoid Response of the Mouse Mammary Tumor Virus Promoter through Smad and GA-Binding Proteins J. Virol., March 1, 2004; 78(5): 2201 - 2211. [Abstract] [Full Text] [PDF]
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J. L. Carey, L. M. Sasur, H. Kawakubo, V. Gupta, B. Christian, P. M. Bailey, and S. Maheswaran Mutually Antagonistic Effects of Androgen and Activin in the Regulation of Prostate Cancer Cell Growth Mol. Endocrinol., March 1, 2004; 18(3): 696 - 707. [Abstract] [Full Text] [PDF]
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H. Sowa, H. Kaji, M. F. Iu, T. Tsukamoto, T. Sugimoto, and K. Chihara Parathyroid Hormone-Smad3 Axis Exerts Anti-apoptotic Action and Augments Anabolic Action of Transforming Growth Factor {beta} in Osteoblasts J. Biol. Chem., December 26, 2003; 278(52): 52240 - 52252. [Abstract] [Full Text] [PDF]
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K. Wu, Y. Yang, C. Wang, M. A. Davoli, M. D'Amico, A. Li, K. Cveklova, Z. Kozmik, M. P. Lisanti, R. G. Russell, et al. DACH1 Inhibits Transforming Growth Factor-{beta} Signaling through Binding Smad4 J. Biol. Chem., December 19, 2003; 278(51): 51673 - 51684. [Abstract] [Full Text] [PDF]
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 M. Fu, M. Rao, C. Wang, T. Sakamaki, J. Wang, D. Di Vizio, X. Zhang, C. Albanese, S. Balk, C. Chang, et al. Acetylation of Androgen Receptor Enhances Coactivator Binding and Promotes Prostate Cancer Cell Growth Mol. Cell. Biol., December 1, 2003; 23(23): 8563 - 8575. [Abstract] [Full Text] [PDF]
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 L. Liu, R. Santora, J. N. Rao, X. Guo, T. Zou, H. M. Zhang, D. J. Turner, and J.-Y. Wang Activation of TGF-{beta}-Smad signaling pathway following polyamine depletion in intestinal epithelial cells Am J Physiol Gastrointest Liver Physiol, November 1, 2003; 285(5): G1056 - G1067. [Abstract] [Full Text] [PDF]
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G. Li, S. Wang, and T. D. Gelehrter Identification of Glucocorticoid Receptor Domains Involved in Transrepression of Transforming Growth Factor-{beta} Action J. Biol. Chem., October 24, 2003; 278(43): 41779 - 41788. [Abstract] [Full Text] [PDF]
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 X. Luo, J. Xu, and N. Chegini The Expression of Smads in Human Endometrium and Regulation and Induction in Endometrial Epithelial and Stromal Cells by Transforming Growth Factor-{beta} J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 4967 - 4976. [Abstract] [Full Text] [PDF]
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 C. J. Loy, K. S. Sim, and E. L. Yong Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions PNAS, April 15, 2003; 100(8): 4562 - 4567. [Abstract] [Full Text] [PDF]
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L. Choy and R. Derynck Transforming Growth Factor-beta Inhibits Adipocyte Differentiation by Smad3 Interacting with CCAAT/Enhancer-binding Protein (C/EBP) and Repressing C/EBP Transactivation Function J. Biol. Chem., March 7, 2003; 278(11): 9609 - 9619. [Abstract] [Full Text] [PDF]
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 J. Xu, A. R. Beyer, W. H. Walker, and E. A. McGee Developmental and Stage-Specific Expression of Smad2 and Smad3 in Rat Testis J Androl, March 1, 2003; 24(2): 192 - 200. [Abstract] [Full Text] [PDF]
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 W.-C. Chou, V. Prokova, K. Shiraishi, U. Valcourt, A. Moustakas, M. Hadzopoulou-Cladaras, V. I. Zannis, and D. Kardassis Mechanism of a Transcriptional Cross Talk between Transforming Growth Factor-beta -regulated Smad3 and Smad4 Proteins and Orphan Nuclear Receptor Hepatocyte Nuclear Factor-4 Mol. Biol. Cell, March 1, 2003; 14(3): 1279 - 1294. [Abstract] [Full Text] [PDF]
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Y. Okada, Y. Fujii, J. P. Moore Jr., and S. J. Winters Androgen Receptors in Gonadotrophs in Pituitary Cultures from Adult Male Monkeys and Rats Endocrinology, January 1, 2003; 144(1): 267 - 273. [Abstract] [Full Text] [PDF]
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M. A. Rao, H. Cheng, A. N. Quayle, H. Nishitani, C. C. Nelson, and P. S. Rennie RanBPM, a Nuclear Protein That Interacts with and Regulates Transcriptional Activity of Androgen Receptor and Glucocorticoid Receptor J. Biol. Chem., December 6, 2002; 277(50): 48020 - 48027. [Abstract] [Full Text] [PDF]
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H.-Y. Kang, K.-E. Huang, S. Y. Chang, W.-L. Ma, W.-J. Lin, and C. Chang Differential Modulation of Androgen Receptor-mediated Transactivation by Smad3 and Tumor Suppressor Smad4 J. Biol. Chem., November 8, 2002; 277(46): 43749 - 43756. [Abstract] [Full Text] [PDF]
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 Y. S. Lee, H.-J. Kim, H. J. Lee, J. W. Lee, S.-Y. Chun, S.-K. Ko, and K. Lee Activating Signal Cointegrator 1 Is Highly Expressed in Murine Testicular Leydig Cells and Enhances the Ligand-Dependent Transactivation of Androgen Receptor Biol Reprod, November 1, 2002; 67(5): 1580 - 1587. [Abstract] [Full Text] [PDF]
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J. Reid, I. Murray, K. Watt, R. Betney, and I. J. McEwan The Androgen Receptor Interacts with Multiple Regions of the Large Subunit of General Transcription Factor TFIIF J. Biol. Chem., October 18, 2002; 277(43): 41247 - 41253. [Abstract] [Full Text] [PDF]
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 J. Skillington, L. Choy, and R. Derynck Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes J. Cell Biol., October 14, 2002; 159(1): 135 - 146. [Abstract] [Full Text] [PDF]
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M. Sharma, W. W. Chuang, and Z. Sun Phosphatidylinositol 3-Kinase/Akt Stimulates Androgen Pathway through GSK3beta Inhibition and Nuclear beta -Catenin Accumulation J. Biol. Chem., August 16, 2002; 277(34): 30935 - 30941. [Abstract] [Full Text] [PDF]
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T. Yamamoto, F. Saatcioglu, and T. Matsuda Cross-Talk between Bone Morphogenic Proteins and Estrogen Receptor Signaling Endocrinology, July 1, 2002; 143(7): 2635 - 2642. [Abstract] [Full Text] [PDF]
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M. Fu, C. Wang, J. Wang, X. Zhang, T. Sakamaki, Y. G. Yeung, C. Chang, T. Hopp, S. A. W. Fuqua, E. Jaffray, et al. Androgen Receptor Acetylation Governs trans Activation and MEKK1-Induced Apoptosis without Affecting In Vitro Sumoylation and trans-Repression Function Mol. Cell. Biol., May 15, 2002; 22(10): 3373 - 3388. [Abstract] [Full Text] [PDF]
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C. A. Heinlein and C. Chang Androgen Receptor (AR) Coregulators: An Overview Endocr. Rev., April 1, 2002; 23(2): 175 - 200. [Abstract] [Full Text] [PDF]
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D. Tomic, S.G. Brodie, C. Deng, R.J. Hickey, J.K. Babus, L.H. Malkas, and J.A. Flaws Smad 3 May Regulate Follicular Growth in the Mouse Ovary Biol Reprod, April 1, 2002; 66(4): 917 - 923. [Abstract] [Full Text] [PDF]
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A. Moustakas, S. Souchelnytskyi, and C.-H. Heldin Smad regulation in TGF-{beta} signal transduction J. Cell Sci., March 14, 2002; 114(24): 4359 - 4369. [Abstract] [Full Text] [PDF]
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E. Holter, N. Kotaja, S. Makela, L. Strauss, S. Kietz, O. A. Janne, J.-A. Gustafsson, J. J. Palvimo, and E. Treuter Inhibition of Androgen Receptor (AR) Function by the Reproductive Orphan Nuclear Receptor DAX-1 Mol. Endocrinol., March 1, 2002; 16(3): 515 - 528. [Abstract] [Full Text] [PDF]
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R. Pero, F. Lembo, E. A. Palmieri, C. Vitiello, M. Fedele, A. Fusco, C. B. Bruni, and L. Chiariotti PATZ Attenuates the RNF4-mediated Enhancement of Androgen Receptor-dependent Transcription J. Biol. Chem., January 25, 2002; 277(5): 3280 - 3285. [Abstract] [Full Text] [PDF]
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 M. E. Grossmann, H. Huang, and D. J. Tindall Androgen Receptor Signaling in Androgen-Refractory Prostate Cancer J Natl Cancer Inst, November 21, 2001; 93(22): 1687 - 1697. [Abstract] [Full Text] [PDF]
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D. Liu, B. L. Black, and R. Derynck TGF-beta inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3 Genes & Dev., November 15, 2001; 15(22): 2950 - 2966. [Abstract] [Full Text] [PDF]
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T. Matsuda, T. Yamamoto, A. Muraguchi, and F. Saatcioglu Cross-talk between Transforming Growth Factor-beta and Estrogen Receptor Signaling through Smad3 J. Biol. Chem., November 9, 2001; 276(46): 42908 - 42914. [Abstract] [Full Text] [PDF]
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J. E. Chipuk, S. C. Cornelius, N. J. Pultz, J. S. Jorgensen, M. J. Bonham, S.-J. Kim, and D. Danielpour The Androgen Receptor Represses Transforming Growth Factor-beta Signaling through Interaction with Smad3 J. Biol. Chem., January 4, 2002; 277(2): 1240 - 1248. [Abstract] [Full Text] [PDF]
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D.-J. Jung, S.-Y. Na, D. S. Na, and J. W. Lee Molecular Cloning and Characterization of CAPER, a Novel Coactivator of Activating Protein-1 and Estrogen Receptors J. Biol. Chem., January 4, 2002; 277(2): 1229 - 1234. [Abstract] [Full Text] [PDF]
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D. L. Segev, Y. Hoshiya, M. Hoshiya, T. T. Tran, J. L. Carey, A. E. Stephen, D. T. MacLaughlin, P. K. Donahoe, and S. Maheswaran Mullerian-inhibiting substance regulates NF-kappa B signaling in the prostate in vitro and in vivo PNAS, January 8, 2002; 99(1): 239 - 244. [Abstract] [Full Text] [PDF]
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,25-dihydroxyvitamin D3 (VDR), 10-7 M ß-estradiol (ER), and 10-8 M triiodo-thyronine (T3).
