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

Abstract

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 androgenresponsive elements in the promoters of androgen-regulated genes. AR-mediated transcription is regulated by many ARinteracting proteins such as ARA 70 (AR-associated proteins) (12) and ARA 160 (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)(18)(19)(20), is under androgenic control. TGF-␤ signals through a cooperative interaction with two cell surface serine/threonine kinase receptors, . TGF-␤ first associates with constitutively active dimeric T␤RII, which then recruits and activates T␤RI kinase by transphosphorylation at a juxtamembrane glycine-serine repeat (21,26). With the help of Smad anchor for receptor activation (27), phosphorylated T␤RI 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

Differential expression of steroid receptors in prostate tissues before and after radiation therapy for prostatic adenocarcinoma

“…Three (ER-a, ER-b and AR) of 4 steroid receptors, which we found in the current study to have higher expression after RT, were found previously to have an ability to inactivate TGF-b antiproliferative signaling pathway by inhibition of Smad3. [35][36][37][38][39] Interestingly, ER-b was the only steroid receptor to be upregulated in prostate adenocarcinoma epithelial cells after RT. We have demonstrated previously that ER-b expression is associated positively with Gleason score and primary Gleason grade with higher levels expressed in higher grade tumors.…”

Cited2 modulates TGF-β-mediated upregulation of MMP9

“…Purification of GST fusions, in vitro transcription/translation, and GST pull-down assay Purification of GST-fusion proteins and GST pull-down assay have been described previously (Chipuk et al, 2002). Briefly, cDNA from wild type of Cited2 (aa1-269), Cited2 C-terminal deletion mutant (aa 1-199), wild type of Smad3 (aa1-425), and Smad3 deletion mutant (aa 1-225) were subcloned into pcDNA3.1(À)B and in vitro transcribed and translated using TNT Coupled Reticulocyte Lysate Systems (Promega).…”

CBL enhances breast tumor formation by inhibiting tumor suppressive activity of TGF-β signaling

“…Flag-Smad3 domain constructs and constitutively active HA-TbRI ca plasmids constructs were described previously. 21,30 Anti-Myc, HA, CBL, CBL-b, GFP, p15 INK4b p21 Cip1 and PAI-I antibodies were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. Anti-atubulin and Flag M2 antibody was from Sigma-Aldrich, St Louis, MO, USA.…”

“…As anticipated, TbRI ca phosphorylated Smad3 and enhanced the interaction between Smad3 and CBL (Figure 4b), suggesting that TGF-b signaling triggers the association between CBL and Smad3. To confirm this result, we examined the ability of an inactive form of Smad3 mutant (AAVA, serine amino acids replaced with alanine) or a constitutively active Smad3-like mutant (DDVD, serine amino acids replaced with aspartic acid) in C-terminal SSXS motif of Smad3 20,21 to interact with CBL. CBL/Smad3 interaction was mostly abrogated by the inactive form of Smad3 AAVA mutant, but greatly enhanced by Smad3 DDVD mutant (Figure 4c).…”