The androgen receptor represses transforming growth factor-β signaling through interaction with Smad3

In the prostate, androgens negatively regulate the expression of transforming growth factor-beta (TGF-beta) ligands and receptors and Smad activation through unknown mechanisms. We show that androgens (dihydrotestosterone and R1881) down-regulate TGF-beta1-induced expression of TGF-beta1, c-Fos, and Egr-1 in the human prostate adenocarcinoma cell line, LNCaP. Moreover, 5alpha-dihydrotestosterone (DHT) inhibits TGF-beta1 activation of three TGF-beta1-responsive promoter constructs, 3TP-luciferase, AP-1-luciferase, and SBE4(BV)-luciferase, in LNCaP cells either with or without enforced expression of TGF-beta receptors (TbetaRI and TbetaRII). Similarly, DHT inhibits the activation of Smad-binding element (SBE)4(BV)-luciferase by either constitutively activated TbetaRI (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-beta1, 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-beta transcriptional responses through selectively repressing the binding of Smad3 to SBE.