Figure 1.

Androgen inhibits TGF-β1-induced Smad3 activation and Smad3-mediated TGF-β responses in NRP154, rat prostatic epithelial cell line. NRP-154 cells were infected for 2 h with AdMax adenoviral for expression of AR (AdMax-AR, 1:500), and after 24 h the infected cells were treated ± 1 nm DHT for 48 h (A) or 1–48 h (B) before TGF-β1 (1 h) (0.5 to 10 ng/ml) (A) or 10 ng/ml (B) treatment. Cell lysates were then analyzed for expression or total Smads 2, 3, and 4 as well as active Smads [phospho-Smad2 (Ser465/467) and phospho-Smad3 (Ser423/425)] by Western blot. C and D, Effect of DHT on suppression of phospho-Smad (Ser423/425) in NRP-154/AR28 (clone 28 cells stably overexpressing AR) ± doxycyclin (2 μg/ml) (C), and in NRP-152 cells infected 2 h with 1:500 AdMax-AR (NRP-152+AR) or control virus (D); DHT and TGF-β treatments were as in panel A. E, NRP-154+AR cells were pretreated with DHT for 24 h and then treated with TGF-β1 48 h TGF-β1 (10 ng/ml) after which lysates were examined for levels of cyclin D3 and Rb phosphorylation by Western blot analysis. Data shown are representative of two or three independent experiments (A–D). In cases in which multiple bands appeared, the correct bands are indicated by the arrows (→ or ←), as confirmed by use of Smad short hairpin RNAs (29) or overexpression of AR (in panel C).