FIGURE 1.

DHT-induced Erk1/2 signaling occurs via MMP-mediated trans-activation of EGFR and is regulated by paxillin. A, serum-starved LnCAP cells were preincubated with vehicle (0.1% DMSO), 100 nm flutamide (AR inhibitor), 20 μm AG1478 (EGFR inhibitor), 5 μm Erlotinib (EGFR inhibitor), 20 μm PP2 (Src inhibitor), or 20 μm galardin (MMP inhibitor) for 30 min before stimulation with ethanol (Media) or 25 nm DHT for 30 min. Western blots of whole-cell extracts were performed for total and phosphorylated Erk1/2 (tERK1/2, pERK1/2). B, A431 cells cultured in DMEM/F-12 (1:1) medium (Invitrogen) containing 10% FBS and 1% penicillin-streptomycin were serum-starved overnight and then stimulated with medium from DHT (25 nm), DHT (25 nm) + galardin (20 μm), or vehicle (0.1% ethanol)-treated LnCAP cells for 60 min. As controls, A431 cells were stimulated with DHT (25 nm) or media alone (no cells). Thereafter, A431 cells were isolated for Western blot analysis to detect phosphorylated and total EGFR (pEGFR, tEGFR). C, serum-starved LnCAP cells were preincubated with vehicle (0.1% DMSO), 20 μm AG1478 (EGFR inhibitor), or 20 μm PP2 (Src inhibitor) for 30 min before stimulation with ethanol (Media) or 25 nm DHT for 30 min. Western blots of whole-cell extracts were performed for Src (tSrc, pSrc) or EGFR (tEGFR, pEGFR). D and E, LnCAP and PC3 cells were treated with non-targeting (Nsp) or paxillin-specific (Pax) siRNAs for 72 h followed by stimulation with 25 nm DHT (D) or 20 ng/ml EGF (E) for the indicated times. Western blots were performed for total and phosphorylated Erk1/2, Akt (tAkt and pAkt) or total paxillin (Pax). All experiments were performed at least three times with identical results.