Abstract
Although transcriptional effects of androgens have been extensively studied, mechanisms regulating transcription-independent (nongenomic) androgen actions are poorly understood. Previously, we have shown that paxillin, a multidomain adaptor protein, is a critical regulator of testosterone-induced MAPK-signaling during Xenopus oocyte maturation. Here we examine the nongenomic effects of dihydrotestosterone (DHT) in prostate cancer cells, focusing on how paxillin mediates Erk signaling and downstream physiologic actions. We show that in LnCAP cells DHT functions as a growth factor that indirectly activates the EGF-receptor (EGFR) via androgen receptor binding and matrix metalloproteinase-mediated release of EGFR ligands. Interestingly, siRNA-mediated knockdown of paxillin expression in androgen-dependent LnCAP cells as well as in androgen-independent PC3 cells abrogates DHT- and/or EGF-induced Erk signaling. Furthermore, EGFR-induced Erk activation requires Src-mediated phosphorylation of paxillin on tyrosines 31/118. In contrast, paxillin is not required for PKC-induced Erk signaling. However, Erk-mediated phosphorylation of paxillin on serines 83/126/130 is still needed for both EGFR and PKC-mediated cellular proliferation. Thus, paxillin serves as a specific upstream regulator of Erk in response to receptor-tyrosine kinase signaling but as a general regulator of downstream Erk actions regardless of agonist. Importantly, Erk-mediated serine phosphorylation of paxillin is also required for DHT-induced prostate-specific antigen mRNA expression in LnCAP cells as well as EGF-induced cyclin D1 mRNA expression in PC3 cells, suggesting that paxillin may regulate prostate cancer proliferation by serving as a liaison between extra-nuclear kinase signaling and intra-nuclear transcriptional signals. Thus, paxillin may prove to be a novel diagnostic or therapeutic target in prostate cancer.
Highlights
Androgen receptors (ARs).2 Genomic actions involve binding of androgens to androgen receptors (ARs), which translocate to the nucleus, bind to androgen-response elements, and alter gene expression
DHT-induced Erk Signaling Occurs via Matrix Metalloproteinase (MMP)-mediated Transactivation of the EGFR—Because androgens induce Erk1/2 signaling in Paxillin Regulation of Nongenomic Androgen Actions (PCa) cell lines (3, 10 –12), we examined the underlying mechanism by which DHT activates Erk1/2 in LnCAP cells
This study reveals a remarkable conservation of paxillin function from frog germ cells to human PCa cells
Summary
Cell Lines and Culture—LnCAP and PC3 cell lines were obtained from ATCC and cultured in RPMI 1640 medium (Invitrogen) containing 10% FBS and 1% penicillin-streptomycin. A431 cells (ATCC) were cultured in DMEM/F-12 (1:1) medium (Invitrogen) containing 10% FBS and 1% penicillin-streptomycin, serum-starved overnight, and stimulated with medium from DHT-, DHT ϩ galardin-, or vehicle (0.1% ethanol)treated LnCAP cells for 60 min. After 72 h, cells were treated overnight with serum-free, phenol redfree RPMI 1640 media and stimulated with 0.1% ethanol/ DMSO (vehicle), 25 nM DHT (Steraloids), 20 ng/ml EGF (BD Biosciences), or 100 nM PMA (Sigma) for the times indicated for Western blots or 24 h for MTT assays. After 48 h, cells were treated overnight with serum-free, phenol red-free RPMI 1640 media and stimulated with the indicated ligands for 30 min for Western blots or 24 h for MTT assays. A value of p Յ 0.05 was considered significant
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