Cross-talk between α1d-adrenergic receptor (α1d-AR) and Transient Receptor Potential Vanilloid 1 (TRPV1) triggers the proliferation of PC-3 prostate cancer cells

Growing evidence supports the role of α1-ARs in the direct mitogenic effect of catecholamines on prostate cancer cell growth. We have previously reported the expression of α1D-AR on PC-3 prostate cancer cells and the ability of noradrenalin (NA) to stimulate PC-3 cell proliferation in a α1D-AR-dependent manner (Quaglia et al., 2005). In addition, TRPV1 expression was also found in normal prostate tissue and prostate cancer cells (Sanchez et al., 2006). Aim of this study was to investigate the relationship between α1D-AR and TRPV1 receptors and the involvement of TRPV1 in NA-induced proliferation of PC3 cells. By using confocal microscopy we found a co-localization of α1D-AR and TRPV1 receptors in the membrane and cytosol of PC3. Moreover, a band of 70 kDa likely corresponding to the α1D-AR, in PC3 lysates, immunoprecipitated with an anti-TRPV1 antibody, was found. Treatment of PC3 cells with NA strongly stimulated proton release, calcium influx and cell proliferation that were completely reverted by WS433 and CPZ, respectively α1D-AR and TRPV1 antagonists,when utilized in combination. We also evaluated the ability of NA to induce calcium influx in α1D-AR or TRPV1 silenced PC3 cells. α1D-AR silencing reverted the early (up to 50 sec) calcium overload whereas TRPV1 knock-down resulted in a marked reduction of delayed (50-180 sec) calcium increase. To further address the role of α1D-AR/TRPV1 receptor, PC3 cells were double silenced and then treated with NA. Results demonstrated that the NA-induced increase of survival and proliferation is totally abrogated in α1D-AR/TRPV1 silenced cells. Finally, we investigated the signalling pathways involved in NA stimulation evaluating proton release and cell viability in PC3 cells treated with NA in the presence of Phospholipase C (PLC), Protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) inhibitors. Our data showed that PLC and PKC inhibitors markedly reduce (about 50-60%) the NA-induced effects, whereas ERK inhibitor displays a less intensive activity (20-30%). By western blot analysis, we also evidenced that NA stimulates, in a time dependent manner, ERK and PKC substratephosphorylation that is completely inhibited by WS433 and partially reverted by CPZ. Overall our results demonstrated that a functional and structural cross-talk between α1D-AR and TRPV1 receptors controls the NA-induced proliferation of prostate cancer cells.