| Abstract
| - Thrombin and trypsin induce cell signaling through a subclass of G-protein-coupled receptorscalled the protease-activated receptors (PARs). In many cells, PAR signaling results in the activation ofRhoA and other members of the Rho family of small GTPases which are involved in cytoskeletalreorganization. The expression of PARs and their role in the activation of Rho GTPases in prostate cancercells are not clearly known. FACS analysis demonstrated that the androgen-dependent LNCaP cells expressPAR1, PAR2, and PAR4 but not PAR3. Stimulation with thrombin and trypsin resulted in the rapidactivation of RhoA in a dose-dependent manner with an EC50 of 1.0 and 5 nM, respectively. Activationof RhoA was enhanced by, but not dependent on, the presence of 1 nM dihydrotestosterone. Inhibition ofthe proteolytic properties of thrombin by hirudin and trypsin by diisopropyl fluorophosphate abolishedthe observed RhoA activation. Stimulation with 150 μM PAR-activating peptides TFFLRN (PAR1),SLIGKV (PAR2), and AYPGKF (PAR4) demonstrated that PAR1 and PAR2 mediated protease-activatedRhoA signaling. Fluorescent microscopy studies showed that LNCaP cells treated with either thrombin(10 nM) or trypsin (10 nM) developed an increased number of filopodia, stress fibers, and focal adhesionsrelative to untreated cells. These observations represent the first report of PAR signaling in prostate cancercells as well as the ability of PAR2 to mediate RhoA activation. Since the activation of RhoA is importantfor cytoskeletal reorganization, we postulate that PAR-mediated RhoA activation may be a major signalingpathway in the biology of prostate cancer.
|
