Expression of protease-activated receptor-2 by osteoblasts

Osteoblasts express protease-activated receptor-1 (PAR-1), which is activat ed by thrombin or by synthetic peptides corresponding to the new "tethered ligand" N-terminus of PAR-1 created by receptor cleavage. Both thrombin and human PAR-l activating peptide stimulate an elevation of [Ca2+](i) in the human SaOS-2 osteoblast-like cell line, but the peptide stimulates receptor -mediated Ca+ entry, whereas thrombin does not. Stimulation of proliferatio n in rat primary osteoblast-like cells is greater in response to rat PAR-1- activating peptide than to thrombin, Because the PAR-1-activating peptides are now known to activate PAR-2, the current study was undertaken to invest igate whether osteoblasts express this receptor and, if so, whether this co uld account for the observed discrepancies between responses of osteoblasts to thrombin and to PAR-1-activating peptides, Reverse transcriptase-polyme rase chain reaction (RT-PCR) and immunocytochemical studies demonstrated ex pression of PAR-2 by primary cultures of rat calvarial osteoblast-like cell s. In immunohistochemical studies of embryonic mouse bones, osteoblasts sho wed positive staining for the presence of PAR-2. Activators of PAR-2 includ e trypsin, mast cell tryptase, gingipain-R, and synthetic peptides correspo nding to the PAR-2 tethered ligand sequence. Treatment of primary rat osteo blast-like cells with rat PAR-2-activating peptide (SLIGRL), or SaOS-2 cell s with human PAR-2-activating peptide (SLIGKV), caused a dose-dependent inc rease in [Ca2+](i). Trypsin or gingipain-R also induced an increase in intr acellular calcium concentration, and caused reciprocal cross desensitizatio n. Activators of PAR-2 caused a sharp peak in [Ca2+](i) followed by a susta ined plateau; [Ca2+](i) returned to baseline levels upon treatment with eth yleneglycol tetraacetic acid (EGTA), Treatment of rat osteoblastlike cells in vitro with SLIGRL did not affect thymidine incorporation or endogenous a lkaline phosphatase activity. The results presented here demonstrate that o steoblasts express PAR-2, and that such expression is able to account for t he observed discrepancies between thrombin and PAR-1-activating peptides in their ability to evoke calcium entry, but not proliferative responses, (C) 2000 by Elsevier Science Inc, ALL rights reserved.