PAR2 response in fluid flow-stimulated chondrocytes

Osteoarthritis (OA) is a heterogeneous musculoskeletal disease. We have compelling evidence that proteinaseactivated receptor-2 (PAR2) is a mediator of OA initiation/progression, integrating matrix deregulation and tissue remodeling/infl ammatory damage. PAR2 offers a potentially novel therapeutic target in OA. The onset of OA has mechanical “damage” as a common denominator and we have observed elevated of PAR2 in articular chondrocytes during the OA mouse destabilization of the medial meniscus (DMM) model and in human OA cartilage. PAR2 was also found in osteocytes. Whilst wild type animals exhibit rapid osteophyte formation, PAR2-defi cient (PAR2−/−) mice are substantially protected from this process (osteophyte formation = 92.3% in WT, vs. 45.5% in PAR2−/− mice, and the latter were signifi cantly smaller, WT = 2.50 ± 0.27, PAR2−/− = 0.41 ± 0.19 μm3). Osteosclerosis was signifi cant by day 14 in WT (P = 0.023) and remained so at day 28 (P = 0.019) whilst PAR2−/− mice showed no signifi cant osteosclerosis at either day 14 or 28, potentially indicating a slower response to the mechanical changes induced by DMM surgery. This led us to question whether PAR2 may play a role in mechanotransduction. Murine osteocyte-like cells (MLOY4) were stimulated with 7 dyn/cm2 of fl uid fl ow shear stress (FSS) and human chondrocyte-like cells (SW1353) with physiological (5 dyn/cm2) and pathophysiological (20 dyn/cm2) FSS for 1 h. During FSS, MLOY4s were administered 10 nM PAR2 activating peptide SLIGRL or control reverse peptide. Immunofl uorescence and western blotting showed intrinsic PAR2 presence in MLOY4s, which did not change after FSS stimulation. FSS increased COX2 gene expression 4-fold (P < 0.01). SLIGRL in static conditions induced a 2-fold increase in IL-6 expression, which was reduced to static control levels after FSS (P < 0.01). No other parameters were signifi cant. SW1353s did not contain intrinsic PAR2 under static conditions, yet a marked increase was noted upon stimulation with FSS (mean grey value ± SD: static control, comparable to background, = 92 ± 5.6, 5 dyn = 226 ± 23, 20 dyn = 191 ± 22, P < 0.001, 1way ANOVA). This FSS-induced increase in PAR2 suggests that altered biomechanical loading may initiate PAR2 mediated mechanisms ultimately leading to cartilage degradation, which could have implications for human disease.