Cathepsin S Causes Inflammatory Pain via Biased Agonism of PAR2 and TRPV4

Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR(2)) at R-36 down arrow S-37 and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR(2) at distinct sites are biased agonists that also induce inflammation and pain is unexplored. Cathepsin S (Cat-S) is a lysosomal cysteine protease of antigen-presenting cells that is secreted during inflammation and which retains activity at extracellular pH. We observed that Cat-S cleaved PAR(2) at E-56 down arrow T-57, which removed the canonical tethered ligand and prevented trypsin activation. In HEK and KNRK cell lines and in nociceptive neurons of mouse dorsal root ganglia, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand-stimulated PAR(2) coupling to G alpha s and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca2+, activate ERK1.2, recruit beta-arrestins, or induce PAR(2) endocytosis. Cat-S caused PAR(2)-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus laevis oocytes, HEK cells and nociceptive neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Intraplantar injection of Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR(2) or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR(2) antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR(2) in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR(2) that causes PAR(2)- and TRPV4-dependent inflammation and pain. They expand the role of PAR(2) as a mediator of protease-driven inflammatory pain.