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 (PAR2) at R36/S37 and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR2 at distinct sites induce inflammation and pain by a biased signaling mechanism is unknown. We observed that cathepsin S (Cat-S) cleaved PAR2 at E56/T57. In cell lines and nociceptive neurons, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand stimulated PAR2 coupling to Gs and formation of cAMP. Unlike trypsin, Cat-S did not mobilize intracellular Ca2+, activate ERK1/2, recruit ß-arrestins, or induce PAR2 endocytosis. Cat-S caused PAR2-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus oocytes, HEK cells and neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR2 or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR2 antagonists suppressed formalin-induced inflammation and pain. Our results reveal a novel mechanism of Cat-S-activation of PAR2, and expand the role of PAR2 as a mediator of protease-driven inflammatory pain.