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↑S³⁷and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR₂at 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 PAR₂at E⁵⁶↑T⁵⁷, 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 PAR₂coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca²⁺, activate ERK1/2, recruit β-arrestins, or induce PAR₂endocytosis. Cat-S caused PAR₂-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 PAR₂or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR₂antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR₂in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR₂that causes PAR₂- and TRPV4-dependent inflammation and pain. They expand the role of PAR2 as a mediator of protease-driven inflammatory pain.