Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

Proteases that cleave protease-activated receptor-2 (PAR 2 ) at Arg 36 ↓Ser 37 reveal a tethered ligand that binds to the cleaved receptor. PAR 2 activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR 2 at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR 2 -biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR 2 , causes inflammation and pain by activating PAR 2 and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR 2 at Ala 66 ↓Ser 67 and Ser 67 ↓Val 68 . Elastase stimulated PAR 2 -dependent cAMP accumulation and ERK1/2 activation, but not Ca 2+ mobilization, in KNRK cells. Elastase induced PAR 2 coupling to Gα s but not Gα q in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK2) or β-arrestin to PAR 2 , consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR 2 -dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR 2 -dependent cAMP formation and ERK1/2 phosphorylation, and a PAR 2 - and TRPV4-mediated influx of extracellular Ca 2+ in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR 2 - and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR 2 causes Gα s -dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR 2 as a mediator of protease-driven inflammation and pain. Background: Proteases cleave protease-activated receptor-2 (PAR 2 ), which activates transient receptor potential (TRP) ion channels to cause inflammation and pain. Results: Neutrophil elastase cleaves PAR 2 , resulting in Gα s -mediated cAMP formation, transient receptor potential vanilloid 4 (TRPV4) activation, and sensitization of nociceptive neurons, inflammation, and pain. Conclusion: Elastase causes PAR 2 - and TRPV4-mediated inflammation and pain. Significance: PARs and TRP channels mediate responses to diverse proteases.

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