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(362) down arrow 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(662) down arrow Ser(67) and Ser(672) down arrow Val(68). Elastase stimulated PAR(2)-dependent cAMP accumulation and ERK1/2 activation, but not Ca2+ mobilization, in KNRK cells. Elastase induced PAR(2) coupling to G alpha(s) but not G alpha(q) in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK2) or beta-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)-dependentc AMP formation and ERK1/2 phosphorylation, and a PAR(2)- and TRPV4-mediated influx of extracellular Ca2+ 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 G alpha(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.