Neutrophil elastase activates protease-activated receptor-2 (PAR2) and transient receptor potential vanilloid 4 (TRPV4) to cause inflammation and pain
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Proteases that cleave protease-activated receptor-2 (PAR2)at Arg36 ? Ser37 reveal a tethered ligand that binds to the cleaved receptor. PAR2 activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR2 at noncanonical sites can trigger distinct signaling cascades, the functional importance of the PAR2-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR2, causes inflammation and pain by activating PAR2 and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR2 at Ala66 ? Ser67 and Ser67 ? Val68. Elastasestimulated PAR2-dependent cAMP accumulation and ERK1/2 activation, but not Ca2+ mobilization, in KNRK cells. Elastase induced PAR2 coupling to Goas but not Goaq in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK2) or ?-arrestin to PAR2, consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR2-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclaseand protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR2-dependent cAMP formationand ERK1/2 phosphorylation, and a PAR2- 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 PAR2- and TRPV4-mediated mechanisms. Thus, the elastasebiased agonism of PAR2 causes Gas-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 PAR2 as a mediator of protease-driven inflammation and pain.