title: Cathepsin S causes inflammatory pain via biased agonism of PAR2 and TRPV4.
abstract: Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR2) at R(36)↓S(37) and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR2 at 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 PAR2 at E(56)↓T(57), 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 PAR2 coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca(2+), activate ERK1/2, recruit β-arrestins, or induce PAR2 endocytosis. 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 PAR2 or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR2 antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR2 in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR2 that causes PAR2- and TRPV4-dependent inflammation and pain. They expand the role of PAR2 as a mediator of protease-driven inflammatory pain.
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville 3052, Australia.
authors.forename: Peishen
authors.initials: P
authors.lastname: Zhao
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville 3052, Australia.
authors.forename: TinaMarie
authors.initials: T
authors.lastname: Lieu
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville 3052, Australia.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Barlow
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville 3052, Australia.
authors.forename: Matthew
authors.initials: M
authors.lastname: Metcalf
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville 3052, Australia.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Veldhuis
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville 3052, Australia.
authors.forename: Dane D
authors.initials: DD
authors.lastname: Jensen
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville 3052, Australia.
authors.forename: Martina
authors.initials: M
authors.lastname: Kocan
authors.affiliation: Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
authors.forename: Silvia
authors.initials: S
authors.lastname: Sostegni
authors.affiliation: Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Haerteis
authors.affiliation: Medivir AB, 141 Huddinge, Sweden.
authors.forename: Vera
authors.initials: V
authors.lastname: Baraznenok
authors.affiliation: Medivir AB, 141 Huddinge, Sweden.
authors.forename: Ian
authors.initials: I
authors.lastname: Henderson
authors.affiliation: Medivir AB, 141 Huddinge, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Lindström
authors.affiliation: Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queen's University, Kingston, Ontario N7L 3N6, Canada.
authors.forename: Raquel
authors.initials: R
authors.lastname: Guerrero-Alba
authors.affiliation: Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queen's University, Kingston, Ontario N7L 3N6, Canada.
authors.forename: Eduardo E
authors.initials: EE
authors.lastname: Valdez-Morales
authors.affiliation: Division of Neurology, Department of Medicine, Duke University, Durham, North Carolina 27710.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Liedtke
authors.affiliation: School of Medical Sciences and Health Innovations Research Institute, RMIT University, Bundoora 3083, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: McIntyre
authors.affiliation: Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queen's University, Kingston, Ontario N7L 3N6, Canada.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Vanner
authors.affiliation: Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Korbmacher
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville 3052, Australia,; Department of Pharmacology, University of Melbourne, Melbourne 3010, Australia, and; ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville 3052, Australia. Electronic address: Nigel.Bunnett@Monash.edu.
authors.forename: Nigel W
authors.initials: NW
authors.lastname: Bunnett
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006930:Hyperalgesia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D007249:Inflammation / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D010146:Pain / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D044464:Receptor, PAR-2 / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D050916:TRPV Cation Channels / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D044464:Receptor, PAR-2; D050916:TRPV Cation Channels; C416986:TRPV4 protein, human; C486576:Trpv4 protein, mouse; D002403:Cathepsins; C028217:cathepsin S; D000262:Adenylyl Cyclases
keywords: G Protein-coupled Receptor (GPCR); Inflammation; Pain; Protease; Protease-activated Receptors; Transient Receptor Potential Channels (TRP Channels)