title: Protease-activated receptor 2 (PAR2) protein and transient receptor potential vanilloid 4 (TRPV4) protein coupling is required for sustained inflammatory signaling.
abstract: G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR(2)), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR(2) regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR(2) agonist stimulated a transient increase in [Ca(2+)](i). TRPV4 expression led to a markedly sustained increase in [Ca(2+)](i). Removal of extracellular Ca(2+) and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A(2) and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR(2) generates arachidonic acid-derived lipid mediators, such as 5',6'-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR(2)-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR(2) was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR(2) signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR(2)-stimulated neurogenic inflammation. Thus, PAR(2) activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR(2).
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville, Victoria 3052, Australia.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Poole
authors.forename: Silvia
authors.initials: S
authors.lastname: Amadesi
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Veldhuis
authors.forename: Fe C
authors.initials: FC
authors.lastname: Abogadie
authors.forename: TinaMarie
authors.initials: T
authors.lastname: Lieu
authors.forename: William
authors.initials: W
authors.lastname: Darby
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Liedtke
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lew
authors.forename: Peter
authors.initials: P
authors.lastname: McIntyre
authors.forename: Nigel W
authors.initials: NW
authors.lastname: Bunnett
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D000090762:Cytochrome P-450 CYP2J2; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation; D008297:Male; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010146:Pain; D064801:Phospholipase A2 Inhibitors; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D044464:Receptor, PAR-2 / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D050916:TRPV Cation Channels / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D064801:Phospholipase A2 Inhibitors; D044464:Receptor, PAR-2; D043562:Receptors, G-Protein-Coupled; D050916:TRPV Cation Channels; C416986:TRPV4 protein, human; D014443:Tyrosine; D003577:Cytochrome P-450 Enzyme System; D000090762:Cytochrome P-450 CYP2J2; D002118:Calcium