The Experts below are selected from a list of 283185 Experts worldwide ranked by ideXlab platform
David P. Fairlie - One of the best experts on this subject based on the ideXlab platform.
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protease activated Receptor 2 controls myelin development resiliency and repair
Glia, 2017Co-Authors: Hyesook Yoon, David P. Fairlie, Maja Radulovic, Grant Walters, Alex R Paulsen, Kristen L Drucker, Phillip Starski, Isobel A. ScarisbrickAbstract:Oligodendrocytes are essential regulators of axonal energy homeostasis and electrical conduction and emerging target cells for restoration of neurological function. Here we investigate the role of protease activated Receptor 2 (PAR2), a unique protease activated G protein-coupled Receptor, in myelin development and repair using the spinal cord as a model. Results demonstrate that genetic deletion of PAR2 accelerates myelin production, including higher proteolipid protein (PLP) levels in the spinal cord at birth and higher levels of myelin basic protein and thickened myelin sheaths in adulthood. Enhancements in spinal cord myelin with PAR2 loss-of-function were accompanied by increased numbers of Olig2- and CC1-positive oligodendrocytes, as well as in levels of cyclic adenosine monophosphate (cAMP), and extracellular signal related kinase 1/2 (ERK1/2) signaling. Parallel promyelinating effects were observed after blocking PAR2 expression in purified oligodendrocyte cultures, whereas inhibiting adenylate cyclase reversed these effects. Conversely, PAR2 activation reduced PLP expression and this effect was prevented by brain derived neurotrophic factor (BDNF), a promyelinating growth factor that signals through cAMP. PAR2 knockout mice also showed improved myelin resiliency after traumatic spinal cord injury and an accelerated pattern of myelin regeneration after focal demyelination. These findings suggest that PAR2 is an important controller of myelin production and regeneration, both in the developing and adult spinal cord.
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Repurposing Registered Drugs as Antagonists for Protease-Activated Receptor 2
Journal of Chemical Information and Modeling, 2015Co-Authors: Weijun Xu, Jonathan Y. Suen, Kai-chen Wu, Yuhong Jiang, David P. FairlieAbstract:Virtual screening of a drug database identified Carvedilol, Loratadine, Nefazodone and Astemizole as PAR2 antagonists, after ligand docking and molecular dynamics simulations using a PAR2 homology model and a putative binding mode of a known PAR2 ligand. The drugs demonstrated competitive binding and antagonism of calcium mobilization and ERK1/2 phosphorylation in CHO-hPAR2 transfected cells, while inhibiting IL-6 secretion in PAR2 expressing MDA-MB-231 breast cancer cells. This research highlights opportunities for GPCR hit-finding from FDA-approved drugs.
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an antagonist of human protease activated Receptor 2 attenuates par2 signaling macrophage activation mast cell degranulation and collagen induced arthritis in rats
The FASEB Journal, 2012Co-Authors: Rinkjan Lohman, Jacky Y. Suen, Adam J Cotterell, David A Vesey, Grant D Barry, David P. FairlieAbstract:Multiple serine proteases exert proinflammatory actions by signaling through Protease-Activated Receptor-2 (PAR2) on the cell surface. Although inhibitors of individual proteases are anti-inflammatory, we sought to discover whether the first potent antagonist of their common target PAR2 might be beneficial in treating chronic arthritis-like inflammatory disease. Using a fluorescence assay, a novel compound, GB88, was shown to antagonize PAR2-induced intracellular Ca2+ release in human monocyte-derived macrophages, being 1000 times more potent than a control compound, ENMD-1068 (IC50 1.6±0.5 μM vs. 1.2±0.4 mM, respectively). In Wistar rats, GB88 was orally bioavailable (F=55%, Tmax 4 h, Cmax 1.7 μM, 10 mg/kg). GB88 inhibited the acute paw edema induced in Wistar rats by intraplantar λ-carrageenan or PAR2 agonists 2-furoyl-LIGRLO-NH2 or mast cell β-tryptase, without inhibiting proteolytic activity of tryptase in vitro. In the chronic collagen-induced model of arthritis in rats, GB88 (10 mg/kg) was disease m...
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an antagonist of human protease activated Receptor 2 attenuates par2 signaling macrophage activation mast cell degranulation and collagen induced arthritis in rats
The FASEB Journal, 2012Co-Authors: Rinkjan Lohman, Jacky Y. Suen, Adam J Cotterell, David A Vesey, Grant D Barry, Ligong Liu, David P. FairlieAbstract:Multiple serine proteases exert proinflammatory actions by signaling through Protease-Activated Receptor-2 (PAR2) on the cell surface. Although inhibitors of individual proteases are anti-inflammatory, we sought to discover whether the first potent antagonist of their common target PAR2 might be beneficial in treating chronic arthritis-like inflammatory disease. Using a fluorescence assay, a novel compound, GB88, was shown to antagonize PAR2-induced intracellular Ca(2+) release in human monocyte-derived macrophages, being 1000 times more potent than a control compound, ENMD-1068 (IC(50) 1.6 ± 0.5 μM vs. 1.2 ± 0.4 mM, respectively). In Wistar rats, GB88 was orally bioavailable (F=55%, T(max) 4 h, C(max) 1.7 μM, 10 mg/kg). GB88 inhibited the acute paw edema induced in Wistar rats by intraplantar λ-carrageenan or PAR2 agonists 2-furoyl-LIGRLO-NH(2) or mast cell β-tryptase, without inhibiting proteolytic activity of tryptase in vitro. In the chronic collagen-induced model of arthritis in rats, GB88 (10 mg/kg) was disease modifying and ameliorated pathological and histopathological changes (edema, pannus formation, synovial hyperplasia, collagen degradation, macrophage invasion, mast cell degranulation) compared to untreated arthritic controls. The results suggest that an orally active PAR2 antagonist is effective in treating chronic arthritis in rats through inhibiting macrophage infiltration, mast cell degranulation, and β-tryptase-PAR2 signaling in joint inflammation.
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antagonism of protease activated Receptor 2 protects against experimental colitis
Journal of Pharmacology and Experimental Therapeutics, 2012Co-Authors: Rinkjan Lohman, Jacky Y. Suen, Adam J Cotterell, Anh Do, David A Vesey, David P. FairlieAbstract:Many trypsin-like serine proteases like β-tryptase are involved in the pathogenesis of colitis and inflammatory bowel diseases. Inhibitors of individual proteases show limited efficacy in treating such conditions, but also likely disrupt digestive and defensive functions of proteases. Here we investigate whether masking their common target, protease activated Receptor 2 (PAR2), is an effective therapeutic strategy for treating acute and chronic experimental colitis in rats. A novel PAR2 antagonist (GB88) was evaluated for blockade of intracellular calcium release in colonocytes, and for anti-inflammatory activity in acute (PAR2 agonistµinduced) versus chronic (TNBS-induced) models of colitis in Wistar rats. Disease progression (disease activity index, weight loss, mortality) and post-mortem colonic histopathology (inflammation, bowel wall thickness, myeloperoxidase) were measured. PAR2 and tryptase co-localization was investigated using immunohistochemistry. GB88 was a more potent antagonist of PAR2 activation in colonocytes than a reported compound ENMD-1068 (IC50 8 µM vs 5 mM). Acute colonic inflammation induced in rats by PAR2 agonist SLIGRL-NH2 was inhibited by oral administration of GB88 (10 mg/kg), with markedly reduced oedema, mucin depletion, PAR2 Receptor internalisation and mastocytosis. Chronic TNBS-induced colitis in rats was ameliorated by GB88 (10mg/kg/day/p.o.), which reduced mortality and pathology (including colon obstruction, ulceration, wall thickness, myeloperoxidase release) more effectively than the clinically used drug sulfasalazine (100 mg/kg/day/p.o.). These disease-modifying properties for the PAR2 antagonist in both acute and chronic experimental colitis strongly support a pathogenic role for PAR2 and PAR2-activating proteases, and therapeutic potential for PAR2 antagonism, in inflammatory diseases of the colon.
Nigel W Bunnett - One of the best experts on this subject based on the ideXlab platform.
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protein kinase d and gβγ subunits mediate agonist evoked translocation of protease activated Receptor 2 from the golgi apparatus to the plasma membrane
Journal of Biological Chemistry, 2016Co-Authors: Dane D Jensen, Tinamarie Lieu, Peishen Zhao, Stephen Vanner, Nestor N Jimenezvargas, Daniel P Poole, Marina Gerges, Holly R Yeatman, Meritxell Canals, Nigel W BunnettAbstract:Abstract Agonist-evoked endocytosis of G protein-coupled Receptors has been extensively studied. The mechanisms by which agonists stimulate mobilization and plasma membrane translocation of G protein-coupled Receptors from intracellular stores are unexplored. Protease-Activated Receptor-2 (PAR2) traffics to lysosomes, and sustained protease signaling requires mobilization and plasma membrane trafficking of PAR2 from Golgi stores. We evaluated the contribution of protein kinase D (PKD) and Gβγ to this process. In HEK293 and KNRK cells, the PAR2 agonists trypsin and 2-furoyl-LIGRLO-NH2 activated PKD in the Golgi apparatus, where PKD regulates protein trafficking. PAR2 activation induced translocation of Gβγ, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between Gγ-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and Gβγ (gallein) prevented PAR2-stimulated activation of PKD. CRT0066101, PKD1 siRNA, and gallein all inhibited recovery of PAR2-evoked Ca2+ signaling. PAR2 with a photoconvertible Kaede tag was expressed in KNRK cells to examine Receptor translocation from the Golgi apparatus to the plasma membrane. Irradiation of the Golgi region (405 nm) induced green-red photo-conversion of PAR2-Kaede. Trypsin depleted PAR2-Kaede from the Golgi apparatus and repleted PAR2-Kaede at the plasma membrane. CRT0066101 inhibited PAR2-Kaede translocation to the plasma membrane. CRT0066101 also inhibited sustained protease signaling to colonocytes and nociceptive neurons that naturally express PAR2 and mediate protease-evoked inflammation and nociception. Our results reveal a major role for PKD and Gβγ in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases.
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neutrophil elastase activates protease activated Receptor 2 par2 and transient Receptor potential vanilloid 4 trpv4 to cause inflammation and pain
Journal of Biological Chemistry, 2015Co-Authors: Peishen Zhao, Tinamarie Lieu, Nicholas Barlow, Silvia Sostegni, Stephen Vanner, Christoph Korbmacher, Wolfgang Liedtke, Silke Haerteis, Nestor N Jimenezvargas, Nigel W BunnettAbstract: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 non-canonical 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. Elastase stimulated PAR2-dependent cAMP accumulation and ERK1/2 activation, but not Ca2+ mobilization, in KNRK cells. Elastase induced PAR2 coupling to Gαs but not Gαq in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled Receptor kinase-2 (GRK2) or β-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 cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR2-dependent cAMP formation and 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 elastase-biased agonism of PAR2 causes Gαs-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.
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endosomal deubiquitinating enzymes control ubiquitination and down regulation of protease activated Receptor 2
Journal of Biological Chemistry, 2009Co-Authors: Burcu Hasdemir, Graeme S. Cottrell, Jane E Murphy, Nigel W BunnettAbstract:The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled Receptor Protease-Activated Receptor 2 (PAR2), which is required for postendocytic sorting of activated Receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR2 deubiquitination and its importance in trafficking and signaling of endocytosed PAR2 are unknown. We report that Receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR2 ubiquitination and trapped the Receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR2. Trapping PAR2 in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR2 with β-arrestin2 or the duration of PAR2-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR2 but not for regulating PAR2 dissociation from β-arrestin2 or PAR2-mediated ERK2 activation.
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c cbl mediates ubiquitination degradation and down regulation of human protease activated Receptor 2
Journal of Biological Chemistry, 2005Co-Authors: Claire Jacob, Graeme S. Cottrell, Eileen F Grady, Daphne Gehringer, Fabien Schmidlin, Nigel W BunnettAbstract:Mechanisms that arrest G-protein-coupled Receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by Protease-Activated Receptor 2 (PAR(2)), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR(2) is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR(2) was mono-ubiquitinated. Immunofluorescence indicated that activated PAR(2) translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR(2) lacking intracellular lysine residues (PAR(2)Delta14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR(2) Delta14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR(2) stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR(2) at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR(2) ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR(2) was degraded, and recovery of agonist responses required synthesis of new Receptors, lysine mutation and dominant negative c-Cbl impeded Receptor ubiquitination and degradation and allowed PAR(2) to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR(2) to irrevocably terminate signaling by this and perhaps other GPCRs.
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protease activated Receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway
Journal of Immunology, 2002Co-Authors: Fabien Schmidlin, Nigel W Bunnett, Silvia Amadesi, Pierangelo Geppetti, Karim Dabbagh, David Lewis, Patrick G Knott, Paul R Gater, Claude Bertrand, Mary E StevensAbstract:Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated Receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 micro g/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.
Silvia Amadesi - One of the best experts on this subject based on the ideXlab platform.
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protease activated Receptor 2 par2 protein and transient Receptor potential vanilloid 4 trpv4 protein coupling is required for sustained inflammatory signaling
Journal of Biological Chemistry, 2013Co-Authors: Daniel P Poole, Tinamarie Lieu, Wolfgang Liedtke, Silvia Amadesi, William Darby, Peter Mcintyre, Nicholas A Veldhuis, Fe C Abogadie, Michael J LewAbstract: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).
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Protease-Activated Receptor 2, Dipeptidyl Peptidase I, and Proteases Mediate Clostridium difficile Toxin A Enteritis
Gastroenterology, 2007Co-Authors: Graeme S. Cottrell, Stella Pikios, Brett R. Murphy, Paul J. Wolters, J. Adam Willardsen, Eric Camerer, Shaun R. Coughlin, Silvia Amadesi, George H Caughey, Anders PetersonAbstract:Background & Aims: We studied the role of Protease-Activated Receptor 2 (PAR2) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. Methods: We injected TxA into ileal loops in PAR2 or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR2 and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 Receptor antagonist SR140333. Results: TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR2 deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%–28% and tissue and fluid myeloperoxidase by 31%–71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR2 and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca2+ responses to PAR2 AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. Conclusions: PAR2 and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR2 and up-regulates PAR2 and activating proteases, and PAR2 causes inflammation by neurogenic mechanisms.
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protease activated Receptor 2 sensitizes the transient Receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice
The Journal of Physiology, 2007Co-Authors: Andrew Douglas Grant, Graeme S. Cottrell, Silvia Amadesi, Marcello Trevisani, Paola Nicoletti, Serena Materazzi, Christophe Altier, Nicolas Cenac, Gerald W Zamponi, Francisco BautistacruzAbstract:Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave Protease-Activated Receptor 2 (PAR2) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR2-mediated mechanical hyperalgesia requires sensitization of the ion channel transient Receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR2, substance P (SP) and calcitonin gene-related peptide (CGRP), mediators of pain transmission. In PAR2-expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR2 agonist enhanced Ca2+ and current responses to the TRPV4 agonists phorbol ester 4alpha-phorbol 12,13-didecanoate (4alphaPDD) and hypotonic solutions. PAR2-agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cbeta and protein kinases A, C and D inhibited PAR2-induced sensitization of TRPV4 Ca2+ signals and currents. 4alphaPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR2 agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4alphaPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR2 agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR2 activates a second messenger to sensitize TRPV4-dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.
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mast cell tryptase controls paracellular permeability of the intestine role of protease activated Receptor 2 and beta arrestins
Journal of Biological Chemistry, 2005Co-Authors: Claire Jacob, Graeme S. Cottrell, Silvia Amadesi, Eileen F Grady, Pingchang Yang, Dalila Darmoul, Toshiyuki Saito, Anne Marie Coelho, Pamela Singh, Mary H PerdueAbstract:Tight junctions between intestinal epithelial cells prevent ingress of luminal macromolecules and bacteria and protect against inflammation and infection. During stress and inflammation, mast cells mediate increased mucosal permeability by unknown mechanisms. We hypothesized that mast cell tryptase cleaves Protease-Activated Receptor 2 (PAR2) on colonocytes to increase paracellular permeability. Colonocytes expressed PAR2 mRNA and responded to PAR2 agonists with increased [Ca2+]i. Supernatant from degranulated mast cells increased [Ca2+]i in colonocytes, which was prevented by a tryptase inhibitor, and desensitized responses to PAR2 agonist, suggesting PAR2 cleavage. When applied to the basolateral surface of colonocytes, PAR2 agonists and mast cell supernatant decreased transepithelial resistance, increased transepithelial flux of macromolecules, and induced redistribution of tight junction ZO-1 and occludin and perijunctional F-actin. When mast cells were co-cultured with colonocytes, mast cell degranulation increased paracellular permeability of colonocytes. This was prevented by a tryptase inhibitor. We determined the role of ERK1/2 and of beta-arrestins, which recruit ERK1/2 to PAR2 in endosomes and retain ERK1/2 in the cytosol, on PAR2-mediated alterations in permeability. An ERK1/2 inhibitor abolished the effects of PAR2 agonist on permeability and redistribution of F-actin. Down-regulation of beta-arrestins with small interfering RNA inhibited PAR2-induced activation of ERK1/2 and suppressed PAR2-induced changes in permeability. Thus, mast cells signal to colonocytes in a paracrine manner by release of tryptase and activation of PAR2. PAR2 couples to beta-arrestin-dependent activation of ERK1/2, which regulates reorganization of perijunctional F-actin to increase epithelial permeability. These mechanisms may explain the increased epithelial permeability of the intestine during stress and inflammation.
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protease activated Receptor 2 sensitizes the capsaicin Receptor transient Receptor potential vanilloid Receptor 1 to induce hyperalgesia
The Journal of Neuroscience, 2004Co-Authors: Silvia Amadesi, Graeme S. Cottrell, Marcello Trevisani, Pierangelo Geppetti, Jingjiang Nie, Nathalie Vergnolle, Eileen F Grady, Chiara Manni, James A Mcroberts, Helena S EnnesAbstract:Inflammatory proteases (mast cell tryptase and trypsins) cleave Protease-Activated Receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient Receptor potential vanilloid Receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.
Peter Mcintyre - One of the best experts on this subject based on the ideXlab platform.
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a functional kinase short interfering ribonucleic acid screen using protease activated Receptor 2 dependent opening of transient Receptor potential vanilloid 4
Assay and Drug Development Technologies, 2017Co-Authors: William Darby, Megan S Grace, Kaylene J Simpson, Owen L Woodman, Peter McintyreAbstract:Abstract Protease-Activated Receptor 2 (PAR2) is a proinflammatory G-protein coupled Receptor (GPCR) that is activated by inflammatory proteases, and its activation initiates signaling pathways that modulate the nonselective cation channel transient Receptor potential vanilloid-4 (TRPV4). PAR2-dependent opening of TRPV4 has been attributed to kinase activation, but the identity of the responsible enzymes is unknown. Deciphering the signaling pathways involved in the PAR2-dependent opening of TRPV4 may yield new targets for pain treatment. This study has identified specific kinases that are involved in opening TRPV4, using a selective screen of short interfering ribonucleic acid (siRNA) SMARTpools, which individually targeted all human kinases, in human embryonic kidney 293 (HEK293) cells that stably express inducible TRPV4. This screen is unique because it uses a real-time assay measuring intracellular calcium with Fura-2AM dye. From the primary screen, subsequent confirmation screen, and on-target messen...
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protease activated Receptor 2 par2 protein and transient Receptor potential vanilloid 4 trpv4 protein coupling is required for sustained inflammatory signaling
Journal of Biological Chemistry, 2013Co-Authors: Daniel P Poole, Tinamarie Lieu, Wolfgang Liedtke, Silvia Amadesi, William Darby, Peter Mcintyre, Nicholas A Veldhuis, Fe C Abogadie, Michael J LewAbstract: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).
Tinamarie Lieu - One of the best experts on this subject based on the ideXlab platform.
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protein kinase d and gβγ mediate sustained nociceptive signaling by biased agonists of protease activated Receptor 2
Journal of Biological Chemistry, 2019Co-Authors: Peishen Zhao, Tinamarie Lieu, Nestor N Jimenezvargas, Dane D Jensen, Luke A Pattison, Rocco Latorre, Josue Obed Jaramillo, Cintya Lopezlopez, Daniel P Poole, Stephen VannerAbstract:Proteases sustain hyperexcitability and pain by cleaving Protease-Activated Receptor-2 (PAR2) on nociceptors through distinct mechanisms. Whereas trypsin induces PAR2 coupling to Gαq, Gαs, and β-arrestins, cathepsin-S (CS) and neutrophil elastase (NE) cleave PAR2 at distinct sites and activate it by biased mechanisms that induce coupling to Gαs, but not to Gαq or β-arrestins. Because proteases activate PAR2 by irreversible cleavage, and activated PAR2 is degraded in lysosomes, sustained extracellular protease-mediated signaling requires mobilization of intact PAR2 from the Golgi apparatus or de novo synthesis of new Receptors by incompletely understood mechanisms. We found here that trypsin, CS, and NE stimulate PAR2-dependent activation of protein kinase D (PKD) in the Golgi of HEK293 cells, in which PKD regulates protein trafficking. The proteases stimulated translocation of the PKD activator Gβγ to the Golgi, coinciding with PAR2 mobilization from the Golgi. Proteases also induced translocation of a photoconverted PAR2-Kaede fusion protein from the Golgi to the plasma membrane of KNRK cells. After incubation of HEK293 cells and dorsal root ganglia neurons with CS, NE, or trypsin, PAR2 responsiveness initially declined, consistent with PAR2 cleavage and desensitization, and then gradually recovered. Inhibitors of PKD, Gβγ, and protein translation inhibited recovery of PAR2 responsiveness. PKD and Gβγ inhibitors also attenuated protease-evoked mechanical allodynia in mice. We conclude that proteases that activate PAR2 by canonical and biased mechanisms stimulate PKD in the Golgi; PAR2 mobilization and de novo synthesis repopulate the cell surface with intact Receptors and sustain nociceptive signaling by extracellular proteases.
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protease activated Receptor 2 in endosomes signals persistent pain of irritable bowel syndrome
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Nestor N Jimenezvargas, Tinamarie Lieu, Peishen Zhao, Dane D Jensen, Luke A Pattison, Rocco Latorre, Joel Castro, Luigi Aurelio, Giang T LeAbstract:Once activated at the surface of cells, G protein-coupled Receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of Protease-Activated Receptor-2 (PAR2), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR2-dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR2, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR2, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR2-dependent hyperexcitability of nociceptors, and PAR2 association with β-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR2. A cholestanol-conjugated PAR2 antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR2 signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR2 antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.
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protein kinase d and gβγ subunits mediate agonist evoked translocation of protease activated Receptor 2 from the golgi apparatus to the plasma membrane
Journal of Biological Chemistry, 2016Co-Authors: Dane D Jensen, Tinamarie Lieu, Peishen Zhao, Stephen Vanner, Nestor N Jimenezvargas, Daniel P Poole, Marina Gerges, Holly R Yeatman, Meritxell Canals, Nigel W BunnettAbstract:Abstract Agonist-evoked endocytosis of G protein-coupled Receptors has been extensively studied. The mechanisms by which agonists stimulate mobilization and plasma membrane translocation of G protein-coupled Receptors from intracellular stores are unexplored. Protease-Activated Receptor-2 (PAR2) traffics to lysosomes, and sustained protease signaling requires mobilization and plasma membrane trafficking of PAR2 from Golgi stores. We evaluated the contribution of protein kinase D (PKD) and Gβγ to this process. In HEK293 and KNRK cells, the PAR2 agonists trypsin and 2-furoyl-LIGRLO-NH2 activated PKD in the Golgi apparatus, where PKD regulates protein trafficking. PAR2 activation induced translocation of Gβγ, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between Gγ-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and Gβγ (gallein) prevented PAR2-stimulated activation of PKD. CRT0066101, PKD1 siRNA, and gallein all inhibited recovery of PAR2-evoked Ca2+ signaling. PAR2 with a photoconvertible Kaede tag was expressed in KNRK cells to examine Receptor translocation from the Golgi apparatus to the plasma membrane. Irradiation of the Golgi region (405 nm) induced green-red photo-conversion of PAR2-Kaede. Trypsin depleted PAR2-Kaede from the Golgi apparatus and repleted PAR2-Kaede at the plasma membrane. CRT0066101 inhibited PAR2-Kaede translocation to the plasma membrane. CRT0066101 also inhibited sustained protease signaling to colonocytes and nociceptive neurons that naturally express PAR2 and mediate protease-evoked inflammation and nociception. Our results reveal a major role for PKD and Gβγ in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases.
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neutrophil elastase activates protease activated Receptor 2 par2 and transient Receptor potential vanilloid 4 trpv4 to cause inflammation and pain
Journal of Biological Chemistry, 2015Co-Authors: Peishen Zhao, Tinamarie Lieu, Nicholas Barlow, Silvia Sostegni, Stephen Vanner, Christoph Korbmacher, Wolfgang Liedtke, Silke Haerteis, Nestor N Jimenezvargas, Nigel W BunnettAbstract: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 non-canonical 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. Elastase stimulated PAR2-dependent cAMP accumulation and ERK1/2 activation, but not Ca2+ mobilization, in KNRK cells. Elastase induced PAR2 coupling to Gαs but not Gαq in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled Receptor kinase-2 (GRK2) or β-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 cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR2-dependent cAMP formation and 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 elastase-biased agonism of PAR2 causes Gαs-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.
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protease activated Receptor 2 par2 protein and transient Receptor potential vanilloid 4 trpv4 protein coupling is required for sustained inflammatory signaling
Journal of Biological Chemistry, 2013Co-Authors: Daniel P Poole, Tinamarie Lieu, Wolfgang Liedtke, Silvia Amadesi, William Darby, Peter Mcintyre, Nicholas A Veldhuis, Fe C Abogadie, Michael J LewAbstract: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).
