Myeloid Protease-Activated Receptor-2 Contributes to Influenza A Virus Pathology in Mice.

Background: Innate immune responses to influenza A virus (IAV) infection are initiated in part by toll-like receptor 3 (TLR3). TLR3-dependent signaling induces an antiviral immune response and an NFκB-dependent inflammatory response. Protease-activated receptor 2 (PAR2) inhibits the antiviral response and enhances the inflammatory response. PAR2 deficiency protected mice during IAV infection. However, the PAR2 expressing cell-types contributing to IAV pathology in mice and the mechanism by which PAR2 contributes to IAV infection is unknown. Methods: IAV infection was analyzed in global (Par2-/- ), myeloid (Par2fl/fl;LysMCre+) and lung epithelial cell (EpC) Par2 deficient (Par2fl/fl ;SPCCre+) mice and their respective controls (Par2+/+ and Par2fl/fl). In addition, the effect of PAR2 activation on polyinosinic-polycytidylic acid (poly I:C) activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM). Lastly, we determined the effect of PAR2 inhibition in wild-type (WT) mice. Results: After IAV infection, Par2-/- and mice with myeloid Par2 deficiency exhibited increased survival compared to infected controls. The improved survival was associated with reduced proinflammatory mediators and reduced cellular infiltration in bronchoalveolar lavage fluid (BALF) of Par2-/- and Par2fl/fl;LysMCre+ 3 days post infection (dpi) compared to infected control mice. Interestingly, Par2fl/fl;SPCCre+ mice showed no survival benefit compared to Par2fl/fl . In vitro studies showed that Par2-/- BMDM produced less IL6 and IL12p40 than Par2+/+ BMDM after poly I:C stimulation. In addition, activation of PAR2 on Par2+/+ BMDM increased poly I:C induction of IL6 and IL12p40 compared to poly I:C stimulation alone. Importantly, PAR2 inhibition prior to IAV infection protect WT mice. Conclusion: Global Par2 or myeloid cell but not lung EpC Par2 deficiency was associated with reduced BALF inflammatory markers and reduced IAV-induced mortality. Our study suggests that PAR2 may be a therapeutic target to reduce IAV pathology.

PAR2 Deficiency Induces Mitochondrial ROS Generation and Dysfunctions, Leading to the Inhibition of Adipocyte Differentiation.

Protease-activated receptor 2 (PAR2) is a member of G-protein-coupled receptors and affects ligand-modulated calcium signaling. Although PAR2 signaling promotes obesity and adipose tissue inflammation in high fat- (HF-) fed conditions, its role in adipocyte differentiation under nonobesogenic conditions needs to be elucidated. Here, we used several tissues and primary-cultured adipocytes of mice lacking PAR2 to study its role in the development of adipose tissues. C57BL/6J mice with PAR2 deficiency exhibited a mild lipodystrophy-like phenotype in a chow diet-fed condition. When adipocyte differentiation was examined using primary-cultured preadipocytes, PAR2 deficiency led to a notable decrease in adipocyte differentiation and related protein expression, and PAR2 agonist treatment elevated adipocyte differentiation. Regarding the mechanism, PAR2-deficient preadipocytes exhibited impaired mitochondrial energy consumption. Further studies indicated that calcium-related signaling pathways for mitochondrial biogenesis are disrupted in the adipose tissues of PAR2-deficient mice and PAR2-deficient preadipocytes. Also, a PAR2 antagonist elevated mitochondrial reactive oxygen species and reduced the MitoTracker fluorescent signal in preadipocytes. Our studies revealed that PAR2 is important for the development of adipose tissue under basal conditions through the regulation of mitochondrial biogenesis and adipocyte differentiation.

Deficiency of protease-activated receptor (PAR) 1 and PAR2 exacerbates collagen-induced arthritis in mice via differing mechanisms.

OBJECTIVES: Protease-activated receptor (PAR) 1 and PAR2 have been implicated in RA, however their exact role is unclear. Here, we detailed the mechanistic impact of these receptors on the onset and development of inflammatory arthritis in murine CIA and antigen-induced arthritis (AIA) models. METHODS: CIA or AIA was induced in PAR1 or PAR2 gene knockout (KO) and matched wild type mice. The onset and development of arthritis was monitored clinically and histologically. Immune cells, cytokines and MMPs were detected by ELISA, zymography, flow cytometry, western blot or immunohistochemistry. RESULTS: In CIA, PAR1KO and PAR2KO exacerbated arthritis, in opposition to their effects in AIA. These deficient mice had high plasma levels of IL-17, IFN-γ, TGF-ß1 and MMP-13, and lower levels of TNF-α; T cells and B cells were higher in both KO spleen and thymus, and myeloid-derived suppressor cells were lower only in PAR1KO spleen, when compared with wild type cells. Th1, Th2 and Th17 cells were lower in PAR1KO spleens cells, whereas Th1 and Th2 cells were lower and Th17 cells higher in both KO thymus cells, when compared with wild type cells. PAR1KO synovial fibroblasts proliferated faster and produced the most abundant MMP-9 amongst three type cells in the control, lipopolysaccharides or TNF stimulated conditions. CONCLUSION: This is the first study demonstrated that deficiency of PAR1 or PAR2 aggravates inflammatory arthritis in CIA. Furthermore, the protective functions of PAR1 and PAR2 in CIA likely occur via differing mechanisms involving immune cell differentiation and cytokines/MMPs.

Protease-activated receptor 2 deficiency in hematopoietic lineage protects against myocardial infarction through attenuated inflammatory response and fibrosis.

Ischaemic heart disease is one of the leading causes of death. Protease-activated receptor 2 (PAR2) is widely expressed within the cardiovascular system and is known to mediate inflammatory processes in various immunocytes, such as macrophages, mastocytes and neutrophils. Here, we investigated whether activating macrophage PAR2 modulates cardiac remodelling in a murine model of myocardial infarction. Myocardial infarction was produced by the permanent ligation of the left anterior descending coronary artery (LAD) in C57BL/6J background wild-type (WT) mice transplanted with bone marrow from WT or PAR2 knockout (PAR2 KO) mice. Hematopoietic deficiency of PAR2 had improvement of left ventricular systolic dysfunction and dilatation and decreased fibrosis deposition in remote zone at 1 week after LAD ligation. Inactivation of PAR2 also led to less recruitment of macrophages in myocardium, which was accompanied by decreased expression of pro-inflammatory cytokines. Furthermore, cultured cardiac fibroblasts (CFs) were activated and showed a fibrotic phenotype after being co-cultured in medium containing PAR2-activating macrophage, which enhances interferon-beta (INF-ß) expression. The beneficial effects of macrophages with INF-ß neutralisation or PAR2-deletion ameliorates the JAK/STAT3 pathway in CFs, which might be attributed to CF activation. These data suggest that macrophage-derived IFN-ß plays a crucial role in adverse cardiac remodelling after myocardial infarction, at least in part, through a PAR2-dependent mechanism.

PAR2 controls cholesterol homeostasis and lipid metabolism in nonalcoholic fatty liver disease.

OBJECTIVE: Increases in hepatic and plasma cholesterol occur in patients with nonalcoholic fatty liver disease (NAFLD), although the reason for this is not well understood. We investigated whether Protease-Activated Receptor 2 (PAR2) plays a role in cholesterol and lipid homeostasis in NAFLD. METHODS: Human liver biopsies (n = 108) were quantified for PAR2 expression from NAFLD cases randomly selected and stratified by liver fibrosis stage, the primary predictor for clinical outcomes, while controlling for age, gender, and BMI between fibrosis groups. Demographic data and laboratory studies on plasma samples were obtained within 6 months of liver biopsy. Wild-type and PAR2-KO (C57BL/6 F2rl1-/-) mice were fed either normal or high fat diet for 16 weeks and plasma and liver assayed for lipids and soluble metabolites. RESULTS: Severity of NAFLD and plasma cholesterol levels significantly correlated with hepatocyte PAR2 expression in NAFLD patients. Conversely, PAR2 deficiency in mice resulted in reduced expression of key hepatic genes involved in cholesterol synthesis, a 50% drop in plasma and total liver cholesterol, and induced a reverse cholesterol transport system that culminated in 25% higher fecal bile acid output. PAR2-deficient mice exhibited enhanced fatty acid ß-oxidation with a ketogenic shift and an unexpected increase in liver glycogenesis. Mechanistic studies identified Gi-Jnk1/2 as key downstream effectors of protease-activated PAR2 in the regulation of lipid and cholesterol homeostasis in liver. CONCLUSIONS: These data indicate that PAR2 may be a new target for the suppression of plasma cholesterol and hepatic fat accumulation in NAFLD and related metabolic conditions.

Protease-activated receptor 2 does not contribute to renal inflammation or fibrosis in the obstructed kidney.

AIM: Protease-activated receptor 2 (PAR2) has been implicated in the development of renal inflammation and fibrosis. In particular, activation of PAR2 in cultured tubular epithelial cells induces extracellular signal-regulated kinase signalling and secretion of fibronectin, C-C Motif Chemokine Ligand 2 (CCL2) and transforming growth factor-ß1 (TGF-ß1), suggesting a role in tubulointerstitial inflammation and fibrosis. We tested this hypothesis in unilateral ureteric obstruction (UUO) in which ongoing tubular epithelial cell damage drives tubulointerstitial inflammation and fibrosis. METHODS: Unilateral ureteric obstruction surgery was performed in groups (n = 9/10) of Par2-/- and wild type (WT) littermate mice which were killed 7 days later. Non-experimental mice were controls. RESULTS: Wild type mice exhibited a 5-fold increase in Par2 messenger RNA (mRNA) levels in the UUO kidney. In situ hybridization localized Par2 mRNA expression to tubular epithelial cells in normal kidney, with a marked increase in Par2 mRNA expression by tubular cells, including damaged tubular cells, in WT UUO kidney. Tubular damage (tubular dilation, increased KIM-1 and decreased α-Klotho expression) and tubular signalling (extracellular signal-regulated kinase phosphorylation) seen in WT UUO were not altered in Par2-/- UUO. In addition, macrophage infiltration, up-regulation of M1 (NOS2) and M2 (CD206) macrophage markers, and up-regulation of pro-inflammatory molecules (tumour necrosis factor, CCL2, interleukin-36α) in WT UUO kidney were unchanged in Par2-/- UUO. Finally, the accumulation of α-SMA+ myofibroblasts, deposition of collagen IV and expression of pro-fibrotic factors (CTGF, TGF-ß1) were not different between WT and Par2-/- UUO mice. CONCLUSION: Protease-activated receptor 2 expression is substantially up-regulated in tubular epithelial cells in the obstructed kidney, but this does not contribute to the development of tubular damage, renal inflammation or fibrosis.

PAR2-Mediated cAMP Generation Suppresses TRPV4-Dependent Ca2+ Signaling in Alveolar Macrophages to Resolve TLR4-Induced Inflammation.

Alveolar macrophages (AMs), upon sensing pathogens, trigger host defense by activating toll-like receptor 4 (TLR4), but the counterbalancing mechanisms that deactivate AM inflammatory signaling and prevent lethal edema, the hallmark of acute lung injury (ALI), remain unknown. Here, we demonstrate the essential role of AM protease-activating receptor 2 (PAR2) in rapidly suppressing inflammation to prevent long-lasting injury. We show that thrombin, released during TLR4-induced lung injury, directly activates PAR2 to generate cAMP, which abolishes Ca2+ entry through the TRPV4 channel. Deletion of PAR2 and thus the accompanying cAMP generation augments Ca2+ entry via TRPV4, causing sustained activation of the transcription factor NFAT to produce long-lasting TLR4-mediated inflammatory lung injury. Rescuing thrombin-sensitive PAR2 expression or blocking TRPV4 activity in PAR2-null AMs restores their capacity to resolve inflammation and reverse lung injury. Thus, activation of the thrombin-induced PAR2-cAMP cascade in AMs suppresses TLR4 inflammatory signaling to reinstate tissue integrity.

Protease-activated receptor 2 deficiency mediates cardiac fibrosis and diastolic dysfunction.

AIMS: Heart failure with preserved ejection fraction (HFpEF) and pathological cardiac aging share a complex pathophysiology, including extracellular matrix remodelling (EMR). Protease-activated receptor 2 (PAR2) deficiency is associated with EMR. The roles of PAR1 and PAR2 have not been studied in HFpEF, age-dependent cardiac fibrosis, or diastolic dysfunction (DD). METHODS AND RESULTS: Evaluation of endomyocardial biopsies from patients with HFpEF (n = 14) revealed that a reduced cardiac PAR2 expression was associated with aggravated DD and increased myocardial fibrosis (r = -0.7336, P = 0.0028). In line, 1-year-old PAR2-knockout (PAR2ko) mice suffered from DD with preserved systolic function, associated with an increased age-dependent α-smooth muscle actin expression, collagen deposition (1.7-fold increase, P = 0.0003), lysyl oxidase activity, collagen cross-linking (2.2-fold increase, P = 0.0008), endothelial activation, and inflammation. In the absence of PAR2, the receptor-regulating protein caveolin-1 was down-regulated, contributing to an augmented profibrotic PAR1 and transforming growth factor beta (TGF-ß)-dependent signalling. This enhanced TGF-ß/PAR1 signalling caused N-proteinase (ADAMTS3) and C-proteinase (BMP1)-related increased collagen I production from cardiac fibroblasts (CFs). PAR2 overexpression in PAR2ko CFs reversed these effects. The treatment with the PAR1 antagonist, vorapaxar, reduced cardiac fibrosis by 44% (P = 0.03) and reduced inflammation in a metabolic disease model (apolipoprotein E-ko mice). Patients with HFpEF with upstream PAR inhibition via FXa inhibitors (n = 40) also exhibited reduced circulating markers of fibrosis and DD compared with patients treated with vitamin K antagonists (n = 20). CONCLUSIONS: Protease-activated receptor 2 is an important regulator of profibrotic PAR1 and TGF-ß signalling in the heart. Modulation of the FXa/FIIa-PAR1/PAR2/TGF-ß-axis might be a promising therapeutic approach to reduce HFpEF.

Endothelial cells in the innate response to allergens and initiation of atopic asthma.

Protease-activated receptor 2 (PAR-2), an airway epithelial pattern recognition receptor (PRR), participates in the genesis of house dust mite-induced (HDM-induced) asthma. Here, we hypothesized that lung endothelial cells and proangiogenic hematopoietic progenitor cells (PACs) that express high levels of PAR-2 contribute to the initiation of atopic asthma. HDM extract (HDME) protease allergens were found deep in the airway mucosa and breaching the endothelial barrier. Lung endothelial cells and PACs released the Th2-promoting cytokines IL-1α and GM-CSF in response to HDME, and the endothelium had PAC-derived VEGF-C-dependent blood vessel sprouting. Blockade of the angiogenic response by inhibition of VEGF-C signaling lessened the development of inflammation and airway remodeling in the HDM model. Reconstitution of the bone marrow in WT mice with PAR-2-deficient bone marrow also reduced airway inflammation and remodeling. Adoptive transfer of PACs that had been exposed to HDME induced angiogenesis and Th2 inflammation with remodeling similar to that induced by allergen challenge. Our findings identify that lung endothelium and PACs in the airway sense allergen and elicit an angiogenic response that is central to the innate nonimmune origins of Th2 inflammation.

Protease-Activated Receptor-2 Plays a Critical Role in Vascular Inflammation and Atherosclerosis in Apolipoprotein E-Deficient Mice.

BACKGROUND: The coagulation system is closely linked with vascular inflammation, although the underlying mechanisms are still obscure. Recent studies show that protease-activated receptor (PAR)-2, a major receptor of activated factor X, is expressed in both vascular cells and leukocytes, suggesting that PAR-2 may contribute to the pathogenesis of inflammatory diseases. Here we investigated the role of PAR-2 in vascular inflammation and atherogenesis. METHODS: We generated apolipoprotein E-deficient ( ApoE-/-) mice lacking systemic PAR-2 expression ( PAR-2-/- ApoE-/-). ApoE-/- mice, which lack or express PAR-2 only in bone marrow (BM) cells, were also generated by BM transplantation. Atherosclerotic lesions were investigated after 20 weeks on a Western-type diet by histological analyses, quantitative reverse transcription polymerase chain reaction, and Western blotting. In vitro experiments using BM-derived macrophages were performed to confirm the proinflammatory roles of PAR-2. The association between plasma activated factor X level and the severity of coronary atherosclerosis was also examined in humans who underwent coronary intervention. RESULTS: PAR-2-/- ApoE-/- mice showed reduced atherosclerotic lesions in the aortic arch ( P<0.05) along with features of stabilized atherosclerotic plaques, such as less lipid deposition ( P<0.05), collagen loss ( P<0.01), macrophage accumulation ( P<0.05), and inflammatory molecule expression ( P<0.05) compared with ApoE-/- mice. Systemic PAR2 deletion in ApoE-/-mice significantly decreased the expression of inflammatory molecules in the aorta. The results of BM transplantation experiments demonstrated that PAR-2 in hematopoietic cells contributed to atherogenesis in ApoE-/- mice. PAR-2 deletion did not alter metabolic parameters. In vitro experiments demonstrated that activated factor X or a specific peptide agonist of PAR-2 significantly increased the expression of inflammatory molecules and lipid uptake in BM-derived macrophages from wild-type mice compared with those from PAR-2-deficient mice. Activation of nuclear factor-κB signaling was involved in PAR-2-associated vascular inflammation and macrophage activation. In humans who underwent coronary intervention, plasma activated factor X level independently correlated with the severity of coronary atherosclerosis as determined by Gensini score ( P<0.05) and plaque volume ( P<0.01). CONCLUSIONS: PAR-2 signaling activates macrophages and promotes vascular inflammation, increasing atherosclerosis in ApoE-/- mice. This signaling pathway may also participate in atherogenesis in humans.

Hepatocyte-secreted DPP4 in obesity promotes adipose inflammation and insulin resistance.

Obesity-induced metabolic disease involves functional integration among several organs via circulating factors, but little is known about crosstalk between liver and visceral adipose tissue (VAT). In obesity, VAT becomes populated with inflammatory adipose tissue macrophages (ATMs). In obese humans, there is a close correlation between adipose tissue inflammation and insulin resistance, and in obese mice, blocking systemic or ATM inflammation improves insulin sensitivity. However, processes that promote pathological adipose tissue inflammation in obesity are incompletely understood. Here we show that obesity in mice stimulates hepatocytes to synthesize and secrete dipeptidyl peptidase 4 (DPP4), which acts with plasma factor Xa to inflame ATMs. Silencing expression of DPP4 in hepatocytes suppresses inflammation of VAT and insulin resistance; however, a similar effect is not seen with the orally administered DPP4 inhibitor sitagliptin. Inflammation and insulin resistance are also suppressed by silencing expression of caveolin-1 or PAR2 in ATMs; these proteins mediate the actions of DPP4 and factor Xa, respectively. Thus, hepatocyte DPP4 promotes VAT inflammation and insulin resistance in obesity, and targeting this pathway may have metabolic benefits that are distinct from those observed with oral DPP4 inhibitors.

PAR2 (Protease-Activated Receptor 2) Deficiency Attenuates Atherosclerosis in Mice.

OBJECTIVE: PAR2 (protease-activated receptor 2)-dependent signaling results in augmented inflammation and has been implicated in the pathogenesis of several autoimmune conditions. The objective of this study was to determine the effect of PAR2 deficiency on the development of atherosclerosis. APPROACH AND RESULTS: PAR2 mRNA and protein expression is increased in human carotid artery and mouse aortic arch atheroma versus control carotid and aortic arch arteries, respectively. To determine the effect of PAR2 deficiency on atherosclerosis, male and female low-density lipoprotein receptor-deficient (Ldlr-/-) mice (8-12 weeks old) that were Par2+/+ or Par2-/- were fed a fat- and cholesterol-enriched diet for 12 or 24 weeks. PAR2 deficiency attenuated atherosclerosis in the aortic sinus and aortic root after 12 and 24 weeks. PAR2 deficiency did not alter total plasma cholesterol concentrations or lipoprotein distributions. Bone marrow transplantation showed that PAR2 on nonhematopoietic cells contributed to atherosclerosis. PAR2 deficiency significantly attenuated levels of the chemokines Ccl2 and Cxcl1 in the circulation and macrophage content in atherosclerotic lesions. Mechanistic studies using isolated primary vascular smooth muscle cells showed that PAR2 deficiency is associated with reduced Ccl2 and Cxcl1 mRNA expression and protein release into the supernatant resulting in less monocyte migration. CONCLUSIONS: Our results indicate that PAR2 deficiency is associated with attenuation of atherosclerosis and may reduce lesion progression by blunting Ccl2- and Cxcl1-induced monocyte infiltration.

A T cell-specific knockout reveals an important role for protease-activated receptor 2 in lymphocyte development.

Activation of protease-activated receptor-2 (PAR2) expressed by T cells has been linked to the bone loss associated with periodontitis. We generated PAR2 conditional-null mice and crossed these with mice expressing Cre recombinase under control of the Lck proximal promoter, to produce T cell-specific PAR2-null mice in order to further study the cellular mechanism involved in periodontitis. Here we report that efficient deletion of PAR2 in thymocytes isolated from T cell-specific PAR2-null mice resulted in thymic and splenic hypoplasia and a reduction in the cells of the cortex and a loss of distinction between the cortex and the medulla of the thymus. FACS analysis confirmed significant reductions in CD4 and CD8 double negative (DN3 and DN4) sub-populations, as well as double positive and single positive T cells, in T cell-specific PAR2-null mice compared to Cre expressing PAR2 wild-type mice. The proportion of annexin V positive and propidium iodide negative cells was increased in CD4 and CD8 double negative, double positive and single positive T cells from T cell-specific PAR2-null mice. No change in the proportion of Ki67 positive cells was observed in sections of thymus from T cell-specific PAR2-null mice, suggesting that the depletion of T cell sub-populations in T cell-specific PAR2-null mice resulted from increased apoptosis rather than reduced proliferation. Together, these results demonstrate that PAR2 plays an important and previously unrecognised anti-apoptotic role in T cell development and suggest that the PAR2 conditional-null mouse will be an important resource for determining tissue and cell specific effects of PAR2.

Precision in the design of an experimental study deflects the significance of proteinase-activated receptor 2 expression in scrapie-inoculated mice.

Proteinase-activated receptor 2 (PAR2) is suspected to modulate the pathogenesis of various neurodegenerative conditions. We previously described delayed onset of clinical symptoms and prolonged survival of PAR2-deficient mice after intracerebral inoculation with prions. Here we report the results from a refined blinded study that aimed to investigate the effects of PAR2 deletion on scrapie pathogenesis after peripheral infection. This study failed to confirm that PAR2 deficiency impacts on the length of the incubation period, with PAR2-/- and PAR2+/+ littermates developing scrapie at the same time. To clarify the discrepancy between the two observations, we repeated the intracerebral inoculation study while utilizing our refined protocol, which aimed to limit possible sources of experimental bias. The study again failed to confirm the significant effect of PAR2 expression on the course of prion infection. Our report emphasizes and discusses the importance of unbiased experimental design and the selection of proper genetic controls when using genetically altered animal models for prion pathogenesis studies.

Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease-activated receptor-2.

BACKGROUND AND PURPOSE: Diverse proteases cleave protease-activated receptor-2 (PAR2) on primary sensory neurons and epithelial cells to evoke pain and inflammation. Trypsin and tryptase activate PAR2 by a canonical mechanism that entails cleavage within the extracellular N-terminus revealing a tethered ligand that activates the cleaved receptor. Cathepsin-S and elastase are biased agonists that cleave PAR2 at different sites to activate distinct signalling pathways. Although PAR2 is a therapeutic target for inflammatory and painful diseases, the divergent mechanisms of proteolytic activation complicate the development of therapeutically useful antagonists. EXPERIMENTAL APPROACH: We investigated whether the PAR2 antagonist GB88 inhibits protease-evoked activation of nociceptors and protease-stimulated oedema and hyperalgesia in rodents. KEY RESULTS: Intraplantar injection of trypsin, cathespsin-S or elastase stimulated mechanical and thermal hyperalgesia and oedema in mice. Oral GB88 or par2 deletion inhibited the algesic and proinflammatory actions of all three proteases, but did not affect basal responses. GB88 also prevented pronociceptive and proinflammatory effects of the PAR2-selective agonists 2-furoyl-LIGRLO-NH2 and AC264613. GB88 did not affect capsaicin-evoked hyperalgesia or inflammation. Trypsin, cathepsin-S and elastase increased [Ca(2+) ]i in rat nociceptors, which expressed PAR2. GB88 inhibited this activation of nociceptors by all three proteases, but did not affect capsaicin-evoked activation of nociceptors or inhibit the catalytic activity of the three proteases. CONCLUSIONS AND IMPLICATIONS: GB88 inhibits the capacity of canonical and biased protease agonists of PAR2 to cause nociception and inflammation.

Coagulation Factor Xa and Protease-Activated Receptor 2 as Novel Therapeutic Targets for Diabetic Nephropathy.

OBJECTIVE: The role of hypercoagulability in the pathogenesis of diabetic nephropathy (DN) remains elusive. We recently reported the increased infiltration of macrophages expressing tissue factor in diabetic kidney glomeruli; tissue factor activates coagulation factor X (FX) to FXa, which in turn stimulates protease-activated receptor 2 (PAR2) and causes inflammation. APPROACH AND RESULTS: Here, we demonstrated that diabetes mellitus increased renal FX mRNA, urinary FXa activity, and FX expression in glomerular macrophages. Administration of an oral FXa inhibitor, edoxaban, ameliorated DN with concomitant reductions in the expression of PARs (Par1 and Par2) and of proinflammatory and profibrotic genes. Diabetes mellitus induced PAR2, and lack of Par2 ameliorated DN. FXa or PAR2 agonist increased inflammatory cytokines in endothelial cells and podocytes in vitro. CONCLUSIONS: We conclude that enhanced FXa and PAR2 exacerbate DN and that both are promising targets for preventing DN. Alleviating inflammation is probably more important than inhibiting coagulation per se when treating kidney diseases using anticoagulants.

Neutrophil elastase induces inflammation and pain in mouse knee joints via activation of proteinase-activated receptor-2.

BACKGROUND AND PURPOSE: Neutrophil elastase plays a crucial role in arthritis. Here, its potential in triggering joint inflammation and pain was assessed, and whether these effects were mediated by proteinase-activated receptor-2 (PAR2). EXPERIMENTAL APPROACH: Neutrophil elastase (5 µg) was injected into the knee joints of mice and changes in blood perfusion, leukocyte kinetics and paw withdrawal threshold were assessed. Similar experiments were performed in animals pretreated with the neutrophil elastase inhibitor sivelestat, the PAR2 antagonist GB83, the p44/42 MAPK inhibitor U0126 and in PAR2 receptor knockout (KO) mice. Neutrophil elastase activity was also evaluated in arthritic joints by fluorescent imaging and sivelestat was assessed for anti-inflammatory and analgesic properties. KEY RESULTS: Intra-articular injection of neutrophil elastase caused an increase in blood perfusion, leukocyte kinetics and a decrease in paw withdrawal threshold. Sivelestat treatment suppressed this effect. The PAR2 antagonist GB83 reversed neutrophil elastase-induced synovitis and pain and these responses were also attenuated in PAR2 KO mice. The MAPK inhibitor U0126 also blocked neutrophil elastase-induced inflammation and pain. Active neutrophil elastase was increased in acutely inflamed knees as shown by an activatable fluorescent probe. Sivelestat appeared to reduce neutrophil elastase activity, but had only a moderate anti-inflammatory effect in this model. CONCLUSIONS AND IMPLICATIONS: Neutrophil elastase induced acute inflammation and pain in knee joints of mice. These changes are PAR2-dependent and appear to involve activation of a p44/42 MAPK pathway. Blocking neutrophil elastase, PAR2 and p44/42 MAPK activity can reduce inflammation and pain, suggesting their utility as therapeutic targets.

Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2.

Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbe Enterococcus faecalis. Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) from E. faecalis V583 and E. faecalis lacking the gelatinase gene (gelE). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutant E. faecalis strains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2(-/-)) mice were used to investigate the role of this receptor in E. faecalis-induced permeability. Gelatinase (GelE) purified from E. faecalis V583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2(-/-) mice by fecal supernatants from ulcerative colitis patients was assessed. Secreted E. faecalis proteins induced permeability in epithelial cell monolayers, which was reduced in the absence of gelE or by blocking PAR2 activity. Secreted E. faecalis proteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2(-/-) mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2(-/-) mice. Our investigations demonstrate that GelE from E. faecalis can regulate enteric epithelial permeability via PAR2.

TMPRSS2, a novel membrane-anchored mediator in cancer pain.

More than half of all cancer patients have significant pain during the course of their disease. The strategic localization of TMPRSS2, a membrane-bound serine protease, on the cancer cell surface may allow it to mediate signal transduction between the cancer cell and its extracellular environment. We show that TMPRSS2 expression is not only dramatically increased in the primary cancers of patients but TMPRSS2 immunopositivity is also directly correlated with cancer pain severity in these patients. TMPRSS2 induced proteolytic activity, activated trigeminal neurons, and produced marked mechanical hyperalgesia when administered into the hind paw of wild-type mice but not PAR2-deficient mice. Coculture of human cancer cells with murine trigeminal neurons demonstrated colocalization of TMPRSS2 with PAR2. These results point to a novel role for a cell membrane-anchored mediator in cancer pain, as well as pain in general.

Protease-activated receptor 2 activation is sufficient to induce the transition to a chronic pain state.

Protease-activated receptor type 2 (PAR2) is known to play an important role in inflammatory, visceral, and cancer-evoked pain based on studies using PAR2 knockout (PAR2(-/-)) mice. We have tested the hypothesis that specific activation of PAR2 is sufficient to induce a chronic pain state through extracellular signal-regulated kinase (ERK) signaling to protein synthesis machinery. We have further tested whether the maintenance of this chronic pain state involves a brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (trkB)/atypical protein kinase C (aPKC) signaling axis. We observed that intraplantar injection of the novel highly specific PAR2 agonist, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at), evokes a long-lasting acute mechanical hypersensitivity (median effective dose ∼12 pmoles), facial grimacing, and causes robust hyperalgesic priming as revealed by a subsequent mechanical hypersensitivity and facial grimacing to prostaglandin E2 (PGE2) injection. The promechanical hypersensitivity effect of 2-at is completely absent in PAR2(-/-) mice as is hyperalgesic priming. Intraplantar injection of the upstream ERK inhibitor, U0126, and the eukaryotic initiation factor (eIF) 4F complex inhibitor, 4EGI-1, prevented the development of acute mechanical hypersensitivity and hyperalgesic priming after 2-at injection. Systemic injection of the trkB antagonist ANA-12 similarly inhibited PAR2-mediated mechanical hypersensitivity, grimacing, and hyperalgesic priming. Inhibition of aPKC (intrathecal delivery of ZIP) or trkB (systemic administration of ANA-12) after the resolution of 2-at-induced mechanical hypersensitivity reversed the maintenance of hyperalgesic priming. Hence, PAR2 activation is sufficient to induce neuronal plasticity leading to a chronic pain state, the maintenance of which is dependent on a BDNF/trkB/aPKC signaling axis.