Discovery of Protease-activated receptor 2 antagonists derived from phenylalanine for the treatment of breast cancer.

Protease-activated receptor 2 (PAR2) has garnered attention as a potential therapeutic target in breast cancer. PAR2 is implicated in the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) via G protein and beta-arrestin pathways, contributing to the proliferation and metastasis of breast cancer cells. Despite the recognized role of PAR2 in breast cancer progression, clinically effective PAR2 antagonists remain elusive. To address this unmet clinical need, we synthesized and evaluated a series of novel compounds that target the orthosteric site of PAR2. Using in silico docking simulations, we identified compound 9a, an optimized derivative of compound 1a ((S)-N-(1-(benzylamino)-1-oxo-3-phenylpropan-2-yl)benzamide), which exhibited enhanced PAR2 antagonistic activity. Subsequent molecular dynamics simulations comparing 9a with the partial agonist 9d revealed that variations in ligand-induced conformational changes and interactions dictated whether the compound acted as an antagonist or agonist of PAR2. The results of this study suggest that further development of 9a could contribute to the advancement of PAR2 antagonists as potential therapeutic agents for breast cancer.

Punicalagin suppresses inflammation in ventilator-induced lung injury through protease-activated receptor-2 inhibition-induced inhibition of NLR family pyrin domain containing-3 inflammasome activation.

Punicalagin is recorded to be a potent anti-inflammatory drug, while its effect on inflammation existing in ventilator-induced lung injury (VILI) requires further verification. Rats were pretreated with punicalagin, followed by VILI modeling. Lung histopathological examination was performed with hematoxylin-eosin staining accompanied by the lung injury score. The lung wet/dry (W/D) weight ratio and total bronchoalveolar lavage fluid (BALF) protein level were measured. After transfection with protease-activated receptor-2 (PAR2) overexpression plasmids, mouse alveolar epithelial MLE-12 cells were treated with punicalagin and then subjected to cyclic stretching. Punicalagin's cytotoxicity to MLE-12 cells were measured by MTT assay. The levels of inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6), PAR2, NLR family pyrin domain containing-3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC) in the BALF, lung tissues or cells were analyzed by enzyme-linked immune-sorbent assay (ELISA), qRT-PCR or/and western blot. Punicalagin treatment attenuated VILI-induced lung histopathological changes and counteracted VILI-induced increases in the lung injury score, W/D weight ratio and total protein level in BALF. Also, punicalagin treatment counteracted in vivo VILI/cyclic stretching-induced increases in the levels of PAR2, inflammatory cytokines, NLRP3, and ASC. PAR2 overexpression potentiated the cyclic stretching-induced effects, while punicalagin treatment revoked this PAR2 overexpression-induced potentiation effect. In turn, PAR2 overexpression partly resisted the punicalagin treatment-induced counteractive effects on the cyclic stretching-induced effects. Punicalagin suppresses inflammation in VILI through PAR2 inhibition-induced inhibition of NLRP3 inflammasome activation.

PAR2 blockade reverses osimertinib resistance in non-small-cell lung cancer cells via attenuating ERK-mediated EMT and PD-L1 expression.

Osimertinib, as the third-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs), is a first-line molecularly targeted drug for non-small cell lung cancer (NSCLC). However, the emergence of therapeutic resistance to osimertinib markedly impairs its efficiency and efficacy, leading to the failure of clinical applications. Novel molecular targets and drugs are urgently needed for reversing osimertinib resistance in NSCLC. Protease-activated receptor 2 (PAR2) that belongs to a subfamily of G protein-coupled receptors can stimulate the transactivation of EGFR to regulate multiple cellular signalling, actively participating in tumour progression. This study firstly discovered that PAR2 expression was notably enhanced when NSCLC cells became resistant to osimertinib. A PAR2 inhibitor facilitated osimertinib to attenuate EGFR transactivation, ERK phosphorylation, EMT and PD-L1 expression which were associated to osimertinib resistance. The combination of the PAR2 inhibitor and osimertinib also notably blocked cell viability, migration, 3D sphere formation and in vivo tumour growth whereas osimertinib itself lost such inhibitory effects in osimertinib-resistant NSCLC cells. Importantly, this reversal effect of PAR2 blockade was uncovered to depend on ERK-mediated EMT and PD-L1, since inhibition of ß-arrestin or ERK, which could be modulated by PAR2, sensitized osimertinib to prevent EMT, PD-L1 expression and consequently overcame osimertinib resistance. Thus, this study demonstrated that PAR2 antagonism could limit ERK-mediated EMT and immune checkpoints, consequently attenuating EGFR transactivation and reactivate osimertinib. It suggested that PAR2 may be a novel drug target for osimertinib resistance, and PAR2 inhibition may be a promising strategy candidate for reversing EGFR-TKI resistance in NSCLC.

Pharmacological blockade of protease-Activated Receptor 2 improves airway remodeling and lung inflammation in experimental allergic asthma

Abstract Protease-activated receptors (PARs) are metabotropic G-protein-coupled receptors that are activated via proteolytic cleavage of a specific sequence of amino acids in their N-terminal region. PAR2 has been implicated in mediating allergic airway inflammation. This study aims to study the effect of PAR2 antagonist ENMD1068in lung inflammation and airway remodeling in experimental asthma. Allergic lung inflammation was induced in sensitized BALB/c mice through intranasal instillations of ovalbumin (OVA), and mice were pretreated with ENMD1068 1 hour before each OVA challenge. Bronchoalveolar lavage fluid (BALF) was collected, and the lungs were removed at different time intervals after OVA challenge to analyze inflammation, airway remodeling and airway hyperresponsiveness. Ovalbumin promoted leukocyte infiltration into BALF in a PAR2-dependent manner. ENMD1068 impaired eosinophil peroxidase (EPO) and myeloperoxidase (MPO) activity in the lung parenchyma into BALF and reduced the loss of dynamic pulmonary compliance, lung resistance in response to methacholine, mucus production, collagen deposition and chemokine (C-C motif) ligand 5 expression compared to those in OVA-challenged mice. We propose that proteases released after an allergen challenge may be crucial to the development of allergic asthma in mice, and PAR2 blockade may be useful as a new pharmacological approach for the treatment of airway allergic diseases.

Trypsin induces an aversive response in zebrafish by PAR2 activation in keratinocytes.

Previously we have shown that trypsin, a protein typically involved in digestion, is released from gills of both fresh and saltwater fishes into surrounding water under stress or injury. We have also shown that each species produces trypsin with different specific activities. In this report, using zebrafish as a model, we identified that trypsin induces an aversive response in zebrafish larvae and adult zebrafish. Since Protease-Activated Receptor 2 (PAR2) responds to trypsin, we tested whether the aversive response is dependent on the activation of PAR2 located on the zebrafish skin cells. Zebrafish larvae treated separately with neomycin and zinc sulfate also showed aversive response indicating neuromast, and olfactory cells are not involved in this aversion. Cultured keratinocytes from zebrafish showed a response to trypsin. Zebrafish larvae subjected to knockdown of par2a also exhibited reduced escape response. Similarly, par2a-deficient mutant larvae displayed no response to trypsin. Since it has been shown that stress activates PAR2 and sends signals to the brain as shown by the increased c-fos expression, we tested c-fos expression in adult zebrafish brains after trypsin treatment of adults and found enhanced c-fos expression by qRT-PCR. Taken together, our results show that the trypsin activates PAR2 on keratinocytes signaling the brain, and this pathway of trypsin-induced escape response will provide a unique communication mechanism in zebrafish. Furthermore, since PAR2 activation also occurs in pain/pruritus sensing, this model might be useful in elucidating components of signaling pathways in pain/pruritus.

Protease-Activated Receptor 2 (PAR-2) Antagonist AZ3451 Mitigates Oxidized Low-Density Lipoprotein (Ox-LDL)-Induced Damage and Endothelial Inflammation.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction plays an important role in the initiation and development of cardiovascular diseases, especially atherosclerosis (AS). Protease-activated receptor 2 (PAR-2) is a receptor for inflammatory proteases. However, the biological function of PAR-2 in endothelial cells and the pathophysiological process of AS are still unknown. In the current study, we found that treatment with ox-LDL increased the gene and protein expressions of PAR-2 in EA.hy926 endothelial cells. Interestingly, we found that antagonism of PAR-2 with its specific antagonist AZ3451 could ameliorate ox-LDL-induced lactate dehydrogenase (LDH) release. Treatment with AZ3451 considerably improved the mitochondrial function by restoring the mitochondrial membrane potential and increasing the levels of intracellular adenosine triphosphate (ATP). Also, we found that AZ3451 attenuated ox-LDL-induced expression and production of pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8). Treatment with AZ3451 also mitigated the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Notably, our results demonstrated that the presence of AZ3451 alleviated ox-LDL-induced expression of the endothelial cell adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1). Mechanistically, we found that AZ3451 attenuated ox-LDL-induced activation of nuclear factor-κB (NF-κB) by reducing the levels of intracellular NF-κB p65 and the luciferase activity of NF-κB promoter. Based on these findings, we conclude that PAR-2 might become a novel therapeutic target for the treatment of AS.

Antagonism of Protease Activated Receptor-2 by GB88 Reduces Inflammation Triggered by Protease Allergen Tyr-p3.

The occurrence of allergic diseases induced by aeroallergens has increased in the past decades. Among inhalant allergens, mites remain the important causal agent of allergic diseases. Storage mites- Tyrophagus putrescentiae are found in stored products or domestic environments. Major allergen Tyr-p3 plays a significant role in triggering IgE-mediated hypersensitivity. However, its effects on pulmonary inflammation, internalization, and activation in human epithelium remain elusive. Protease-activated receptors (PARs) are activated upon cleavage by proteases. A549 cells were used as an epithelial model to examine the PAR activation by Tyr-p3 and therapeutic potential of PAR-2 antagonist (GB88) in allergic responses. Enzymatic properties and allergen localization of Tyr-p3 were performed. The release of inflammatory mediators, phosphorylation of mitogen-activated protein kinase (MAPK), and cell junction disruptions were evaluated after Tyr-p3 challenge. Enzymatic properties determined by substrate digestion and protease inhibitors indicated that Tyr-p3 processes a trypsin-like serine protease activity. The PAR-2 mRNA levels were significantly increased by nTyr-p3 but inhibited by protease inhibitors or GB88. Protease allergen of nTyr-p3 significantly increased the levels of pro-inflammatory cytokines (IL-6 and TNF-α), chemokine (IL-8), and IL-1ß in epithelial cells. nTyr-p3 markedly increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and MAP kinase. When cells were pretreated with GB88 then added nTyr-p3, the phosphorylated ERK1/2 did not inhibit by GB88. GB88 increased ERK1/2 phosphorylation in human epithelium cells. GB88 is able to block PAR-2-mediated calcium signaling which inhibits the nTyr-p3-induced Ca2+ release. Among the pharmacologic inhibitors, the most effective inhibitor of the nTyr-p3 in the induction of IL-8 or IL-1ß levels was GB88 followed by SBTI, MAPK/ERK, ERK, and p38 inhibitors. Levels of inflammatory mediators, including GM-CSF, VEGF, COX-2, TSLP, and IL-33 were reduced by treatment of GB88 or SBTI. Further, GB88 treatment down-regulated the nTyr-p3-induced PAR-2 expression in allergic patients with asthma or rhinitis. Tight junction and adherens junction were disrupted in epithelial cells by nTyr-p3 exposure; however, this effect was avoided by GB88. Immunostaining with frozen sections of the mite body showed the presence of Tyr-p3 throughout the intestinal digestive system, especially in the hindgut around the excretion site. In conclusion, our findings suggest that Tyr-p3 from domestic mites leads to disruption of the airway epithelial barrier after inhalation. Proteolytic activity of Tyr-p3 causes the PAR-2 mRNA expression, thus leading to the release of numerous inflammatory mediators. Antagonism of PAR2 activity suggests GB88 as the therapeutic potential for anti-inflammation medicine, especially in allergy development triggered by protease allergens.

Bone marrow derived mast cells injected into the osteoarthritic knee joints of mice induced by sodium monoiodoacetate enhanced spontaneous pain through activation of PAR2 and action of extracellular ATP.

Conditions that resemble osteoarthritis (OA) were produced by injection of sodium monoiodoacetate (MIA) into the knee joints of mice. Bone marrow derived mast cells (BMMCs) injected into the OA knee joints enhanced spontaneous pain. Since no spontaneous pain was observed when BMMCs were injected into the knee joints of control mice that had not been treated with MIA, BMMCs should be activated within the OA knee joints and release some pain-inducible factors. Protease activated receptor-2 (PAR2) antagonist (FSLLRY-NH2) almost abolished the pain-enhancing effects of BMMCs injected into the OA knee joints, suggesting that tryptase, a mast cell protease that is capable of activating PAR2, should be released from the injected BMMCs and enhance pain through activation of PAR2. When PAR2 agonist (SLIGKV-NH2) instead of BMMCs was injected into the OA knee joints, it was also enhanced pain. Apyrase, an ATP degrading enzyme, injected into the OA knee joints before BMMCs suppressed the pain enhanced by BMMCs. We showed that purinoceptors (P2X4 and P2X7) were expressed in BMMCs and that extracellular ATP stimulated the release of tryptase from BMMCs. These observations suggest that ATP may stimulate degranulation of BMMCs and thereby enhanced pain. BMMCs injected into the OA knee joints stimulated expression of IL-1ß, IL-6, TNF-α, CCL2, and MMP9 genes in the infrapatellar fat pads, and PAR2 antagonist suppressed the stimulatory effects of BMMCs. Our study suggests that intermittent pain frequently observed in OA knee joints may be due, at least partly, to mast cells through activation of PAR2 and action of ATP, and that intraarticular injection of BMMCs into the OA knee joints may provide a useful experimental system for investigating molecular mechanisms by which pain is induced in OA knee joints.

Protease-activated receptor-2 ligands reveal orthosteric and allosteric mechanisms of receptor inhibition.

Protease-activated receptor-2 (PAR2) has been implicated in multiple pathophysiologies but drug discovery is challenging due to low small molecule tractability and a complex activation mechanism. Here we report the pharmacological profiling of a potent new agonist, suggested by molecular modelling to bind in the putative orthosteric site, and two novel PAR2 antagonists with distinctly different mechanisms of inhibition. We identify coupling between different PAR2 binding sites. One antagonist is a competitive inhibitor that binds to the orthosteric site, while a second antagonist is a negative allosteric modulator that binds at a remote site. The allosteric modulator shows probe dependence, more effectively inhibiting peptide than protease activation of PAR2 signalling. Importantly, both antagonists are active in vivo, inhibiting PAR2 agonist-induced acute paw inflammation in rats and preventing activation of mast cells and neutrophils. These results highlight two distinct mechanisms of inhibition that potentially could be targeted for future development of drugs that modulate PAR2.

The development of proteinase-activated receptor-2 modulators and the challenges involved.

Protease-activated receptor-2 (PAR2) has been extensively studied since its discovery in the mid-1990. Despite the advances in understanding PAR2 pharmacology, it has taken almost 25 years for the first inhibitor to reach clinical trials, and so far, no PAR2 antagonist has been approved for human use. Research has employed classical approaches to develop a wide array of PAR2 agonists and antagonists, consisting of peptides, peptoids and antibodies to name a few, with a surge in patent applications over this period. Recent breakthroughs in PAR2 structure determination has provided a unique insight into proposed PAR2 ligand binding sites. Publication of the first crystal structures of PAR2 resolved in complex with two novel non-peptide small molecule antagonists (AZ8838 and AZ3451) revealed two distinct binding pockets, originally presumed to be allosteric sites, with a PAR2 antibody (Fab3949) used to block tethered ligand engagement with the peptide-binding domain of the receptor. Further studies have proposed orthosteric site occupancy for AZ8838 as a competitive antagonist. One company has taken the first PAR2 antibody (MEDI0618) into phase I clinical trial (NCT04198558). While this first-in-human trial is at the early stages of the assessment of safety, other research into the structural characterisation of PAR2 is still ongoing in an attempt to identify new ways to target receptor activity. This review will focus on the development of novel PAR2 modulators developed to date, with an emphasis placed upon the advances made in the pharmacological targeting of PAR2 activity as a strategy to limit chronic inflammatory disease.

The PAR2 inhibitor I-287 selectively targets Gαq and Gα12/13 signaling and has anti-inflammatory effects.

Protease-activated receptor-2 (PAR2) is involved in inflammatory responses and pain, therefore representing a promising therapeutic target for the treatment of immune-mediated inflammatory diseases. However, as for other GPCRs, PAR2 can activate multiple signaling pathways and those involved in inflammatory responses remain poorly defined. Here, we describe a new selective and potent PAR2 inhibitor (I-287) that shows functional selectivity by acting as a negative allosteric regulator on Gαq and Gα12/13 activity and their downstream effectors, while having no effect on Gi/o signaling and ßarrestin2 engagement. Such selective inhibition of only a subset of the pathways engaged by PAR2 was found to be sufficient to block inflammation in vivo. In addition to unraveling the PAR2 signaling pathways involved in the pro-inflammatory response, our study opens the path toward the development of new functionally selective drugs with reduced liabilities that could arise from blocking all the signaling activities controlled by the receptor.

Targeting of protease activator receptor-2 (PAR-2) antagonist FSLLRY-NH2 as an asthma adjuvant therapy.

Asthma is a chronic inflammatory and multifactorial respiratory tract disease. It affects over 18 million adults and 6 million children in the USA with Puerto Ricans showing the highest prevalence (12%-19%). This airways illness can be triggered by an environmental stimulus such as grass pollen, fungi spores, cockroaches allergens, dust mites metabolic compounds, and importantly, by environmental proteases such as trypsin and tryptase. Because of the pivotal role of proteases in the onset of asthma pathophysiology, we focused this study on the serine Protease Activated Receptor-2 (PAR-2), a G-protein-coupled receptor widely expressed in cells across the respiratory tract. Herein, we measured the activation of PAR-2 on primary pulmonary bronchial/tracheal epithelial cells, human small airway epithelial cells, lung bronchial smooth muscle cells (with and without asthma). We tested human-derived eosinophils from 61 Puerto Rican participants (33 asthmatic and 28 non-asthmatic). As surrogate of PAR-2 activation or inhibition we used intracellular calcium mobilization assay. We hypothesized that following exposure of the PAR-2 agonist (AC264613), the studied human primary cell types will increase the mobilization of intracellular calcium levels. In contrast, we expected a decrease of the intracellular calcium levels upon exposure to a PAR-2 antagonist (FSLLRY-NH2). The Puerto Rican-derived eosinophils were analyzed for the proinflammatory markers MAPK/PI3K using flow cytometry (n = 8). As expected, the PAR-2 agonist significantly increased the activation of PAR-2 on the bronchial/tracheal epithelial cells, bronchial smooth muscle cells and human small airway epithelial cells (P = .01). The PAR-2 antagonist significantly decreased the intracellular calcium levels of these lung primary down to undetectable levels (P = .01). Remarkably, the asthmatic-derived eosinophils showed a striking 300% increase of intracellular calcium mobilization suggesting a severe response to the PAR-2 agonist stimuli in asthmatics. In contrast, there were no significant changes between groups after adding the PAR-2 antagonist. Our outcomes revealed that PAR-2 antagonist effectively inhibited the studied primary cells, expecting to decrease the immune response of eosinophils. Most importantly, our results reveal a promising role for the PAR-2 antagonist in targeting bronchial/tracheal epithelial cells, human small airway epithelial cells and bronchial smooth muscle cells with the potential to oblige an asthma adjuvant therapy.

Topical doxycycline monohydrate hydrogel 1% targeting proteases/PAR2 pathway is a novel therapeutic for atopic dermatitis.

Atopic Dermatitis (AD) is characterized by skin barrier disruption and an aberrant immune response. Doxycycline is tetracycline antibiotics broadly used systemically to treat inflammatory dermatologic conditions. Several studies have shown doxycycline has anti-inflammatory and pro-healing properties, mainly by blocking tissue proteolytic activity. It is our hypothesis that daily application of a novel doxycycline topical formulation in AD subjects will reduce severity of the disease, by blocking cutaneous proteases activity and restoring skin barrier function and inflammation. To test this hypothesis, we performed a proof of concept, open-label clinical study. Subjects enrolled in the study (n = 15) applied NanoDOX® Hydrogel 1% daily for 4 weeks on a chosen eczematous area. Investigational drug was well tolerated, and no local or systemic adverse events due to investigational drug were reported. Notably, a significant clinical improvement was observed based on a modified Eczema Area & Severity Index (EASI) score of the treated area from start of treatment to 14 and 28 days post-treatment (P < .001). A significant improvement of pruritus was also observed (P = .02). This proof of concept clinical trial is first to explore the impact of a non-systemic doxycycline treatment on AD patients. Our results provide evidence to investigate novel AD treatment strategies targeting cutaneous proteases activity.

Pain responses to protease-activated receptor-2 stimulation in the spinal cord of naïve and arthritic rats.

There is strong evidence showing that the activation of peripheral proteinase-activated receptors type 2 (PAR-2) can initiate hyperalgesic and inflammatory responses in the joint. However, to date, there is no report of functional spinal PAR-2 receptors in arthritis models. The primary aim of this study was to evaluate the activity of PAR-2 receptors at the spinal cord by using a potent agonist (FLIGRL) in naïve animals, and an antagonist (GB83) in different models of joint pain. Saline or FLIGRL (10 nmol) were injected intrathecally in naïve animals and nociceptive behaviour was evaluated over a 24 h time period by von Frey hair algesiometry. Paw withdrawal threshold decreased from 3 to 24 h and this allodynic effect was blocked by GB83 (90 nmol; i.p.). Acute inflammatory joint pain was induced by injecting 0.5 % kaolin/carrageenan (50 µL each) into the right knee joint of male Wistar rats (24 h recovery). Chronic inflammatory joint pain was modelled by intraarticular injection of Freund's complete adjuvant (FCA; 50 µL; 7 days recovery) or chronic osteoarthritis pain by sodium monoiodoacetate (MIA; 3 mg; 14 days recovery). Animals were then treated with either intrathecal vehicle or 10 nmol of GB83 (10 µL); joint pain was evaluated throughout the subsequent 3 h period. The acute inflammatory pain induced by kaolin/carrageenan was not affected by treatment with GB83. Conversely, both chronic arthritis models demonstrated increased hind paw withdrawal threshold after spinal injection of the PAR-2 antagonist. Based on these results, spinal PAR-2 receptors are involved in joint nociceptive processing in chronic but not acute arthritic conditions.

A role for mast cells and mast cell tryptase in driving neutrophil recruitment in LPS-induced lung inflammation via protease-activated receptor 2 in mice.

OBJECTIVE: This study aims to investigate the role of protease-activated receptor (PAR) 2 and mast cell (MC) tryptase in LPS-induced lung inflammation and neutrophil recruitment in the lungs of C57BL/6 mice. METHODS: C57BL/6 mice were pretreated with the PAR2 antagonist ENMD-1068, compound 48/80 or aprotinin prior to intranasal instillation of MC tryptase or LPS. Blood leukocytes, C-X-C motif chemokine ligand (CXCL) 1 production leukocytes recovered from bronchoalveolar lavage fluid (BALF), and histopathological analysis of the lung were evaluated 4 h later. Furthermore, we performed experiments to determine intracellular calcium signaling in RAW 264.7 cells stimulated with LPS in the presence or absence of a protease inhibitor cocktail or ENMD-1068 and evaluated PAR2 expression in the lungs of LPS-treated mice. RESULTS: Pharmacological blockade of PAR2 or inhibition of proteases reduced neutrophils recovered in BALF and LPS-induced calcium signaling. PAR2 blockade impaired LPS-induced lung inflammation, PAR2 expression in the lung and CXCL1 release in BALF, and increased circulating blood neutrophils. Intranasal instillation of MC tryptase increased the number of neutrophils recovered in BALF, and MC depletion with compound 48/80 impaired LPS-induced neutrophil migration. CONCLUSION: Our study provides, for the first time, evidence of a pivotal role for MCs and MC tryptase in neutrophil migration, lung inflammation and macrophage activation triggered by LPS, by a mechanism dependent on PAR2 activation.

Down-regulation of protease-activated receptor 2 ameliorated osteoarthritis in rats through regulation of MAPK/NF-κB signaling pathway in vivo and in vitro.

Recently, protease-activated receptor 2 (PAR2) has been proved to be involved in the inflammatory response including osteoarthritis (OA). In the present study, we found that PAR2 antagonist could remarkably improve the pathological condition of OA rats in vivo. In addition, we also found that PAR2 antagonist could suppress the production of inflammatory factors (TNF-α and Cox-2), decrease the levels of MMP-1 and MMP-13, and restrain the levels of P62 proteins and aggravate the expression of LC3-II both in vivo and in vitro. Besides, in vitro, PAR2 antagonist could increase the proliferation and colony formation of chondrocytes induced with IL-1ß. Moreover, PAR2 antagonist could decrease the expression of expressions of p-p38, p-IκBα and p-NF-κB in vitro. However, PAR2 agonist exhibited the opposite effects. Furthermore, SB203580, a p38 MAPK inhibitor, could remarkably promote the proliferation of chondrocytes induced with IL-1ß, could alleviate the production of TNF-α and Cox-2, could down-regulate the protein expressions of MMP-1 and MMP-13, and could decrease the expression of P62 and increase the expressions of LC3-II of chondrocytes induced with IL-1ß. Importantly, SB203580 could reverse the effects of PAR2 agonist on the functions of chondrocytes induced with IL-1ß. Taken together, the present data suggest that down-regulation of PAR2 can ameliorate OA through inducing autophagy via regulation of MAPK/NF-κB signaling pathway in vivo and in vitro, and PAR2 can be considered as a potential candidate to treat OA.

Dual blockade of protease-activated receptor 1 and 2 additively ameliorates diabetic kidney disease.

Protease-activated receptors (PARs) are coagulation protease targets, and they increase expression of inflammatory cytokines and chemokines in various diseases. Of all PARs, previous reports have shown that PAR1 or PAR2 inhibition is protective against diabetic glomerular injury. However, how PAR1 and PAR2 cooperatively contribute to diabetic kidney disease (DKD) pathogenesis and whether dual blockade of PARs is more effective in DKD remain elusive. To address this issue, male type I diabetic Akita mice heterozygous for endothelial nitric oxide synthase were used as a model of DKD. Mice (4 mo old) were divided into four treatment groups and administered vehicle, PAR1 antagonist (E5555, 60 mg·kg-1·day-1), PAR2 antagonist (FSLLRY, 3 mg·kg-1·day-1), or E5555 + FSLLRY for 4 wk. The results showed that the urinary albumin creatinine ratio was significantly reduced when both PAR1 and PAR2 were blocked with E5555 + FSLLRY compared with the vehicle-treated group. Dual blockade of PAR1 and PAR2 by E5555 + FSLLRY additively ameliorated histological injury, including mesangial expansion, glomerular macrophage infiltration, and collagen type IV deposition. Marked reduction of inflammation- and fibrosis-related gene expression in the kidney was also observed. In vitro, PAR1 and PAR2 agonists additively increased mRNA expression of macrophage chemoattractant protein 1 or plasminogen activator inhibitor-1 in human endothelial cells. Changes induced by the PAR1 agonist were blocked by a NF-κB inhibitor, whereas those of the PAR2 agonist were blocked by MAPK and/or NF-κB inhibitors. These findings suggest that PAR1 and PAR2 additively contribute to DKD pathogenesis and that dual blockade of both could be a novel therapeutic option for treatment of patients with DKD.

FSLLRY-NH2 Improves Neurological Outcome After Cardiac Arrest in Rats.

AIM: To evaluate the effect of FSLLRY-NH2, a protease-activated receptor 2 (PAR2) inhibitor, on neurocognitive impairment and hippocampal neuronal degeneration in the setting of asphyxial cardiac arrest (ACA)-induced global cerebral ischemia (GCI) in rats. MATERIAL AND METHODS: A total of 43 Sprague-Dawley male rats were used. Shams and rats resuscitated from 9 minutes of ACA were randomized to two separate experiments including time course and short-term neurological outcomes. FSLLRY-NH2 (50 microgram [µg] per rat) was administered intranasally at 1 hour postresuscitation. Neurological function and hippocampal neuronal degeneration were evaluated after ACA. RESULTS: Significant neurological function decline and hippocampal neuron degeneration were observed in ACA animals as compared with the shams. Treatment with FSLLRY-NH2 significantly improved neurological outcome and reduced the number of degenerating hippocampal neurons after ACA. CONCLUSION: Targeting PAR2 may be a novel therapeutic approach in the management of neurological dysfunction after cardiac arrest-associated ischemic injury.

PAR2 Mediates Itch via TRPV3 Signaling in Keratinocytes.

Animal studies have suggested that transient receptor potential ion channels and G-protein coupled receptors play important roles in itch transmission. TRPV3 gain-of-function mutations have been identified in patients with Olmsted syndrome, which is associated with severe pruritus. However, the mechanisms causing itch remain poorly understood. Here, we show that keratinocytes lacking TRPV3 impair the function of protease-activated receptor 2 (PAR2), resulting in reduced neuronal activation and scratching behavior in response to PAR2 agonists. Moreover, we show that TRPV3 and PAR2 were upregulated in skin biopsies from patients and mice with atopic dermatitis, whereas their inhibition attenuated scratching and inflammatory responses in mouse atopic dermatitis models. These results reveal a previously unrecognized link between TRPV3 and PAR2 in keratinocytes to convey itch information and suggest that a blockade of PAR2 or TRPV3 individually or both may serve as a potential approach for antipruritic therapy in atopic dermatitis.

N-acetylcysteine (NAC) alleviates the peripheral neuropathy associated with liver cirrhosis via modulation of neural MEG3/PAR2/ NF-Ò¡B axis.

BACKGROUND AND AIM: Oxidative stress (OS) is accused in pathogenesis of many diseases, including liver cirrhosis by many mechanisms. One of them is the disturbance of long non coding maternally expressed 3 (MEG3)/protease activated receptor 2 (PAR2) downstream pathway. We aimed to investigate the role of this axis in cirrhotic neuropathy and whether an antioxidant compound such as N-acetylcysteine (NAC) could improve the peripheral nerve function through repression of MEG3/PAR2. METHODS: Thirty Wistar rats were used and divided into 5 groups; naive, thiacetamide (TAA) (200 mg/kg 3 times/week. i.p. for 8 weeks) and TAA+NAC (50 or 100 or 200 mg/kg/day) groups. Von Frey (VF) test for mechanical nociceptive responses, hepatic& neural MEG3, NF-Ò¡B and neural PAR2 expression by PCR, histological studies for liver and sciatic nerve together with the dorsopedal skin thickness were done. RESULTS: TAA induced significant decrease in liver function, negative VF test, an increase in the expression of hepatic& neural MEG3, NF-Ò¡B and neural PAR2. The histological studies showed cirrhotic changes with atrophy of the sciatic nerve and the dorsal skin. NAC improved the liver function together with reversal of the neural: functional, biochemical and histological changes in a dose dependent manner. CONCLUSIONS: NAC could improve the peripheral neuropathy in cirrhotic rat through suppression of MEG3/PAR2 expression.