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.

Protease Activated Receptors and Arthritis.

The catabolic and destructive activity of serine proteases in arthritic joints is well known; however, these enzymes can also signal pain and inflammation in joints. For example, thrombin, trypsin, tryptase, and neutrophil elastase cleave the extracellular N-terminus of a family of G protein-coupled receptors and the remaining tethered ligand sequence then binds to the same receptor to initiate a series of molecular signalling processes. These protease activated receptors (PARs) pervade multiple tissues and cells throughout joints where they have the potential to regulate joint homeostasis. Overall, joint PARs contribute to pain, inflammation, and structural integrity by altering vascular reactivity, nociceptor sensitivity, and tissue remodelling. This review highlights the therapeutic potential of targeting PARs to alleviate the pain and destructive nature of elevated proteases in various arthritic conditions.

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.

House dust mite-treated PAR2 over-expressor mouse: A novel model of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a complex skin disease involving causative effects from both intrinsic and extrinsic sources. Murine models of the disease often fall short in one of these components and, as a result, do not fully encapsulate these disease mechanisms. OBJECTIVE: We aimed to determine whether the protease-activated receptor 2 over-expressor mouse (PAR2OE) with topical house dust mite (HDM) application is a more comprehensive and clinically representative AD model. METHODS: Following HDM extract application to PAR2OE mice and controls, AD clinical scoring, itching behaviour, skin morphology and structure, barrier function, immune cell infiltration and inflammatory markers were assessed. Skin morphology was analysed using haematoxylin and eosin staining, and barrier function was assessed by transepidermal water loss measurements. Immune infiltrate was characterised by histological and immunofluorescence staining. Finally, an assessment of AD-related gene expression was performed using quantitative RT-PCR. RESULTS: PAR2OE mice treated with HDM displays all the characteristic clinical symptoms including erythema, dryness and oedema, skin morphology, itch and inflammation typically seen in patients with AD. There is a significant influx of mast cells (P < .01) and eosinophils (P < .0001) into the dermis of these mice. Furthermore, the PAR2OE + HDM mice exhibit similar expression patterns of key differentially expressed genes as seen in human AD. CONCLUSION: The PAR2OE + HDM mouse presents with a classic AD pathophysiology and is a valuable model in terms of reproducibility and overall disease representation to study the condition and potential therapeutic approaches.

Store-operated calcium entry (SOCE) contributes to phosphorylation of p38 MAPK and suppression of TNF-α signalling in the intestinal epithelial cells.

Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gαq/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca2+ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca2+-dependent early phase p38 MAPK phosphorylation and extracellular Ca2+-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca2+ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.

Exogenous Activation of Protease-Activated Receptor 2 Attenuates Cutaneous Vasodilatation and Sweating in Older Men Exercising in the Heat.

BACKGROUND: Protease-activated receptor 2 (PAR2) exists in the cutaneous vasculature and eccrine sweat glands. We previously showed that in young habitually active men, exogenous PAR2 activation via the agonist SLIGKV-NH2 had no effect on heat loss responses of cutaneous vasodilatation and sweating during rest or exercise in the heat. However, ageing is associated with altered mechanisms governing these responses. Thus, the effect of exogenous PAR2 activation on cutaneous vasodilatation and sweating in older individuals may differ from that in young adults. METHODS: Local cutaneous vascular conductance (CVC) and sweat rate were measured in 9 older males (62 ± 4 years) at four forearm skin sites treated with the following: (1) lactated Ringer solution (control), (2) 0.05 mM, (3) 0.5 mM, or (4) 5 mM SLIGKV-NH2. Measurements were performed while participants rested in a non-heat-stress environment (25°C) for ∼60 min and an additional 50 min thereafter in the heat (40°C). Participants then performed 50 min of cycling at a fixed metabolic heat load of 200 W/m2 (to maintain the same thermal drive for heat loss between participants) followed by a 30-min recovery. RESULTS: CVC during non-heat-stress resting was elevated from the control site with 5 mM SLIGKV-NH2 (p ≤ 0.05), but this response was not observed during ambient heat exposure. By contrast, 5 mM SLIGKV-NH2 lowered CVC during the early stage (10 and 20 min) of exercise compared to the control site (all p ≤ 0.05). Although sweating during non-heat-stressed and heat-stressed resting was not affected by any dose of SLIGKV-NH2, it was reduced with all SLIGKV-NH2 doses relative to the control site during and following exercise (all p ≤ 0.05). CONCLUSION: We show that while exogenous PAR2 activation induces cutaneous vasodilatation at rest under non-heat-stressed conditions, it attenuates cutaneous vasodilatation and sweating during and following an exercise-induced heat stress in older men.

Role of protease-activated receptor 2 in regulation of renin synthesis and secretion in mice.

It has been reported that the serine protease kallikrein stimulates and that aprotinin, a protease inhibitor, inhibits renal renin secretion. Since direct stimulation of the protease-activated receptor (PAR) 2 increases renin secretion in isolated perfused mouse kidneys, we hypothesized that activation of PAR2 receptors by serine proteases could be involved in the synthesis and secretion of renin in vivo. We therefore determined the response of plasma renin concentration (PRC) to acute intraperitoneal administration of the PAR2 agonist SLIGRL, isoproterenol, hydralazine, furosemide, losartan, or lipopolysaccharide in conscious wild-type (WT) and Par2-deficient mice. Basal PRC was not different in Par2-deficient mice compared with WT mice. All six acute treatments caused significant increases of PRC in both WT and Par2-deficient mice. The response was significantly lower only in endotoxin-treated Par2-deficient mice. Chronic treatment with losartan, low salt intake, the combination of both, or furosemide caused an increase of PRC and renin mRNA in WT mice, whereas a high salt intake caused a decrease. Alterations in PRC and renal renin mRNA expression were not different between WT and Par2 -/- mice in response to chronic treatments. Par2-deficiency did not alter furosemide-induced diuresis and natriuresis. Systolic blood pressure responses to chronic treatments were not different between WT and Par2 -/- mice. In conclusion, deficiency of Par2 receptors does not alter renin secretion and renin gene expression modulated by a variety of typical maneuvers. However, activation of Par2 receptors by serine proteases seems to be of importance for renin secretion in the context of inflammation.

Dermatophagoides farinae Upregulates the Effector Functions of Eosinophils through αMß2-Integrin and Protease-Activated Receptor-2.

BACKGROUND: Even in subjects who are not sensitized to house dust mite (HDM), allergic symptoms can be aggravated by exposure to dust, suggesting that innate immune responses may be involved in these processes. Since eosinophils express pattern recognition receptors, HDM may directly upregulate eosinophil functions through these re ceptors. The objective of this study was to examine whether Dermatophagoides farinae (Df), a representative HDM, or Der f 1, a major allergen of Df, modifies the effector functions of eosinophils. METHODS: Eosinophils isolated from the blood of healthy donors or allergic patients were stimulated with Df extract or Der f 1, and their adhesion to recombinant human intercellular adhesion molecule (ICAM)-1 was measured using eosinophil peroxidase assays. Generation of the eosinophil superoxide anion (O2-) was examined based on the superoxide dismutase-inhibitable reduction of cytochrome C. Eosinophil-derived neurotoxin (EDN) concentrations in cell media were measured by ELISA as a marker of degranulation. RESULTS: Df extract or Der f 1 directly induced eosinophil adhesion to ICAM-1, O2- generation, and EDN release. Anti-αM- or anti-ß2-integrin antibodies or protease-activated receptor (PAR)-2 antagonists suppressed the eosinophil adhesion, O2- generation, and EDN release induced by Df extract or Der f 1. Eosinophils from allergic patients showed higher adhesion to ICAM-1 than those from healthy donors. CONCLUSIONS: These findings suggested that Df extract and Der f 1 directly activate eosinophil functions through αMß2-integrin and PAR-2. Eosinophil activation by HDM may play roles in the aggravation of allergic symptoms, not only in HDM-sensitized patients, but also in nonsensitized patients.

Association of PAR-2 Gene Polymorphisms with the Inflammatory Response and Susceptibility to Knee Osteoarthritis in the Chinese Han Population.

OBJECTIVE: To investigate the relationship between single nucleotide polymorphisms (SNPs) of protease-activated receptor 2 (PAR-2) and the susceptibility to knee osteoarthritis (KOA) and synovial expression of inflammatory factors in the Chinese Han population. METHODS: Three hundred fifty KOA patients (KOA group) and 345 healthy volunteers (control group) were recruited for the study. Five milliliters of venous blood was taken from each subject to detect the PAR-2 rs1529505, rs631465, and rs2242991 locus genotypes. The expression of PAR-2 mRNA in the synovial tissue and the levels of matrix metalloproteinase (MMP-1), MMP-9, interleukin (IL)-6, and IL-1ß in the joint effusion were detected in 205 KOA patients who had undergone joint replacement surgery. RESULTS: The PAR-2 rs1529505 T allele and the rs2242991 G allele carriers had a higher risk of KOA (p < 0.001). The severity of KOA in patients with the PAR-2 rs1529505 and rs2242991 mutations were higher than in the wild-type controls (p < 0.05). The expression levels of the PAR-2 mRNA in wild types, heterozygotes, and homozygotes of the rs1529505 and rs2242991 loci increased in turn (p < 0.001). The levels of MMP-1, MMP-9, IL-6, and IL-1ß in the synovial fluid of the PAR-2 rs1529505 and rs2242991 locus mutants were distinctly higher than those with the wild-type alleles (p < 0.01). There was no correlation between the rs631465 SNP and susceptibility to KOA, severity of KOA, nor levels of PAR-2 mRNA, MMP-1, MMP-9, IL-6, and IL-1ß. CONCLUSIONS: The PAR-2 SNPs rs1529505 and rs2242991 are associated with the susceptibility to KOA. KOA is more severe in patients harboring the T and G alleles of these two SNPs, respectively. The levels of inflammatory factors in synovial tissue and blood are higher than those in wild-type patients.

Plasma kallikrein modulates immune cell trafficking during neuroinflammation via PAR2 and bradykinin release.

Blood-brain barrier (BBB) disruption and transendothelial trafficking of immune cells into the central nervous system (CNS) are pathophysiological hallmarks of neuroinflammatory disorders like multiple sclerosis (MS). Recent evidence suggests that the kallikrein-kinin and coagulation system might participate in this process. Here, we identify plasma kallikrein (KK) as a specific direct modulator of BBB integrity. Levels of plasma prekallikrein (PK), the precursor of KK, were markedly enhanced in active CNS lesions of MS patients. Deficiency or pharmacologic blockade of PK renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by a remarkable reduction of BBB disruption and CNS inflammation. In vitro analysis revealed that KK modulates endothelial cell function in a protease-activated receptor-2-dependent manner, leading to an up-regulation of the cellular adhesion molecules Intercellular Adhesion Molecule 1 and Vascular Cell Adhesion Molecule 1, thereby amplifying leukocyte trafficking. Our study demonstrates that PK is an important direct regulator of BBB integrity as a result of its protease function. Therefore, KK inhibition can decrease BBB damage and cell invasion during neuroinflammation and may offer a strategy for the treatment of MS.

Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome.

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 ß-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.

Impairment of Protease-Activated Receptor 2-Induced Relaxation of Aortas of Aged Spontaneously Hypertensive Rat.

Hypertension is one of the most prevalent diseases worldwide and can cause harmful complications within the vascular system. Further research on vascular responsiveness to different ligands and diverse receptors in various arteries is required to understand the mechanisms underlying the development of these vascular complications. Here, we investigated the vasorelaxant effect of the protease-activated receptor 2 (PAR2) agonist 2-furoyl-LIGRLO-amide (2-Fly) and two commonest agents, namely endothelium-dependent dilator acetylcholine (ACh) and endothelium-independent dilator sodium nitroprusside (SNP), on the thoracic aorta isolated from aged spontaneously hypertensive rats (SHR) (age, 52±1 weeks). The effects of these agents were compared between aortas isolated from SHR and age-matched normotensive Wistar Kyoto (WKY) rats. Compared with the WKY group, in the SHR group, 2-Fly-induced relaxation was impaired, ACh-induced relaxation was slightly decreased at low concentrations, and SNP-induced relaxation was similar. In addition, 2-Fly-induced aortic relaxation was largely decreased by a PAR2 antagonist (FSLLRY), endothelial denudation, and a nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NNA) but not by an Akt inhibitor. These results suggested that PAR2-induced relaxations of aortas of aged SHR was impaired, and this impaired aortic relaxation may be attributed to decreased NO bioavailability rather than altered NO sensitivity unrelated to the Akt activity.

Prostanoid-dependent bladder pain caused by proteinase-activated receptor-2 activation in mice: Involvement of TRPV1 and T-type Ca2+ channels.

We studied the pronociceptive role of proteinase-activated receptor-2 (PAR2) in mouse bladder. In female mice, intravesical infusion of the PAR2-activating peptide, SLIGRL-amide (SL), caused delayed mechanical hypersensitivity in the lower abdomen, namely 'referred hyperalgesia', 6-24 h after the administration. The PAR2-triggered referred hyperalgesia was prevented by indomethacin or a selective TRPV1 blocker, and restored by a T-type Ca2+ channel blocker. In human urothelial T24 cells, SL caused delayed prostaglandin E2 production and COX-2 upregulation. Our data suggest that luminal PAR2 stimulation in the bladder causes prostanoid-dependent referred hyperalgesia in mice, which involves the activation of TRPV1 and T-type Ca2+ channels.

Protease activated-receptor 2 is necessary for neutrophil chemorepulsion induced by trypsin, tryptase, or dipeptidyl peptidase IV.

Compared to neutrophil chemoattractants, relatively little is known about the mechanism neutrophils use to respond to chemorepellents. We previously found that the soluble extracellular protein dipeptidyl peptidase IV (DPPIV) is a neutrophil chemorepellent. In this report, we show that an inhibitor of the protease activated receptor 2 (PAR2) blocks DPPIV-induced human neutrophil chemorepulsion, and that PAR2 agonists such as trypsin, tryptase, 2f-LIGRL, SLIGKV, and AC55541 induce human neutrophil chemorepulsion. Several PAR2 agonists in turn block the ability of the chemoattractant fMLP to attract neutrophils. Compared to neutrophils from male and female C57BL/6 mice, neutrophils from male and female mice lacking PAR2 are insensitive to the chemorepulsive effects of DPPIV or PAR2 agonists. Acute respiratory distress syndrome (ARDS) involves an insult-mediated influx of neutrophils into the lungs. In a mouse model of ARDS, aspiration of PAR2 agonists starting 24 h after an insult reduce neutrophil numbers in the bronchoalveolar lavage (BAL) fluid, as well as the post-BAL lung tissue. Together, these results indicate that the PAR2 receptor mediates DPPIV-induced chemorepulsion, and that PAR2 agonists might be useful to induce neutrophil chemorepulsion.

Melanin Transferred to Keratinocytes Resides in Nondegradative Endocytic Compartments.

Melanin transfer from melanocytes to keratinocytes and subsequent accumulation in the supranuclear region is a critical process in skin pigmentation and protection against UVR. We have previously proposed that the main mode of transfer between melanocytes and keratinocytes is through exo/endocytosis of the melanosome core, termed melanocore. In this study, we developed an in vitro uptake assay using melanocores secreted by melanocytes. We show that the uptake of melanocores, but not melanosomes, by keratinocytes is protease-activated receptor-2-dependent. Furthermore, we found that the silencing of the early endocytic regulator Rab5b, but not the late endocytic regulators Rab7a or Rab9a, significantly impairs melanocore uptake by keratinocytes. After uptake, we observed that melanin accumulates in compartments that are positive for both early and late endocytic markers. We found that melanin does not localize to either highly degradative or acidic organelles, as assessed by LysoTracker and DQ-BSA staining, despite the abundance of these types of organelles within keratinocytes. Therefore, we propose that melanocore uptake leads to storage of melanin within keratinocytes in hybrid endocytic compartments that are not highly acidic or degradative. By avoiding lysosomal degradation, these specialized endosomes may allow melanin to persist within keratinocytes for long periods.

Protease-activated receptor-2 suppresses interleukin (IL)-10 expression in B cells via upregulating Bcl2L12 in patients with allergic rhinitis.

BACKGROUND AND AIMS: The function of interleukin (IL)-10-producing B cells (B10 cell) is compromised in patients with allergic diseases. Protease-activated receptor (PAR)-2 has immunoregulatory functions. This study aimed to elucidate the role of PAR2 in the suppression of IL-10 expression in peripheral B cells. METHODS: Peripheral blood B cells were collected from patients with allergic rhinitis (AR). A correlation between the expression of Bcl2-like protein 12 (Bcl2L12) and IL-10 in the B cells was analyzed. An AR mouse model was developed. RESULTS: We observed that the expression of IL-10 was lower in the peripheral B cells from patients with airway allergy. A negative correlation was identified between the expression of IL-10 and PAR2 in B cells. Activation of PAR2 of B cells increased the expression of Bcl2L12 and suppression of LPS-induced IL-10 expression, which were inhibited by knocking down the Bcl2L12 gene. Treating B cells from AR patients with Bcl2L12-shRNA-carrying liposomes reversed the capability of IL-10 expression and the immunosuppressive function. Administration of Bcl2L12 shRNA-carrying liposomes attenuated experimental AR in mice. CONCLUSIONS: Activation of PAR2 inhibits the expression of IL-10 in B cells, which can be reversed by treating B cells with Bcl2L12 shRNA-carrying liposomes. The data suggest that regulation of Bcl2L12 may be a novel approach in the treatment for AR.

Biased Signaling by Agonists of Protease Activated Receptor 2.

Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer, and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH2) triggered PAR2-mediated calcium release (EC50 2 µM) but not ERK1/2 phosphorylation (EC50 > 100 µM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist (EC50 40 nM, Ca2+; EC50 2 µM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH2) was an ERK-biased agonist (EC50 2 nM, ERK1/2; EC50 80 nM, Ca2+). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing, and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.

[Research progress on protease-activated receptor 2 in pathogenesis of osteoarthritis].

Objective: To review the research progress on protease-activated receptor 2 (PAR-2) in the pathogenesis of osteoarthritis (OA). Methods: The relevant literature about the mechanism of PAR-2 in the occurrence and development of OA in recent years was extensively reviewed and comprehensively analyzed. Results: Abnormal activation of PAR-2 plays an important role in responses to occurrence and development of OA. Through regulating production and releasing of a variety of cytokines (such as inflammatory factors, metabolic factors, pain factors, etc.), the PAR-2 can involve in pathophysiological progression of OA articular cartilage, subchondral bone, and synovial membrane, as well as occurrence and transmission of pain. Conclusion: PAR-2 participation in the development of OA has been confirmed. However, since PAR-2 is complicated and widespread, it is necessary to study the specific role of PAR-2 and the interaction between various signal pathways in the progression of OA, and to elucidate the potential pathophysiological mechanisms of PAR-2 participating in the process of OA, in the hope of exploring the new targets for the effective control of OA.

Proteinase activated receptor-2 counterbalances the vascular effects of endothelin-1 in fibrotic tight-skin mice.

BACKGROUND AND PURPOSE: The majority of the severe vascular complications in fibrosis are a consequence of a deregulated activity of mediators controlling vasomotor tone. One of the most important of these mediators is endothelin-1 (ET-1). Here, we have investigated the role of proteinase-activated receptor 2 (PAR2) in the vascular dysfunction in a model of fibrosis, using tight-skin (Tsk) mice. EXPERIMENTAL APPROACH: Aortas were collected from Tsk, transgenic over-expressing PAR2 (TgPAR2), PAR2 deficient (PAR2-/- ) or the corresponding WT mice. Histological and immunohistochemistry analysis for α-smooth muscle actin, PAR2 and ET-1 receptors were performed on aorta sections. Vascular responses to phenylephrine, ET-1 and PAR2 activating peptide (PAR2-AP) were assessed on aortic rings. KEY RESULTS: In aortas from Tsk mice, responses to phenylephrine were reduced, contractions to ET-1 were increased and vasorelaxation to PAR2-AP was enhanced. These alterations matched changes observed in whole vessel architecture such as vascular fibre re-organization, increased collagen deposition and enhanced α-smooth muscle actin expression. Expression of both ETA receptors and PAR2 was enhanced in Tsk mice. Antagonism of PAR2 potentiated vascular effects of ET-1, whereas antagonism of ETA receptors increased vasorelaxation induced by PAR2-AP. In TgPAR2 mice, responses to ET-1 and ET-1 plasma levels were reduced. Conversely, PAR2-/- mice showed enhanced ET-1 induced contraction in aortic rings and higher circulating ET-1 levels. CONCLUSIONS AND IMPLICATIONS: Our data show that PAR2 counterbalanced enhanced contractions to ET-1 in aortas from Tsk mice. PAR2 could represent a possible target for novel drugs in the treatment of vascular complications in fibrosis. LINKED ARTICLES: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc.