Expression Of Proteinase-activated Receptors Research Articles

Involvement of PAR2 in inflammatory mediator release from human blood eosinophils

Proteinase Activated Receptors (PARs) are the members of G-protein-coupled receptor family and can be cleaved by certain serine proteases to expose a tethered ligand domain, which binds and activates the receptors to initiate multiple signaling cascades. There is some evidence that certain proteases may regulate target cells by activating PARs. There are many studies, in which PARs play important roles in inflammation. One study indicated that PAR2 inhibition and deletion significantly suppressed the degree of inflammation due to decreased IL-6 and IL-1 levels. Another study also showed that PARs activation could mediate reactive oxygen species production and MAPK signaling leading to alveolar inflammation. In addition, platelet-derived CAPN1 can trigger the vascular inflammation associated with diabetes via cleavage of PAR1 and the release of TNF from the endothelial cell surface, and sarsasapogenin may alleviate diabetic nephropathy by the downregulation of PAR1. Another Phellodendron amurense bark extract can suppress the particulate matter-induced Ca2+ influx caused by direct action upon PAR2, alleviating inflammation and maintaining homeostatic levels of cell adhesion components. There are also other two antagonists of I-287 and GB88, which can reduce the PAR2-mediated inflammatory reaction. In this study, we tested expression of PARs and IL-5, IL-6, RANTES and ECP release from human blood eosinophils using different enzymes and PAR agonists. The expression of PARs was assessed in human blood eosinophils by flow cytometry and RT-PCR, and the levels of cytokine and eosinophil cationic protein (ECP) in the cultured supernatants were determined with ELISA kits. Flow cytometry shows that human eosinophils express PAR2 protein and do not express PAR1, PAR3 and PAR4 proteins. RT-PCR analysis revealed expression of PAR2 and PAR3 genes in human eosinophils. Tryptase, trypsin and elastase can induce significant IL-5, IL-6 and ECP release. Trypsin and elastase may also stimulate RANTES secretion, but tryptase cannot induce the RANTES secretion. Tryptase, trypsin and elastase-induced cytokine and ECP release from human blood eosinophils most likely occurs via activation of PAR2.

Expression of Proteinase-activated Receptor 2 (PAR2) as a Correlate of Concern in Triple-negative Breast Cancer (TNBC).

Triple-negative breast cancer (TNBC), a highly aggressive cancer with poor outcome and lacking specific diagnostic, prognostic, or targeted therapeutic strategies, constitutes roughly 20% of all breast cancer cases. TNBC cells lack receptors for estrogen, progesterone, and human epidermal growth factor. The effort continues to find a suitable correlate that could serve as a TNBC biomarker, or as therapeutic target, or both. A retrospective study was performed with 88 TNBC and 74 non-TNBC patients who had undergone mastectomy/lumpectomy with axillary clearance for carcinoma breast. Immunohistochemical staining was carried out for levels of proteinase-activated receptor 2 (PAR2), encoded by F2RL1 gene, and staining scores were calculated, based on intensity and percentage positivity. PAR2 levels were markedly upregulated in TNBC patients, compared with patients with other breast cancer subtypes. Amongst different non-TNBC subtypes, higher expression was noted in luminal B (88.8%) and HER2+ (100%), compared with luminal A (52.5%). PAR2 levels were significantly high in TNBC patients with age more than 40 years than corresponding patients of non-TNBC group (P=0.0017). Furthermore, there was a statistically significant increase in levels of PAR2 expression in lymph node negative (P=0.0096) and early stage (P=0.005) of TNBC versus non-TNBC patients. PAR2 staining of ductal carcinoma in situ and invasive ductal carcinoma revealed lower expression in invasive component. Our data suggest that PAR2 levels constitute a correlate of concern for TNBC, tying in with a recent report that higher levels of F2RL1 gene expression correlate with poorer disease-free, as well as overall survival in TNBCs.

Expression of Proteinase-Activated Receptor 2 During Colon Volvulus in the Horse.

Large colon volvulus in horses is associated with a poor prognosis, especially when ischemic-reperfusion injury of the affected intestinal tract develops. Proteinase-activated receptor 2 (PAR2) plays an important role in the pathogenesis of inflammation in the gastrointestinal tract. The aim of this study was to evaluate the distribution and expression of PAR2 in colonic pelvic flexure of horses spontaneously affected by large colon volvulus (CVH group). Eight horses admitted for severe abdominal colon volvolus and which underwent surgery were included. Colon samples were collected after enterotomy. Data previously obtained from healthy horses were used as a control group. Histologic evaluation was carried out to grade the severity of the colon lesions. Immunofluorescence, western blot and quantitative polymerase chain reaction (RT-qPCR) were carried out on colon samples to evaluate PAR2 expression. In addition, the transcriptional profile of cytokines and chemokines was evaluated using RT2 Profiler™ PCR Array Horse Cytokines & Chemokines. Three out of the eight patients were euthanised due to clinical deterioration. Immunostaining for PAR2 was observed in the enterocytes, intestinal glands and neurons of the submucosal and myenteric plexi. In the CVH horses, the expression of PAR2 mesenger RNA (mRNA) did not differ significantly from that of the healthy animals; western blots of the mucosa of the colon tracts showed a clear band of the expected molecular weight for PAR2 (~44 kDa) and a band smaller than the expected molecular weight for PAR2 (25kDa), suggesting its activation. The gene expressions for C-X-C motif ligand 1 (CXCL1); interleukin 8 (IL8), macrophage inflammatory protein 2 beta (MIP-2BETA) were upregulated in the colic horses as compared with the colons of the healthy horses. Therefore, in the present study, the expression and activation of PAR2 in the colons of horses in the presence of an inflammatory reaction like that occurring in those with spontaneous colon volvulus was confirmed.

The Role of PAR2 in TGF-β1-Induced ERK Activation and Cell Motility.

Background: Recently, the expression of proteinase-activated receptor 2 (PAR2) has been shown to be essential for activin receptor-like kinase 5 (ALK5)/SMAD-mediated signaling and cell migration by transforming growth factor (TGF)-β1. However, it is not known whether activation of non-SMAD TGF-β signaling (e.g., RAS–RAF–MEK–extracellular signal-regulated kinase (ERK) signaling) is required for cell migration and whether it is also dependent on PAR2. Methods: RNA interference was used to deplete cells of PAR2, followed by xCELLigence technology to measure cell migration, phospho-immunoblotting to assess ERK1/2 activation, and co-immunoprecipitation to detect a PAR2–ALK5 physical interaction. Results: Inhibition of ERK signaling with the MEK inhibitor U0126 blunted the ability of TGF-β1 to induce migration in pancreatic cancer Panc1 cells. ERK activation in response to PAR2 agonistic peptide (PAR2–AP) was strong and rapid, while it was moderate and delayed in response to TGF-β1. Basal and TGF-β1-dependent ERK, but not SMAD activation, was blocked by U0126 in Panc1 and other cell types indicating that ERK activation is downstream or independent of SMAD signaling. Moreover, cellular depletion of PAR2 in HaCaT cells strongly inhibited TGF-β1-induced ERK activation, while the biased PAR2 agonist GB88 at 10 and 100 µM potentiated TGF-β1-dependent ERK activation and cell migration. Finally, we provide evidence for a physical interaction between PAR2 and ALK5. Our data show that both PAR2–AP- and TGF-β1-induced cell migration depend on ERK activation, that PAR2 expression is crucial for TGF-β1-induced ERK activation, and that the functional cooperation of PAR2 and TGF-β1 involves a physical interaction between PAR2 and ALK5.

Proteinase-activated receptor 2 distribution and expression in equine small intestine tracts following herniation through the epiploic foramen

Proteinase-activated receptor 2 (PAR2) is a G-protein-coupled receptor for trypsin and mast cell tryptase; it is highly expressed at the intestinal level with multiple functions, such as epithelial permeability and intestinal motility. The aim of the study was to evaluate the distribution and expression of proteinase-activated receptor 2 in the small intestine during herniation through epiploic foramen. In this prospective clinical study, eight horses admitted for colic and which underwent exploratory laparotomy were considered. During surgery, the jejunum or the ileum was sampled by enterectomy. Morphological examination (histology, PAR2 immunohistochemistry) and molecular biology analysis (western blot and quantitative polymerase chain reaction) were carried out on the resected intestinal samples. The Marginal Injured Tracts (MITs) and Central Injury Tracts (CITs) were defined as the oral and caudal marginal segments of the resected bowel tract and as the geometric centre of the intestinal ischaemic lesion length, respectively.The PAR2 immunoreactivity was particularly evident in the epithelial cells, with higher immunoreactivity in the MIT rather than in the CIT. Moreover, a different immune localisation was observed in the MITs at the cell membrane level and in the CITs in the cytoplasm. No statistical difference was observed in PAR2 mRNA and protein (44kDa) expression between the MIT and the CIT. The PAR2 protein content in the intestinal tracts which were removed from horses with herniation was lower when compared with the control animals.This study provided data concerning the PAR2 presence and distribution in horses with intestinal herniation through the epiploic foramen.

Expression of proteinase-activated receptor (PAR)-2 in monocytes from allergic patients and potential molecular mechanism.

Serine proteases play an important role in inflammation via PARs. However, little is known of expression levels of PARs on monocytes of allergic patients, and influence of serine proteases and PARs on TNF-α secretion from monocytes. Using quantitative real-time PCR (qPCR) and flowcytometry techniques, we observed that the expression level of PAR-2 in monocytes of patients with allergic rhinitis and asthma was increased by 42.9 and 38.2%. It was found that trypsin, thrombin, and tryptase induced up to 200, 320, and 310% increase in TNF-α release from monocytes at 16h, respectively. PAR-1 agonist peptide, SFLLR-NH2, and PAR-2 agonist peptide tc-LIGRLO-NH2 provoked up to 210 and 240% increase in release of TNF-α. Since SCH 79797, a PAR-1 antagonist, and PD98059, an inhibitor of ERK inhibited thrombin- and SFLLR-NH2-induced TNF-α release, the action of thrombin is most likely through a PAR-1- and ERK-mediated signaling mechanism. Similarly, because FSLLRN-NH2, an inhibitor of PAR-2 diminished tryptase- and tc-LIGRLO-NH2-induced TNF-α release, the action of tryptase appears PAR-2 dependent. Moreover, in vivo study showed that both recombinant cockroachmajor allergens Per a 1 and Per a 7 provoked upregulation of PAR-2 and PAR-1 expression on CD14+ cells in OVA-sensitized mouse peritoneum. In conclusion, increased expression of PAR-2 in monocytes of AR and asthma implicates that PAR-2 likely play a role in allergy. PAR-2- and PAR-1-mediated TNF-α release from monocytes suggests that these unique protease receptors are involved in the pathogenesis of inflammation.

Proteinase activated-receptors-associated signaling in the control of gastric cancer.

Gastric cancer (GC) is the fourth most common cancer in the world and the second cause of cancer-related death. Gastric carcinogenesis is a multifactorial process, in which environmental and genetic factors interact to activate multiple intracellular signals thus leading to uncontrolled growth and survival of GC cells. One such a pathway is regulated by proteinase activated-receptors (PARs), seven transmembrane-spanning domain G protein-coupled receptors, which comprise four receptors (i.e., PAR-1, PAR-2, PAR-3, and PAR-4) activated by various proteases. Both PAR-1 and PAR-2 are over-expressed on GC cells and their activation triggers and/or amplifies intracellular pathways, which sustain gastric carcinogenesis. There is also evidence that expression of either PAR-1 or PAR-2 correlates with depth of wall invasion and metastatic dissemination and inversely with the overall survival of patients. Consistently, data emerging from experimental models of GC suggest that both these receptors can be important targets for therapeutic interventions in GC patients. In contrast, PAR-4 levels are down-regulated in GC and correlate inversely with the aggressiveness of GC, thus suggesting a negative role of this receptor in the control of GC. In this article we review the available data on the expression and role of PARs in GC and discuss whether manipulation of PAR-driven signals may be useful for interfering with GC cell behavior.

Cross‐talk between toll‐like receptor 4 (TLR4) and proteinase‐activated receptor 2 (PAR2) is involved in vascular function

Proteinase-activated receptors (PARs) and toll-like receptors (TLRs) are involved in innate immune responses. The aim of this study was to evaluate the possible cross-talk between PAR(2) and TLR4 in vessels in physiological condition and how it varies following stimulation of TLR4 by using in vivo and ex vivo models. Thoracic aortas were harvested from both naïve and endotoxaemic rats for in vitro studies. Arterial blood pressure was monitored in anaesthetized rats in vivo. LPS was used as a TLR4 agonist while PAR(2) activating peptide (AP) was used as a PAR(2) agonist. Aortas harvested from TLR4(-/-) mice were also used to characterize the PAR(2) response. PAR(2) , but not TLR4, expression was enhanced in aortas of endotoxaemic rats. PAR(2) AP-induced vasorelaxation was increased in aortic rings of LPS-treated rats. TLR4 inhibitors, curcumine and resveratrol, reduced PAR(2) AP-induced vasorelaxation and PAR(2) AP-induced hypotension in both naïve and endotoxaemic rats. Finally, in aortic rings from TLR4(-/-) mice, the expression of PAR(2) was reduced and the PAR(2) AP-induced vasodilatation impaired compared with those from wild-type mice and both resveratrol and curcumine were ineffective. Cross-talk between PAR(2) and TLR4 contributes to vascular homeostasis.

Effects of tacrolimus on the expression of proteinase activated receptor 2 in lesions of atopic dermatitis

Objective To detect the expression of proteinase activated receptor 2 (PAR-2) in the skin of patients with atopic dermatitis (AD) and to evaluate the effects of tacrolimus on the expression.Methods Six patients with acute moderate or severe AD were enrolled in this study and topically treated with tacrolimus 0.1％ ointment twice daily for 3 weeks.Tissue samples were obtained from the lesions and non-lesional skin at least 10 cm away from the lesions before and after the 3-week treatment.Skin specimens from 6 normal human controls served as the control.Patients were evaluated at the baseline,1 and 3 weeks after the beginning of treatment for clinical symptoms and signs by visual analogue scale (VAS),eczema area and severity index (EASI) and investigator's global assessment (IGA).The expression of PAR-2 in tissue specimens were determined by immunohistochemistry.Results PAR-2 was expressed throughout the whole epidermis,especially in the granular layer,hair follicles,sweat glands,endothelial cells and nerve fiber-like structures.Before treatment,the expression level (mean optical density) of PAR-2 in keratinocytes was 4339.6 ± 115.8 in lesional skin of AD patients,significantly higher than that in non-lesional skin (4189.0 t 228.9,t =2.85,P ＜0.05) and in normal skin (3864.0 ± 237.3,t =4.31,P ＜ 0.05).After the 3-week treatment with tacrolimus ointment,the expression level of PAR-2 significant decreased to 3942.4 ± 176.6 in keratinocytes from lesional skin of patients with AD (t =4.55,P ＜ 0.05).The expression level of PAR-2 was positively correlated with VAS score for itch,EASI and IGA score in the patients.Conclusions The expression of PAR-2 is enhanced in keratinocytes of lesions from AD patients,and is positively correlated with itch and lesion severity.Topical tacrolimus may suppress the overexpression of PAR-2 in keratinocytes in lesional skin of AD. Key words: Dermatitis, atopic; Receptor, PAR-2; Tacrolimus; Keratinocyte

Expression of proteinase-activated receptor 1-4 (PAR 1-4) in human cancer

Proteinase activated receptors (PAR 1-4) are membrane receptors with a unique way of activation by proteinases like thrombin, trypsin and matrix metalloproteinases which lead to a specific cellular response. To evaluate the significance of expression and co-expression of PAR in cancer we performed a survey on published data. A Pubmed literature search on "PAR, thrombin, cancer" was performed and 46 publications were selected for systematic review based on the availability of information on tumor type, material type, detection method and specification of positive cases. PAR-1 was found in 77.3% of malignant samples (n = 678), PAR-2 in 79.5% (n = 592), PAR-3 in 12.6% (n = 87) and PAR-4 in 54.9% (n = 153). PAR-1 and -2 were present in adenocarcinomas, melanomas, osteosarcomas, glioblastomas, meningiomas, leukaemias and squamous cell carcinomas. Presence of PAR-3 was limited to kidney and liver cancer. The data on PAR-4 expression was inconclusive. Those studies analysing PAR-1 and PAR-2 reported coexpression of the two receptors. PAR-1 and -2 are widely expressed in human tumors suggesting an important role in tumorigenesis and providing potential targets for therapy. PAR-3 and PAR-4 are less frequently detectable, their expression and potential role in tumorigenesis require further investigation.

Effects of pinaverium bromide on the development of visceral hypersensitivity and expression of proteinase activated-receptor 2

Objective To investigate the effect of pinaverium bromide on the development of visceral hypersensitivity and the expression of proteinase-activated-receptor 2(PAR2)in rats with water avoidance stress (WAS).Methods The Wistar rats were randomly divided into 3 groups with 12 each.The control group was submitted to sham-WAS and the group A1 was submitted to WAS.The rats in group A2 were submitted to WAS and received pinaverium bromide treatment (30 mg·kg~(-1)·d~(-1)).The stool pellets were counted during WAS and sham-WAS.The magnitude of visceromotor response (VMR) during colorectal distention was used to measure the changes of visceral hypersensitivity.The PAR2 expression was detected by immunofluorescence staining and Western blotting.Results Increased stool pellets were found in group A1 compared to the control group (8.10±2.75 vs.4.13±1.36,P<0.001).Pinaverium bromide treatment decreased stool pellets during WAS (5.87±1.78,P<0.001).The magnitude of VMR (60 mmHg) was higher in group Al compared to the control group (8.64±2.88 vs.3.80±1.04,P<0.001).Pinaverium bromidetreatment could decrease the magnitude of VMR effectively after WAS (6.09±0.77,P=0.02).The expression of PAR2 in group Al was significantly higher than that in the control group (rAU:0.70±0.06 vs.0.32±0.04;M:279.08±100.91 vs.77.16±10.74,P<0.001).The increased PAR2 expression induced by WAS could be inhibited by pinaverium bromide (rAU:0.53±0.06,IA:180.59±25.75,P<0.05).Conclusions Pinaverium bromide may decrease the expression of PAR2 and inhibit the development of visceral hypersensitivity in WAS rats. Key words: Visceral hypersensitivity; Pinaverium bromide; Proteinase activated-receptor 2

Expression and functionality of proteinase-activated receptors (PARs) on primary human lung mast cells

Introduction: Mast cell (MC) activation is known to promote anaphylactic reactions as well as the development of allergic and non-allergic inflammatory diseases. Apart from reacting to cross-linking of IgE-receptors by allergen, MC can be activated through a variety of alternative mechanisms. There is initial evidence that also proteinase-activated receptors (PARs) could have a role. PARs belong to the G protein-coupled receptor family and comprise four members (PAR-1 to 4). While the expression of functional PAR-2 on human skin MC as well as mRNA expression for PAR-1, PAR-3 and PAR-4 has been reported recently, so far nothing is known about the expression and the potential function(s) of PARs on primary human lung MC. We, therefore, isolated primary MC from human lung tissue and examined the expression and functionality of PARs. Methods: Human primary lung MC were isolated by a three-step procedure including enzymatic lung tissue digestion, density gradient centrifugation, and immunoaffinity purification by using anti-CD117 and anti-CD203c antibodies. Results: Using &gt;95% pure MC we could show mRNA expression for PAR-1, PAR-2, PAR-3 and PAR-4. PAR-specific antibodies revealed the expression of PAR-1 on the cell membrane and of PAR-2 being located intracellularly, whereas PAR-3 and PAR-4 were not detected by the antibodies used. PAR-activating peptides (PAR-APs) selective for PAR-1, PAR-2 and PAR-4 were able to induce elevation of intracellular free Ca2+-ion concentration ([Ca2+]i) in these cells. Moreover, PAR-1-AP and PAR-4-AP, but not PAR-2-AP stimulated MC degranulation as determined by the release of β-hexosaminidase. Stimulation with PAR-1-selective ligands led to downregulation of PAR-1. Looking for the effect of natural PAR-ligands, we tested proteinases from blood (thrombin, factor Xa, plasmin), from leukocytes (elastase, cathepsin G, proteinase 3), trypsin, and also MC-specific proteinases (chymase, tryptase). Out of these, only thrombin (PAR-1, and -4-ligand) and trypsin (PAR-2-ligand) induced Ca2+-signalling whereas none induced degranulation. Discussion: Our results demonstrate the functional expression of PAR-1, PAR-2 and PAR-4 in primary human lung MC. However, the induction of Ca2+-signalling in the absence of a degranulation response to thrombin and trypsin suggests, that natural ligands for these receptors may act differently from synthetic APs and moreover are likely to stimulate alternative functions in human primary lung MC.

Met receptor tyrosine kinase transactivation is involved in proteinase-activated receptor-2-mediated hepatocellular carcinoma cell invasion

The expression of proteinase-activated receptor (PAR)(2) in human hepatocellular carcinoma (HCC) was established by reverse transcription-polymerase chain reaction, confocal immunofluorescence and electron microscopy in permanent cell lines, primary HCC cell cultures and HCC tumor tissue. Stimulation of HCC cells with trypsin and the PAR(2)-selective activating peptide, 2-furoyl-LIGRLO-NH(2), increased cell invasion across Matrigel. Both effects were blocked by a PAR(2)-selective pepducin antagonist peptide (pal-PAR(2)) and by PAR(2) silencing with specific small interfering RNA (siRNA). PAR(2)-initiated HCC cell invasion was also blocked by inhibiting the hepatocyte growth factor receptor (Met receptor tyrosine kinase) with the receptor-targeted kinase inhibitors, SU 11274 and PHA 665752, or by downregulation of Met with specific siRNA. The involvement of Met in PAR(2)-mediated HCC invasive signaling was further supported by the finding that treatment of HCC cells with trypsin or the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated Met activation-phosphorylation. In addition, Met-dependent stimulation of p42/p44 mitogen-activated protein Kinases was found to be critical for the PAR(2)-Met receptor tyrosine kinase-invasive signaling axis in HCC cells. Our study establishes an important link between the PAR(2) and Met receptor tyrosine kinase signaling in promoting HCC cell invasion.

Relationship between expression of PARs and infection with H. pylori in the stomach of gastric cancer patients

Helicobacter pylori (H. pylori) is involved in the pathogenesis of gastric ulcer and cancer, and might secret proteinases. Given emerging roles of proteinase‐activated receptors (PARs) in the gastric mucosa, we investigated the correlation between expression of PARs and H. pylori infection in the stomach isolated from gastric cancer patients. The extent of H. pylori infection was evaluated as regional protein levels of ureases A and B, and analyzed in relation to expression levels of PAR1 and PAR2 in the corresponding regions. In the cancer area, expression of PAR1, but not PAR2, was positively correlated with protein levels of either urease, whereas no such a correlation was observed in the non‐cancer area. There was no significant difference in levels of any proteins between the non‐cancer and cancer areas. Together, expression of PAR1, but not PAR2, appears to be associated with H. pylori infection in gastric cancer tissues. It is likely that the increased expression of PAR1 might be involved in the development of H. pylori‐related gastric cancer.

Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs)

The prostate is a site of high expression of serine proteinases including members of the kallikrein-related peptidase (KLK) family, as well as other secreted and membrane-anchored serine proteinases. It has been known for some time that members of this enzyme family elicit cellular responses by acting directly on cells. More recently, it has been recognised that for serine proteinases with specificity for cleavage after arginine and lysine residues (trypsin-like or tryptic enzymes) these cellular responses are often mediated by cleavage of members of the proteinase-activated receptor (PAR) family--a four member sub-family of G protein-coupled receptors. Here, we review the expression of PARs in prostate, the ability of prostatic trypsin-like KLKs and other prostate-expressed tryptic enzymes to cleave PARs, as well as the prostate cancer-associated consequences of PAR activation. In addition, we explore the dysregulation of trypsin-like serine proteinase activity through the loss of normal inhibitory mechanisms and potential interactions between these dysregulated enzymes leading to aberrant PAR activation, intracellular signalling and cancer-promoting cellular changes.

Thrombin-Stimulated Proliferation of Cultured Human Synovial Fibroblasts Through Proteolytic Activation of Proteinase-Activated Receptor-1

We examined the mechanism of thrombin on proliferation of synovial fibroblasts obtained from rheumatoid arthritis (RA). Thrombin concentration-dependently induced proliferation of synovial fibroblasts. Proliferation in response to thrombin (10 U /ml) was completely blocked by hirudin. TP367 and TP508, peptides corresponding to 2 noncatalytic regions of thrombin, failed to induce cell proliferation. Thrombin did not induce the production of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) in synovial fibroblasts. Expression of proteinase-activated receptor (PAR)-1 and PAR-3 mRNAs was observed in synovial fibroblasts. Thrombin and PAR-1 agonist peptide (AP), but not PAR-3 AP, induced intracellular calcium mobilization. PAR-1 AP induced cell proliferation whereas PAR-3 AP and PAR-4 AP had no effect on proliferation. Pertussis toxin (PTX), a Giα protein inhibitor; wortmannin, a PI (phosphatidylinositol) 3-kinase inhibitor; and PD98059, a specific MEK [mitogen-activated protein (MAK) kinase kinase] inhibitor, inhibited the thrombin-induced cell proliferation. Furthermore, the proliferation of synovial fibroblasts was suppressed by U-73122, a PLC (phospholipase C) inhibitor; 2-APB, an antagonist of InsP3 (inositol 1,4,5-triphosphate) receptor; and GF-109203X, a PKC (protein kinase C) inhibitor. These results suggest that thrombin induces the proliferation of RA synovial fibroblasts through the activation of PAR-1, leading to the PTX-sensitive G proteins – PI3 kinase pathway and PTX-insensitive G proteins – PLC (InsP3 receptor) Ca2+-PKC branch.

Agonists of proteinase‐activated receptor‐2 affect transendothelial migration and apoptosis of human neutrophils

Skin is the first barrier preventing microorganism invasion in host. Wounds destroy this defense barrier and, without an appropriate care, may lead to sepsis. Neutrophil activation and immigration plays an important role at the inflammatory stage of wound healing. Neutrophils are known to express proteinase-activated receptors (PARs), which can be activated by serine proteases, also by enzymes involved in wound healing. We previously reported that PAR(2) agonists up-regulate cell adhesion molecule expression and cytokine production by human neutrophils. Here, we demonstrate that PAR(2) agonists (serine proteases as well as synthetic peptides) reduce transendothelial migration of neutrophils and prolong their life in vitro. Synthetic PAR(2) agonist also enhanced protective interferon (IFN)gamma-induced FcgammaRI expression at neutrophil cell surface. Of note, IFNgamma is a cytokine, which was used in clinical trials to reactivate human neutrophil functions during sepsis. Moreover, we observed a significant increase of PAR(2) expression on cell surface of neutrophils from septic patients as compared with healthy volunteers. Together, our results indicate that PAR(2) may be involved in the pathophysiology of neutrophil-endothelial interactions during wound healing or later during sepsis in humans, potentially by affecting neutrophil apoptosis, transendothelial migration and Fcgamma receptor-mediated phagocytosis.

Proteinase-activated receptors in the lower urinary tract

Proteinase-activated receptors (PARs) are G-protein-coupled receptors that convert specific extracellular proteolytic activity into intracellular signals, and have been suggested to play diverse roles in the body. In this review, evidence for the roles of PARs in bladder contractility and inflammation are presented. The role of PARs in prostate cancer is also reviewed. The existing literature in this area can be difficult to interpret due to the many nonspecific actions of the pharmacological tools employed. Although there are reports that PAR activators can cause contraction of bladder smooth muscle, further pharmacological and molecular studies are required to define roles for these receptors in bladder contractility. While structural studies suggest that roles for PARs in bladder inflammation are likely, few functional investigations have been performed. The significance of the expression of PARs on sensory nerves innervating the bladder and changes in receptor expression in inflammatory disease models are fascinating areas for future research. Finally, it seems probable that PARs, particularly PAR1, may play important roles in the growth and metastasis of prostate cancers.

The Roles of Proteinase-Activated Receptors in the Vascular Physiology and Pathophysiology

Proteinase-activated receptors (PARs) belong to a family of G protein-coupled receptors, thus mediating the cellular effects of proteinases. In the vascular system, thrombin and other proteinases in the coagulation-fibrinolysis system are considered to be the physiologically relevant agonists, whereas PARs are among the most important mechanisms mediating the interaction between the coagulation-fibrinolysis system and the vascular wall. Under physiological conditions, PARs are mainly expressed in endothelial cells, and participate in the regulation of vascular tone, mostly by inducing endothelium-dependent relaxation. PARs in endothelial cells are also suggested to contribute to a proinflammatory phenotypic conversion and an increase in the permeability of vascular lesions. In smooth muscle cells, PARs mediate contraction, migration, proliferation, hypertrophy, and production of the extracellular matrix, thereby contributing to the development of vascular lesions and the pathophysiology of such vascular diseases as atherosclerosis. However, the expression of PARs in the smooth muscle of normal arteries is limited. The upregulation of PARs in the smooth muscle is thus considered to be a key step for PARs to participate in the pathogenesis of vascular lesions. Elucidating the molecular mechanism regulating the PARs expression is therefore important to develop new strategies for the prevention and treatment of vascular diseases.

Induction of IL‐6 release from human T cells by PAR‐1 and PAR‐2 agonists

Proteinase-activated receptors (PAR) have been recognized as playing an important role in inflammation and immune response. However, little is known of the expression and function of PAR on human T cells. In this study, the expression of PAR on highly purified human T cells was determined and the secretion of IL-6 from cultured T cells in response to serine proteinases and agonist peptides of PAR was examined. The results showed that T cells express PAR-1, PAR-2 and PAR-3 proteins and genes. Thrombin, trypsin and tryptase, but not elastase, were able to stimulate concentration-dependent secretion of IL-6 from T cells following a 16 h incubation period. The specific inhibitors of thrombin, trypsin and tryptase inhibited the actions of these proteinases on T cells, indicating that the enzymatic activity is essential for their actions. Agonist peptides of PAR SFLLR-NH2, TFLLRN-NH2 and SLIGKV-NH2, but not TFRGAP-NH2, GYPGQV-NH2 and AYPGKF-NH2, are also capable of inducing IL-6 release from T cells. In conclusion, induction of IL-6 secretion from T cells by thrombin, trypsin and tryptase is probably through the activation of PAR, suggesting that serine proteinases are involved in the regulation of immune response of the body.