PAR-1 Activation Research Articles

Protease-activated receptor 4: a critical participator in inflammatory response.

Protease-activated receptors (PARs) are G protein-coupled receptors of which four members PAR1, PAR2, PAR3, and PAR4 have been identified, characterized by a typical mechanism of activation involving various related proteases. The amino-terminal sequence of PARs is cleaved by a broad array of proteases, leading to specific proteolytic cleavage which forms endogenous tethered ligands to induce agonist-biased PAR activation. The biological effect of PARs activated by coagulation proteases to regulate hemostasis and thrombosis plays an enormous role in the cardiovascular system, while PAR4 can also be activated by trypsin, cathepsin G, the activated factor X of the coagulation cascade, and trypsin IV. Irrespective of its role in thrombin-induced platelet aggregation, PAR4 activation is believed to be involved in inflammatory lesions, as show by investigations that have unmasked the effects of PAR4 on neutrophil recruitment, the regulation of edema, and plasma extravasation. This review summarizes the roles of PAR4 in coagulation and other extracellular protease pathways, which activate PAR4 to participate in normal regulation and disease.

Unexpected anti-hypertrophic responses to low-level stimulation of protease-activated receptors in adult rat cardiomyocytes.

Activators of protease-activated receptors PAR-1 and PAR-2 such as thrombin and synthetic hexapeptides promote hypertrophy of isolated neonatal cardiomyocytes at pathological concentrations. Since PAR-activating proteases often show dual actions at low vs. high concentrations, the potential hypertrophic effects of low-level PAR activation were examined. In H9c2 cardiomyoblasts, messenger RNA (mRNA) expression of the hypertrophic marker atrial natriuretic peptide (ANP) was significantly increased only by higher concentrations of thrombin, trypsin or the synthetic PAR-2 agonist SLIGRL. The dual PAR-1/PAR-2 agonist SFLLRN did not influence basal ANP mRNA expression in H9c2 cells. Low concentration of thrombin or trypsin (up to 0.1U/mL) or of the synthetic ligands SFLLRN and SLIGRL (1μM); however, all suppressed ANP mRNA expression stimulated by angiotensin II (Ang II). The PAR-1 selective ligand TFLLRN exerted a comparable effect as SFLLRN. In adult rat cardiomyocytes, protein synthesis determined by [(3)H]phenylalanine incorporation was not increased by various PAR agonists at concentrations tenfold lower than conventionally used to study PAR function in vitro (10μM for SFLLRN or SLIGRL, 0.1U/mL for thrombin or trypsin). The positive control endothelin-1 (ET-1, 60 nM) however significantly increased protein synthesis in adult rat cardiomyocytes. Addition of low concentrations of PAR agonists to cardiomyocytes treated with ET-1 or Ang II suppressed [(3)H]phenylalanine incorporation induced by the hypertrophic stimuli. The inhibitory effect of SFLLRN effect was partially reversed by the PAR-1 antagonist RWJ56110. These findings suggest that physiological concentrations of PAR activators may suppress hypertrophy, in contrast to the pro-hypertrophic effects evident at high concentrations. PAR-1 and PAR-2 may dynamically control cardiomyocyte growth, with the net effect critically dependent upon local agonist concentrations. The precise significance of proposed concept of bimodal PAR function in cardiomyocytes remains to be defined, particularly in vivo where hemodynamic and other regulatory factors may counteract or mask the direct cellular actions described here.

Systemic and localized seminal plasma hypersensitivity patients exhibit divergent immunologic characteristics

Systemic and localized seminal plasma hypersensitivity patients exhibit divergent immunologic characteristics

Sphingosine-1-phosphate induces thrombin receptor PAR-4 expression to enhance cell migration and COX-2 formation in human monocytes.

Thrombin is not only a central factor in blood coagulation but also stimulates inflammatory processes, including monocyte responses, via activation of PARs. The signaling lipid S1P is a major determinant of monocyte function. Here, we established an interaction between S1P and human monocyte responses to thrombin. S1P induced PAR-1 and PAR-4 mRNA and total protein expression in human monocytes and U937 cells in a concentration (0.1-10 μM)- and time (1-24 h)-dependent manner, respectively. However, only PAR-4 cell-surface expression was increased significantly by S1P, whereas PAR-1 remained unaffected. This response was associated with activation of the Akt, Erk, and p38 pathway and induction of COX-2 but not COX-1. PAR-4-mediated induction of COX-2 was prevented by the PI3K inhibitor LY (10 μM). Preincubation of human monocytes with S1P (1 μM; 16 h) resulted in an enhanced chemotaxis toward thrombin or to selective AP for PAR-4 but not PAR-1. Furthermore, down-regulation of PAR-4 transcription with siRNA attenuated the chemotactic response to thrombin and AP4. In conclusion, S1P enhances monocyte responses to thrombin via up-regulation of PAR-4 expression, which promotes cell migration and COX-2 abundance. This mechanism may facilitate monocyte recruitment to sites of vessel injury and inflammation.

Cathepsin S signals via PAR2 and generates a novel tethered ligand receptor agonist.

Protease-activated receptor-2 is widely expressed in mammalian epithelial, immune and neural tissues. Cleavage of PAR2 by serine proteases leads to self-activation of the receptor by the tethered ligand SLIGRL. The contribution of other classes of proteases to PAR activation has not been studied in detail. Cathepsin S is a widely expressed cysteine protease that is upregulated in inflammatory conditions. It has been suggested that cathepsin S activates PAR2. However, cathepsin S activation of PAR2 has not been demonstrated directly nor has the potential mechanism of activation been identified. We show that cathepsin S cleaves near the N-terminus of PAR2 to expose a novel tethered ligand, KVDGTS. The hexapeptide KVDGTS generates downstream signaling events specific to PAR2 but is weaker than SLIGRL. Mutation of the cathepsin S cleavage site prevents receptor activation by the protease while KVDGTS retains activity. In conclusion, the range of actions previously ascribed to cysteine cathepsins in general, and cathepsin S in particular, should be expanded to include molecular signaling. Such signaling may link together observations that had been attributed previously to PAR2 or cathepsin S individually. These interactions may contribute to inflammation.

Proteinase-activated receptor 2 blockade impairs CCL11- or allergen-induced eosinophil recruitment in experimental pleurisy

Although proteinase-activated receptor (PAR)-2 has been implicated in inflammatory diseases, its role in regulating eosinophil recruitment in response to chemoattractants remains unclear. Here, we investigated the role of PAR-2 and PAR-2-activating Mast Cell (MC) tryptase on chemokine C–C motif ligand (CCL)11- and antigen-induced eosinophil recruitment to the pleural cavity of BALB/c mice. The PAR-2-activating peptide H–Ser–Leu–Ile–Gly–Arg–Leu–NH2 (SLIGRL–NH2) induced eosinophil recruitment whereas PAR-2 blockade inhibited ovalbumin (OVA)- or CCL11-induced eosinophil recruitment. Moreover, OVA and CCL11 induced PAR-2 expression in pleural leukocytes, and the MC tryptase inhibitor APC 366 ([N-(1-hydroxy-2-napthoyl)-l-arginyl-l-prolinamide hydrochloride]) abolished CCL11-induced eosinophil recruitment. These results suggest a pro inflammatory effect of PAR-2 and support a role for MC tryptase mediating eosinophil migration via PAR-2 signaling. Taken together, our results suggest that PAR-2 activation through endogenous MC tryptase activity could be required, at least partially, to mediate CCL11-induced eosinophil migration.

204 Modulation of Protease Activated Receptor Gene Expression in Endothelial Cells

IntroductionProtease activated receptors (PARs) form a distinct group of G-protein coupled receptors with key roles in several pathophysiological processes including inflammation and atherothrombotic disease. This study aimed to investigate the...

Abstract 556: Soluble TREM-Like Transcript-1 Differentially Responds to PAR1 and PAR4 Signaling: A Possible New Role for sTLT-1 in Angiogenesis?

Platelets have been shown to play important roles in angiogenesis due in part to the differential release of proteins from their α-granules. For example PAR-1 receptor activation in platelets releases α-granules containing pro-angiogenic molecules, while PAR-4 receptor activation promotes the release of anti-angiogenic molecules. Studies from our laboratory reported the identification of the Triggering Receptor Expressed in Myeloid cells (TREM)-Like 1 transcript (TLT)-1, a protein preferentially expressed in platelets and megakaryocytes. TLT-1 is stored in platelet α-granules with p-selectin and has a soluble extracellular fragment that is released upon platelet activation. Comparative immunofluorescence (IF) to p-selectin shows that only ~50% of the TLT-1 colocalized with p-selectin suggesting that TLT-1 may be differentially stored and dispersed from α-granules. To address this possibility we compared colocalization of TLT-1 to VEGF and endostatin, two proteins that are stored segregated into granules differentially. IF staining demonstrated greater colocalization with pro-angiogenic VEGF than anti-angiogenic endostatin. We then hypothesized that pro- or anti-angiogenic PAR-1 & PAR-4 signaling would differentially affect TLT-1 dispersion in platelets. To test this, purified human platelets were stimulated with a PAR-1 or a PAR-4 agonist and TLT-1 was monitored by IF. Results showed that PAR-1 activation caused a greater TLT-1 dissemination throughout the platelet than PAR-4. Supernatants of platelets activated by PAR-1 showed an increased amount of sTLT-1 compared to those activated by PAR-4 agonist. Our results suggest that TLT-1 is associated to platelet release of pro-angiogenic α-granules. We next addressed the question if sTLT-1 has an effect on endothelial cells. Soluble TLT-1 was added to primary endothelial cells 30 minutes and cells were evaluated for changes in size and actin polymerization. Studies by IF showed an increase in the size and actin polymerization of cells, suggesting it promotes a pro-angiogenic morphology. Ongoing experiments are addressing if sTLT-1 affects tube formation and migration of endothelial cells. These results could shed light into TLT-1 properties and its possible new role in angiogenesis.

Salmon and king crab trypsin stimulate interleukin-8 and matrix metalloproteinases via protease-activated receptor-2 in the skin keratinocytic HaCaT cell line

Occupational skin symptoms are prevalent among the workers of the seafood processing industry. In this study we investigate the role of salmon (Salmo salar) and king crab trypsin (Paralithodes camtschaticus) as inducers of inflammation in skin via secretion of inflammatory mediators. Human skin keratinocytes (HaCaT cells) were exposed to purified salmon and king crab trypsin. We observed that salmon trypsin enhanced the secretion of IL-8 and MMP-2 and crab trypsin enhanced the secretion of IL-8, MMP-2 and MMP-9 in a dose dependent manner. As protease activated receptors (PAR)-2 in skin are known to play an important role in physiology and pathology, we explored the involvement of these receptors in mediating the release of interleukin (IL)-8 and matrix metalloproteinase (MMP)-2 and -9 subsequent to exposure of skin keratinocytes to salmon and crab trypsin. In addition we observed that salmon and crab trypsin exhibit individual differences in stimulating the release of these inflammatory mediators. Finally, using specific small interfering RNA (siRNA) against PAR-2, we confirmed that the increase in secretion of IL-8, MMP-2 and MMP-9 in skin keratinocytes following exposure to salmon and crab trypsin was mediated via activation of PAR-2. These results suggest that exposure to proteases from the seafood may lead to inflammatory reactions in skin.

PAR1 antagonists inhibit thrombin-induced platelet activation whilst leaving the PAR4-mediated response intact

Thrombin-induced platelet activation is initiated by PAR1 and PAR4 receptors. Vorapaxar, a PAR1 antagonist, has been assessed in patients with acute coronary syndromes (ACS) and stable atherosclerotic disease in addition to standard-of-care treatment. In clinical trials, vorapaxar has been observed to reduce the frequency of ischaemic events in some subgroups though in others has increased the frequency of bleeding events. Among patients undergoing CABG surgery, which is associated with excess thrombin generation, bleeding was not increased. The aim of these studies was to investigate the effects of selective PAR1 antagonism on thrombin-induced platelet activation in patients receiving vorapaxar or placebo in the TRACER trial and to explore the roles of PAR1 and PAR4 in thrombin-induced platelet activation in healthy volunteers. ACS patients receiving vorapaxar or placebo in the TRACER trial were studied at baseline and 4 hours, 1 and 4 months during drug administration. Thrombin-induced calcium mobilisation in platelet-rich plasma was assessed by flow cytometry. In vitro studies were performed in healthy volunteers using the PAR1 antagonist SCH79797 or PAR4 receptor desensitisation. Vorapaxar treatment significantly inhibited thrombin-induced calcium mobilisation, leaving a residual, delayed response. These findings were consistent with calcium mobilisation mediated via the PAR4 receptor and were reproduced in vitro using SCH79797. PAR4 receptor desensitization, in combination with SCH79797, completely inhibited thrombin-induced calcium mobilisation confirming that the residual calcium mobilisation was mediated via PAR4. In conclusion vorapaxar selectively antagonises the PAR1-mediated component of thrombin-induced platelet activation, leaving the PAR4-mediated response intact, which may explain why vorapaxar is well tolerated in patients undergoing CABG surgery since higher thrombin levels in this setting may override the effects of PAR1 antagonism through PAR4 activation, thus preserving haemostasis. Further assessment may be warranted.

Tryptase inhibitor APC 366 prevents hepatic fibrosis by inhibiting collagen synthesis induced by tryptase/protease-activated receptor 2 interactions in hepatic stellate cells.

Protease-activated receptor (PAR) 2 is a G-protein-coupled receptor that is activated by mast cell tryptase. PAR-2 activation augments profibrotic pathways through the induction of extracellular matrix proteins. PAR-2 is widely expressed in hepatic stellate cells (HSCs), but the role of tryptase/PAR-2 interaction in liver fibrosis is unclear. We studied the development of bile duct ligation (BDL)-induced hepatic fibrosis in rats treated with mast cell tryptase inhibitor APC 366, and showed that APC 366 reduced hepatic fibrosis scores, collagen content and serum biochemical parameters. Reduced fibrosis was associated with decreased expression of PAR-2 and α-smooth muscle actin (α-SMA). Our findings demonstrate that mast cell tryptase induces PAR-2 activation to augment HSC proliferation and promote hepatic fibrosis in rats. Treatment with tryptase antagonists may be a novel therapeutic approach to prevent fibrosis in patients with chronic liver disease.

The physical association of the P2Y12 receptor with PAR4 regulates arrestin-mediated Akt activation.

It is now well accepted that protease activated receptor (PAR) 1 and PAR4 have differential roles in platelet activation. PAR4, a low-affinity thrombin receptor in human platelets, participates in sustained platelet activation in a P2Y12-dependent manner; however, the mechanisms are not defined. Our previous studies demonstrated that thrombin induces the association of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to coregulate Akt signaling after PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4 but not PAR1 by bioluminescent resonance energy transfer when the receptors were coexpressed in human embryonic kidney 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. By using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant form of PAR4, "PAR4SFT," by replacing LGL194-196 at the base of transmembrane domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT supported only 8.74% of PAR4-P2Y12 interaction, abolishing P2Y12-dependent arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux when expressed independently; however, coexpression of increasing concentrations of PAR4SFT, together with PAR4 potentiated PAR4-mediated calcium flux, suggested that PAR4 act as homodimers to signal to Gq-coupled calcium responses. In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4 with P2Y12 and contributes to Gq-coupled calcium responses. PAR4-P2Y12 association supports arrestin-mediated sustained signaling to Akt. Hence, PAR4-P2Y12 dimerization is likely to be important for the PAR4-P2Y12 dependent stabilization of platelet thrombi.

Regulation of intestinal epithelial apoptosis by protease‐activated receptor 2 involves EGFR‐transactivation and is associated with Mcl‐1 upregualtion (151.1)

Colonic biopsies from inflamed colon have higher levels of epithelial apoptosis than biopsies from healthy individuals. Therapies that inhibit intestinal epithelial apoptosis may decrease the inflammation observed in inflammatory bowel disease. We previously showed that signaling by PAR2, a GPCR activated by serine proteases, delayed IFN‐γ & TNF‐α‐induced apoptosis in colonic epithelial (HT‐29) cells. The observed decrease in the cleavage of caspase‐8, 9, 3 and PARP following the activation of PAR2 by 2‐furoyl‐LIGRLO (2fLI) was shown to be dependent on the activities of PI3K and ERK1/2. In this study we found that selective inhibition of EGFR with AG1478 blocked the phosphorylation of PI3K and ERK1/2. Pre‐treatment with AG1478 prior to the addition of IFN‐γ & TNF‐α also blocked 2fLI‐induced intestinal epithelial survival, as determined by PARP cleavage. Modulation of BCL‐2 family members was then investigated due to their known function in regulating mitochondrial permeability and apoptosis. Protein expression of pro‐apoptotic BAK, BAX and BIM were not altered by either cytokine or 2fLI treatment. Likewise, no change in expression of anti‐apoptotic BCL‐XL was observed. Treatment with 2fLI stimulated phosphorylation and inactivation of the apoptotic BH3‐only protein, BAD. Knockdown of BAD with siRNA, however, did not alter 2fLI‐stimulated intestinal survival. Finally, 2fLI stimulated a rapid increase in anti‐apoptotic MCL‐1 expression. PAR2 signaling reduces epithelial apoptosis in response to inflammatory cytokines via EGFR transactivation and possibly by regulation of MCL‐1 expression. Our findings may represent a mechanism whereby proteases facilitate epithelial cell survival and colonic healing after inflammation.Grant Funding Source: Supported by the Crohn’s and Colitis Foundation of Canada

Protease‐activated receptor ‐3 signals independent of protease‐activated receptor‐1 to regulate endothelial nitric oxide synthase in human endothelial cells (1097.1)

Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites Thr495 and Ser1177. The eNOS/NO pathway is responsible for vascular smooth muscle cell relaxation and disruption of this pathway leads to endothelial dysfunction, laminar shear stress and vascular inflammation, a precursor to cellular induced hypertension. Using human coronary artery endothelial cells (HCAECs) as a model, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide ligand, TFRGAP. We hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS via site‐ specific phosphorylation and induce pro‐inflammation via the NFkB pathway. Analysis using eNOS specific antibodies showed that 500uM of TFRGAP lead to the phosphorylation of the positive site of eNOS‐Ser1177 and the negative site ‐Thr495, 30 seconds and 15 minutes respectively. Pretreatment of HCAECs with 10uM SCH79797, a selective PAR‐1 inhibitor, did not block the increase in phosphorylation at both eNOS regulatory sites stimulated by TFRGAP. Similar results where found in a stable PAR‐1 deficient cell line of HCAECs which confirmed the signaling potential of PAR‐3 using thrombin and the PAR‐3 activating peptide. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site‐specific phosphorylation that is independent of PAR‐1 activation. In additional studies cytokine production was measured using an ELISA after PAR‐3 activation. Data from these studies will lead to a better understanding of endothelial dysfunction and vascular inflammation while identifying potential drug targets for cardiovascular diseases.Grant Funding Source: The project [poster] described was supported by CTSA award No. UL1TR000445

Protease-Activated Receptor-2 Activation Contributes to House Dust Mite-Induced IgE Responses in Mice

Aeroallergens such as house dust mite (HDM), cockroach, and grass or tree pollen are innocuous substances that can induce allergic sensitization upon inhalation. The serine proteases present in these allergens are thought to activate the protease-activated receptor (PAR)-2, on the airway epithelium, thereby potentially inducing allergic sensitization at the expense of inhalation tolerance. We hypothesized that the proteolytic activity of allergens may play an important factor in the allergenicity to house dust mite and is essential to overcome airway tolerance. Here, we aimed to investigate the role of PAR-2 activation in allergic sensitization and HDM-induced allergic airway inflammation. In our study, Par-2 deficient mice were treated with two different HDM extracts containing high and low serine protease activities twice a week for a period of 5 weeks. We determined airway inflammation through quantification of percentages of mononuclear cells, eosinophils and neutrophils in the bronchial alveolar lavage fluid and measured total IgE and HDM-specific IgE and IgG1 levels in serum. Furthermore, Th2 and pro-inflammatory cytokines including IL-5, IL-13, Eotaxin-1, IL-17, KC, Chemokine (C-C motif) ligand 17 (CCL17) and thymic stromal lymphopoietin (TSLP), were measured in lung tissue homogenates. We observed that independent of the serine protease content, HDM was able to induce elevated levels of eosinophils and neutrophils in the airways of both wild-type (WT) and Par-2 deficient mice. Furthermore, we show that induction of pro-inflammatory cytokines by HDM exposure is independent of Par-2 activation. In contrast, serine protease activity of HDM does contribute to enhanced levels of total IgE, but not HDM-specific IgE. We conclude that, while Par-2 activation contributes to the development of IgE responses, it is largely dispensable for the HDM-induced induction of pro-inflammatory cytokines and airway inflammation in an experimental mouse model of HDM-driven allergic airway disease.

Environmental changes could enhance the biological effect of Hop J pollens on human airway epithelial cells

Environmental changes could enhance the biological effect of Hop J pollens on human airway epithelial cells

Himbacine-derived thrombin receptor antagonists: c7-spirocyclic analogues of vorapaxar.

We have synthesized several C7-spirocyclic analogues of vorapaxar and evaluated their in vitro activities against PAR-1 receptor. Some of these analogues showed activities and rat plasma levels comparable to vorapaxar. Compound 5c from this series showed excellent PAR-1 activity (K i = 5.1 nM). We also present a model of these spirocyclic compounds docked to the PAR-1 receptor based on the X-ray crystal structure of vorapaxar bound to PAR-1 receptor. This model explains some of the structure-activity relationships in this series.

Inhibition of airway epithelial-to-mesenchymal transition and fibrosis by kaempferol in endotoxin-induced epithelial cells and ovalbumin-sensitized mice

Chronic airway remodeling is characterized by structural changes within the airway wall, including smooth muscle hypertrophy, submucosal fibrosis and epithelial shedding. Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis, which can be induced by TGF-β. In the in vitro study, we investigated whether 1–20 μM kaempferol inhibited lipopolysaccharide (LPS)-induced bronchial EMT in BEAS-2B cells. The in vivo study explored demoting effects of 10–20 mg/kg kaempferol on airway fibrosis in BALB/c mice sensitized with ovalbumin (OVA). LPS induced airway epithelial TGF-β1 signaling that promoted EMT with concurrent loss of E-cadherin and induction of α-smooth muscle actin (α-SMA). Nontoxic kaempferol significantly inhibited TGF-β-induced EMT process through reversing E-cadherin expression and retarding the induction of N-cadherin and α-SMA. Consistently, OVA inhalation resulted in a striking loss of epithelial morphology by displaying myofibroblast appearance, which led to bronchial fibrosis with submucosal accumulation of collagen fibers. Oral administration of kaempferol suppressed collagen deposition, epithelial excrescency and goblet hyperplasia observed in the lung of OVA-challenged mice. The specific inhibition of TGF-β entailed epithelial protease-activated receptor-1 (PAR-1) as with 20 μM kaempferol. The epithelial PAR-1 inhibition by SCH-79797 restored E-cadherin induction and deterred α-SMA induction, indicating that epithelial PAR-1 localization was responsible for resulting in airway EMT. These results demonstrate that dietary kaempferol alleviated fibrotic airway remodeling via bronchial EMT by modulating PAR1 activation. Therefore, kaempferol may be a potential therapeutic agent targeting asthmatic airway constriction.

Tissue Kallikrein Mediates Pro-Inflammatory Pathways and Activation of Protease-Activated Receptor-4 in Proximal Tubular Epithelial Cells

Tissue kallikrein (KLK1) expression is up-regulated in human diabetic kidney tissue and induced by high glucose (HG) in human proximal tubular epithelial cells (PTEC). Since the kallikrein-kinin system (KKS) has been linked to cellular inflammatory process in many diseases, it is likely that KLK1 expression may mediate the inflammatory process during the development of diabetic nephropathy. In this study, we explored the role of KLK1 in tubular pro-inflammatory responses under the diabetic milieu. Recombinant KLK1 stimulated the production of inflammatory cytokines in PTEC via the activation of p42/44 and p38 MAPK signaling pathways. Molecular knockdown of endogenous KLK1 expression by siRNA transfection in PTEC attenuated advanced glycation end-products (AGE)-induced IL-8 and ICAM-1 productions in vitro. Interestingly, exposure of PTEC to KLK1 induced the expression of protease-activated receptors (PARs). There was a 2.9-fold increase in PAR-4, 1.4-fold increase in PAR-1 and 1.2-fold increase in PAR-2 mRNA levels. Activation of PAR-4 by a selective agonist was found to elicit the pro-inflammatory and pro-fibrotic phenotypes in PTEC while blockade of the receptor by specific antagonist attenuated high glucose-induced IL-6, CCL-2, CTGF and collagen IV expression. Calcium mobilization by the PAR-4 agonist in PTEC was desensitized by pretreatment with KLK1. Consistent with these in vitro findings, there was a markedly up-regulation of tubular PAR-4 expression in human diabetic renal cortical tissues. Together, these results suggest that up-regulation of KLK1 in tubular epithelial cells may mediate pro-inflammatory pathway and PAR activation during diabetic nephropathy and provide a new therapeutic target for further investigation.

Proteinase-activated receptor-1 (PAR1) and PAR2 mediate relaxation of guinea pig internal anal sphincter

Activation of proteinase-activated receptor-1 (PAR1) and PAR2 stimulates contraction of the rat but relaxation of the guinea pig colon. The aim of the present study was to investigate PAR effects on internal anal sphincter (IAS) motility. We measured relaxation of isolated muscle strips from the guinea pig IAS caused by PAR agonists using isometric transducers. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the existence of PAR. In the IAS, thrombin and PAR1 peptide agonists TFLLR-NH2 and SFLLRN-NH2 evoked moderate to marked relaxation in a concentration-dependent manner. In addition, trypsin and PAR2 peptide agonists 2-furoyl-LIGRLO-NH2, SLIGRL-NH2 and SLIGKV-NH2 produced relaxation. In contrast, both PAR1 and PAR2 inactive control peptides did not elicit relaxation. Furthermore, the selective PAR1 antagonist vorapaxar and PAR2 antagonist GB 83 specifically inhibited thrombin and trypsin-induced relaxations, respectively. RT-PCR revealed the presence of PAR1 and PAR2 in the IAS. This indicates that PAR1 and PAR2 mediate the IAS relaxation. The relaxant responses of TFLLR-NH2 and trypsin were attenuated by N(omega)-Nitro-L-arginine (L-NNA), indicating involvement of NO. These responses were not affected by tetrodotoxin, implying that the PAR effects are not neurally mediated. On the other hand, PAR4 agonists GYPGKF-NH2, GYPGQV-NH2 and AYPGKF-NH2 did not cause relaxation or contraction, suggesting that PAR4 is not involved in the sphincter motility. Taken together, these results demonstrate that both PAR1 and PAR2 mediate relaxation of the guinea pig IAS through the NO pathway. PAR1 and PAR2 may regulate IAS tone and might be potential therapeutic targets for anal motility disorders.