Protease-activated receptors and myocardial infarction

Protease-activated receptors (PARs) are widely expressed within the heart. They are activated by a myriad of proteases, including coagulation proteases. In vitro studies showed that activation of PAR-1 and PAR-2 on cardiomyocytes induced hypertrophy. In addition, PAR-1 stimulation on cardiac fibroblasts induced proliferation. Genetic and pharmacologic approaches have been used to investigate the role of the different PARs in cardiac ischemia/reperfusion (I/R) injury. In mice and rats, PAR-1 is reported to play a role in inflammation, infarct size, and remodeling after cardiac I/R injury. However, there are notable differences between the effect of a deficiency in PAR-1 and inhibition of PAR-1. For instance, inhibition of PAR-1 reduced infarct size whereas there was no effect of a deficiency of PAR-1. These differences maybe due to off-target effects of the inhibitor or PAR-4 compensation of PAR-1 deficiency. Similarly, a deficiency of PAR-2 was associated with reduced cardiac inflammation and improved heart function after I/R injury, whereas pharmacologic activation of PAR-2 was found to be protective due to increased vasodilatation. These differences maybe due to different signaling responses induced by an endogenous protease versus an exogenous agonist peptide. Surprisingly, PAR-4 deficiency resulted in increased cardiac injury and increased mortality after I/R injury. In contrast, a pharmacological study indicated that inhibition of PAR-4 was cardioprotective. It is possible that the major cellular target of the PAR-4 inhibitor is platelets, which have been shown to contribute to inflammation in the injured heart, whereas PAR-4 signaling in cardiomyocytes may be protective. These discrepant results between genetic and pharmacological approaches indicate that further studies are needed to determine the role of different PARs in the injured heart.     

Protease-activated receptors: a means of converting extracellular proteolysis into intracellular signals

Protease-activated receptors (PARs) mediate cellular responses to a variety of extracellular proteases. The four known PARs constitute a subgroup of the family of seven-transmembrane domain G protein-coupled receptors and activate intracellular signalling pathways typical for this family of receptors. Activation of PARs involves proteolytic cleavage of the extracellular domain, resulting in formation of a new N terminus, which acts as a tethered ligand. PAR-1, -3, and -4 are relatively selective for activation by thrombin whereas PAR-2 is activated by a variety of proteases, including trypsin and tryptase. Recent studies in mice genetically incapable of expressing specific PARs have defined roles for PAR-1 in vascular development, and for PAR-3 and -4 in platelet activation, which plays a fundamental role in blood coagulation. PAR-1 has also been implicated in a variety of other biological processes including inflammation, and brain and muscle development. Responses mediated by PAR-2 include contraction of intestinal smooth muscle, epithelium-dependent smooth muscle relaxation in the airways and vasculature, and potentiation of inflammatory responses. The area of PAR research is rapidly expanding our understanding of how cells communicate and control biological functions, in turn increasing our knowledge of disease processes and providing potential targets for therapeutic intervention.     

Protease activated receptors in cardiovascular function and disease

Recent studies have shown that a novel class of protease activated receptors (PARs), which are composed of seven transmembrane G protein-coupled domains, are activated by serine proteases such as thrombin, trypsin and tryptase. Although four types (PAR 1, PAR 2, PAR 3 and PAR 4) of this class of receptors have been identified, their discrete physiological and pathological roles are still being unraveled. Extracellular proteolytic activation of PARs results in the cleavage of specific sites in the extracellular domain and formation of a new N-terminus which functions as a tethered ligand. The newly formed tethered ligand binds intramolecularly to an exposed site in the second transmembrane loop and triggers G-protein binding and intracellular signaling. Recent studies have shown that PAR-1, PAR-2 and PAR-4 have been involved in vascular development and a variety of other biological processes including apoptosis and remodeling. The use of animal model systems, mainly transgenic mice and synthetic tethered ligand domains, have contributed enormously to our knowledge of molecular signaling and the regulatory properties of various PARs in cardiomyocytes. This review focuses on the role of PARs in cardiovascular function and disease.     

Thrombin signaling in the brain: the role of protease-activated receptors

Signaling by the protease thrombin has started to be appreciated in cell biology, especially since the gene for protease-activated receptor-1 (PAR-1) has been cloned. Apart from the central role of thrombin in blood coagulation and wound healing, thrombin also regulates cellular functions in a large variety of cells through PAR-1, PAR-3 and PAR-4. Receptors are activated by a proteolytic cleavage mechanism via G protein-coupled signaling pathways. Accumulating evidence shows that thrombin changes the morphology of neurons and astrocytes, induces glial cell proliferation, and even exerts, depending on the concentration applied, either cytoprotective or cytotoxic effects on neural cells. These effects may be mediated, through either distinct or overlapping signal transduction cascades, by activation of PARs. This review focuses on the underlying signaling events initiated by thrombin in neuronal and glial cells, to summarize our understanding of the intracellular signaling machinery linking thrombin receptors to their potential physiological and pathological functions in the CNS.     

Effects of thrombin,PAR-1 activating peptide and a PAR-1 antagonist on umbilical artery resistance in vitro

Background  

The non-thrombotic effects of thrombin in cardiovascular tissues, as mediated via the protease activated receptors (PARs), and particularly PAR-1, have been the focus of much recent research. The aims of this study were to evaluate the effects of thrombin, a specific PAR-1 activating peptide (PAR1-AP), and a PAR-1 antagonist on human umbilical artery tone in vitro.     

Altered expression and in vivo lung function of protease-activated receptors during influenza A virus infection in mice

Protease-activated receptors (PARs) are widely distributed in human airways, and recent evidence indicates a role for PARs in the pathophysiology of inflammatory airway disease. To further investigate the role of PARs in airway disease, we determined the expression and function of PARs in a murine model of respiratory tract viral infection. PAR-1, PAR-2, PAR-3, and PAR-4 mRNA and protein were expressed in murine airways, and confocal microscopy revealed colocalization of PAR-2 and cyclooxygenase (COX)-2 immunostaining in basal tracheal epithelial cells. Elevated levels of PAR immunostaining, which was particularly striking for PAR-1 and PAR-2, were observed in the airways of influenza A/PR-8/34 virus-infected mice compared with sham-infected mice. Furthermore, increased PAR-1 and PAR-2 expression was associated with significant changes in in vivo lung function responses. PAR-1 agonist peptide potentiated methacholine-induced increases in airway resistance in anesthetized sham-infected mice (and in indomethacin-treated, virus-infected mice), but no such potentiation was observed in virus-infected mice. PAR-2 agonist peptide transiently inhibited methacholine-induced bronchoconstriction in sham-infected mice, and this effect was prolonged in virus-infected mice. These findings suggest that during viral infection, the upregulation of PARs in the airways is coupled to increased activation of COX and enhanced generation of bronchodilatory prostanoids.     

Mesotrypsin, a brain trypsin, activates selectively proteinase-activated receptor-1, but not proteinase-activated receptor-2, in rat astrocytes

Proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, which are activated by serine proteases, such as trypsin, play pivotal roles in the CNS. Mesotrypsin (trypsin IV) has been identified as a brain-specific trypsin isoform. However, its potential physiological role concerning PAR activation in the brain is largely unknown. Here, we show for the first time that mesotrypsin, encoded by the PRSS3 (proteinase, serine) gene, evokes a transient and pronounced Ca(2+) mobilization in both primary rat astrocytes and retinal ganglion RGC-5 cells, suggesting a physiological role of mesotrypsin in brain cells. Mesotrypsin mediates Ca(2+) responses in rat astrocytes in a concentration-dependent manner, with a 50% effective concentration (EC(50)) value of 25 nm. The maximal effect of mesotrypsin on Ca(2+) mobilization in rat astrocytes is much higher than that observed in 1321N1 human astrocytoma cells, indicating that the activity of mesotrypsin is species-specific. The pre-treatment of cells with thrombin or the PAR-1-specific peptide TRag (Ala-pFluoro-Phe-Arg-Cha-HomoArg-Tyr-NH(2), synthetic thrombin receptor agonist peptide), but not the PAR-2-specific peptide, reduces significantly the mesotrypsin-induced Ca(2+) response. Treatment with the PAR-1 antagonist SCH79797 confirms that mesotrypsin selectively activates PAR-1 in rat astrocytes. Unlike mesotrypsin, the two other trypsin isoforms, cationic and anionic trypsin, activate multiple PARs in rat astrocytes. Therefore, our data suggest that brain-specific mesotrypsin, via the regulation of PAR-1, is likely to be involved in multiple physiological/pathological processes in the brain.     

Protease activated receptors in cardiovascular function and disease

Recent studies have shown that a novel class of protease activated receptors (PARs), which are composed of seven transmembrane G protein-coupled domains, are activated by serine proteases such as thrombin, trypsin and tryptase. Although four types (PAR 1, PAR 2, PAR 3 and PAR 4) of this class of receptors have been identified, their discrete physiological and pathological roles are still being unraveled. Extracellular proteolytic activation of PARs results in the cleavage of specific sites in the extracellular domain and formation of a new N-terminus which functions as a tethered ligand. The newly formed tethered ligand binds intramolecularly to an exposed site in the second transmembrane loop and triggers G-protein binding and intracellular signaling. Recent studies have shown that PAR-1, PAR-2 and PAR-4 have been involved in vascular development and a variety of other biological processes including apoptosis and remodeling. The use of animal model systems, mainly transgenic mice and synthetic tethered ligand domains, have contributed enormously to our knowledge of molecular signaling and the regulatory properties of various PARs in cardiomyocytes. This review focuses on the role of PARs in cardiovascular function and disease. (Mol Cell Biochem 263: 227–239, 2004)     

Protease Activated Receptors 1 and 2 Correlate Differently with Breast Cancer Aggressiveness Depending on Tumor ER Status

Experimental models implicate protease activated receptors (PARs) as important sensors of the proteolytic tumor microenvironment during breast cancer development. However, the role of the major PARs, PAR-1 and PAR-2, in human breast tumors remains to be elucidated. Here, we have investigated how PAR-1 and PAR-2 protein expression correlate with established clinicopathological variables and patient outcome in a well-characterized cohort of 221 breast cancer patients. Univariable and multivariable hazard ratios (HR) were estimated by the Cox proportional hazards model, distant disease-free survival (DDFS) and overall survival by the Kaplan–Meier method, and survival in different strata was determined by the log-rank test. Associations between PARs and clinicopathological variables were analyzed using Pearson’s χ²-test. We find that PAR-2 associates with DDFS (HR = 3.1, P = 0.003), whereas no such association was found with PAR-1 (HR = 1.2, P = 0.6). Interestingly, the effect of PAR-2 was confined to the ER-positive sub-group (HR = 5.5, P = 0.003 vs. HR = 1.2 in ER-negative; P = 0.045 for differential effect), and PAR-2 was an independent prognostic factor specifically in ER-positive tumors (HR = 3.9, P = 0.045). On the contrary, PAR-1 correlated with worse prognosis specifically in the ER-negative group (HR = 2.6, P = 0.069 vs. HR = 0.5, P = 0.19 in ER-positive; P = 0.026 for differential effect). This study provides novel insight into the respective roles of PAR-1 and PAR-2 in human breast cancer and suggests a hitherto unknown association between PARs and ER signaling that warrants further investigation.     

Proteinase-activated receptors: novel mechanisms of signaling by serine proteases

Although serineproteases are usually considered to act principally as degradativeenzymes, certain proteases are signaling molecules that specificallyregulate cells by cleaving and triggering members of a new family ofproteinase-activated receptors (PARs). There are three members of thisfamily, PAR-1 and PAR-3, which are receptors for thrombin, and PAR-2, areceptor for trypsin and mast cell tryptase. Proteases cleave withinthe extracellular NH₂-terminus oftheir receptors to expose a newNH₂-terminus. Specific residueswithin this tethered ligand domain interact with extracellular domainsof the cleaved receptor, resulting in activation. In common with many Gprotein-coupled receptors, PARs couple to multiple G proteins andthereby activate many parallel mechanisms of signal transduction. PARsare expressed in multiple tissues by a wide variety of cells, wherethey are involved in several pathophysiological processes, includinggrowth and development, mitogenesis, and inflammation. Because thecleaved receptor is physically coupled to its agonist, efficientmechanisms exist to terminate signaling and prevent uncontrolledstimulation. These include cleavage of the tethered ligand, receptorphosphorylation and uncoupling from G proteins, and endocytosis andlysosomal degradation of activated receptors.

     

Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression

Kallikrein-related peptidase 4 (KLK4) is one of the 15 members of the human KLK family and a trypsin-like, prostate cancer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasma membrane primarily by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. Here we show, using Ca(2+) flux assays, that KLK4 signals via both PAR-1 and PAR-2 but not via PAR-4. Dose-response analysis over the enzyme concentration range 0.1-1000 nM indicated that KLK4-induced Ca(2+) mobilization via PAR-1 is more potent than via PAR-2, whereas KLK4 displayed greater efficacy via the latter PAR. We confirmed the specificity of KLK4 signaling via PAR-2 using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressing and small interfering RNA-mediated receptor knockdown cell lines. Consistently, confocal microscopy analyses indicated that KLK4 initiates loss of PAR-2 from the cell surface and receptor internalization. Immunohistochemical analysis indicated the co-expression of agonist and PAR-2 in primary prostate cancer and bone metastases, suggesting that KLK4 signaling via this receptor will have pathological relevance. These data provide insight into KLK4-mediated cell signaling and suggest that signals induced by this enzyme via PARs may be important in prostate cancer.     

Protease-activated receptors in the musculoskeletal system

Protease-activated receptors (PARs) mediate cellular responses to a subset of extracellular proteases, including blood coagulation factors and proteases produced by inflammatory cells. Cells in bone, cartilage and muscle exhibit cell type-specific expression patterns and functional responses for the different PARs. Activators of PAR-1 include thrombin, and activators of PAR-2 include trypsin and tryptase; PARs-3 and -4 are also receptors for thrombin. Thrombin stimulates PAR-1-mediated proliferative responses in osteoblasts, chondrocytes and myoblasts, and in developing muscle, PAR-1 activation by thrombin appears to mediate activity-dependent polyneuronal synapse reduction. In bone, activation of PAR-2 leads to inhibition of osteoblast-mediated osteoclast differentiation induced by hormones or cytokines, and in muscle, PAR-2 activation leads to stimulation of myoblast proliferation. Although there is some evidence for a role for PARs expressed by cells of the musculoskeletal system at specific stages of development, their major role appears to be in protecting the tissues from the destructive effects of inflammation and promoting regeneration. This review discusses the regulation of cell function in the musculoskeletal system by receptor-mediated responses to proteases. Expression patterns of PARs, the circumstances in which PAR activators are likely to be present, functional responses of PAR activation, and responses to thrombin for which receptors have not yet been identified are considered.     

Localization of Protease-Activated Receptors -1 and -2 in Human Mast Cells: Indications for an Amplified Mast Cell Degranulation Cascade

Protease-activated receptors (PARs) belong to a family of G-coupled seven transmembrane receptors that are activated by a proteolytic cleavage of their N-termini. Recent studies suggest the involvement of protease-activated receptors-1 and -2 (PAR-1, PAR-2) activators in mast cell de-granulation in various physiological and pathophysiological processes in inflammatory responses. Although PAR-1 and PAR-2 activating proteases, thrombin and tryptase, have been associated with mast cell activation, PAR-1 and PAR-2 have not been localized within these cells. We describe here the localization of PAR-1 and PAR-2 in mast cells from various normal human tissues using im-munohistochemical and double immunofluorescence techniques. The presence of these receptors on the membrane may explain the actions of accessible extracellular thrombin and tryptase for mast cell activation. In addition to the membrane labeling, these receptors are also localized on the membrane of the intracellular tryptase-positive granules, which may function to sustain further mast cell degranulation upon exocytosis. The localization of these two receptors in mast cells suggests a novel mechanism for controlling mast cell activation through regulation of PARI and PAR-2.     

Gastrointestinal functions of proteinase-activated receptors

Proteinase-activated receptors (PARs) are a family of G-protein-coupled-seven-trans-membrane-domain receptors, consisting of four family members. PARs, especially PAR-1, a thrombin receptor, and PAR-2, a receptor for trypsin, tryptase and coagulation factors VIIa and Xa, are abundantly distributed throughout the gastrointestinal tract. PAR-2, but not other PARs, induces salivary and pancreatic exocrine secretion. Both PAR-2 and PAR-1 play protective roles in the gastric mucosa, modulating a variety of gastric functions. However, the mechanisms underlying the mucosal protection caused by PAR-2 and PAR-1 are entirely different. In the intestinal mucosa, PAR-2 appears to play a dual role, being pro- and anti-inflammatory. PAR-1, PAR-2 and also PAR-4 modulate the motility of the smooth muscle in the gastrointestinal tract including the esophageal muscularis mucosae, producing contraction and/or relaxation upon activation. Thus, PARs, especially PAR-1 and PAR-2, play extensive roles in modulating the gastrointestinal functions.     

Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.