Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs).Although thrombin is a recognized physiological activator of PAR 1 and PAR 4 , the endogenous enzymes responsible for activating PAR 2 in settings other than the gastrointestinal system, where trypsin can activate PAR 2 , are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR 1 , PAR 2 , and PAR 4 ; 2) PAR-dependent calcium signaling responses in cells expressing PAR 1 , PAR 2 , and PAR 4 and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR 4 -dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR 1 , PAR 2 , and PAR 4 and to disarm/inhibit PAR 1 . Although hK5 and -6 were also able to activate PAR 2 , they failed to cause PAR 4 -dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR 2 -mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR 1 , PAR 2 , and PAR 4 . Proteinase-activated receptors (PAR 1-4 )3 compose a unique family of four G-protein-coupled cell surface receptors for certain proteinases (1-9). Proteolytic cleavage within the extracellular N terminus reveals a tethered ligand that binds to the extracellular receptor domains to initiate cell signaling (5, 6, 9, 10). Proteinases that activate PARs include coagulation factors, enzymes from inflammatory cells, and proteinases from epithelial cells and neurons. These enzymes, generated and released during injury and inflammation, can cleave and activate PARs on many cell types from a variety of species (humans, rats, and mice) to regulate the critically important processes of hemostasis, inflammation, pain, and tissue repair. Other proteinases that cleave PARs downstream of the N-terminal tethered ligand sequence disable the receptors for further proteolytic activation, thus abrogating PAR signaling. It is of considerable interest to identify the proteinases that activate and/or disable PARs, in view of the emerging role that these receptors can play in diseases such as asthma, arthritis, inflammatory bowel disease, and cancer (5-7).In some systems, the proteinases that activate PARs have been established. For example, in the circulatory system, PAR 1 , PAR 3 , and PAR 4 are recognized physiological targets for thrombin (4), which does not efficiently acti...

Citrobacter rodentium is a bacterial pathogen that causes a murine infectious colitis equivalent to enterohemorrhagic Escherichia coli infection in humans. Colonic luminal fluid from C. rodentiuminfected mice, but not from sham-infected mice, contains active serine proteinases that can activate proteinase-activated receptor-2 (PAR2). We have identified granzyme A and murine trypsins to be present in C. rodentium-infected luminal fluid, as determined by mass spectrometry and Western blot analysis. Inflammatory indices (colonic mucosa macroscopic damage score, increased intestinal wall thickness, granulocyte infiltration, and bacterial translocation from the colonic lumen to peritoneal organs) were all increased in C. rodentium-infected mice, compared with shaminfected mice. Soybean trypsin inhibitor-treated wild-type mice and untreated PAR2-deficient (PAR2 ؊/؊ ) mice (compared with their wild-type littermates) both had substantially reduced levels of C. rodentium-induced inflammation. These data point to an important role for both pathogen-induced host serine proteinases and PAR2 in the setting of infectious colitis.colitis ͉ inflammation ͉ trypsin ͉ granzyme

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Pseudomonas aeruginosa, a major lung pathogen in cystic fibrosis (CF) patients, secretes an elastolytic metalloproteinase (EPa) contributing to bacterial pathogenicity. Proteinase-activated receptor 2 (PAR2), implicated in the pulmonary innate defense, is activated by the cleavage of its extracellular N-terminal domain, unmasking a new N-terminal sequence starting with SLIGKV, which binds intramolecularly and activates PAR2. We show that EPa cleaves the N-terminal domain of PAR2 from the cell surface without triggering receptor endocytosis as trypsin does. As evaluated by measurements of cytosolic calcium as well as prostaglandin E(2) and interleukin-8 production, this cleavage does not activate PAR2, but rather disarms the receptor for subsequent activation by trypsin, but not by the synthetic receptor-activating peptide, SLIGKV-NH(2). Proteolysis by EPa of synthetic peptides representing the N-terminal cleavage/activation sequences of either human or rat PAR2 indicates that cleavages resulting from EPa activity would not produce receptor-activating tethered ligands, but would disarm PAR2 in regard to any further activating proteolysis by activating proteinases. Our data indicate that a pathogen-derived proteinase like EPa can potentially silence the function of PAR2 in the respiratory tract, thereby altering the host innate defense mechanisms and respiratory functions, and thus contributing to pathogenesis in the setting of a disease like CF.