Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues.

The serine proteases thrombin and trypsin are among many factors that malignant cells secrete into the extracellular space to mediate metastatic processes such as cellular invasion, extracellular matrix degradation, angiogenesis, and tissue remodeling. The degree of protease secretion from malignant cells has been correlated to their metastatic potential. Protease activated receptors (PAR)-1 and -2, which are activated by thrombin and trypsin respectively, have not been extensively characterized in human tumors in situ. We investigated the presence of PAR-1 and PAR-2 in human normal, benign and malignant tissues using immunohistochemistry and in situ hybridization. Our results demonstrate PAR-1 and PAR-2 expression in the tumor cells, mast cells, macrophages, endothelial cells, and vascular smooth muscle cells of the metastatic tumor microenvironment. Most notably, an up-regulation of PAR-1 and PAR-2 observed in proliferating, smooth muscle actin (SMA)-positive stromal fibroblasts surrounding the carcinoma cells was not observed in normal or benign conditions. Furthermore, in vitro studies using proliferating, SMA-positive, human dermal fibroblasts, and scrape-wounded human dermal fibroblasts demonstrated the presence of PAR-1 and PAR-2 not detected in quiescent, SMA-negative cultures. PAR-1 and PAR-2 in the cells forming the tumor microenvironment suggest that these receptors mediate the signaling of secreted thrombin and trypsin in the processes of cellular metastasis.

Protease-activated receptor-2 (PAR-2): structure-function study of receptor activation by diverse peptides related to tethered-ligand epitopes.

Protease-activated receptor-2 (PAR-2) is a tethered-ligand, G-protein-coupled receptor that is activated by proteolytic cleavage or by small peptides derived from its cleaved N-terminal sequence, such as SLIGRL-NH2. To assess specific PAR activity, we developed an immortalized murine PAR-1 (-/-) cell line transfected with either human PAR-2 or PAR-1. A "directed" library of more than 100 PAR agonist peptide analogues was synthesized and evaluated for PAR-2 and PAR-1 activity to establish an in-depth structure-function profile for specific action on PAR-2. The most potent agonist peptides (EC50 = 2-4 microM) had Lys at position 6, Ala at position 4, and pFPhe at position 2; however, these also exhibited potent PAR-1 activity (EC50 = 0.05-0.35 microM). We identified SLIARK-NH2 and SL-Cha-ARL-NH2 as relatively potent, highly selective PAR-2 agonists with EC50 values of 4 microM. Position 1 did not tolerate basic, acidic, or large hydrophobic amino acids. N-Terminal capping by acetyl eliminated PAR-2 activity, although removal of the amino group reduced potency by just 4-fold. At position 2, substitution of Leu by Cha or Phe gave equivalent PAR-2 potency, but this modification also activated PAR-1, whereas Ala, Asp, Lys, or Gln abolished PAR-2 activity; at position 3, Ile and Cha were optimal, although various amino acids were tolerated; at position 4, Ala or Cha increased PAR-2 potency 2-fold, although Cha introduced PAR-1 activity; at position 5, Arg or Lys could be replaced successfully by large hydrophobic amino acids. These results with hexapeptide C-terminal amides that mimic the native PAR-2 ligand indicate structural modes for obtaining optimal PAR-2 activity, which could be useful for the design of PAR-2 antagonists.

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor.

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH(2), RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

Cardiovascular responses mediated by protease-activated receptor-2 (PAR-2) and thrombin receptor (PAR-1) are distinguished in mice deficient in PAR-2 or PAR-1.

We developed mice deficient in protease-activated receptor-2 (PAR-2) or PAR-1 to explore the pathophysiological functions of these receptors. In this report, we evaluated mean arterial pressure and heart rate (HR) changes in response to PAR-1 or PAR-2 activation in anesthetized wild-type (WT), PAR-1-deficient (PAR-1(-/-)), and PAR-2-deficient (PAR-2(-/-)) mice. In WT mice, TFLLRNPNDK, a PAR-1 selective activating peptide, caused hypotension and HR decreases at 1 micromol/kg. TFLLRNPNDK also caused secondary hypertension following L-NAME pretreatment. These responses were absent in PAR-1(-/-) mice. In WT mice, SLIGRL, a PAR-2 selective activating peptide, caused hypotension without changing HR at 0.3 micromol/kg. SLIGRL did not induce hypertension following Nomega-nitrol-arginine-methyl ester-HCl (L-NAME). The response to SLIGRL was absent in PAR-2(-/-) mice. SFLLRN, a nonselective receptor activating peptide caused hypotension and HR decreases in WT mice at 0.3 micromol/kg, as well as secondary hypertension following L-NAME. SFLLRN still induced hypotension in PAR-1(-/-) mice, but HR decrease and secondary hypertension following L-NAME were absent. The hypotensive and bradycardic responses to SFLLRN and TFLLRNPNDK in PAR-2(-/-) mice were accentuated compared with WT mice. By using mouse strains deficient in either PAR-1 or PAR-2, we confirmed the in vivo specificity of TFLLRNPNDK and SLIGRL as respective activating peptides for PAR-1 and PAR-2, and the distinct hemodynamic responses mediated by activation of PAR-1 or PAR-2. Moreover, the accentuated response to PAR-1 activation in PAR-2-deficient mice suggests a compensatory response and potential receptor cross-talk.

Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues.

PAR-2 is a second member of a novel family of G-protein-coupled receptors characterized by a proteolytic cleavage of the amino terminus, thus exposing a tethered peptide ligand that autoactivates the receptor. The physiological and/or pathological role(s) of PAR-2 are still unknown. This study provides tissue-specific cellular localization of PAR-2 in normal human tissues by immunohistochemical techniques. A polyclonal antibody, PAR-2C, was raised against a peptide corresponding to the amino terminal sequence SLIGKVDGTSHVTGKGV of human PAR-2. Significant PAR-2 immunoreactivity was detected in smooth muscle of vascular and nonvascular origin and stromal cells from a variety of tissues. PAR-2 was also present in endothelial and epithelial cells independent of tissue type. Strong immunolabeling was observed throughout the gastrointestinal tract, indicating a possible function for PAR-2 in this system. In the CNS, PAR-2 was localized to many astrocytes and neurons, suggesting involvement of PAR-2 in neuronal function. A role for PAR-2 in the skin was further supported by its immunolocalization in the epidermis. PAR-2C antibody exemplifies an important tool to address the physiological role(s) of PAR-2.

Protease-activated receptor-2 (PAR-2) is present in the rat hippocampus and is associated with neurodegeneration.

Protease-activated receptor-2 (PAR-2) is a seven-transmembrane G protein-coupled receptor that possesses a structure and activation mechanism similar to those of the thrombin receptor. It is activated by low concentrations of trypsin (300 pM) and a synthetic hexapeptide [sequence of serine, leucine, isoleucine, glycine, arginine, leucine (SLIGRL), the rodent PAR-2 "tethered ligand"] representing the first six amino acids following the putative PAR-2 cleavage site. Previous studies have indicated that alpha-thrombin and SFLLRN (synthetic hexapeptide sequence of serine, phenylalanine, leucine, leucine, arginine, asparagine; the human thrombin receptor "tethered ligand") induce neurite retraction and neurotoxicity. Because of the strong similarities between thrombin receptor and PAR-2, we have proposed that PAR-2 may also participate in neurodegeneration. In the present study, we used reverse transcriptase polymerase chain reaction and immunocytochemistry to provide the first evidence that PAR-2 is present in the rat hippocampus. Moreover, we found SLIGRL to be toxic to hippocampal neurons in a concentration-dependent manner (> or = 100 microM). Calcium signaling studies were performed to aid in determining the mechanism by which PAR-2 activation is neurotoxic.

Biological consequences of thrombin receptor deficiency in mice.

The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetycholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2. Our results indicate that ThrR deficiency has a strong impact on fetal development; however. ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.

Selective inhibition of N-formylpeptide-induced neutrophil activation by carbamate-modified peptide analogues.

Stimulation of the leukocyte N-formylpeptide receptor (FPR) induces chemotaxis, cell adhesion, free radical release, and degranulation, responses associated with infection and inflammation. Under conditions where continuous activation of the receptor prevails, neutrophil-dependent tissue damage ensues. Antagonists of the FPR have potential for use as diagnostic and therapeutic agents. Hence, we have synthesized and evaluated a series of amino-terminal carbamate analogues of the peptide Met-Leu-Phe (MLF) in order to determine the structural requirements for imparting agonist or antagonist activity at the human neutrophil FPR. Peptides were evaluated in three in vitro assays: receptor binding, superoxide anion release, and cell adhesion. Unbranched carbamates (methoxycarbonyl, ethoxycarbonyl, and n-butyloxycarbonyl) resulted in agonist activity, whereas branched carbamates (iso-butyloxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl) were antagonists. The peptide antagonists were more potent inhibitors of superoxide anion release than cell adhesion by 4-7-fold. When iso-butyloxycarbonyl-MLF (i-Boc-MLF) was further modified at the carboxy terminus with Lys, antagonist potency was retained but without functional selectivity. Further C-terminal modification with the radionuclide linker diethylenetriaminepentaacetic acid did not alter the potency of i-Boc-MLFK. These results indicate that the switch from agonist to antagonist activity can be achieved by modifying the overall size and shape of the amino-terminal group; that modifications at both the amino and carboxy termini can alter the functional selectivity of the peptide; and that modifications can be tolerated at the carboxy terminus to allow for development of an antagonist for diagnostic applications.

Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders.

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases.

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca2+]i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca2+]i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca2+]i studies. The kinetic profile for [Ca2+]i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca2+]i in guinea pigs, had no effect in rat, and increased [Ca2+]i in all other species. Staurosporine inhibited SFLLRN-induced [Ca2+]i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca2+]i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.

Inhibition of chemotactic peptide-induced neutrophil adhesion to vascular endothelium by cAMP modulators.

An early event associated with neutrophil-dependent tissue damage involves the adhesion of neutrophils to the vascular endothelium and the subsequent release of oxygen-derived free radicals and granule constituents. Elevations in intracellular cAMP are known to inhibit free radical release but not lysosomal enzyme release. The role of cAMP in FMLP-induced neutrophil adhesion was examined in this study by using an in vitro model of neutrophil-endothelial cell adhesion. FMLP stimulated a time- and concentration-dependent increase in human neutrophil adhesion to HUVEC. FMLP-mediated adhesion was inhibited by a diverse group of cAMP modulators: forskolin, isoproterenol, phosphodiesterase IV inhibitors (rolipram and Ro 20-1724), but not phosphodiesterase III inhibitors (milrinone and bemoradan). Endogenous adenosine has previously been shown to mediate FMLP-induced increases in cAMP enhanced in the presence of Ro 20-1724. In this study, adenosine deaminase prevented the inhibitory effects observed with rolipram and Ro 20-1724, implicating endogenous adenosine as a co-modulator of inhibition. FMLP stimulated neutrophil shape change and the surface expression of the beta 2 integrins CD11b/CD18 and CD11a/CD18. Both these responses were inhibited by rolipram but not bemoradan. With the use of 4,4'-diisothiocyanostilbene-2,2'disulfonic acid, we showed that mobilization of the intracellular pool of CD11b/CD18 paralleled adhesion. We conclude that neutrophil-endothelial cell adhesion is attenuated by elevating neutrophil intracellular cAMP and that inhibition of neutrophil CD11b/CD18 surface expression by cAMP accounts for this observed inhibition of adhesion.