PGE2 and PAR-1 in pulmonary fibrosis: a case of biting the hand that feeds you?

MOST MEMBRANE RECEPTORS in mammalian systems are seventransmembrane domain G protein-coupled receptors. Proteinase (or protease)-activated receptors (PARs) are a novel family of G protein-coupled receptors. Unlike other G protein-coupled receptors that require ligand binding for activation, PARs are activated upon cleavage of their extracellular NH2 terminus by serine proteases such as thrombin, trypsin, and tryptase (16). PAR activation is a key player in many physiological responses: platelet aggregation, regulation of vascular tone, increased vascular permeability, granulocyte chemotaxis, Cl secretion in the intestinal epithelium, bone resorption, apoptosis (neurons, tumorigenic cell lines, and intestinal epithelium), and intestinal permeability (6, 16). As a result of these actions, PARs are now valuable therapeutic targets in various disease states, including genetic disorders (e.g., 5q syndrome, refractory anemia), thrombosis and vascular remodeling, cancer, neurological disorders (e.g., Alzheimer’s disease) and diseases as a result of brain trauma, and inflammatory diseases (e.g., psoriasis, asthma, Crohn’s disease) (16). There are four members in the PAR family: PAR-1 through PAR-4. PAR-1 and PAR-3 are dominantly activated by thrombin and are, therefore, essentially thrombin receptors in mammals and humans (12, 22). PAR-2 is insensitive to thrombin but activated by trypsin and trypsin-like enzymes such as mast cell tryptase (4, 7, 17), whereas PAR-4 seems to be activated by both thrombin and trypsin (24). PARs are not only widely expressed in different species (e.g., rat, mouse, Xenopus laevis, bovine, and human) but are also expressed ubiquitously in a variety of (mammalian and human) tissue and cell types, including lung fibroblasts, airway smooth muscle and epithelium, platelets, osteoblasts, connective tissue, vascular smooth muscle and endothelium, keratinocytes, stomach, intestine, kidney, neurons, astrocytes, and skeletal muscle. Although most of the organs and tissues in mammals and humans express functional PARs, distribution of different isoforms of PARs (i.e., PAR-1, -2, -3, and -4) can be cellular and tissue specific (or quantitatively different). Similar to other G protein-coupled receptors, topology of PAR shows that the receptor is composed of an extracellular NH2 terminus, seven transmembrane segments, and a cytoplasmic COOH terminus. The extracellular NH2 termini of PARs contain the protease cleavage sites for different serine proteases. For example, a putative thrombin cleavage site (LDPR/S) has been identified in the NH2 terminus of PAR-1. Thrombin-mediated proteolytic cleavage generates a new tethered ligand (SFLLRN) that interacts with the extracellular loop-2 of the receptor, activating the receptor and its downstream signal transduction cascades. The protease cleavage sites for PAR-2, PAR-3, and PAR-4 are SKGR/S, LPIK/T, and PAPR/G, respectively. The tethered ligand domains are SLIGKV for PAR-2, TERGAP for activating PAR-3, and GYPGQV for activating PAR-4. In addition to the protease cleavage sites, the NH2 terminal DKYEPF hirudin-like domain in PAR-1 also plays an important role in facilitating the interaction of thrombin with the receptor. The extracellular loop-2, where the tethered ligand and receptor interaction occurs, is composed of 24 amino acids that are conserved among different species. The cytoplasmic COOH terminus of PAR-1 also contains sequences that are involved in desensitization and intracellular signaling (16). Based on the PAR activation mechanism and the sequences of the tethered ligand domains and the extracellular loop-2, many synthetic PAR-activating peptides have been designed and demonstrated to be able to activate PARs without cleavage of the NH2 terminus. For example, the thrombin receptor activating peptide (TRAP) is able to interact with the extracellular loop-2 of PAR-1 via its SFLLRN domain, activates the receptor, and stimulates downstream signaling cascades. Although many different PAR-activating peptides (which include the sequences of the tethered ligand domains) are all effective in activating the receptors, the composition of amino acids and the threedimensional structure of the peptides significantly alter the efficacy and potency for activation of the receptors (16). Similar to other G protein-coupled receptors, the downstream signaling cascades of PARs include many kinases and intracellular messengers, such as 1) Gq/11-mediated increase in cytoplasmic free Ca ([Ca ]cyt) by inositol 1,4,5-trisphosphate and diacylglycerol and activation of PKC and calmodulin kinases (CaMKII and CaMKIV); 2) G12/13-mediated activation Rho/Rho kinase and c-Jun NH2-terminal kinase; and 3) G12 mediated Ras/MAPK and PKB. The systemic, tissue, and cellular effects of the serine protease-mediated PAR activation include platelet aggregation, cell proliferation, prostanoid synthesis and release, cytokine production, smooth muscle contraction and mitogenesis, endothelial release of von Willebrand factor and nitric oxide, inhibition of keratinocyte differentiation, neuronal apoptosis, and procollagen production (1, 6, 16, 18). In airway smooth muscle cells and in pulmonary vascular fibroblasts, PAR-1-mediated activation of p70 and PKB is related to phosphatidylinositol 3-kinase. PKB and P70 are two important regulators of cell survival and proliferation (3, 13, 15, 23). Thrombin-mediated activation of PAR-1 has been shown to stimulate the tyrosine phosphorylation of the growth factor receptors, activate MAPK cascade, stimulate transactivation of growth factor receptors, promote cell survival, and enhance mitogenesis. Classically, serine proteases (e.g., thrombin, trypsin, mast cell tryptase) play important roles in diverse biological functions, including clot formation and wound healing (16). Originally identified as a key mediator of the coagulation Address for reprint requests and other correspondence: J. X.-J. Yuan, Dept. of Medicine, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0725 (E-mail: [email protected]). Am J Physiol Lung Cell Mol Physiol 288: L789–L792, 2005; doi:10.1152/ajplung.00016.2005.