Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California
MOST MEMBRANE RECEPTORS in mammalian systems are seven-transmembrane 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 three-dimensional 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 Ca2+ ([Ca2+]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 p70s6k and PKB is related to phosphatidylinositol 3-kinase. PKB and P70s6k 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 cascade and a potent activator of platelet aggregation, thrombin has been identified as a physiological activator of PAR-1 and PAR-2. More recently, studies have shown that thrombin may also contribute to the development of pulmonary fibrosis and acute lung injury (9, 10), primarily via PAR-1 activation. In a similar study, pharmacological activation of PAR-1 and PAR-2 by agonist peptides, or TRAP, has also been linked to the production of cytokines (IL-6, IL-8) in inflamed lungs (2). In the vasculature itself, thrombin has effects on all cell types that make up the arterial wall. In the tunica intima, thrombin triggers 1) endothelial cell rounding (increasing permeability), 2) endothelial cell migration, and 3) endothelial production of cytokines, growth factors, and matrix proteins (14, 20). At a more basic level, thrombin signaling appears to involve PAR-1 (and PAR-4) activation, elevating [Ca2+]cyt levels and causing ERK phosphorylation, and enhancing the expression of early growth response 1 and c-Fos, two immediate early genes that have been implicated in inflammation and tissue remodeling in response to injury (14). In support of this finding, another study has suggested that activated protein C, which activates PAR-1, can inhibit hypoxia-induced human brain endothelial cell apoptosis (5), casting PAR-1 activation in a neuroprotective role. In the tunica media, thrombin enhances smooth muscle proliferation (11, 21) and causes endothelium-dependent relaxation (8), the latter being coupled to activation of PAR-1 or a PAR-1-like receptor in human pulmonary arteries.
Although PAR-1 activation by thrombin promotes pulmonary fibrosis due to fibroblast proliferation and differentiation, there is evidence that PAR-1 and PAR-2 activation may also stimulate airway epithelial cell production of PGE2, an antifibrotic mediator (2). In the report from Sokolova et al., one of the current articles in focus (Ref. 19, see p. L793 in this issue), the authors examine the cyclical relationship between PAR activation and PGE2 production in thrombin-induced pulmonary fibroblast activation. Their data show that, although PAR-2 is upregulated, PAR-1 and -3 are unchanged in fibrotic fibroblasts, suggesting that PAR-2 may be important in the development of fibroproliferative disorders. In thrombin- and trypsin-treated cells, PAR-1 activation caused transient cathepsin-regulated Ca2+ increases in lung fibroblasts. Both PAR-1 and -3 activation (by thrombin and thrombin receptor agonist, respectively) dose dependently enhanced PGE2 release via cyclooxygenase-2 (COX-2) production. This increase in PGE2 was diminished in thrombin-treated fibrotic fibroblasts. Finally, thrombin-induced PAR-1 upregulation is significantly reduced by PGE2 application, the latter acting via E-prostanoid 2 (EP2) receptors and cAMP production. Therefore, PGE2 synthesis, in addition to its role in limiting cell proliferation and collagen synthesis, serves a novel protective role in preventing lung fibroblast activation by thrombin, further repressing lung fibrosis.
Using tissue samples from patients undergoing diagnostic open lung biopsy or pneumonectomia for tumor resection, Sokolova et al. (19) provide compelling evidence that 1) normal lung fibroblasts express PAR-1, PAR-2, and PAR-3; 2) PAR-2 is significantly upregulated in fibrotic fibroblasts compared with normal fibroblasts, although the relative expression level in both normal and fibrotic fibroblasts is lower than in PAR-1 and PAR-3; 3) treatment of human lung fibroblasts with thrombin, TRAP, and trypsin all increased [Ca2+]cyt, indicating functional expression of PARs; 4) thrombin-mediated rise in [Ca2+]cyt enhances PGE2 synthesis and release by upregulating COX-2 expression; and 5) the thrombin- or TRAP-mediated PGE2 release is significantly inhibited in fibrotic fibroblasts compared with normal fibroblasts.
As shown in Fig. 1, thrombin-mediated proteolytic cleavage via the cleavage site facilitates the tethered ligand domain to interact with the extracellular loop-2 and activates the receptor (e.g., PAR-1). PAR activating peptides (e.g., TRAP), which include the various tethered ligand domains, can also activate the receptor by directly interacting with the extracellular loop-2 (without cleavage of the NH2 terminus). The downstream signal transduction pathways vary in different cells and tissues. One of the critical pathways is the Gq/11-mediated increase in [Ca2+]cyt that not only activates CaMKII (or CaMKIV) and PKC but upregulates mRNA and protein expression of COX-2 (see Ref. 19). The increased amount of COX-2 enzyme and the Ca2+-mediated increase in COX-2 enzymatic activity both enhance the production of PGH2 and PGE2 (via the PGE synthase) and release of PGE2 (Fig. 1). Extracellular or intercellular PGE2 then activates the EP2 via an autocrine mechanism and increases cytoplasmic levels of cAMP.
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In fibroblasts from patients with fibrosis, the authors (19) show that the mRNA expression of PAR-2 was upregulated, whereas the thrombin- (and TRAP-) mediated PGE2 release is attenuated. The increased PAR-2 would enhance the thrombin-mediated proliferative or fibrotic effects on pulmonary fibroblasts, whereas the inhibited PGE2 would significantly facilitate or enhance the thrombin-mediated fibrotic effects. One of the important findings in this study is that inhibited negative feedback pathway, the thrombin-mediated PGE2 release, and cAMP/PKA-mediated inhibitory effects on fibroblast proliferation and PAR expression may play a critical role in the development of pulmonary fibrosis.
The observations from this study also suggest an important concept: a pathogenic or etiological mechanism of a disease (e.g., pulmonary fibrosis) always includes a "triggering process" that mediates the transition from a normal to a diseased phenotype, accompanied by a positive- and a negative-feedback pathway that "ensure and control" the development (and progression) of the disease. Regulation or modulation of the feedback pathways not only plays an important role in the pathogenesis but also plays a pivotal role in developing therapeutic approaches for the disease. Searching for the sequences of events that are responsible for the phenotypical transition and progression of normal fibroblasts to fibrotic fibroblasts is important and may involve multiple factors and pathways.
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