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

Carmelle V. Remillard and Jason X.-J. Yuan

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{beta}{gamma}-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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Fig. 1. Schematic diagram depicting the potential mechanisms involved in thrombin-mediated fibroblast proliferation and the PGE2-mediated negative feedback effect on the fibrotic effects of thrombin and other serine proteases. Multiple mechanisms promote or enhance (circled plus sign) pulmonary fibroblast proliferation and migration, whereas only prostanoid E2 (EP2) receptor stimulation acts as a negative feedback regulator implicating downregulation of proteinase (or protease)-activated receptor (PAR) expression and function by PGE2 production. G, 7-transmembrane domain G protein; TRAP, thrombin receptor activating peptide; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; ROK, Rho kinase; JNK, c-Jun NH2-terminal kinase; p70, p70s6k; CaMK, calmodulin kinase II and IV; COX-2, cyclooxygenase-2. Ac, adenylyl cyclase; circled minus sign, inhibition.

 
Although cAMP can directly activate or inhibit certain types of ion channels in the plasma membrane, it exerts its effects mainly by activating cAMP-dependent protein kinase or PKA. The increased cAMP and activated PKA by PGE2 and EP2 receptor activation not only "directly" inhibit thrombin-mediated fibroblast proliferation but also "indirectly" downregulate the mRNA expression of PARs (e.g., PAR-1, -2, -3) (Fig. 1) (19). This negative feedback effect of thrombin on PAR expression via PGE2/EP2 receptor/cAMP-PKA cascade may play an important role in controlling the proliferative or fibrotic effects of thrombin and other serine proteases in normal lung fibroblasts.

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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This work was supported by National Heart, Lung, and Blood Institute Grants HL-64945, HL-54043, HL-66012, and HL-69758.


    ACKNOWLEDGMENTS
 
The authors apologize for the investigators whose work is not included in the figure or cited in this work because of page limitations.


    FOOTNOTES
 

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: xiyuan{at}ucsd.edu)


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 REFERENCES
 

  1. Andersen H, Greenberg DL, Fujikawa K, Xu W, Chung DW, and Davie EW. Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity. Proc Natl Acad Sci USA 96: 11189–11193, 1999.[Abstract/Free Full Text]
  2. Asokananthan N, Graham PT, Fink J, Knight DA, Bakker AJ, McWilliam AS, Thompson PJ, and Stewart GA. Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells. J Immunol 168: 3577–3585, 2002.[Abstract/Free Full Text]
  3. Belham CM, Scott PH, Twomey DP, Gould GW, Wadsworth RM, and Plevin R. Evidence that thrombin-stimulated DNA synthesis in pulmonary arterial fibroblasts involves phosphatidylinositol 3-kinase-dependent p70 ribosomal S6 kinase activation. Cell Signal 9: 109–116, 1997.[CrossRef][ISI][Medline]
  4. Böhm SK, Kong W, Brömme D, Smeekens SP, Anderson DC, Connolly A, Kahn M, Nelken NA, Coughlin SR, Payan DG, and Bunnett NW. Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2. Biochem J 314: 1009–1016, 1996.[ISI][Medline]
  5. Cheng T, Liu D, Griffin JH, Fernández JA, Castellino F, Rosen ED, Fukudome K, and Zlokovic BV. Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective. Nat Med 9: 338–342, 2003.[CrossRef][ISI][Medline]
  6. Flynn AN and Buret AG. Proteinase-activated receptor 1 and cell apoptosis. Apoptosis 9: 729–737, 2004.[CrossRef][ISI][Medline]
  7. Fox MT, Harriott P, Walker B, and Stone SR. Identification of potential activators of proteinase-activated receptor-2. FEBS Lett 417: 267–269, 1997.[CrossRef][ISI][Medline]
  8. Hamilton JR, Moffatt JD, Frauman AG, and Cocks TM. Protease-activated receptor 1 but not PAR2 or PAR4 mediates endothelium-dependent relaxation to thrombin and trypsin in human pulmonary arteries. J Cardiovasc Pharmacol 38: 108–119, 2001.[CrossRef][ISI][Medline]
  9. Hernández-Rodríguez NA, Cambrey AD, Chambers RC, Gray AJ, McAnulty RJ, Laurent GJ, Harrison NK, Southcott AM, duBois RM, Black CM, and Scully MF. Role of thrombin in pulmonary fibrosis. Lancet 346: 1071–1073, 1995.[CrossRef][ISI][Medline]
  10. Hoffmann H, Siebeck M, Spannagl M, Weis M, Geiger R, Jochum M, and Fritz H. Effect of recombinant hirudin, a specific inhibitor of thrombin, on endotoxin-induced intravascular coagulation and acute lung injury in pigs. Am Rev Respir Dis 142: 782–788, 1990.[ISI][Medline]
  11. Hoshi S, Goto M, Koyama N, Nomoto K, and Tanaka H. Regulation of vascular smooth muscle cell proliferation by nuclear factor-{kappa}B and its inhibitor, I-{kappa}B. J Biol Chem 275: 883–889, 2000.[Abstract/Free Full Text]
  12. Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, Timmons C, Tram T, and Coughlin SR. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386: 502–506, 1997.[CrossRef][ISI][Medline]
  13. Johanson SO, Naccache PA, and Crouch MF. A p85 subunit-independent p110{alpha} PI 3-kinase colocalizes with p70 S6 kinase on actin stress fibers and regulates thrombin-stimulated stress fiber formation in Swiss 3T3 cells. Exp Cell Res 248: 223–233, 1999.[CrossRef][ISI][Medline]
  14. Kataoka H, Hamilton JR, McKemy DD, Camerer E, Zheng YW, Cheng A, Griffin C, and Coughlin SR. Protease-activated receptors 1 and 4 mediate thrombin signaling in endothelial cells. Blood 102: 3224–3231, 2003.[Abstract/Free Full Text]
  15. Krymskaya VP, Penn RB, Orsini MJ, Scott PH, Plevin RJ, Walker TR, Eszterhas AJ, Amrani Y, Chilvers ER, and Panettieri RA Jr. Phosphatidylinositol 3-kinase mediates mitogen-induced human airway smooth muscle cell proliferation. Am J Physiol Lung Cell Mol Physiol 277: L65–L78, 1999.[Abstract/Free Full Text]
  16. Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, and Plevin GD. Proteinase-activated receptors. Pharmacol Rev 53: 245–282, 2001.[Abstract/Free Full Text]
  17. Nystedt S, Emilsson K, Wahlestedt C, and Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 91: 9208–9212, 1994.[Abstract/Free Full Text]
  18. Shapiro MJ, Trejo J, Zeng D, and Coughlin SR. Role of the thrombin receptor's cytoplasmic tail in intracellular trafficking. Distinct determinants for agonist-triggered versus tonic internalization and intracellular localization. J Biol Chem 271: 32874–32880, 1996.[Abstract/Free Full Text]
  19. Sokolova E, Grishina Z, Bühling F, Welte T, and Reiser G. Protease-activated receptor-1 in human lung fibroblasts mediates a negative feedback downregulation via prostaglandin E2. Am J Physiol Lung Cell Mol Physiol 288: L793–L802, 2005.
  20. Tiruppathi C, Freichel M, Vogel SM, Paria BC, Mehta D, Flockerzi V, and Malik AB. Impairment of store-operated Ca2+ entry in TRPC4–/– mice interferes with increase in lung microvascular permeability. Circ Res 91: 70–76, 2002.[Abstract/Free Full Text]
  21. Tokunou T, Ichiki T, Takeda K, Funakoshi Y, Iino N, and Takeshita A. cAMP response element-binding protein mediates thrombin-induced proliferation of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 21: 1764–1769, 2001.[Abstract/Free Full Text]
  22. Vu TK, Hung DT, Wheaton VI, and Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64: 1057–1068, 1991.[CrossRef][ISI][Medline]
  23. Walker TR, Moore SM, Lawson MF, Panettieri RA Jr, and Chilvers ER. Platelet-derived growth factor-BB and thrombin activate phosphoinositide 3-kinase and protein kinase B: role in mediating airway smooth muscle proliferation. Mol Pharmacol 54: 1007–1015, 1998.[Abstract/Free Full Text]
  24. Xu WF, Andersen H, Whitmore TE, Presnell SR, Yee DP, Ching A, Gilbert T, Davie EW, and Foster DC. Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci USA 95: 6642–6646, 1998.[Abstract/Free Full Text]




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