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Factor Xa Stimulates Proinflammatory and Profibrotic Responses in Fibroblasts via Protease-Activated Receptor-2 Activation

2008; Elsevier BV; Volume: 172; Issue: 2 Linguagem: Inglês

10.2353/ajpath.2008.070347

1525-2191

Keren Borensztajn, Jurriën Stiekema, S.W.R. Nijmeijer, Pieter H. Reitsma, Maikel P. Peppelenbosch, C. Arnold Spek,

Venous Thromboembolism Diagnosis and Management

Coagulation proteases have been suggested to play a role in the pathogenesis of tissue remodeling and fibrosis. We therefore assessed the proinflammatory and fibroproliferative effects of coagulation protease factor (F)Xa. We show that FXa elicits a signaling response in C2C12 and NIH3T3 fibroblasts. FXa-induced ERK1/2 phosphorylation was dependent on protease-activated receptor (PAR)-2 cleavage because desensitization with a PAR-2 agonist (trypsin) but not a PAR-1 agonist (thrombin) abolished FXa-induced signal transduction and PAR-2 siRNA abolished FXa-induced ERK1/2 phosphorylation. The PAR-2-dependent cellular effects of FXa led to fibroblast proliferation, migration, and differentiation into myofibroblasts, as demonstrated by the expression of α-smooth muscle actin and desmin, followed by the secretion of the cytokines monocyte chemotactic protein-1 and interleukin-6 as well as the expression of the fibrogenic proteins transforming growth factor-β and fibronectin. To assess the relevance of FXa-induced proliferation and cell migration, we examined the effect of FXa in a wound scratch assay. Indeed, FXa facilitated wound healing in a PAR-2- and ERK1/2-dependent manner. Taken together, these results support the notion that, beyond its role in coagulation, FXa-dependent PAR-2 cleavage might play a role in the progression of tissue fibrosis and remodeling. Coagulation proteases have been suggested to play a role in the pathogenesis of tissue remodeling and fibrosis. We therefore assessed the proinflammatory and fibroproliferative effects of coagulation protease factor (F)Xa. We show that FXa elicits a signaling response in C2C12 and NIH3T3 fibroblasts. FXa-induced ERK1/2 phosphorylation was dependent on protease-activated receptor (PAR)-2 cleavage because desensitization with a PAR-2 agonist (trypsin) but not a PAR-1 agonist (thrombin) abolished FXa-induced signal transduction and PAR-2 siRNA abolished FXa-induced ERK1/2 phosphorylation. The PAR-2-dependent cellular effects of FXa led to fibroblast proliferation, migration, and differentiation into myofibroblasts, as demonstrated by the expression of α-smooth muscle actin and desmin, followed by the secretion of the cytokines monocyte chemotactic protein-1 and interleukin-6 as well as the expression of the fibrogenic proteins transforming growth factor-β and fibronectin. To assess the relevance of FXa-induced proliferation and cell migration, we examined the effect of FXa in a wound scratch assay. Indeed, FXa facilitated wound healing in a PAR-2- and ERK1/2-dependent manner. Taken together, these results support the notion that, beyond its role in coagulation, FXa-dependent PAR-2 cleavage might play a role in the progression of tissue fibrosis and remodeling. Fibroblasts comprise the most abundant cell type in connective tissues and are central players in organ homeostasis. Their main function is to maintain the structural integrity of connective tissue by secreting collagen and fibronectin, which are principal components of the extracellular matrix (ECM). On injury, the fibrotic response orchestrated by fibroblasts is a critical component of tissue restoration and wound healing. Fibroblasts are frequently exposed to coagulation proteases after vascular damage. As fibroblasts constitutively express tissue factor, vascular injury exposes tissue factor to circulating factor (F)VII.1Furie B Furie BC The molecular basis of blood coagulation.Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (980) Google Scholar After activation, FVIIa converts FX into its active form, FXa, which subsequently converts prothrombin into thrombin leading to the formation of a fibrin clot.On the action of cytokines and growth factors secreted by monocytes, fibroblasts migrate into the wound and subsequently proliferate and concomitantly differentiate into myofibroblasts2Tomasek JJ Gabbiani G Hinz B Chaponnier C Brown RA Myofibroblasts and mechano-regulation of connective tissue remodelling.Nat Rev Mol Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3066) Google Scholar, 3Li Y Foster W Deasy BM Chan Y Prisk V Tang Y Cummins J Huard J Transforming growth factor-beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis.Am J Pathol. 2004; 164: 1007-1019Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar that develop ultrastructural and phenotypic characteristics of smooth muscle (SM) cells. The neoexpression of α-SM actin (α-SMA), typical of SM cells located in the vessel wall, is the hallmark of myofibroblastic cells.4Darby I Skalli O Gabbiani G Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing.Lab Invest. 1990; 63: 21-29PubMed Google Scholar Myofibroblasts, which have enhanced metabolic activity as compared to undifferentiated fibroblasts, synthesize components of the new ECM, thereby restoring the structure and function of the injured tissue. In addition, myofibroblasts amplify the healing process by secreting inflammatory mediators.5Diegelmann RF Evans MC Wound healing: an overview of acute, fibrotic and delayed healing.Front Biosci. 2004; 9: 283-289Crossref PubMed Scopus (1444) Google ScholarFibroblast migration, proliferation, and differentiation in combination with enhanced ECM synthesis require tight regulation. The escape to self-limiting control results in a broad range of pathologies characterized by fibrotic disorders attributable to excessive fibroblast proliferation and deregulated ECM deposition.5Diegelmann RF Evans MC Wound healing: an overview of acute, fibrotic and delayed healing.Front Biosci. 2004; 9: 283-289Crossref PubMed Scopus (1444) Google Scholar, 6Christen T Verin V Bochaton-Piallat M Popowski Y Ramaekers F Debruyne P Camenzind E van Eys G Gabbiani G Mechanisms of neointima formation and remodeling in the porcine coronary artery.Circulation. 2001; 103: 882-888Crossref PubMed Scopus (144) Google Scholar Eventually, the accumulation of connective tissue compromises organ function and often leads to premature death. Additionally, vascular remodeling, which is characterized by deregulated fibroproliferative responses represents a common theme of many cardiovascular abnormalities. Mechanistic studies suggest that transforming growth factor (TGF)-β is the most potent cytokine inducing ECM synthesis by myofibroblasts.7Border WA Noble NA Transforming growth factor beta in tissue fibrosis.N Engl J Med. 1994; 331: 1286-1292Crossref PubMed Scopus (2994) Google ScholarStrikingly, many of the tissue fibrosis and remodeling disorders are associated with activation of the coagulation cascade. Persistent fibrin deposition has been observed in renal fibrotic disease.8Eneström S Druid H Rammer L Fibrin deposition in the kidney in post-ischaemic renal damage.Br J Exp Pathol. 1988; 69: 387-394PubMed Google Scholar In lung fibrosis patients, thrombin levels but also zymogen FX levels are increased together with enhanced fibrin deposition.9Idell S Coagulation, fibrinolysis, and fibrin deposition in acute lung injury.Crit Care Med. 2003; 31: S213-S220Crossref PubMed Google Scholar Abundant fibrin deposition is also observed in atherosclerosis,10Spronk HM van der Voort D Ten Cate H Blood coagulation and the risk of atherothrombosis: a complex relationship.Thromb J. 2004; 2: 12Crossref PubMed Scopus (92) Google Scholar and patients with a thrombus after coronary angioplasty (which is known to contain thrombin and FXa11Prager NA Abendschein DR McKenzie CR Eisenberg PR Role of thrombin compared with factor Xa in the procoagulant activity of whole blood clots.Circulation. 1995; 92: 962-967Crossref PubMed Scopus (104) Google Scholar) at the site of vascular injury are at higher risk of restenosis.12Bauters C Lablanche JM McFadden EP Hamon M Bertrand ME Relation of coronary angioscopic findings at coronary angioplasty to angiographic restenosis.Circulation. 1995; 92: 2473-2479Crossref PubMed Scopus (61) Google ScholarThe identification of protease-activated receptors (PARs), a family of G-protein-coupled receptors, constitutes a potential link between activation of the coagulation cascade and the progression of tissue remodeling or fibrotic disease. Indeed, besides its properties in blood coagulation, FXa and thrombin induce intracellular signaling via proteolytic cleavage of PARs. PAR-1, -3, and -4 are cleaved by thrombin,13Coughlin SR Protease-activated receptors in vascular biology.Thromb Haemost. 2001; 86: 298-307PubMed Google Scholar whereas FXa can activate both PAR-1 and PAR-2.14Riewald M Kravchenko VV Petrovan RJ O'Brien PJ Brass LF Ulevitch RJ Ruf W Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1.Blood. 2001; 97: 3109-3116Crossref PubMed Scopus (174) Google ScholarAlthough the role of thrombin-induced PAR-1 activation in tissue repair has been well documented,15Bogatkevich GS Tourkina E Silver RM Ludwicka-Bradley A Thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the proteolytically activated receptor-1 and a protein kinase C-dependent pathway.J Biol Chem. 2001; 276: 45184-45192Crossref PubMed Scopus (161) Google Scholar, 16Zhang A Liu X Cogan JG Fuerst MD Polikandriotis JA Kelm Jr, RJ Strauch AR YB-1 coordinates vascular smooth muscle alpha-actin gene activation by transforming growth factor beta1 and thrombin during differentiation of human pulmonary myofibroblasts.Mol Biol Cell. 2005; 16: 4931-4940Crossref PubMed Scopus (46) Google Scholar less is known about the contribution of FXa. In addition, the involvement of PAR-2 in wound healing and fibrotic disease has only recently begun to unfold. For instance, α-SMA and PAR-2 expression are correlated in a renal interstitial fibrosis model.17Xiong J Zhu Z Liu J Wang Y Li Z Role of protease activated receptor-2 expression in renal interstitial fibrosis model in mice.J Huazhong Univ Sci Technolog Med Sci. 2005; 25: 523-526Crossref PubMed Google Scholar Pulmonary PAR-2 is highly expressed in acute and chronic lung injury, which suggests that PAR-2 may participate in inflammation and fibroproliferation.18Cederqvist K Haglund C Heikkila P Hollenberg MD Karikoski R Andersson S High expression of pulmonary proteinase-activated receptor 2 in acute and chronic lung injury in preterm infants.Pediatr Res. 2005; 57: 831-836Crossref PubMed Scopus (39) Google Scholar PAR-2 furthermore maintains pancreatic fibrosis through increased proliferation and collagen production in pancreatic stellate cells.19Masamune A Kikuta K Satoh M Suzuki N Shimosegawa T Protease-activated receptor-2-mediated proliferation and collagen production of rat pancreatic stellate cells.J Pharmacol Exp Ther. 2005; 312: 651-658Crossref PubMed Scopus (64) Google Scholar Finally, IgA nephropathy biopsies revealed the presence of PAR-2.20Grandaliano G Pontrelli P Cerullo G Monno R Ranieri E Ursi M Loverre A Gesualdo L Schena FP Protease-activated receptor-2 expression in IgA nephropathy: a potential role in the pathogenesis of interstitial fibrosis.J Am Soc Nephrol. 2003; 14: 2072-2083Crossref PubMed Scopus (71) Google ScholarInterestingly, FXa triggers signaling pathways involved in the regulation of cell growth and ECM deposition: it stimulates proliferation of fibroblasts and SM cells21Borensztajn KS Bijlsma MF Groot AP Bruggemann LW Versteeg HH Reitsma PH Peppelenbosch MP Spek CA Coagulation factor Xa drives tumour cells into apoptosis through BH3-only protein Bim up-regulation.Exp Cell Res. 2007; 313: 2622-2633Crossref PubMed Scopus (23) Google Scholar, 22Blanc-Brude OP Archer F Leoni P Derian C Bolsover S Laurent GJ Chambers RC Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation.Exp Cell Res. 2005; 304: 16-27Crossref PubMed Scopus (79) Google Scholar, 23Koo BH Chung KH Hwang KC Kim DS Factor Xa induces mitogenesis of coronary artery smooth muscle cell via activation of PAR-2.FEBS Lett. 2002; 523: 85-89Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 24Rauch BH Millette E Kenagy RD Daum G Clowes AW Thrombin- and factor Xa-induced DNA synthesis is mediated by transactivation of fibroblast growth factor receptor-1 in human vascular smooth muscle cells.Circ Res. 2004; 94: 340-345Crossref PubMed Scopus (72) Google Scholar and induces the expression of interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP-1),25Bachli EB Pech CM Johnson KM Johnson DJ Tuddenham EG McVey JH Factor Xa and thrombin, but not factor VIIa, elicit specific cellular responses in dermal fibroblasts.J Thromb Haemost. 2003; 1: 1935-1944Crossref PubMed Scopus (49) Google Scholar and TGF-β.20Grandaliano G Pontrelli P Cerullo G Monno R Ranieri E Ursi M Loverre A Gesualdo L Schena FP Protease-activated receptor-2 expression in IgA nephropathy: a potential role in the pathogenesis of interstitial fibrosis.J Am Soc Nephrol. 2003; 14: 2072-2083Crossref PubMed Scopus (71) Google Scholar Moreover, FXa induces ERK1/2 phosphorylation in several cell lines, both murine and human, such as SM cells,26Millette E Rauch BH Defawe O Kenagy RD Daum G Clowes AW Platelet-derived growth factor-BB-induced human smooth muscle cell proliferation depends on basic FGF release and FGFR-1 activation.Circ Res. 2005; 96: 172-179Crossref PubMed Scopus (100) Google Scholar, 27Koo BH Kim DS Factor Xa induces mitogenesis of vascular smooth muscle cells via autocrine production of epiregulin.J Biol Chem. 2003; 278: 52578-52586Crossref PubMed Scopus (21) Google Scholar cancer cells,14Riewald M Kravchenko VV Petrovan RJ O'Brien PJ Brass LF Ulevitch RJ Ruf W Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1.Blood. 2001; 97: 3109-3116Crossref PubMed Scopus (174) Google Scholar, 21Borensztajn KS Bijlsma MF Groot AP Bruggemann LW Versteeg HH Reitsma PH Peppelenbosch MP Spek CA Coagulation factor Xa drives tumour cells into apoptosis through BH3-only protein Bim up-regulation.Exp Cell Res. 2007; 313: 2622-2633Crossref PubMed Scopus (23) Google Scholar, 28Morris DR Ding Y Ricks TK Gullapalli A Wolfe BL Trejo J Protease-activated receptor-2 is essential for factor VIIa and Xa-induced signaling, migration, and invasion of breast cancer cells.Cancer Res. 2006; 66: 307-314Crossref PubMed Scopus (174) Google Scholar endothelial cells,29Camerer E Kataoka H Kahn M Lease K Coughlin SR Genetic evidence that protease-activated receptors mediate factor Xa signaling in endothelial cells.J Biol Chem. 2002; 277: 16081-16087Crossref PubMed Scopus (142) Google Scholar mesangial cells,30Tanaka M Arai H Liu N Nogaki F Nomura K Kasuno K Oida E Kita T Ono T Role of coagulation factor Xa and protease-activated receptor 2 in human mesangial cell proliferation.Kidney Int. 2005; 67: 2123-2133Crossref PubMed Scopus (33) Google Scholar and fibroblasts.22Blanc-Brude OP Archer F Leoni P Derian C Bolsover S Laurent GJ Chambers RC Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation.Exp Cell Res. 2005; 304: 16-27Crossref PubMed Scopus (79) Google Scholar, 31Versteeg HH Hoedemaeker I Diks SH Stam JC Spaargaren M van Bergen En Henegouwen PM van Deventer SJ Peppelenbosch MP Factor VIIa/tissue factor-induced signaling via activation of Src-like kinases, phosphatidylinositol 3-kinase, and Rac.J Biol Chem. 2000; 275: 28750-28756Crossref PubMed Scopus (96) Google Scholar The phosphorylation of ERK1/2 is widely used as a surrogate marker for PAR-1 and PAR-2 activation.32Ossovskaya VS Bunnett NW Protease-activated receptors: contribution to physiology and disease.Physiol Rev. 2004; 84: 579-621Crossref PubMed Scopus (914) Google Scholar Moreover, the activation of ERK1/2 has been closely associated with cellular proliferation,33Dunn C Wiltshire C MacLaren A Gillespie DA Molecular mechanism and biological functions of c-Jun N-terminal kinase signalling via the c-Jun transcription factor.Cell Signal. 2002; 14: 585-593Crossref PubMed Scopus (168) Google Scholar, 34Meloche S Pouyssegur J The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition.Oncogene. 2007; 26: 3227-3239Crossref PubMed Scopus (835) Google Scholar protein synthesis,35Padmasekar M Nandigama R Wartenberg M Schluter KD Sauer H The acute phase protein alpha2-macroglobulin induces rat ventricular cardiomyocyte hypertrophy via ERK1,2 and PI3-kinase/Akt pathways.Cardiovasc Res. 2007; 75: 118-128Crossref PubMed Scopus (52) Google Scholar and more recently wound healing.36Fitsialos G Chassot AA Turchi L Dayem MA LeBrigand K Moreilhon C Meneguzzi G Busca R Mari B Barbry P Ponzio G Transcriptional signature of epidermal keratinocytes subjected to in vitro scratch wounding reveals selective roles for ERK1/2, p38, and phosphatidylinositol 3-kinase signaling pathways.J Biol Chem. 2007; 282: 15090-15102Crossref PubMed Scopus (96) Google Scholar Taken together, these data support the hypothesis that FXa is involved in the pathogenesis of tissue fibrosis and remodeling. Therefore, we studied FXa-induced intracellular signaling and determined the potential consequence on proinflammatory and profibrotic responses.Materials and MethodsCell CultureGrowth-arrested fibroblasts NIH3T3 (CRL-1658) and myoblasts C2C12 (CRL-1772) were purchased from American Type Culture Collection (Manassas, VA), and human dermal fibroblasts were obtained from Promocell (Heidelberg, Germany). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). Unless stated otherwise, cells were washed twice with phosphate-buffered saline (PBS), serum-starved for 4 hours, and subsequently stimulated as described.AntibodiesPrimary antibodies against α-actin, β-actin, desmin, TGF-β, fibronectin, PAR-1 (H-111), and PAR-2 (SAM11) were from Santa Cruz Biotechnology, Santa Cruz, CA. Phospho-FAK, phospho-p42/p44 MAP kinase, and phospho-Src antibodies were purchased from Cell Signaling Technology (Beverly, MA).ReagentsHuman FXa and thrombin were obtained from Kordia (Leiden, the Netherlands). Tick anticoagulant peptide (TAP) was kindly provided by Dr. Georges Vlasuk (Corvas International Inc., San Diego, CA). Hirudin was purchased from Calbiochem (San Diego, CA). All experiments involving 24-hour incubation with FXa included hirudin to block any thrombin signaling as described.14Riewald M Kravchenko VV Petrovan RJ O'Brien PJ Brass LF Ulevitch RJ Ruf W Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1.Blood. 2001; 97: 3109-3116Crossref PubMed Scopus (174) Google Scholar Control experiments demonstrated that hirudin alone had no effect in our assays. ERK1/2 inhibitor U0126 was purchased from Cell Signaling Technology. Phalloidin-fluorescein isothiocyanate and phalloidin-tetramethyl-rhodamine isothiocyanate were purchased from Sigma (St. Louis, MO). Cell tracker green CMFDA (5-chloromethylfluorescein diacetate) was obtained from Molecular Probes (Eugene, OR). PP1 was from Biomol (Plymouth Meeting, PA). PAR-1 cleavage-blocking monoclonal antibody ATAP2 was obtained from Santa Cruz Biotechnology and used as previously reported.37Feistritzer C Lenta R Riewald M Protease-activated receptors-1 and -2 can mediate endothelial barrier protection: role in factor Xa signaling.J Thromb Haemost. 2005; 3: 2798-2805Crossref PubMed Scopus (73) Google Scholar Predesigned PAR-2 (no. 158457) and control siRNA (no. 4611) was obtained from Ambion Inc. (Austin, TX). One U/ml FXa corresponds to 174 nmol/L.Western BlotCells were lysed in Laemmli lysis buffer and incubated for 5 minutes at 95°C, and whole cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to an immobilon-P polyvinylidene difluoride membrane (Millipore, Billerica, MA). Membranes were incubated overnight at 4°C with primary antibodies. All secondary antibodies were horseradish peroxidase-conjugated from DakoCytomation (Glostrup, Denmark). Blots were imaged using Lumilight Plus ECL substrate from Roche (Basel, Switzerland) on a GeneGnome imager (Syngene, Cambridge, UK). Densitometry was performed in Photoshop 7.0 (Adobe Systems, San Jose, CA) using the histogram function in a selected area of constant size for each band. Background was subtracted and values for the protein of interest were corrected for those of β-actin.ImmunofluorescenceCells were grown on coverslips at 20 to 30% confluence. After pretreatment (when indicated), and subsequent stimulation with 0.75 U/ml FXa or PBS for the indicated time periods, cells were washed with PBS and fixed with 3.7% formaldehyde for 20 minutes at room temperature. Fixed cells were permeabilized and blocked in PBS/0.1% Triton X-100 (PBS-T) supplemented with 10% FCS for 1 hour. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (200 ng/ml, Roche) in PBS-T for 30 minutes. The actin cytoskeleton was stained with 10 μg/ml of phalloidin-tetramethyl-rhodamine isothiocyanate in PBS-T supplemented with 1% bovine serum albumin. For imaging FAK or Src phosphorylation, cells were blocked as for phalloidin staining but subsequently incubated with the appropriate antibody and subsequent fluorescein isothiocyanate-conjugated secondary antibody (both in 3% bovine serum albumin/PBS-T). After staining, the cells were washed and mounted in Mowiol/DABCO aqueous mounting medium (Vector Laboratories, Burlingame, CA). Images were visualized using an epifluorescence microscope (Leica DMRA, Wetzlar, Germany) and captured on a cooled charge-coupled camera (KX Series; Apogee, Auburn, CA) operated by ImagePro Plus software (Media Cybernetics, Silver Spring, MD).Proliferation Assay (MTT)Cells seeded at a density of 104/cm2 in 96-well plates in DMEM supplemented with 1% FCS, were (if indicated) pretreated with TAP (200 nmol/L), hirudin (100 nmol/L), or the ERK1/2 inhibitor U0126 (10 μmol/L), after which they were stimulated with FXa (1 U/ml) or PBS as a control. Cell survival was determined at the indicated intervals using a MTT assay as described before.38Versteeg HH Spek CA Richel DJ Peppelenbosch MP Coagulation factors VIIa and Xa inhibit apoptosis and anoikis.Oncogene. 2004; 23: 410-417Crossref PubMed Scopus (87) Google ScholarTransfectionsTransfections of predesigned siRNAs were performed using HiPerfect kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For transfections in six-well plates, 75 ng of siRNA was used at a 1:3 ratio of RNA/HiPerfect reagent. Cells were incubated with transfection complexes for 16 hours, after which fresh medium was added for 6 hours preceding further experimentation.Cell Migration AssayCells were grown to 70% confluence in six-well plates and before experimentation labeled for 1 hour with 10 μmol/L CellTracker Green in serum-free medium. The dye was fixed by a 1-hour incubation in medium with 10% FCS. Subsequently, cells were washed and detached with 2 mmol/L ethylenediaminetetraacetic acid in PBS. Next, cells were resuspended in serum-free medium and transferred to 8-μm pore size HTS FluoroBlok cell culture inserts (BD Falcon, Franklin Lakes, NJ). FXa (1 U/ml) or PBS (control), were added to the bottom well and cell migration was assessed as described before.39Bijlsma MF Borensztajn KS Roelink H Peppelenbosch MP Spek CA Sonic hedgehog induces transcription-independent cytoskeletal rearrangement and migration regulated by arachidonate metabolites.Cell Signaling. 2007; 19: 2596-2604Crossref PubMed Scopus (80) Google Scholar Briefly, fluorescence values representing the number of cells on the bottom side of the insert were read during 30 cycles (each cycle comprising four readings spanning 2 minutes) at 37°C on a Series 4000 CytoFluor multiwell plate reader (Perseptive Biosystems, Framingham, MA). The raw fluorescence data were corrected for background fluorescence and fading of the fluorophore, and the data were plotted with GraphPad Prism-4 (San Diego, CA).Wound Scratch AssayCells were plated in six-well plates and maintained in DMEM supplemented with 10% FCS. After the cells reached 80 to 90% confluence, a wound was created in the center of the cell monolayer by a sterile plastic pipette tip. Immediately thereafter, the cells were washed with PBS to remove floating cellular debris and reincubated for an additional 18 hours with either serum-free medium (for use as a negative control), DMEM medium supplemented with 10% FCS (as positive control), or serum-free medium containing FXa. When indicated, cells were preincubated with PP1 (10 μmol/L), U0126 (10 μmol/L), or TAP (200 nmol/L) 30 minutes before wounding the cells. The ability of cells to proliferate and migrate into the wound area was assessed after 18 hours by comparing the 0- and 18-hour phase-contrast micrographs of six marked points along the wounded area at each plate. The percentage of nonrecovered wound area was calculated by dividing the nonrecovered area after 18 hours by the initial wound area at 0 time.Cytokine/Chemokine AssayIL-6, MCP-1, interferon-γ, tumor necrosis factor (TNF)-α, and IL-10 were measured using the BD Cytometric bead array mouse inflammation kit (Becton Dickinson, Franklin Lakes, NJ) as described before.40Slofstra SH Cate HT Spek CA Low dose endotoxin priming is accountable for coagulation abnormalities and organ damage observed in the Shwartzman reaction. A comparison between a single-dose endotoxemia model and a double-hit endotoxin-induced Shwartzman reaction.Thromb J. 2006; 4: 13Crossref PubMed Scopus (27) Google Scholar Detection limits were 10 pg/ml.StatisticsStatistical analyses were conducted using GraphPad Prism version 4.00 software. Data are expressed as means ± SEM. Comparisons between two conditions were analyzed using Mann-Whitney t-tests.ResultsExpression of PAR-1 and PAR-2 by NIH3T3 and C2C12 CellsFor efficient signal transduction of FXa, it is essential that its cellular receptors are expressed. Therefore, we assessed the presence of PAR-1 and PAR-2 on the NIH3T3 and C2C12 fibroblast cells used in this study. As shown in Figure 1A, both NIH3T3 (lane 1) and C2C12 (lane 2) cells constitutively expressed PAR-1 and PAR-2.PAR-1 and PAR-2 Expressed by NIH3T3 and C2C12 Are FunctionalWe next determined whether the expression of PAR-1 and PAR-2 was associated with functional responses in both cell lines. As read-out we examined the phosphorylation of ERK1/2, which is widely used as a surrogate marker for PAR-1 and PAR-2 activation.32Ossovskaya VS Bunnett NW Protease-activated receptors: contribution to physiology and disease.Physiol Rev. 2004; 84: 579-621Crossref PubMed Scopus (914) Google Scholar Cells were serum-starved for 4 hours, stimulated with thrombin (1 U/ml corresponding to 10 nmol/L), a specific PAR-1 agonist; trypsin (200 nmol/L), a specific PAR-2 agonist; or PBS as negative control. As shown in Figure 1B for C2C12 cells, stimulation with thrombin (lane 2) or trypsin (lane 3) induced strong phosphorylation of ERK1/2 as compared to the negative control (lane1). Similar results were obtained with NIH3T3 cells (data not shown). Hence, both PAR-1 and PAR-2 are functionally active on the fibroblasts.FXa Elicits Signal Transduction in NIH3T3 and C2C12 CellsTo assess the capacity of FXa to induce intracellular signaling in fibroblasts, we examined FXa-induced phosphorylation of ERK1/2. Figure 1C shows that signaling induced by FXa was already detectable at 0.25 U/ml (lane 2). ERK1/2 phosphorylation strongly increased using 0.5 U/ml FXa (lane 3) and was maximal at 1 U/ml (lane 5). The same results were obtained in NIH3T3 cells (data not shown). To verify the specificity of ERK1/2 phosphorylation induced by FXa, cells were preincubated with TAP, an FXa inhibitor, or hirudin, a thrombin inhibitor. As shown in Figure 1D for C2C12 cells, pretreatment with TAP (lane 2), but not with hirudin (lane 3), almost completely inhibited FXa-induced ERK1/2 phosphorylation. Similar results were obtained with NIH3T3 cells (data not shown). Hence, FXa specifically induces ERK1/2 phosphorylation, which is independent of thrombin formation.FXa Signals via PAR-2 ActivationTo identify the receptor by which FXa induces signaling, we tested whether the activation of PAR-1 is required for FXa signaling. Cells were treated with FXa in the absence or presence of the PAR-1-blocking antibody ATAP2. As shown in Figure 1E, ERK1/2 phosphorylation was strongly induced by FXa (lane 3) independently of the blocking antibody (compare lanes 3 and 6). In contrast, nearly 70% of ERK1/2 phosphorylation induced by thrombin (lane 2) was inhibited by antibody treatment (lane 5). These results suggest that PAR-1 activation is not required for FXa-mediated signaling (in contrast to thrombin-mediated signaling) and that PAR-2 is likely to be involved. To confirm the involvement of PAR-2, we studied FXa-induced signaling in thrombin- or trypsin-desensitized fibroblasts. To validate the experimental set-up, we first verified the specificity of our desensitization experiments. As shown in Figure 1F, thrombin desensitization abolished subsequent stimulation by thrombin (lanes 1 to 4) but not by trypsin (lanes 5 to 8). Reciprocally, trypsin pretreatment prevented subsequent ERK1/2 phosphorylation when the fibroblasts were treated with trypsin (lanes 9 to 12) but not when treated with thrombin (lanes 13 to 16). Because thrombin and trypsin indeed specifically desensitized cells, we next assessed FXa-induced signaling in desensitized cells. As shown in Figure 1F, FXa still induced ERK1/2 phosphorylation in thrombin-desensitized cells (lanes 21 to 24). In contrast, fibroblasts pretreated with trypsin failed to respond to subsequent FXa stimulation (lanes 25 to 28 as compared to lanes 17 to 20), indicating both agonists activate the same receptor (ie, PAR-2). Simi