Thrombin Is a Potent Inducer of Connective Tissue Growth Factor Production via Proteolytic Activation of Protease-activated Receptor-1статья из журнала
Аннотация: The coagulation protease thrombin plays a critical role in hemostasis and exerts pro-inflammatory and pro-fibrotic effects via proteolytic activation of the major thrombin receptor, protease-activated receptor-1 (PAR-1). Connective tissue growth factor (CTGF) is a novel fibroblast mitogen and also promotes extracellular matrix protein production. It is selectively induced by transforming growth factor β (TGF-β) and is thought to be the autocrine agent responsible for mediating its pro-fibrotic effects. CTGF is up-regulated during tissue repair and in fibrotic conditions associated with activation of the coagulation cascade. We therefore hypothesized that coagulation proteases promote the production of CTGF by cells at sites of tissue injury. To begin to address this hypothesis, we assessed the effect of coagulation proteases on fibroblast CTGF expression in vitro, and we show that thrombin, at physiological concentrations, up-regulated CTGF mRNA levels 5-fold relative to base line (p < 0.01) in fetal fibroblasts and 7-fold in primary adult fibroblasts (p < 0.01). These effects were cycloheximide-insensitive and were not blocked with a pan-specific TGF-β-neutralizing antibody. They were further paralleled by a concomitant increase in CTGF protein production and could be mimicked with selective PAR-1 agonists. In addition, fibroblasts derived from PAR-1 knockout mice were unresponsive to thrombin but responded normally to TGF-β1. Finally, factor Xa, which is responsible for activating prothrombin during blood coagulation, exerted similar stimulatory effects. We propose that coagulation proteases and PAR-1 may play a role in promoting connective tissue formation during normal tissue repair and the development of fibrosis by up-regulating fibroblast CTGF expression. The coagulation protease thrombin plays a critical role in hemostasis and exerts pro-inflammatory and pro-fibrotic effects via proteolytic activation of the major thrombin receptor, protease-activated receptor-1 (PAR-1). Connective tissue growth factor (CTGF) is a novel fibroblast mitogen and also promotes extracellular matrix protein production. It is selectively induced by transforming growth factor β (TGF-β) and is thought to be the autocrine agent responsible for mediating its pro-fibrotic effects. CTGF is up-regulated during tissue repair and in fibrotic conditions associated with activation of the coagulation cascade. We therefore hypothesized that coagulation proteases promote the production of CTGF by cells at sites of tissue injury. To begin to address this hypothesis, we assessed the effect of coagulation proteases on fibroblast CTGF expression in vitro, and we show that thrombin, at physiological concentrations, up-regulated CTGF mRNA levels 5-fold relative to base line (p < 0.01) in fetal fibroblasts and 7-fold in primary adult fibroblasts (p < 0.01). These effects were cycloheximide-insensitive and were not blocked with a pan-specific TGF-β-neutralizing antibody. They were further paralleled by a concomitant increase in CTGF protein production and could be mimicked with selective PAR-1 agonists. In addition, fibroblasts derived from PAR-1 knockout mice were unresponsive to thrombin but responded normally to TGF-β1. Finally, factor Xa, which is responsible for activating prothrombin during blood coagulation, exerted similar stimulatory effects. We propose that coagulation proteases and PAR-1 may play a role in promoting connective tissue formation during normal tissue repair and the development of fibrosis by up-regulating fibroblast CTGF expression. protease-activated receptor connective tissue growth factor sterile Dulbecco's modified Eagle's medium human fetal lung fibroblasts newborn calf serum platelet-derived growth factor recombinant tick anticoagulant peptide transforming growth factor β kilobase pair plasminogen activator inhibitor mink lung epithelial cells Thrombin is a pluripotent serine protease that plays a central role in hemostasis following tissue injury by converting soluble plasma fibrinogen into an insoluble fibrin clot and by promoting platelet aggregation. In addition to these procoagulant effects, thrombin also influences a number of cellular responses that play important roles in subsequent inflammatory and tissue repair processes. Thrombin influences the recruitment and trafficking of inflammatory cells and is a potent mitogen for a number of cell types, including endothelial cells, fibroblasts, and smooth muscle cells (reviewed in Ref. 1Dery O. Corvera C.U. Steinhoff M. Bunnett N.W. Am. J. Physiol. 1998; 274: C1429-C1452Crossref PubMed Google Scholar). Thrombin also promotes the production and secretion of extracellular matrix proteins (2Chambers R.C. Dabbagh K. McAnulty R.J. Gray A.J. Blanc Brude O.P. Laurent G.J. Biochem. J. 1998; 333: 121-127Crossref PubMed Scopus (134) Google Scholar, 3Papadimitriou E. Manolopoulos V.G. Hayman G.T. Maragoudakis M.E. Unsworth B.R. Fenton J.W., II Lelkes P.I. Am. J. Physiol. 1997; 272: C1112-C1122Crossref PubMed Google Scholar) and influences connective tissue remodeling processes (4Duhamel-Clerin E. Orvain C. Lanza F. Cazenave J.P. Klein-Soyer C. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1931-1938Crossref PubMed Scopus (85) Google Scholar). There is increasing in vivo evidence that the pro-inflammatory and pro-fibrotic effects of thrombin play an important role in both normal tissue and vascular repair (5Carney D.H. Mann R. Redin W.R. Pernia S.D. Berry D. Heggers J.P. Hayward P.G. Robson M.C. Christie J. Annable C. J. Clin. Invest. 1992; 89: 1469-1477Crossref PubMed Scopus (134) Google Scholar), as well as in a number of pathological conditions associated with acute or persistent activation of the coagulation cascade, including restenosis and neointima formation following vascular injury (6Ragosta M. Barry W.L. Gimple L.W. Gertz S.D. McCoy K.W. Stouffer G.A. McNamara C.A. Powers E.R. Owens G.K. Sarembock I.J. Circulation. 1996; 93: 1194-1200Crossref PubMed Scopus (31) Google Scholar, 7Rade J.J. Schulick A.H. Virmani R. Dichek D.A. Nat. Med. 1996; 2: 293-298Crossref PubMed Scopus (150) Google Scholar), atherosclerosis (8Nelken N.A. Soifer S.J. O'Keefe J. Vu T.K. Charo I.F. Coughlin S.R. J. Clin. Invest. 1992; 90: 1614-1621Crossref PubMed Scopus (296) Google Scholar), pulmonary fibrosis (9Hernandez Rodriguez N.A. Cambrey A.D. Harrison N.K. Chambers R.C. Gray A.J. Southcott A.M. duBois R.M. Black C.M. Scully M.F. McAnulty R.J. Laurent G.J. Lancet. 1995; 346: 1071-1073Abstract Full Text PDF PubMed Scopus (125) Google Scholar), and glomerulonephritis (10Cunningham M.A. Rondeau E. Chen X. Coughlin S.R. Holdsworth S.R. Tipping P.G. J. Exp. Med. 2000; 191: 455-462Crossref PubMed Scopus (183) Google Scholar). Most of the cellular effects elicited by thrombin are mediated via a family of widely expressed G-protein-coupled receptors, termed protease-activated receptors (PARs)1 that are activated by limited proteolytic cleavage of the N-terminal extracellular domain. The newly generated N terminus acts as a tethered ligand and interacts intramolecularly with the body of the receptor to initiate subsequent cell signaling events (11Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2660) Google Scholar). To date, four PARs have been described, of which three (PAR-1, -3, and -4) are activated by thrombin. Synthetic peptides corresponding to the tethered ligand of PAR-1 and PAR-4 act as agonists for these receptors and have been useful tools for invoking the involvement of these receptors in mediating the cellular effects of thrombin. Studies with these agonists, as well as with PAR-1-deficient mice, have led to the conclusion that PAR-1 is the major receptor responsible for mediating most of the pro-inflammatory (10Cunningham M.A. Rondeau E. Chen X. Coughlin S.R. Holdsworth S.R. Tipping P.G. J. Exp. Med. 2000; 191: 455-462Crossref PubMed Scopus (183) Google Scholar, 12Cirino G. Cicala C. Bucci M.R. Sorrentino L. Maraganore J.M. Stone S.R. J. Exp. Med. 1996; 183: 821-827Crossref PubMed Scopus (233) Google Scholar) and pro-fibrotic effects (2Chambers R.C. Dabbagh K. McAnulty R.J. Gray A.J. Blanc Brude O.P. Laurent G.J. Biochem. J. 1998; 333: 121-127Crossref PubMed Scopus (134) Google Scholar, 13Trejo J. Connolly A.J. Coughlin S.R. J. Biol. Chem. 1996; 271: 21536-21541Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 14Dabbagh K. Laurent G.J. McAnulty R.J. Chambers R.C. Thromb. Haemostasis. 1998; 79: 405-409Crossref PubMed Scopus (66) Google Scholar) of thrombin. Once thrombin has interacted with its receptor, it exerts its cellular effects either directly or via the induction and release of secondary mediators, including classical growth factors, pro-inflammatory cytokines, and vasoactive peptides and amines (reviewed in Ref. 1Dery O. Corvera C.U. Steinhoff M. Bunnett N.W. Am. J. Physiol. 1998; 274: C1429-C1452Crossref PubMed Google Scholar). Connective tissue growth factor (CTGF) is a novel potent cysteine-rich heparin-binding growth factor, originally isolated from human umbilical vein endothelial cells (15Bradham D.M. Igarashi A. Potter R.L. Grotendorst G.R. J. Cell Biol. 1991; 114: 1285-1294Crossref PubMed Scopus (807) Google Scholar), that is also highly expressed by fibroblasts (16Igarashi A. Okochi H. Bradham D.M. Grotendorst G.R. Mol. Biol. Cell. 1993; 4: 637-645Crossref PubMed Scopus (642) Google Scholar). It belongs to an emerging family of conserved and modular proteins (known as the CCN family) with diverse biological functions involved in the regulation of cell growth and differentiation. Six members have been described to date and include the gene products of the serum-induced immediate-early genes Cyr 61 (a homolog of CTGF), Fisp12 (the mouse ortholog of human CTGF), Cef10 (the chicken ortholog ofCyr 61), a putative avian oncogene protein Novand Elm1, a mouse gene expressed in low metastatic mouse melanoma cells (17O'Brien T.P. Yang G.P. Sanders L. Lau L.F. Mol. Cell. Biol. 1990; 10: 3569-3577Crossref PubMed Scopus (271) Google Scholar, 18Ryseck R.P. Macdonald-Bravo H. Mattei M.G. Bravo R. Cell Growth Differ. 1991; 2: 225-233PubMed Google Scholar, 19Simmons D.L. Levy D.B. Yannoni Y. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1178-1182Crossref PubMed Scopus (296) Google Scholar, 20Joliot V. Martinerie C. Dambrine G. Plassiart G. Brisac M. Crochet J. Perbal B. Mol. Cell. Biol. 1992; 12: 10-21Crossref PubMed Scopus (276) Google Scholar, 21Hashimoto Y. Shindo-Okada N. Tani M. Nagamachi Y. Takeuchi K. Shiroishi T. Toma H. Yokota J. J. Exp. Med. 1998; 187: 289-296Crossref PubMed Scopus (133) Google Scholar). Recent studies have provided evidence that CTGF may play an important role in promoting connective tissue formation after tissue injury. As well as being a potent fibroblast mitogen and chemoattractant, CTGF stimulates fibroblast procollagen and fibronectin protein production and influences α5 integrin mRNA levelsin vitro (22Frazier K. Williams S. Kothapalli D. Klapper H. Grotendorst G.R. J. Invest. Dermatol. 1996; 107: 404-411Abstract Full Text PDF PubMed Scopus (673) Google Scholar). CTGF mRNA levels are strongly up-regulated in skin wound healing models in vivo (16Igarashi A. Okochi H. Bradham D.M. Grotendorst G.R. Mol. Biol. Cell. 1993; 4: 637-645Crossref PubMed Scopus (642) Google Scholar), and subcutaneous injection of CTGF into newborn mice results in increased connective tissue deposition (22Frazier K. Williams S. Kothapalli D. Klapper H. Grotendorst G.R. J. Invest. Dermatol. 1996; 107: 404-411Abstract Full Text PDF PubMed Scopus (673) Google Scholar). CTGF is also thought to be involved in the development of tissue fibrosis, based on the observation that CTGF expression is increased in skin and internal organ fibrosis (23Igarashi A. Nashiro K. Kikuchi K. Sato S. Ihn H. Fujimoto M. Grotendorst G.R. Takehara K. J. Invest. Dermatol. 1996; 106: 729-733Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 24Ito Y. Aten J. Bende R.J. Oemar B.S. Rabelink T.J. Weening J.J. Goldschmeding R. Kidney Int. 1998; 53: 853-861Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 25Allen J.T. Knight R.A. Bloor C.A. Spiteri M.A. Am. J. Respir. Cell Mol. Biol. 1999; 21: 693-700Crossref PubMed Scopus (167) Google Scholar) and the fibrotic areas of atherosclerotic lesions (26Oemar B.S. Werner A. Garnier J.M. Do D.D. Godoy N. Nauck M. Marz W. Rupp J. Pech M. Luscher T.F. Circulation. 1997; 95: 831-839Crossref PubMed Scopus (290) Google Scholar). There is also growing evidence that CTGF may be the downstream autocrine mediator responsible for mediating some of the cellular effects of TGF-β1, the most fibrogenic mediator characterized to date. CTGF expression by cultured fibroblasts is exclusively induced by TGF-β1, whereas other fibrotic mediators such as PDGF, epidermal growth factor, basic fibroblast growth factor, and insulin-like growth factor-1 have no effect (16Igarashi A. Okochi H. Bradham D.M. Grotendorst G.R. Mol. Biol. Cell. 1993; 4: 637-645Crossref PubMed Scopus (642) Google Scholar, 27Ricupero D.A. Rishikof D.C. Kuang P.P. Poliks C.F. Goldstein R.H. Am. J. Physiol. 1999; 277: L1165-L1171PubMed Google Scholar). This is consistent with the recent characterization of a novel TGF-β response element within the CTGF promoter (28Grotendorst G.R. Okochi H. Hayashi N. Cell Growth Differ. 1996; 7: 469-480PubMed Google Scholar). In addition, CTGF antisense constructs or neutralizing antibodies have been shown to block the effects of TGF-β on fibroblast proliferation and procollagen production (27Ricupero D.A. Rishikof D.C. Kuang P.P. Poliks C.F. Goldstein R.H. Am. J. Physiol. 1999; 277: L1165-L1171PubMed Google Scholar, 29Kothapalli D. Frazier K.S. Welply A. Segarini P.R. Grotendorst G.R. Cell Growth Differ. 1997; 8: 61-68PubMed Google Scholar, 30Duncan M.R. Frazier K.S. Abramson S. Williams S. Klapper H. Huang X. Grotendorst G.R. FASEB J. 1999; 13: 1774-1786Crossref PubMed Scopus (577) Google Scholar), although this was not a universal finding (31Hong H.H. Uzel M.I. Duan C. Sheff M.C. Trackman P.C. Lab. Invest. 1999; 79: 1655-1667PubMed Google Scholar). In this study, we hypothesized that coagulation proteases promote the production of CTGF by cells at sites of tissue injury and repair. To address this hypothesis, we assessed the effect of thrombin on fibroblast CTGF expression in vitro and show for the first time that thrombin, at physiological concentrations, increases both CTGF mRNA levels and protein production via proteolytic activation of the major thrombin receptor, PAR-1. We further show that the coagulation protease factor Xa, responsible for the activation of prothrombin during blood coagulation, exerts similar stimulatory effects. These in vitro findings support a role for coagulation proteases and PAR-1 in promoting early wound healing responses and connective tissue formation by up-regulating the production of CTGF. Our results may further be relevant to a number of fibroproliferative and fibrotic disorders where both thrombin levels and CTGF expression are increased. Human thrombin (catalog number T4393) and recombinant hirudin (catalog number H0393) and cycloheximide were from Sigma. Purified Russell's viper venom-activated human factor Xa was from Calbiochem. Recombinant tick anticoagulant peptide (rTAP) was a kind gift from Dr. Mike Scully (Thrombosis Research Institute, London, UK), originally prepared by Dr. G. Vlasuk (Corvas International, San Diego, CA). Selective PAR-1 agonists, corresponding to the sequence TFLLR, were obtained from by Dr. Robert P. Mecham (University of Washington Medical School, St. Louis, MO). Activated porcine pancreatic TGF-β1 and pan-specific TGF-β neutralizing antibodies were from R & D Systems (Abingdon, Oxon, UK). Sterile Dulbecco's modified Eagle's medium (DMEM), tissue culture supplements, and tissue culture plates were from Life Technologies, Inc. The cDNA probe for human CTGF, encoding the entire open reading frame, was kindly provided inserted into the EcoRI andNotI sites of pBluescript by Dr. Raj Beri (AstraZeneca R & D Charnwood, Loughborough, UK). The cDNA probe for FISP12, encompassing nucleotides 1663–2930, was generated from a plasmid (pBluescript fisp12del) kindly provided by Dr. Joseph A. Lasky (Tulane University, New Orleans, LA). The pBluescript fisp12del plasmid was subcloned from a plasmid (A12/pmexNeo I) originally obtained from Dr. Rolf-Peter Ryseck (Bristol-Myers Squibb Co.) (18Ryseck R.P. Macdonald-Bravo H. Mattei M.G. Bravo R. Cell Growth Differ. 1991; 2: 225-233PubMed Google Scholar). The anti-CTGF antibody was a previously described protein G-purified goat anti-human CTGF IgG (30Duncan M.R. Frazier K.S. Abramson S. Williams S. Klapper H. Huang X. Grotendorst G.R. FASEB J. 1999; 13: 1774-1786Crossref PubMed Scopus (577) Google Scholar) and was kindly provided by Dr. Gary R. Grotendorst (University of Miami School of Medicine, Miami, FL). Human lung fibroblasts (HFL1) were purchased from the American Type Culture Collection (Manassas, VA), and primary human adult lung fibroblasts grown from explant cultures of normal lung tissue were kindly provided by Dr. Robin J. McAnulty in our laboratory. Mouse lung fibroblasts from PAR-1 knockout and corresponding wild type mice were a kind gift from Professor Shaun R. Coughlin (University of California, San Francisco, CA) and have been described previously (13Trejo J. Connolly A.J. Coughlin S.R. J. Biol. Chem. 1996; 271: 21536-21541Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Cells were maintained in DMEM supplemented with penicillin (100 units/ml), streptomycin (100 units/ml), and 5% (v/v) NCS (DMEM, 5% NCS), in a humidified atmosphere containing 10% CO2. Cells were routinely passaged every 6–7 days and tested for mycoplasma infection. There were no noticeable effects on the parameters measured for cells used between passages 14 and 25. Cells were seeded at 2 × 105 cells/ml in 6-cm diameter dishes in DMEM, 5% NCS. Upon reaching visual confluence, cells were quiesced in serum-free DMEM for 16 h and incubated in fresh serum-free DMEM containing thrombin, factor Xa, or the highly selective PAR-1 agonist TFLLR (32Hollenberg M.D. Saifeddine M. Al Ani B. Kawabata A. Can. J. Physiol. Pharmacol. 1997; 75: 832-841Crossref PubMed Scopus (216) Google Scholar). For cycloheximide experiments, cells were preincubated with cycloheximide (25 μg/ml) for 2 h prior to addition to serum-free control media or thrombin. For thrombin and factor Xa proteolytic inhibition experiments, thrombin or factor Xa was incubated with either hirudin (in 2-fold molar excess) or rTAP (in 4-fold molar excess), respectively, and protease-inhibitor complex formation was allowed to proceed for 2 h at 37 °C with shaking, prior to addition to cell cultures. At the end of the incubation, the media were removed, and total RNA was isolated with Trizol reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Five μg of total RNA were mixed with RNA loading buffer containing ethidium bromide (Sigma), heated to 65 °C for 10 min, and electrophoresed on a formaldehyde 1% (w/v) agarose gel. RNA loading and integrity was visualized and quantitated by fluorescent scanning of the gel (Fuji, FLA 3000) prior to transfer to nylon membranes (Hybond N, Amersham Pharmacia Biotech) by Northern transfer and fixation by UV cross-linking. Membranes were hybridized overnight at 65 °C in a rotating hybridization oven in standard Denhardt's containing hybridization solution in the presence of the [32P]dCTP-labeled cDNA probes for either CTGF or FISP12, generated by random priming using an oligolabeling kit (Amersham Pharmacia Biotech). At the end of the hybridization, filters were rinsed at low stringency (2× SSC, 0.1% SDS for 5 min at room temperature, followed by 15 min at 65 °C), once at medium stringency (0.5× SSC, 0.1% SDS for 25 min at 65 °C), and once at high stringency (0.1 × SSC, 0.1% SDS for 5 min at 65 °C). Membranes were exposed to a PhosphorImager storage screen (Fuji) for 2–4 h, and CTGF/FISP12 mRNA levels were quantitated by PhosphorImager analysis (Fuji FLA 3000). Cells were seeded at 5 × 104 cells/ml in 2.4-cm diameter dishes in DMEM, 5% NCS. Upon reaching visual confluence, cells were quiesced in serum-free DMEM for 16 h and incubated with fresh serum-free incubation media with and without thrombin (25 nm) or TGF-β1 (1 ng/ml). After 6 h, the medium was removed, and the monolayer was washed twice with ice-cold phosphate-buffered saline, and cells were lysed by adding 100 μl of Laemmli sample buffer directly to the monolayer followed by scraping with a rubber policeman. The cell lysate was mixed several times to shear DNA, and 25 μl of each were heated for 5 min at 95 °C prior to electrophoresis on a 12% SDS-polyacrylamide gel with a 7% stacking gel for 3 h at 125 V. Separated proteins were transferred onto Hybond-ECL nylon membranes (Amersham Pharmacia Biotech) for 1 h at 25 V. The membrane was blocked with TBST (150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 0.1% Tween 20) containing 5% dry milk for 1 h, and the anti-CTGF antibody was added at a 1:1000 dilution overnight at 4 °C. A horseradish peroxidase-conjugated anti-rabbit IgG (Dako Ltd., Cambridge, UK) was added at a 1:2000 dilution for 1 h followed by three washes in TBST for 15 min. The CTGF band was visualized by enhanced chemiluminescence (ECL) according to the manufacturer's protocol (Amersham Pharmacia Biotech). Membranes were also stripped and reprobed with a rabbit anti-human actin antibody (Sigma) at a 1:2000 dilution for 2 h at room temperature, followed by the same secondary antibody used above. Active TGF-β in conditioned media from HFL1 cells exposed to control media, thrombin (25 nm), or TGF-β1 (0.25–1 ng/ml) in identical serum-free conditions for 90 min, as described above, was assessed using a highly quantitative bioassay, based on the ability of TGF-β to induce plasminogen activator inhibitor-1 (PAI-1) gene expression in mink lung epithelial cells (MLEC) stably transfected with an expression construct containing a truncated TGF-β-responsive PAI-1 promoter fused to a luciferase reporter gene. These cells were a kind gift from Dr. D. B. Rifkin (New York University Medical Center, New York), and the assay was performed as described previously (33Abe M. Harpel J.G. Metz C.N. Nunes I. Loskutoff D.J. Rifkin D.B. Anal. Biochem. 1994; 216: 276-284Crossref PubMed Scopus (677) Google Scholar). Briefly, cells were grown to 75% confluence and incubated with fibroblast conditioned media, naive media, or conditioned media spiked with thrombin and TGF-β1 for 16 h. At the end of the incubation, the media were removed, and the cell layer was washed with cold phosphate-buffered saline, and luciferase activity in cell lysates (passive buffer) was assayed using a luciferase assay kit (Promega, Southampton, UK) according to the manufacturer's instructions, with a luminometer (Turner Designs- 20/20). The data are expressed in relative light units per well. All numerical data are presented as means ± S.E. from four replicate cultures, unless otherwise indicated. Statistical evaluation was performed using an unpaired Student's t test or by one-way analysis of variance using the Neuman-Keuls procedure for multiple group comparisons. The mean values of various parameters were said to be significantly different when the probability of the differences of that magnitude, assuming the null hypothesis to be correct, fell below 5% (i.e. p < 0.05). To determine the potential effect of thrombin on fibroblast CTGF expression, human fetal lung fibroblasts (HFL1) and primary human adult lung fibroblasts were exposed to a single concentration of thrombin (25 nm), and CTGF mRNA levels were assessed by Northern analysis of total cellular RNA at 1.5 h. In both cell lines, thrombin caused a dramatic increase in CTGF mRNA levels, with values increased 5-fold relative to base line for fetal lung fibroblasts and 7-fold for primary adult lung fibroblasts (Fig. 1 A). For comparison, at the same time point, TGF-β1 (1 ng/ml) only induced a small non-significant increase in CTGF mRNA levels in both cell types examined. We next performed detailed thrombin concentration-response experiments in human fetal lung fibroblasts at a range of physiological concentrations, from 10 pm to 500 nm. Fig.1 B shows the results obtained up to 10 nmthrombin after 1.5 h of exposure. Thrombin increased CTGF mRNA levels at concentrations as low as 10 pm with values increased 2.6- fold relative to base line. At 1 nm, the response was maximal with CTGF mRNA levels increased 4-fold. There was no further up-regulation with increasing concentrations of thrombin up to 500 nm, the highest concentration tested. The effect of thrombin on CTGF protein levels was assessed by Western blotting of cell layer extracts after 6 h of exposure to control media, thrombin (25 nm), or TGF-β1 (1 ng/ml) (Fig. 1 C). The anti-CTGF antibody recognized a faint immunoreactive 38-kDa protein in unstimulated fetal and adult fibroblasts as has been previously reported (22Frazier K. Williams S. Kothapalli D. Klapper H. Grotendorst G.R. J. Invest. Dermatol. 1996; 107: 404-411Abstract Full Text PDF PubMed Scopus (673) Google Scholar). The intensity of this band was dramatically increased in cells exposed to thrombin or TGF-β1 for both fetal and adult fibroblasts, with the greatest intensity observed for thrombin-stimulated cells. In order to determine the time course by which thrombin stimulates CTGF mRNA levels, detailed time course experiments were performed with fetal fibroblasts exposed to a single dose of thrombin (25 nm) or TGF-β1 (1 ng/ml) up to 48 h. Combined data for four separate time course experiments are shown in Fig.2 A. Thrombin stimulated CTGF mRNA levels 2-fold relative to media control levels at the earliest time point examined (0.5 h). CTGF mRNA levels were maximally increased by at least 3-fold at 3 and 6 h and then gradually returned to base-line values by 30 h. For comparison, the stimulatory effects obtained with TGF-β1 were not apparent until after 1.5 h. At 3 and 6 h, CTGF mRNA levels were maximally increased and gradually returned to base-line values by 48 h.Figure 2The stimulatory effects of thrombin on CTGF mRNA levels are rapid and occur independently of de novoprotein synthesis and the secretion of active TGF-β. A, time course for the stimulatory effects of thrombin on CTGF mRNA levels. Confluent cultures of HFL1 fibroblasts were quiesced in serum-free conditions and exposed to serum-free control media (DMEM), thrombin (25 nm), or TGF-β1 (1 ng/ml) for incubation times from 0.5 to 48 h. CTGF mRNA levels at each time point were assessed by Northern analysis as described under “Experimental Procedures” and in Fig. 1. The figure shows combined data as means and S.E. for four separate time course experiments performed.B, thrombin stimulates CTGF mRNA levels in a cycloheximide-independent manner. Confluent cultures of HFL-1 fibroblasts were quiesced in serum-free conditions and pre-exposed to cycloheximide (25 μg/ml) for 2 h prior to addition of thrombin (10 nm) or control media. CTGF mRNA levels at 1.5 h were assessed by Northern analysis as described under “Experimental Procedures” and in Fig. 1. The figure shows the 2.4-kb CTGF transcripts and the corresponding ethidium bromide-stained 28 S rRNA bands for a representative experiment (n = 3).C = media control; Thr = thrombin.C, thrombin stimulates CTGF mRNA levels in the presence of TGF-β-neutralizing antibodies. Confluent cultures of HFL-1 fibroblasts were quiesced in serum-free conditions and exposed to serum-free control media (DMEM) or thrombin (25 nm) with and without TGF-β-neutralizing antibodies or isotype-matched control IgG (80 μg/ml). CTGF mRNA levels at 1.5 h were assessed by Northern analysis as described under “Experimental Procedures” and in Fig. 1. The figure shows the 2.4-kb CTGF transcripts and the corresponding ethidium bromide-stained 28 S rRNA bands for a representative experiment (n = 3). Ab = TGF-β-neutralizing antibody; IgG = control antibody.D, thrombin does not induce the secretion or release of active TGF-β. MLEC were exposed to serum-free control media, control media supplemented with either thrombin (25 nm) or TGF-β1 (0.25 ng/ml), or conditioned media from HFL1 fibroblasts exposed to control media, thrombin (25 nm), or TGF-β1 (0.25 ng/ml) for 1.5 h. The last two bars represent MLEC exposed to conditioned media subsequently “spiked” with thrombin (25 nm) or TGF-β1(0.25 ng/ml). MLEC were lysed after 16 h, and luciferase activity was measured as an index of PAI-1 promoter activity as described under “Experimental Procedures.” The bar graph represents the data as means and S.E. for four replicate cultures, expressed in arbitrary relative light units. Where no error bar is shown it is within the column representing the data point. C = media control;Thr = thrombin; p values are calculated against media control activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The time course for the stimulatory effects of thrombin on CTGF mRNA levels suggested that CTGF may be responding to thrombin in a typical immediate-early gene response fashion. To test this, we examined the effect of cycloheximide on the ability of thrombin to stimulate CTGF mRNA levels by fetal fibroblasts. Fig. 2 Bshows a representative experiment where thrombin increased CTGF mRNA levels about 4-fold relative to media control levels. As expected, cycloheximide (250 μg/ml) did not block the stimulatory effects of thrombin on CTGF mRNA levels, indicating that thrombin exerts its stimu
Год издания: 2000
Авторы: Rachel C. Chambers, Patricia Leoni, Olivier Blanc‐Brude, David E. Wembridge, Geoffrey J. Laurent
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Connective Tissue Growth Factor Research, Blood Coagulation and Thrombosis Mechanisms, Coagulation, Bradykinin, Polyphosphates, and Angioedema
Другие ссылки: Journal of Biological Chemistry (PDF)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Открытый доступ: hybrid
Том: 275
Выпуск: 45
Страницы: 35584–35591