Fibroblast Matrix Gene Expression and Connective Tissue Remodeling: Role of Endothelin-1
2001; Elsevier BV; Volume: 116; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1747.2001.01256.x
ISSN1523-1747
AutoresShiwen Xu, Christopher P. Denton, Alan Holmes, Carol M. Black, David Abraham, Michael R. Dashwood, George Bou‐Gharios, Jeremy D. Pearson,
Tópico(s)Dermatological and Skeletal Disorders
ResumoThis study examines endothelin-induced modulation of extracellular matrix synthesis and remodeling by fibroblasts, and its potential role in the pathogenesis of systemic sclerosis (scleroderma). Endothelin-1 promoted fibroblast synthesis of collagen types I and III, but not fibronectin, by a mechanism dependent upon both ETA and ETB receptors. Conversely, endothelin-1 inhibited both protein expression of matrix metalloproteinase 1 and zymographic activity exclusively via ETA receptors. A dual regulatory role for endothelin-1 in transcriptional regulation was suggested by the ability of endothelin-1 to enhance steady-state levels of collagen mRNA and activate the proα2(I) collagen (Col1a2) promoter, but in contrast to reduce matrix metalloproteinase 1 transcript expression and suppress transcription of a human matrix metalloproteinase 1 promoter reporter construct in transient transfection assays. Although endothelin-1 significantly enhanced remodeling of three-dimensional collagen lattices populated by normal fibroblasts, this was not observed for lattices populated by systemic sclerosis fibroblasts. Promotion of matrix remodeling was dependent upon ETA receptor expression and was blocked by specific inhibitors of tyrosine kinases or protein kinase C. Reverse transcriptase polymerase chain reaction, S1 nuclease, and functional cell surface binding studies showed that normal and systemic sclerosis fibroblasts express both ETA and ETB receptors (predominantly ETA), but that ETA receptor mRNA levels and ETA binding sites on fibroblasts cultured from systemic sclerosis skin biopsies are reduced by almost 50%. Endothelin-1 is thus able to induce a fibrogenic phenotype in normal fibroblasts that is similar to that of lesional systemic sclerosis fibroblasts. Moreover, reduced responsiveness to exogenous endothelin-1 in systemic sclerosis suggests that downstream pathways may have already been activated in vivo. These data further implicate dysregulated endothelin-receptor pathways in fibroblasts in the pathogenesis of connective tissue fibrosis. This study examines endothelin-induced modulation of extracellular matrix synthesis and remodeling by fibroblasts, and its potential role in the pathogenesis of systemic sclerosis (scleroderma). Endothelin-1 promoted fibroblast synthesis of collagen types I and III, but not fibronectin, by a mechanism dependent upon both ETA and ETB receptors. Conversely, endothelin-1 inhibited both protein expression of matrix metalloproteinase 1 and zymographic activity exclusively via ETA receptors. A dual regulatory role for endothelin-1 in transcriptional regulation was suggested by the ability of endothelin-1 to enhance steady-state levels of collagen mRNA and activate the proα2(I) collagen (Col1a2) promoter, but in contrast to reduce matrix metalloproteinase 1 transcript expression and suppress transcription of a human matrix metalloproteinase 1 promoter reporter construct in transient transfection assays. Although endothelin-1 significantly enhanced remodeling of three-dimensional collagen lattices populated by normal fibroblasts, this was not observed for lattices populated by systemic sclerosis fibroblasts. Promotion of matrix remodeling was dependent upon ETA receptor expression and was blocked by specific inhibitors of tyrosine kinases or protein kinase C. Reverse transcriptase polymerase chain reaction, S1 nuclease, and functional cell surface binding studies showed that normal and systemic sclerosis fibroblasts express both ETA and ETB receptors (predominantly ETA), but that ETA receptor mRNA levels and ETA binding sites on fibroblasts cultured from systemic sclerosis skin biopsies are reduced by almost 50%. Endothelin-1 is thus able to induce a fibrogenic phenotype in normal fibroblasts that is similar to that of lesional systemic sclerosis fibroblasts. Moreover, reduced responsiveness to exogenous endothelin-1 in systemic sclerosis suggests that downstream pathways may have already been activated in vivo. These data further implicate dysregulated endothelin-receptor pathways in fibroblasts in the pathogenesis of connective tissue fibrosis. fibroblast-populated collagen lattices metalloproteinase 1 Endothelin-1 (ET-1) is a 21 amino acid peptide, first characterized as a potent endothelial cell-derived vasoconstrictor. It is synthesized as an inactive precursor polypeptide (preproendothelin) and processed to mature active peptides (big endothelin and endothelin) by zinc metalloproteinases, known as endothelin-converting enzymes (Shao et al., 1999Shao R. Yan W. Rockay D.C. Regulation of endothelin-1 synthesis by endothelin-converting enzyme-1 during wound healing.J Biol Chem. 1999; 274: 3228-3234Crossref PubMed Scopus (82) Google Scholar). Three isoforms of endothelin are recognized (ET-1, ET-2, ET-3), exhibiting substantial sequence homology although the extent of functional overlap is uncertain. In addition to effects on vascular tone (Barton et al., 1998Barton M. Haudenschild C.C. D'Uscio L.V. Shaw S. Munter K. Luscher T.F. Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficent mice.Proc Natl Acad Sci USA. 1998; 95: 14367-14372https://doi.org/10.1073/pnas.95.24.14367Crossref PubMed Scopus (323) Google Scholar), endothelins modulate survival, growth, and differentiation of a number of different cell types (Rubanyi and Polokoff, 1994Rubanyi G.M. Polokoff M.A. Endothelins. Molecular biology, biochemistry, pharmacology, physiology and pathophysiology.Pharmacol Rev. 1994; 46: 325-415PubMed Google Scholar). Elevated circulating levels of ET-1 have been reported in a wide variety of vascular and inflammatory disease states (Levin, 1995Levin E.R. Endothelins.N Engl J Med. 1995; 333: 356-363Crossref PubMed Scopus (0) Google Scholar), and in the fibrosing connective tissue disease systemic sclerosis (scleroderma) (Vancheeswaran et al., 1994aVancheeswaran R. Azam A. Black C.M. Dashwood M.R. Localisation of endothelin-1 and its binding sites in scleroderma skin.J Rheumatol. 1994; 21: 1268-1276PubMed Google Scholar). Moreover, increased endothelin binding has also been demonstrated by autoradiography in skin and lung biopsies from scleroderma patients (Vancheeswaran et al., 1994bVancheeswaran R. Magoulas T. Efrat G. Wheeler-Jones C. Olsen I. Black C.M. Circulating endothelin-1 levels in systemic sclerosis subsets – a marker of fibrosis or vascular dysfunction?.J Rheumatol. 1994; 21: 1838-1844PubMed Google Scholar;Abraham et al., 1997Abraham D.J. Vancheeswaran R. Dashwood M.R. Pantelides P. Shi-wen X. du Bois R.M. Black C.M. Increased levels of endothelin-1 and differential endothelin type A and B receptor expression in scleroderma-associated fibrotic lung disease.Am J Pathol. 1997; 151: 831-841PubMed Google Scholar) suggesting an overall increase in ET receptor expression in disease. These observations provide evidence that this peptide is involved in the systemic sclerosis pathogenesis, but do not precisely define its role in regulating fibroblast properties, nor the pattern of receptor subtype expression on these cells. Lesional fibroblasts from biopsies of clinically active systemic sclerosis skin demonstrate increased matrix biosynthesis (LeRoy, 1974LeRoy E.C. Increased collagen synthesis by scleroderma skin fibroblasts in vitro: a possible defect in the regulation or activation of the scleroderma fibroblast.J Clin Invest. 1974; 54: 880-889Crossref PubMed Scopus (481) Google Scholar;Jimenez et al., 1986Jimenez S. Feldmenn G. Bashey R. Bienkowski R. Co-ordinate increase in the expression of type II and type III collagen genes in progressive systemic sclerosis fibroblasts.Biochem J. 1986; 237: 837-843Crossref PubMed Scopus (151) Google Scholar) and reduced degradation (Strehlow and Korn, 1998Strehlow D. Korn J.H. Biology of the scleroderma fibroblast.Current Opin Rheumatol. 1998; 10: 572-578Crossref PubMed Scopus (54) Google Scholar). Other fundamental fibroblast properties are also altered, including proliferative capacity (Ichiki et al., 1995Ichiki Y. Smith E. LeRoy E.C. Trojanowska M. Different effects of basic fibroblast growth factor and transforming growth factor-beta on the two platelet-derived growth factor receptors' expression in scleroderma and healthy human dermal fibroblasts.J Invest Dermatol. 1995; 104: 124-127Crossref PubMed Scopus (28) Google Scholar) and downregulation of collagen gene expression, which normally occurs in three-dimensional collagen gel cultures (Ivarsson et al., 1993Ivarsson M. McWhirter A. Black C.M. Rubin K. Impaired regulation of collagen pro-a1 (I) mRNA and change in pattern of collagen-binding integrins on scleroderma fibroblasts.J Invest Dermatol. 1993; 101: 216-221Abstract Full Text PDF PubMed Google Scholar;Shi-Wen et al., 1997Shi-Wen X. Denton C.P. McWhirter A. Bou-Gharios G. Abraham D.J. du Bois R.M. Black C.M. Scleroderma lung fibroblasts exhibit elevated and dysregulated collagen type I biosynthesis.Arthritis Rheum. 1997; 40: 1237-1244Crossref PubMed Scopus (41) Google Scholar). The process by which fibroblasts acquire this altered phenotype is uncertain, but chronic exposure to profibrotic cytokines or growth factors in vivo is postulated. The occurrence of vascular damage and endothelial dysfunction coexisting with, or even preceding, dermal and visceral fibrosis in systemic sclerosis implicates endothelial cell-derived factors (Carvalho et al., 1996Carvalho D. Savage C.O. Black C.M. Pearson J.D. IgG antiendothelial cell autoantibodies from scleroderma patients induce leukocyte adhesion to human vascular endothelial cells in vitro. Induction of adhesion molecule expression and involvement of endothelium-derived cytokines.J Clin Invest. 1996; 97: 111-119Crossref PubMed Scopus (223) Google Scholar;Denton et al., 1996Denton C.P. Shiwen X. Welsh K.I. Pearson J.D. Black C.M. Scleroderma fibroblast phenotype is modulated by endothelial cell co-culture.J Rheumatol. 1996; 23: 633-638PubMed Google Scholar) and makes ET-1 a plausible mediator. We have investigated endothelin-induced modulation of extracellular matrix turnover and remodeling by normal fibroblasts, to address the hypothesis that ET-1 is an activator of fibroblasts with respect to extracellular matrix synthesis, and to delineate the endothelin receptors and signaling pathways involved. Fibroblasts were obtained from biopsies of lesional areas of the skin of patients with early (less than 3 y duration) diffuse cutaneous systemic sclerosis (n = 8) and from age, sex, and anatomical site matched healthy volunteers (n = 5). All patients fulfilled the criteria of the American College of Rheumatology for the classification of systemic sclerosis (1980). None was receiving immunosuppressive medication or corticosteroids at the time of biopsy. Fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco), 100 U per ml penicillin, and 100 mg per ml streptomycin, and cultured in a humidified atmosphere of 5% CO2 in air. Fibroblasts were subcultured at confluence and used between passages 2 and 5. Total collagen type I and III synthesis by cultured fibroblasts was measured in the absence and presence of ET-1 (concentration range 10-8-10-12 M), by inhibition enzyme-linked immunosorbent assay (ELISA) (Shi-Wen et al., 1997Shi-Wen X. Denton C.P. McWhirter A. Bou-Gharios G. Abraham D.J. du Bois R.M. Black C.M. Scleroderma lung fibroblasts exhibit elevated and dysregulated collagen type I biosynthesis.Arthritis Rheum. 1997; 40: 1237-1244Crossref PubMed Scopus (41) Google Scholar). Culture supernatants were collected after incubating the cells in the presence of ascorbate (50 μg per ml) for 48 h. Interstitial collagenase (MMP-1) concentration in culture supernatants in the absence of ascorbate was measured using a commercial ELISA kit (Amersham, Buckinghamshire, U.K.). For inhibition experiments, cells were preincubated in the presence of endothelin receptor antagonist (100-fold molar excess) for 30 min prior to initiation of the assay. The specific receptor antagonists used were as follows: ETA receptor antagonist (ETA-RA) PD 156707 (sodium 2-benzo [1,3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4,5-trimethoxy-benzyl)- but-2-enoate), ETB receptor antagonist (ETB-RA) BQ-788 (N-cis-2,6-dimethylpiperidimocarbonyl-L-gMeLeuD-Nle-ONa), and the mixed ETA/B receptor antagonist (Bosentan) Ro 47-0203 (4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2′-bipyrimidin-4-yl]-ben- zenesulfonamine). For protein studies fibroblasts were grown to confluence in DMEM with 10% fetal bovine serum, serum starved in DMEM containing 0.5% bovine serum albumin (BSA) (Sigma, St. Louis, MO) for 24 h, and then stimulated with ET-1. Culture medium aliquots were adjusted to 20% (vol/vol) ammonium sulfate and incubated at 4°C with rotation overnight. Samples were then centrifuged (14,000g for 30 min) at 4°C, and the pellet was resuspended in Laemmli sample buffer containing β-mercaptoethanol. The cell layer was washed twice with Tris-buffered saline and cells were directly lyzed by the addition of 1 × sample buffer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 12% polyacrylamide gels, and the separated proteins were transferred onto nitrocellulose membranes at 30 V for 90 min. Membranes were blocked by incubation for 1 h with 5% nonfat milk in phosphate-buffered saline (PBS) containing 0.2% Tween-20 and antigens were detected using the following mono-specific antibodies: rabbit antiproα1(I) collagen antibody (directed against the proα1 chain), rabbit anticollagen type I (directed against collagen type I), and rabbit anticollagen type III (all from Fibrogen, CA); mouse monoclonal antibody against human fibronectin (NovoCastra Laboratories, Newcastle-upon-Tyne, U.K.); mouse antihuman MMP-1 (Oncogene Research Products, Boston, MA); goat antihuman actin (Santa Cruz Biotech, CA), all diluted in PBS. Primary antibody was visualized using a species-specific secondary IgG biotin (Vector Labs, Burlingame, CA) conjugate. Resultant antigen-antibody complexes were detected by incubation with ABC reagent (Vector Labs) using the enhanced chemiluminescence substrate kit (Amersham). Films were analyzed by laser scanning densitometry on an Ultroscan XL (LKB-Wallac, U.K.), with correction of densitometric units according to actin signal, as a surrogate for cell number. Gelatin and casein zymography was performed using fibroblast culture supernatants from fibroblast cultures grown in the absence and presence of ET-1 (100 nM) for 48 h. Briefly, culture medium (15 μl) was diluted in SDS loading buffer and applied to precast 10% polyacrylamide gel Zymogram (Novex, Frankfurt, Germany). Following electrophoresis at 125 V, 4°C, gels were washed twice with renaturing buffer at room temperature for 60 min. Zymograms were then transferred into activity buffer and developed at 37°C for between 4 and 12 h. Following fixation and staining with Coomassie Brilliant Blue R-250 (0.25%), the zymograms were destained with 10% (vol/vol) acetic acid. The following promoter reporter DNA constructions were used: a Col1a2-luciferase construction containing -3.5 kb of the human collagen type I alpha 2 gene promoter (from Francesco Ramirez, Mount Sinai School of Medicine, New York); the hFNLuc construction, containing a -1.3 kb fragment of the human fibronectin promoter (from Noelynn Oliver, FibroGen, San Francisco, CA); and a -4.3 kb MMP-1 luciferase plasmid (-4372hMMP1Luc) containing the human MMP-1 promoter fragment (from Constance Brinckerhoff, Dartmouth Medical School, New Hampshire). Transient cell transfection of fibroblasts was carried using FuGene6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's instructions. Briefly, fibroblasts were grown to subconfluence, serum starved for 12 h, and then transfected with the indicated DNA constructions (0.5–1.0 μg) mixed with FuGene6 and medium in a standard 50–100 μl volume. Following transfection, cells were washed and further cultured in medium in the absence and presence of ET-1 (100 nM). After 48 h of incubation the cells were rinsed once with PBS and lyzed in 200 μl of reporter lysis buffer (Promega, Madison, WI). Reporter plasmids and pCMV-βGal, pGL3-promoter, and pTK-renilla-Luc used as internal control for transfection efficiency were transfected in a 5:1 ratio. Luciferase activity was measured by luminometry (Turner Designs, Sunnyvale, CA) using the Dual-Luciferase Reporter Assay System (Promega), and β-galactosidase activity was measured according to the manufacturer's instructions (Tropix, Bedford, MA). Values given are the means ±SEM of triplicate assays from three individual experiments. To study collagen gel contraction, fibroblasts were cultured within three-dimensional collagen lattices (fibroblast-populated collagen lattices, FPCL). These were prepared as previously described (Ivarsson et al., 1993Ivarsson M. McWhirter A. Black C.M. Rubin K. Impaired regulation of collagen pro-a1 (I) mRNA and change in pattern of collagen-binding integrins on scleroderma fibroblasts.J Invest Dermatol. 1993; 101: 216-221Abstract Full Text PDF PubMed Google Scholar;Shi-Wen et al., 1997Shi-Wen X. Denton C.P. McWhirter A. Bou-Gharios G. Abraham D.J. du Bois R.M. Black C.M. Scleroderma lung fibroblasts exhibit elevated and dysregulated collagen type I biosynthesis.Arthritis Rheum. 1997; 40: 1237-1244Crossref PubMed Scopus (41) Google Scholar). In brief, 24-well tissue culture plates (Costar) were precoated with sterile 2% BSA in PBS (2 ml per well) by incubation at 37°C overnight, and were then washed three times with sterile PBS. For FPCL, neutral collagen solution (containing one part of 0.2 M HEPES, pH 8.0, four parts collagen (Vitrogen-100, 3 mg per ml, Celltrix, Santa Clara, CA), and five parts of MCDB 104 medium (Sigma, two times concentrated) was prepared and mixed with fibroblasts that were resuspended in two times MCDB 104 medium, to bring the final concentrations to 80,000 cells and 1.2 mg collagen per ml. The collagen-cell suspension (1 ml) was added to each well and allowed to gel for 1 h. After polymerization, 1 ml of MCDB medium was added to each well, causing detachment of the FPCL from the tissue culture plastic. ET-1 was added at defined concentrations and gel contraction was measured up to 48 h. Endothelin receptor antagonists were used as described above, except that gel contraction was measured at 12 h. To examine the downstream mediators of ET-1 induced gel remodeling, specific inhibitors of signal transduction pathways were employed. Each inhibitor was added to the culture medium prior to seeding fibroblasts. Control experiments using monolayer fibroblast culture and fibroblasts in collagen gel assays demonstrated that these inhibitors had an effective range of 20–300 μM. This range was determined by the retention of cell viability of fibroblasts cultured as monolayers in the presence of increasing concentrations of inhibitors and the observed optimal gel retraction. Blocking experiments were performed with inhibitors at a concentration of 200 μM. Thus, protein kinase C (PKC) activity was inhibited using calphostin C; genistein and herbimycin A were used to block tyrosine kinases and vanadate to inhibit phosphatase activity. Fibroblast viability following exposure to inhibitors was assessed by exclusion of Trypan Blue vital dye (0.4% in phosphate-buffered saline). Rates of gel contraction were measured by ocular micrometry, and results are presented as the percentage of initial gel area or, upon treatment, as the percentage of the retraction rate observed where the retraction rate in the absence of treatment was taken as 100%. Total RNA was isolated from fibroblasts using the isothiocyanate/caesium chloride method (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single step method of RNA isolation by acid-guanidinium-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62306) Google Scholar). Levels of specific transcripts were determined by northern blotting, following separation of RNA on 1% agarose gels containing 2.2 M formaldehyde and capillary transfer to Hybond N+ membrane (Amersham). Filters were hybridized and probed with cDNA fragments specific for proα1(I) and proα1(III) collagen, fibronectin, and MMP-1 mRNAs as previously described (Shi-Wen et al., 1997Shi-Wen X. Denton C.P. McWhirter A. Bou-Gharios G. Abraham D.J. du Bois R.M. Black C.M. Scleroderma lung fibroblasts exhibit elevated and dysregulated collagen type I biosynthesis.Arthritis Rheum. 1997; 40: 1237-1244Crossref PubMed Scopus (41) Google Scholar). A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific probe was used as an internal standard to allow for differences in amounts of RNA. Probes were labeled with [α32P]-dCTP to a specific activity of 109 dpm per μg, using the Megaprime random priming method (Amersham). Levels of transcripts were determined from signal intensities after quantitation performed by Phosphorimager analysis (Molecular Dynamics, Chesham, Buckinghamshire, U.K.) and adjusted relative to GAPDH signal intensity. Experiments in the presence of ET-1 and endothelin receptor antagonist were performed as described above. For RT-PCR, 5 μg RNA was reverse transcribed using the SUPERSCRIPT Choice system (Gibco BRL, Paisley, U.K.) and amplified by nested PCR for ETA and ETB receptor mRNAs as previously described (Pagotto et al., 1995Pagotto U. Arzberger T. Hopfner U. et al.Expression and localization of endothelin-1 and endothelin receptors in human meningiomas. Evidence for a role in tumoral growth.J Clin Invest. 1995; 96: 2017-2025Crossref PubMed Scopus (70) Google Scholar). The PCR consisted of 30 cycles, each cycle consisting of 94°C for 1 min, 45°C for 1 min, and 72°C for 1 min. Specific amplified products (ETA, 299 bp; ETB, 428 bp; and GAPDH, 605 bp) were analyzed on 1.8% agarose gel and stained with ethidium bromide. GAPDH control primers (sense 5′-CTTCACCACCATGGAGAAGG-3′ antisense 5′-AGGGCA-ATGCCAGCCCCAG-3′ positions cDNA 363–968) were used as normalization controls for RNA. Receptor mRNA levels were measured using S1 nuclease analysis as previously described (Abraham et al., 1997Abraham D.J. Vancheeswaran R. Dashwood M.R. Pantelides P. Shi-wen X. du Bois R.M. Black C.M. Increased levels of endothelin-1 and differential endothelin type A and B receptor expression in scleroderma-associated fibrotic lung disease.Am J Pathol. 1997; 151: 831-841PubMed Google Scholar). The PCR fragments for ETA and ETB receptors were cloned into pGEM-T vector (Promega) and sequenced, and S1 probes were generated by restriction enzyme and phosphatase digestion followed by end-labeling with T4 polynucleotide kinase using [γ-32P]-ATP (Abraham et al., 1997Abraham D.J. Vancheeswaran R. Dashwood M.R. Pantelides P. Shi-wen X. du Bois R.M. Black C.M. Increased levels of endothelin-1 and differential endothelin type A and B receptor expression in scleroderma-associated fibrotic lung disease.Am J Pathol. 1997; 151: 831-841PubMed Google Scholar). Probes were hybridized to 20–60 μg total RNA for 16 h at 53°C, after which samples were digested with S1 nuclease (100 U) by incubation at 23°C for 1 h. Following incubation, digestion was terminated by addition of stop buffer (5 mM Tris-HCl, 50 mM ethylenediamine tetraacetic acid, 1.0% SDS, 10 mg tRNA, pH 7.4) and protected fragments were resolved on 8% denaturing polyacrylamide gel. Transcript levels were determined from signal intensities by phosphorimager analysis (Molecular Dynamics), and are presented relative to levels of the control β-actin RNA transcript. Data summarize three scleroderma and three normal fibroblast strains. Receptor binding studies were performed on confluent fibroblasts cultured in 96-well plates. Washing fibroblasts in 50 mM Tris-HCl, pH 7.4, buffer three times at room temperature reduced endogenous peptide levels. Cells were then incubated for 2 h in binding buffer (PBS containing 5 mM MgCl2, 100 kIU per ml aprotinin, and 1% BSA) containing [125I]-ET-1, or the ETA selective ligand ([125I]-PD151242), or the ETB selective ligand ([125I]-BQ3020), over the concentration range 0.3–1000 pM. Non-specific binding was established in the presence of 1 mM unlabeled ET-1. After incubation, cells were rinsed with buffer at 4°C, harvested by treatment with cell lysis buffer for 10 min (0.25 M NaOH containing 0.5% SDS), and counted using a Packard gamma counter. Specific binding was determined by subtracting nonspecific from total binding, and receptor density and affinity [BMAX (sites per cell) and KD] were calculated using GraphPad InPlot software (Graph Pad, San Diego, CA). In order to study ET receptor binding characteristics, confluent normal and systemic sclerosis fibroblasts were incubated (in triplicate) at a fixed concentration (approximate KD value, 150 pM) of specific ligand, and binding kinetics were determined as above. Experiments using radiolabeled ET-1 and receptor selective ligands characterized fibroblast endothelin receptor binding kinetics. Dissociation constant values (KD) were determined using saturation binding studies where the radiolabeled ligands (over a 4-fold log range of concentrations) were incubated with fibroblast monolayers as described above. Values for the number of endothelin receptor binding sites per cell are given as means ±SEM for five individual cell lines. All results are expressed as means ±SEM unless otherwise stated. Student's t test was used for statistical analyses. p-values less than 0.05 were considered statistically significant. Normal dermal fibroblasts treated with ET-1 showed a dose- and time-dependent increase in proα1(I) collagen chain production (Figure 1a). This was apparent at 1 nM and maximal induction was at 100 nM ET-1 (data not shown). Elevated collagen type I production above basal levels was observed from 8 h post-treatment and was maximal at 72 h. ET-1 also significantly enhanced fibroblast production of collagen type III. The induction of type III collagen was dose dependent (data not shown), again reaching maximal levels after 48 h of culture (Figure 1b). Treatment of normal fibroblasts with ET-1 had no significant influence on the production or secretion of fibronectin (Figure 1b). In contrast, there was suppression of MMP-1 secretion, determined by Western blot analysis, in response to ET-1 (Figure 1b). Blocking studies showed that the mixed selective ligand bosentan (ETA and ETB receptor antagonist) blocked ET-1 induced collagen type I synthesis (p <0.01). Neither an ETA selective receptor antagonist (PD156707) nor an ETB receptor antagonist (BQ-788) alone blocked induction of collagen secretion, however, even at concentrations that saturate subtype-specific binding, suggesting that signaling via both receptor types was necessary for this effect (Figure 2a). Similar results were obtained for type III collagen (data not shown). In contrast to the inductive effects of ET-1 on collagen type I and III synthesis, incubation with ET-1 resulted in a significant decrease in MMP-1 expression by normal fibroblasts (p <0.01) (Figure 2b). The suppression of interstitial collagenase by ET-1 was not influenced by the presence of ETB receptor selective antagonists, but could be prevented by the addition of either the mixed (ETA and ETB) or ETA receptor antagonist (Figure 2b). Examination of MMP-1 activity using zymography (data not shown) also confirmed that the influence of ET-1 on MMP-1 was mediated by the ETA receptor.Figure 2ET-1 induced changes in secretion of collagen depend upon both ETA and ETB receptor subtypes. (A) Collagen secretion by normal fibroblasts (n = 6) was increased by incubation with ET-1 (10-7 M) for 48 h. PD 156707 and BQ 788 (10 mM) alone did not prevent the increase in collagen type I synthesis by ET-1. Collagen induction can be effectively abolished by coincubation with the mixed ETA/B receptor antagonist bosentan (10 mM), however. (B) Incubation of fibroblasts with ET-1 resulted in a decrease in interstitial collagenase (MMP-1) production. This suppression could be abolished by coincubation with either the mixed ETA/B receptor antagonist bosentan or the ETA selective receptor antagonist PD 156707. Data presented are means (± SEM) of four independent experiments. *Significant (*p <0.05, Student's unpaired t test) with respect to control values.View Large Image Figure ViewerDownload (PPT) To determine whether changes in extracellular matrix protein levels were due to altered gene expression, collagen α1(I), α1(III), collagenase (MMP-1), and fibronectin genes were studied in fibroblasts grown in monolayers in the presence of ET-1. The expression profiles of these genes in normal fibroblasts are compared with those of lesional systemic sclerosis fibroblasts in Figure 3(a). The latter exhibit increased steady-state levels of transcripts of proα1(I), proα1(III) collagen, and fibronectin, but substantially reduced levels of MMP-1 mRNA (Figure 3a). Normal fibroblasts treated with ET-1 showed a significant upregulation of proα1(I) and proα1(III) collagen mRNA but downregulation of collagenase (MMP-1). Consistent with the protein data, there was little effect on fibronectin mRNA level (Figure 3b). Incubation of normal fibroblasts with ET-1 in the presence of bosentan blocked the inductive influence of ET-1 on proα1(I) and proα1(III) collagen mRNA (Figure 3b). Inhibition of MMP-1 mRNA level by ET-1 could be blocked by an ETA receptor selective antagonist alone (Figure 3b), but both ETA and ETB receptor blockade was necessary to prevent collagen I or III mRNA induction. To explore further the ET-1 mediated changes in steady-state levels of matrix gene transcripts we under
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