Matrix Contraction by Dermal Fibroblasts Requires Transforming Growth Factor-β/Activin-Linked Kinase 5, Heparan Sulfate-Containing Proteoglycans, and MEK/ERK
2005; Elsevier BV; Volume: 167; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)61252-7
ISSN1525-2191
AutoresYunliang Chen, Shiwen Xu, Jonathan van Beek, Laura Kennedy, Marilyn McLeod, Elisabetta Renzoni, George Bou‐Gharios, Sarah A. Wilcox‐Adelman, Paul F. Goetinck, Mark Eastwood, Carol M. Black, David Abraham, Andrew Leask,
Tópico(s)Systemic Sclerosis and Related Diseases
ResumoScarring is characterized by excessive synthesis and contraction of extracellular matrix. Here, we show that fibroblasts from scarred (lesional) areas of patients with the chronic fibrotic disorder diffuse scleroderma [diffuse systemic sclerosis (dSSc)] show an enhanced ability to adhere to and contract extracellular matrix, relative to fibroblasts from unscarred (nonlesional) areas of dSSc patients and dermal fibroblasts from normal, healthy individuals. The contractile abilities of normal and dSSc dermal fibroblasts were suppressed by blocking heparin sulfate-containing proteoglycan biosynthesis or antagonizing transforming growth factor-β receptor type I [activin-linked kinase (ALK5)] or ras/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK). Compared with both normal and nonlesional fibroblasts, lesional dSSc fibroblasts overexpressed the heparin sulfate-containing proteoglycan syndecan 4. We also found that the procontractile signals from transforming growth factor (TGF)-β were integrated through syndecan 4 and MEK/ERK because the ability of TGFβ to induce contraction of dermal fibroblasts was prevented by MEK antagonism. TGFβ could not induce a contractile phenotype or phosphorylate ERK in syndecan 4−/− dermal fibroblasts. These results suggest that integrating TGFβ and ERK signals via syndecan 4 is essential for the contractile ability of dermal fibroblasts. We conclude that antagonizing MEK/ERK, TGFβ1/ALK5, or syndecan 4 may alleviate scarring in chronic fibrotic disease. Scarring is characterized by excessive synthesis and contraction of extracellular matrix. Here, we show that fibroblasts from scarred (lesional) areas of patients with the chronic fibrotic disorder diffuse scleroderma [diffuse systemic sclerosis (dSSc)] show an enhanced ability to adhere to and contract extracellular matrix, relative to fibroblasts from unscarred (nonlesional) areas of dSSc patients and dermal fibroblasts from normal, healthy individuals. The contractile abilities of normal and dSSc dermal fibroblasts were suppressed by blocking heparin sulfate-containing proteoglycan biosynthesis or antagonizing transforming growth factor-β receptor type I [activin-linked kinase (ALK5)] or ras/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK). Compared with both normal and nonlesional fibroblasts, lesional dSSc fibroblasts overexpressed the heparin sulfate-containing proteoglycan syndecan 4. We also found that the procontractile signals from transforming growth factor (TGF)-β were integrated through syndecan 4 and MEK/ERK because the ability of TGFβ to induce contraction of dermal fibroblasts was prevented by MEK antagonism. TGFβ could not induce a contractile phenotype or phosphorylate ERK in syndecan 4−/− dermal fibroblasts. These results suggest that integrating TGFβ and ERK signals via syndecan 4 is essential for the contractile ability of dermal fibroblasts. We conclude that antagonizing MEK/ERK, TGFβ1/ALK5, or syndecan 4 may alleviate scarring in chronic fibrotic disease. The wound healing response to tissue injury requires the de novo synthesis of connective tissue. Normally, the wound healing process is appropriately terminated, and proper organ function is restored. However, it is believed that if the wound healing process continues unabated, fibrosis, a condition characterized by the excessive deposition and contraction of extracellular matrix (ECM), results.1Turck CW Dohlman JG Goetzl EJ Immunological mediators of wound healing and fibrosis.J Cell Physiol Suppl. 1987; 5: 89-93Crossref PubMed Scopus (29) Google Scholar In its less severe forms, fibrosis might consist of merely a disfiguring scar, or in a localized hyperproliferation of fibroblasts, such as a keloid. However, in its most severe forms, excessive scarring can result in fibrotic disease, which is characterized by tissue destruction, organ dysfunction, or death due to systemic organ failure. Currently, there is no effective therapy for fibrotic disease, in part because the underlying cause of these disorders remains elusive. Fibroblasts are generally considered to be major effecter cells in fibrotic disease by contributing to the increased synthesis and contraction of extracellular matrix (ECM) characteristic of these disorders.2Jimenez SA Hitraya E Varga J Pathogenesis of scleroderma.Collagen Rheum Dis Clin North Am. 1996; 22: 647-674Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3Krieg T Heckmann M Regulatory mechanisms of fibroblast activity.Recenti Prog Med. 1989; 80: 594-598PubMed Google Scholar In the wound healing response, fibroblasts proliferate, migrate into the wound and produce increased amounts of ECM. This transformation of fibroblasts to proliferating, excessively matrix-producing cells has been termed fibroblast activation. Activated fibroblasts in the wound are often termed “myofibroblasts,”4Desmouliere A Gabbiani G Myofibroblast differentiation during fibrosis.Exp Nephrol. 1995; 3: 134-139PubMed Google Scholar because they express the procontractile protein α-smooth muscle actin (α-SMA). In fibrotic lesions, α-smooth muscle actin (α-SMA)-positive matrix-producing myofibroblasts persist.4Desmouliere A Gabbiani G Myofibroblast differentiation during fibrosis.Exp Nephrol. 1995; 3: 134-139PubMed Google Scholar Based on data observed in normal fibroblasts induced to express α-SMA in cell culture, these α-SMA-expressing fibroblasts present in fibrotic lesions would be expected to contribute to the excessive contraction and synthesis of extracellular matrix characteristic of the enhanced tensile strength of scar tissue.5Gabbiani G The myofibroblast in wound healing and fibrocontractive diseases.J Pathol. 2003; 200: 500-503Crossref PubMed Scopus (1270) Google Scholar However, the precise extent to which the lesional fibroblast is autonomously activated in chronic fibrotic disease and the phenotypic alterations in the fibroblast correlating with the progression of fibrosis have yet to be fully elucidated. Furthermore, although dysregulated transforming growth factor (TGF)-β receptor signaling has been hypothesized to play a role in chronic fibrotic disease such as scleroderma,6Leask A Abraham DJ Finlay DR Holmes A Pennington D Shi-Wen X Chen Y Venstrom K Dou X Ponticos M Black C Bernabeu C Jackman JK Findell PR Connolly MK Dysregulation of transforming growth factor beta signaling in scleroderma: overexpression of endoglin in cutaneous scleroderma fibroblasts.Arthritis Rheum. 2002; 46: 1857-1865Crossref PubMed Scopus (100) Google Scholar, 7Leask A Abraham DJ TGFβ signaling and the fibrotic response.FASEB J. 2004; 18: 816-827Crossref PubMed Scopus (1982) Google Scholar, 8Pannu J Gore-Hyer E Yamanaka M Smith EA Rubinchik S Dong JY Jablonska S Blaszczyk M Trojanowska M An increased transforming growth factor beta receptor type I: type II ratio contributes to elevated collagen protein synthesis that is resistant to inhibition via a kinase-deficient transforming growth factor beta receptor type II in scleroderma.Arthritis Rheum. 2004; 50: 1566-1577Crossref PubMed Scopus (62) Google Scholar the precise contribution of TGFβ receptors to the pathology of fibrotic disorders remains to be elucidated. Scleroderma [systemic sclerosis (SSc)] is a chronic disease of unknown etiology characterized by microvascular injury, autoimmune inflammatory responses, and severe and often progressive fibrosis.2Jimenez SA Hitraya E Varga J Pathogenesis of scleroderma.Collagen Rheum Dis Clin North Am. 1996; 22: 647-674Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3Krieg T Heckmann M Regulatory mechanisms of fibroblast activity.Recenti Prog Med. 1989; 80: 594-598PubMed Google Scholar, 9Black CM Scleroderma: clinical aspects.J Intern Med. 1993; 234: 115-118Crossref PubMed Scopus (49) Google Scholar, 10Furst DE Clements PJ Hypothesis for the pathogenesis of systemic sclerosis.J Rheumatol Suppl. 1997; 48: 53-57PubMed Google Scholar Because SSc shares similar features to other fibrotic diseases, elucidating the molecular basis of SSc is likely to be beneficial in understanding the nature of fibrotic disease in general. Clinically, SSc is heterogeneous ranging form mild, limited skin sclerosis with minimal organ involvement (limited SSc), to diffuse skin involvement and se-vere fibrosis of multiple internal organs [diffuse SSc (dSSc)].2Jimenez SA Hitraya E Varga J Pathogenesis of scleroderma.Collagen Rheum Dis Clin North Am. 1996; 22: 647-674Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3Krieg T Heckmann M Regulatory mechanisms of fibroblast activity.Recenti Prog Med. 1989; 80: 594-598PubMed Google Scholar, 9Black CM Scleroderma: clinical aspects.J Intern Med. 1993; 234: 115-118Crossref PubMed Scopus (49) Google Scholar, 10Furst DE Clements PJ Hypothesis for the pathogenesis of systemic sclerosis.J Rheumatol Suppl. 1997; 48: 53-57PubMed Google Scholar Mortality of dSSc patients is high and is directly related to the extent of scarring.2Jimenez SA Hitraya E Varga J Pathogenesis of scleroderma.Collagen Rheum Dis Clin North Am. 1996; 22: 647-674Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3Krieg T Heckmann M Regulatory mechanisms of fibroblast activity.Recenti Prog Med. 1989; 80: 594-598PubMed Google Scholar, 9Black CM Scleroderma: clinical aspects.J Intern Med. 1993; 234: 115-118Crossref PubMed Scopus (49) Google Scholar, 10Furst DE Clements PJ Hypothesis for the pathogenesis of systemic sclerosis.J Rheumatol Suppl. 1997; 48: 53-57PubMed Google Scholar Clinically, dSSc skin has been characterized by “lesional” and “nonlesional” (ie, clinically affected and nonaffected) areas based on the physical appearance of appreciable scar tissue.9Black CM Scleroderma: clinical aspects.J Intern Med. 1993; 234: 115-118Crossref PubMed Scopus (49) Google Scholar SSc dermal fibroblasts can be readily isolated and cultured and retain their ability to overexpress type I collagen and connective tissue growth factor.11LeRoy 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 (496) Google Scholar, 12Kahari VM Multimaki P Vuorio E Elevated pro alpha 2(I) collagen mRNA levels in cultured scleroderma fibroblasts result from an increased transcription rate of the corresponding gene.FEBS Lett. 1987; 215: 331-334Crossref PubMed Scopus (55) Google Scholar, 13Abraham DJ Shiwen X Black CM Sa S Xu Y Leask A Tumor necrosis factor alpha suppresses the induction of connective tissue growth factor by transforming growth factor-beta in normal and scleroderma fibroblasts.J Biol Chem. 2000; 275: 15220-15225Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 14Shi-wen X Pennington D Holmes A Leask A Bradham D Beauchamp JR Fonseca C du Bois RM Martin GR Black CM Abraham DJ Autocrine overexpression of CTGF maintains fibrosis: RDA analysis of fibrosis genes in systemic sclerosis.Exp Cell Res. 2000; 259: 213-224Crossref PubMed Scopus (171) Google Scholar Thus examination of the phenotypic—and molecular—difference among normal fibroblasts from healthy individuals and fibroblasts from nonlesional and lesional areas of dSSc patients should yield valuable insights into the molecular nature of scar tissue formation and progression in chronic fibrotic disease in general. Consequently, to begin to understand the molecular nature of scar formation in progressive fibrotic disease, we compare the phenotypes and gene expression profiles of normal, nonlesional, and lesional dSSc dermal fibroblasts. Our results yield new insights into the molecular basis of ECM contraction by fibroblasts and suggest new methods of combating chronic, pathological fibrosis. Briefly, cell culture was performed as previously described.13Abraham DJ Shiwen X Black CM Sa S Xu Y Leask A Tumor necrosis factor alpha suppresses the induction of connective tissue growth factor by transforming growth factor-beta in normal and scleroderma fibroblasts.J Biol Chem. 2000; 275: 15220-15225Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar Dermal fibroblasts from lesional and nonlesional (clinically affected and nonaffected) areas of patients with diffuse SSc (between 12 and 18 months duration) and normal individuals were taken from biopsies of age, sex, and anatomically site-matched volunteers, after informed consent and ethical approval was obtained. All patients fulfilled the criteria of the American College of Rheumatology for the diagnosis of diffuse SSc. Patients were female. None was receiving immunosuppressive medication or corticosteroids at the time of biopsy. Fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies), 100 U/ml penicillin, and 100 mg/ml streptomycin and cultured in a humidified atmosphere of 5% CO2 in air. Fibroblasts were subcultured 1:4 at confluence. Syndecan 4+/+ and syndecan 4−/− dermal fibroblasts were isolated and cultured as previously described.15Wilcox-Adelman SA Denhez F Goetinck PF Syndecan-4 modulates focal adhesion kinase phosphorylation.J Biol Chem. 2002; 277: 32970-32977Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar When appropriate, the MEK inhibitors U0126 (10 μmol/L; Promega, Southampton, United Kingdom) and PD98059 (30 μmol/L; Calbiochem, La Jolla, CA) or the ALK5 inhibitor SB431542 (10 μmol/L; Tocris, Bristol, United Kingdom) was added for the durations indicated. Expression profiling was conducted as previously described.16Shi-wen X Howat SL Renzoni EA Holmes A Pearson JD Dashwood MR Bou-Gharios G Denton CP du Bois RM Black CM Leask A Abraham DJ Endothelin-1 induces expression of matrix-associated genes in lung fibroblasts through MEK/ERK.J Biol Chem. 2004; 279: 23098-23103Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17Shi-wen X Chen Y Denton CP Eastwood M Renzoni E Bou-Gharios G Dashwood MR duBois R Black CM Leask A Abraham DJ Endothelin-1 promotes mechanoregulation and myofibroblast formation in fibroblasts through the ETA receptor via Akt/PI3 kinase: implications for lung fibrosis.Mol Biol Cell. 2004; 15: 2707-2719Crossref PubMed Scopus (321) Google Scholar, 18Renzoni EA Abraham DJ Howat S Shi-Wen X Sestini P Bou-Gharios G Wells AU Veeraraghavan S Nicholson AG Denton CP Leask A Pearson JD Black CM Welsh KI du Bois RM Gene expression profiling reveals novel TGFbeta targets in adult lung fibroblasts.Respir Res. 2004; 5: 24Crossref PubMed Scopus (108) Google Scholar Dermal fibroblasts from four normal individuals and dermal fibroblasts from areas of persistent fibrotic areas of lesions of three dSSc patients were cultured until confluence and serum starved for 18 hours. Fibroblasts were of passage 4. Media were changed and cultured for an additional 8 hours. At the end of the treatment period, total RNA was harvested (Trizol; Life Technologies) and quantified, and integrity was verified by denaturing gel electrophoresis. Equal amounts of identically treated RNA were pooled and reverse transcribed (Life Technologies) into cDNA that was then in vitro transcribed into biotinylated cRNA. The target cRNA was then fragmented and hybridized to the Affymetrix human U133A array (Affymetrix, Santa Clara, CA), following Affymetrix protocol. Hybridization of cRNA to Affymetrix human U133A chips, signal amplification, and data collection were performed using an Affymetrix fluidics station and chip reader. Chip files were scaled to an average intensity of 100 per gene and analyzed using the Affymetrix version 5.0 (MAS5) comparison analysis software. Experiments were performed twice, and fold changes presented in Table 1 are an average of these independent studies. Criteria indicated by Affymetrix were used to determine robust changes in gene expression. Briefly, transcripts were defined as up-regulated in dSSc only when identified as “Present” by the Affymetrix detection algorithm and as significantly increased as determined by the Affymetrix change algorithm, with a change in P value of Threefold) in Normal FibroblastsAffymetrix IDMatrix, cytoskeleton, and adhesion-associated genesAccession no.Fold increase202620_s_atProcollagen-lysine 2 (PLOD2)NM_000935.16.2209663_s_atIntegrin α-7AF072132.15.5204627_s_atIntegrin, β3M359997.4202351_atIntegrin, αVAI3802984.6201389_atIntegrin, α5NM_003461.14.6212158_atSyndecan 2AL5773224.8202071_atSyndecan 4NM_002999.13200859_x_atFilamin A, α (actin-binding protein-280)NM_014000.14.4200974_atActin, α2, smooth muscle, aortaNM_001613.17.1205132_atActin, α, cardiac muscleNM_005159.26.1211126_s_atSmooth muscle LIM proteinU46006.15.9211823_s_atPaxillin βD86862.14.7200931_s_atVinculinNM_014000.14.4200808_s_atZyxinNM_006932.14.6207390_s_atSmoothelinAL0469794.5221748_s_atTensinAL0469794.6210764_s_atCYR61AF003114.15.6209101_atConnective tissue growth factorM92934.16.4 Open table in a new tab Cells were cultured until confluence in DMEM 10% fetal bovine serum (FBS). Cell layers were harvested using 2% sodium dodecyl sulfate (SDS). Proteins were quantified (Bradford; Bio-Rad, Hercules, CA), and equal amounts of protein (25 μg) were subjected to SDS-polyacrylamide gel electrophoresis using 4 to 12% polyacrylamide gels (Invitrogen, Carlsbad, CA). Gels were blotted onto nitrocellulose, and proteins were detected using anti-CCN2 (Santa Cruz, Santa Cruz, CA), anti-moesin, anti-paxillin, anti-vinculin, anti-ezrin (Cell Signaling, Beverly, MA), anti α-SMA (Sigma, St. Louis, MO), anti-syndecan 4, anti-syndecan 2, anti-α4 and anti-β1 integrin (Zymed, South San Francisco, CA), and appropriate horseradish peroxidase-conjugated secondary antibodies (Zymed) and an ECL kit (Amersham, Little Chalfont, United Kingdom). To detect type I collagen, equal amounts of media were precipitated with 30% ammonium sulfate, resuspended in 2% SDS, and subjected to SDS-polyacrylamide gel electrophoresis. Gels were blotted onto nitrocellulose, and type I collagen was detected with an anti-type I collagen antibody (Biodesign, Saco, ME), as described above. For immunofluorescent detection, cells were fixed in 3% paraformaldehyde (15 minutes), and localization of proteins was detected as previously described.19Chen Y Abraham DJ Shi-wen X Pearson JD Black CM Lyons KM Leask A CCN2 (connective tissue growth factor) promotes fibroblast adhesion to fibronectin.Mol Biol Cell. 2004; 15: 5635-5646Crossref PubMed Scopus (151) Google Scholar Densitometry was performed using AlphaEase (Alpha Innotech, San Leandro, CA) as previously described.19Chen Y Abraham DJ Shi-wen X Pearson JD Black CM Lyons KM Leask A CCN2 (connective tissue growth factor) promotes fibroblast adhesion to fibronectin.Mol Biol Cell. 2004; 15: 5635-5646Crossref PubMed Scopus (151) Google Scholar Expression values were calculated relative to baseline, and average ± SD was obtained. Student's paired t-test was performed on the protein expression obtained in dSSc fibroblasts, relative to protein expression in normal fibroblasts (P < 0.05). Fibroblasts were isolated and cultured as described above. Fibroblasts isolated from three normal individuals and lesional areas of three individuals with dSSc were assayed in triplicate. Fibroblasts were used at passage 3. Adhesion assays were performed19Chen Y Abraham DJ Shi-wen X Pearson JD Black CM Lyons KM Leask A CCN2 (connective tissue growth factor) promotes fibroblast adhesion to fibronectin.Mol Biol Cell. 2004; 15: 5635-5646Crossref PubMed Scopus (151) Google Scholar by initially coating wells of 96-well plates overnight at 4°C, with 6 mg/ml fibronectin (Sigma) in 0.5% bovine serum albumin (BSA) and 1× phosphate-buffered saline (PBS). Wells were blocked for 1 hour in 10% BSA in PBS, room temperature. Fibroblasts were harvested with 2 mmol/L ethylenediamine tetraacetic acid in PBS (20 minutes at room temperature), washed twice with DMEM serum-free medium containing 1% BSA (Sigma), and resuspended in the same medium at 2.5 × 105 cells/ml. To detect cell adhesion, an acid phosphatase assay was used, and adherent cells were quantified by incubation with 100 μl of substrate solution (0.1 mol/L sodium acetate, pH 5.5, 10 mmol/L −p-nitophenylphosphate, and 0.1% Triton X-100) for 2 hours at 37°C. The reaction was stopped by the addition of 15 μl of 1 N NaOH/well, and A450 was measured. Comparison of adhesive abilities was performed by using Student's unpaired t-test. A P value <0.05 was considered as statistically significant. Experiments were performed essentially as described previously.17Shi-wen X Chen Y Denton CP Eastwood M Renzoni E Bou-Gharios G Dashwood MR duBois R Black CM Leask A Abraham DJ Endothelin-1 promotes mechanoregulation and myofibroblast formation in fibroblasts through the ETA receptor via Akt/PI3 kinase: implications for lung fibrosis.Mol Biol Cell. 2004; 15: 2707-2719Crossref PubMed Scopus (321) Google Scholar Briefly, 24-well tissue culture plates were pre-coated with BSA. Trypsinized fibroblasts were suspended in MCDB medium (Sigma) and mixed with collagen solution [one part 0.2 mol/L N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 8.0; four parts collagen (Vitrogen-100; 3 μg/ml); and five parts MCDB ×2] yielding a final concentration of 80,000 cells/ml and 1.2 mg/ml collagen. Collagen/cell suspension (1 ml) was added to each well. After polymerization, gels were detached from wells by adding 1 ml of MCDB medium. Contraction of the gel was quantified by loss of gel weight and decrease in gel diameter over a 24-hour period. Comparison of collagen gel contraction was performed by using Student's unpaired t-test. A P value <0.05 was considered statistically significant. For experiments of proteoglycan biosynthesis inhibition, cells were cultured for 4 days in DMEM, 10% FBS with 1 mmol/L α-d-xyloside (4-methylumbelliferyl-α-d-xyloside; Sigma) as a control or 1 mmol/L β-d-xyloside (4-methylumbelliferyl-β-d-xyloside; Sigma), which blocks heparin sulfate side chain formation and proteoglycan biosynthesis.20Carey DJ Rafferty CM Todd MS Effects of inhibition of proteoglycan synthesis on the differentiation of cultured rat Schwann cells.J Cell Biol. 1987; 105: 1013Crossref PubMed Scopus (43) Google Scholar, 21Hamati HF Britton EL Carey DJ Inhibition of proteoglycan synthesis alters extracellular matrix deposition, proliferation, and cytoskeletal organization of rat aortic smooth muscle cells in culture.J Cell Biol. 1989; 108: 2495Crossref PubMed Scopus (71) Google Scholar, 22Rosamond S Brown L Gomez C Braciale TJ Schwartz BD Xyloside inhibits synthesis of the class II-associated chondroitin sulfate proteoglycan and antigen presentation events.J Immunol. 1987; 139: 1946-1951Crossref PubMed Google Scholar The cells were then washed twice in PBS before further experimentation. Inhibitor toxicity was assessed using a cell viability assay as described by the manufacturer (MTT; Roche, Laval, Quebec). Fibroblasts (NIH 3T3; ATCC, Manassas, VA) were transfected using Polyfect (Qiagen, Crawley, United Kingdom) essentially as previously described.6Leask A Abraham DJ Finlay DR Holmes A Pennington D Shi-Wen X Chen Y Venstrom K Dou X Ponticos M Black C Bernabeu C Jackman JK Findell PR Connolly MK Dysregulation of transforming growth factor beta signaling in scleroderma: overexpression of endoglin in cutaneous scleroderma fibroblasts.Arthritis Rheum. 2002; 46: 1857-1865Crossref PubMed Scopus (100) Google Scholar, 13Abraham DJ Shiwen X Black CM Sa S Xu Y Leask A Tumor necrosis factor alpha suppresses the induction of connective tissue growth factor by transforming growth factor-beta in normal and scleroderma fibroblasts.J Biol Chem. 2000; 275: 15220-15225Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 16Shi-wen X Howat SL Renzoni EA Holmes A Pearson JD Dashwood MR Bou-Gharios G Denton CP du Bois RM Black CM Leask A Abraham DJ Endothelin-1 induces expression of matrix-associated genes in lung fibroblasts through MEK/ERK.J Biol Chem. 2004; 279: 23098-23103Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar Briefly, fibroblasts were seeded into 6-well plates and transfected at ∼50% confluence with a generic MEK/ERK-responsive promoter (Clontech, Palo Alto, CA; 0.5 μg/well) with either empty expression vector or expression vector encoding syndecan 4 (15; 1 μg/well). Cells were cotransfected with a CMV promoter-driven β-galactosidase reporter gene (Clontech; 0.25 mg/well) expression from which (Applied Biosystems, Foster City, CA) was used to adjust for differences in transfection efficiencies among wells. After transfection, cells were maintained in DMEM, 0.5% calf serum (Life Technologies) for 48 hours. Reporter gene expression vas determined (Applied Biosystems), normalized for differences in transfection efficiencies, and expressed as average ± SD (Student's paired t-test, P < 0.05). Experiments were performed twice, with six replicates. Specific siRNA recognizing syndecan 4 was purchased through a kit containing a pool of several siRNA (Syndecan 4 SMARTPool; Dharmacon, Denver, CO). A recommended control siRNA labeled with a green fluorescent protein (GFP) tag (cyclophilin B; Dharmacon) was also purchased. Normal and dSSc fibroblasts were transfected using an electroporator and an optimized kit for primary fibroblasts (Nucleofector; Amaxa, Cologne, Germany). Cells were transfected either with control siRNA or control siRNA with syndecan 4 siRNA. Cells were fixed in 4% paraformaldehyde (15 minutes; Sigma) and stained with mouse anti-phospho-ERK (Cell Signaling) and Texas Red anti-mouse (Jackson, West Grove, PA) antibody. Transfected cells were detected by looking for green cells (from the GFP tag of the control siRNA). Scar tissue is characterized by the excessive production of collagen. To begin to investigate the basis of the contribution of the fibroblast in dSSc to scar formation in dSSc, we used Western blot analysis with an anti-type I collagen antibody to compare the abilities of dermal fibroblasts from healthy individuals and dermal fibroblasts from unaffected and affected areas of dSSc patients to produce type I collagen. To our surprise, we found that although dSSc fibroblasts produced elevated levels of type I collagen, nonlesional (clinically unaffected) dSSc fibroblasts produced a level of type I collagen intermediate between lesional (clinically affected) dSSc fibroblasts and normal fibroblasts (Figure 1). Thus, we concluded that substantial differences in type I collagen levels per se were not directly responsible for the outward appearance of scar tissue in dSSc patients. To further address the basis of the contribution of the lesional dermal dSSc fibroblast to scar formation in dSSc, we directly compared the abilities of dermal fibroblasts from healthy individuals and dermal fibroblasts from unaffected and affected areas of dSSc patients to adhere to and contract ECM. To assess the abilities of normal, nonlesional and lesional dermal fibroblasts to adhere to ECM, we coated 96-well plates with fibronectin. Fibroblasts were cultured from three normal individuals and nonlesional and lesional areas of three individuals with diffuse SSc. Cells were detached from tissue culture dishes with ethylenediamine tetraacetic acid and allowed to adhere to fibronectin for 45 minutes. We found that fibroblasts cultured from lesional areas of dSSc patients showed markedly elevated adhesive ability, relative to fibroblasts cultured from normal donors and nonlesional areas of dSSc patients (Figure 2A). We reasoned that because lesional dermal dSSc fibroblasts excessively adhered to ECM, they might be expected to excessively contract ECM, because both of these activities require the formation of focal adhesions between the cytoskeleton and ECM.23Yoneda A Couchman JR Regulation of cytoskeletal organization by syndecan transmembrane proteoglycans.Matrix Biol. 2003; 22: 25-33Crossref PubMed Scopus (145) Google Scholar, 24McGrath JA Eady RA Heparan sulphate proteoglycan and wound healing in skin.J Pathol. 1997; 183: 251-252Crossref PubMed Scopus (16) Google Scholar To compare the abilities of normal, nonlesional, and lesional dermal fibroblasts to contract ECM, we seeded dermal fibroblasts within a collagen matrix and allowed the resultant mixture to polymerize on a tissue culture plate. The solidified gel was then detached from the tissue culture plate and incubated for 24 hours in the presence of 0.5% FBS. After this period, we assessed collagen gel contraction by measuring the size and weight of the contracted gel.17Shi-wen X Chen Y Denton CP Eastwood M Renzoni E Bou-Gharios G Dashwood MR duBois R Black CM Leask A Abraham DJ Endothelin-1 promotes mechanoregulation and myofibroblast formation in fibroblasts through the ETA receptor via Akt/PI3 kinase: implications for lung fibrosis.Mol Biol Cell. 2004; 15: 2707-2719Crossref PubMed Scopus (321) Google Scholar, 25Grinnell F Ho CH Lin YC Skuta G Differences in the regulation of fibroblast contraction of floating versus stressed collagen matrices.J Biol Chem. 1999; 274: 918-923Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar We found that lesional dSSc dermal fibroblasts showed a greatly increased ability, relative to nonlesional dSSc and normal dermal fibroblasts, to contract a collagen matri
Referência(s)