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Altered production of extra-cellular matrix components by muscle-derived Duchenne muscular dystrophy fibroblasts before and after TGF-β1 treatment

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Abstract

To probe pro-fibrotic mechanisms in dystrophic muscle, we isolated primary fibroblasts from Duchenne muscular dystrophy (DMD) and control muscle biopsies and induced transdifferentiation in myofibroblasts by transforming growth factor β1 (TGF-β1) treatment. We compared proliferating activity, soluble collagen production, and transcript and protein levels of decorin, myostatin, TGF-β1, matrix metalloproteinase-1 (MMP-1; interstitial collagenase), MMP-2 (gelatinase), MMP-3 (stromelysin), MMP-7 (matrilysin), and the tissue inhibitors of metalloproteinases inhibitors (TIMPs) 1–4, in fibroblasts and myofibroblasts. Principal differences included a significantly greater proliferation rate and soluble collagen production, a significant upregulation of decorin, myostatin and MMP-7 transcripts and proteins, and a significant downregulation of MMP-1 and TIMP-3 transcripts (with MMP-1 protein being reduced as shown by enzyme-linked immunosorbent assay and TIMP-3 protein apparently being reduced on Western blot), in untreated DMD fibroblasts compared with controls. TGF-β1 transdifferentiation significantly lowered decorin and myostatin and significantly increased TGF-β1 transcript and protein, significantly increased MMP-1 and TIMP-3, and significantly lowered MMP-7 transcript and protein in DMD cells compared with pretreatment controls. The differences between DMD and control fibroblasts showed that DMD fibroblasts had a profibrotic phenotype, accentuated by TGF-β1 treatment. Dystrophin absence itself could exert a direct influence on the homeostasis of the extracellular matrix (ECM) by allowing leakage of cellular components to the extracellular space or by abnormal cellular uptake of extracellular growth factors, cytokines, or enzymes influencing muscle fibroblasts either directly by altering adhesion properties or indirectly by interactions with molecules released into the ECM by muscle or inflammatory cells. The transdifferentiation of muscle fibroblasts might serve as a simplified model of fibrosis for further elucidation of the mechanisms of muscle fibrosis and for testing possible anti-fibrotic agents.

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Abbreviations

ECM:

Extracellular matrix

DMD:

Duchenne muscular dystrophy

α-SMA:

α-Smooth muscle actin

MMPs:

Matrix metalloproteinases

TIMPs:

Tissue inhibitors of metalloproteinases

TGF-β1:

Transforming growth factor β1

References

  • Ahonen M, Baker AH, Kähäri VM (1998) Adenovirus-mediated gene delivery of tissue inhibitor of metalloproteinases-3 inhibits invasion and induces apoptosis in melanoma cells. Cancer Res 58:2310–2315

    CAS  PubMed  Google Scholar 

  • Amour A, Slocombe PM, Webster A, Butler M, Knight CG, Smith BJ, Stephens PE, Shelley C, Hutton M, Knäuper V, Docherty AJ, Murphy G (1998) TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3. FEBS Lett 435:39–44

    Article  CAS  PubMed  Google Scholar 

  • Baker AH, Zaltsman AB, George SJ, Newby AC (1998) Divergent effects of tissue inhibitor of metalloproteinase-1, −2, or −3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis. J Clin Invest 101:1478–1487

    Article  CAS  PubMed  Google Scholar 

  • Bernasconi P, Torchiana E, Confalonieri P, Brugnoni R, Barresi R, Mora M, Cornelio F, Morandi L, Mantegazza R (1995) Expression of transforming growth-factor beta 1 in dystrophic patient muscles correlates with fibrosis. J Clin Invest 96:1137–1144

    Article  CAS  PubMed  Google Scholar 

  • Blake DJ, Weir A, Newey SE, Davies KE (2002) Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol Rev 82:291–329

    CAS  PubMed  Google Scholar 

  • Border WA, Ruoslahti E (1992) Transforming growth factor-beta in disease: the dark side of tissue repair. J Clin Invest 90:1–7

    Article  CAS  PubMed  Google Scholar 

  • Brinckerhoff CE, Ruby PL, Austin SD, Fini ME, White HD (1987) Molecular cloning of human synovial cell collagenase and selection of a single gene from genomic DNA. J Clin Invest 79:542–546

    Article  CAS  PubMed  Google Scholar 

  • Chang HY, Chi JT, Dudoit S, Bondre C, M van de Rijn, Botstein D, Brown PO (2002) Diversity, topographic differentiation, and positional memory in human fibroblasts. Proc Natl Acad Sci USA 99:12877–12882

    Article  CAS  PubMed  Google Scholar 

  • De Luca A, Santra M, Baldi A, Giordano A, Iozzo RV (1996) Decorin-induced growth suppression is associated with up-regulation of p21, an inhibitor of cyclin-dependent kinases. J Biol Chem 271:18961–18965

    Article  PubMed  Google Scholar 

  • Desmoulière A, Geinoz A, Gabbiani F, Gabbiani G (1993) Transforming growth factor-β1 induces α-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 122:103–111

    Article  PubMed  Google Scholar 

  • Desmoulière A, Redard M, Darby I, Gabbiani G (1995) Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am J Pathol 146:56–66

    PubMed  Google Scholar 

  • Desmoulière A, Chaponnier C, Gabbiani G (2005) Tissue repair, contraction, and the myofibroblast. Wound Repair Regen 13:7–12

    Article  PubMed  Google Scholar 

  • Dickson G, Gower HJ, Barton CH, Prentice HM, Elsom VL, Moore SE, Cox RD, Quinn C, Putt W, Walsh FS (1987) Human muscle neural cell adhesion molecule (N-CAM): identification of a muscle-specific sequence in the extracellular domain. Cell 50:1119–1130

    Article  CAS  PubMed  Google Scholar 

  • Eckes B, Zigrino P, Kessler D, Holtkötter O, Shephard P, Mauch C, Krieg T (2000) Fibroblast-matrix interactions in wound healing and fibrosis. Matrix Biol 19:325–332

    Article  CAS  PubMed  Google Scholar 

  • Fadic R, Mezzano V, Alvarez K, Cabrera D, Holmgren J, Brandan E (2006) Increase in decorin and biglycan in Duchenne muscular dystrophy: role of fibroblasts as cell source of these proteoglycans in the disease. J Cell Mol Med 10:758–769

    Article  CAS  PubMed  Google Scholar 

  • Finlay GA, Thannickal VJ, Fanburg BL, Paulson KE (2000) Transforming growth factor-beta 1-induced activation of the ERK pathway/activator protein-1 in human lung fibroblasts requires the autocrine induction of basic fibroblast growth factor. J Biol Chem 275:27650–27656

    CAS  PubMed  Google Scholar 

  • Gabbiani G (2003) The myofibroblast in wound healing and fibrocontractive diseases. J Pathol 200:500–503

    Article  CAS  PubMed  Google Scholar 

  • Gharaee-Kermani M, Hu B, Phan SH, Gyetko MR (2009) Recent advances in molecular targets and treatment of idiopathic pulmonary fibrosis: focus on TGFbeta signaling and the myofibroblast. Curr Med Chem 16:1400–1417

    Article  CAS  PubMed  Google Scholar 

  • Heino J, Ignotz RA, Hemler ME, Crouse C, Massagué J (1989) Regulation of cell adhesion receptors by transforming growth factor-beta. Concomitant regulation of integrins that share a common beta 1 subunit. J Biol Chem 264:380–388

    CAS  PubMed  Google Scholar 

  • Herpel E, Pritsch M, Koch A, Dengler TJ, Schirmacher P, Schnabel PA (2006) Interstitial fibrosis in the heart: differences in extracellular matrix proteins and matrix metalloproteinases in end-stage dilated, ischaemic and valvular cardiomyopathy. Histopathology 48:736–747

    Article  CAS  PubMed  Google Scholar 

  • Hildebrand A, Romaris M, Rasmussen LM, Heinegard D, Twardzik DR, Border WA, Ruoslahti E (1994) Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta. Biochem J 302:527–534

    CAS  PubMed  Google Scholar 

  • Huang CC, Chuang JH, Chou MH, Wu CL, Chen CM, Wang CC, Chen YS, Chen CL, Tai MH (2005) Matrilysin (MMP-7) is a major matrix metalloproteinase upregulated in biliary atresia-associated liver fibrosis. Mod Pathol 18:941–950

    Article  CAS  PubMed  Google Scholar 

  • Ignotz RA, Massagué J (1986) Transforming growth factor-beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Biol Chem 261:4337–4345

    CAS  PubMed  Google Scholar 

  • Iozzo RV (1999) The biology of the small leucine-rich proteoglycans. Functional network of interactive proteins. J Biol Chem 274:18843–18846

    Article  CAS  PubMed  Google Scholar 

  • Iredale JP (2007) Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest 117:539–548

    Article  CAS  PubMed  Google Scholar 

  • Jordana M, Schulman J, McSharry C, Irving LB, Newhouse MT, Jordana G, Gauldie J (1988) Heterogeneous proliferative characteristics of human adult lung fibroblast lines and clonally derived fibroblasts from control and fibrotic tissue. Am Rev Respir Dis 137:579–584

    CAS  PubMed  Google Scholar 

  • Kaar JL, Li Y, Blair HC, Asche G, Koepsel RR, Huard J, Russell AJ (2008) Matrix metalloproteinase-1 treatment of muscle fibrosis. Acta Biomater 4:1411–1420

    Article  CAS  PubMed  Google Scholar 

  • Kissin EY, Lemairem R, Kornm JH, Lafyatis R (2002) Transforming growth factor beta induces fibroblast fibrillin-1 matrix formation. Arthritis Rheum 46:3000–3009

    Article  CAS  PubMed  Google Scholar 

  • Kossakowska AE, Edwards DR, Lee SS, Urbanski LS, Stabbler AL, Zhang CL, Phillips BW, Zhang Y, Urbanski SJ (1998) Altered balance between matrix metalloproteinases and their inhibitors in experimental biliary fibrosis. Am J Pathol 153:1895–1902

    CAS  PubMed  Google Scholar 

  • Kuroda K, Shinkai H (1997) Gene expression of types I and III collagen, decorin, matrix metalloproteinases and tissue inhibitors of metalloproteinases in skin fibroblasts from patients with systemic sclerosis. Arch Dermatol Res 289:567–572

    Article  CAS  PubMed  Google Scholar 

  • Le Bousse-Kerdilès MC, Martyré MC, Samson M (2008) Cellular and molecular mechanisms underlying bone marrow and liver fibrosis: a review. Eur Cytokine Netw 19:69–80

    PubMed  Google Scholar 

  • Li YY, Feng YQ, Kadokami T, McTiernan CF, Draviam R, Watkins SC, Feldman AM (2000) Myocardial extracellular matrix remodeling in transgenic mice overexpressing tumor necrosis factor alpha can be modulated by anti-tumor necrosis factor alpha therapy. Proc Natl Acad Sci USA 97:12746–12751

    Article  CAS  PubMed  Google Scholar 

  • Li ZB, Kollias HD, Wagner KR (2008) Myostatin directly regulates skeletal muscle fibrosis. J Biol Chem 283:19371–19378

    Article  CAS  PubMed  Google Scholar 

  • Liu X, Wu H, Byrne M, Jeffrey J, Krane S, Jaenisch R (1995) A targeted mutation at the known collagenase cleavage site in mouse type I collagen impairs tissue remodeling. J Cell Biol 130:227–237

    Article  CAS  PubMed  Google Scholar 

  • Lorena D, Uchio K, Costa AM, Desmoulière A (2002) Normal scarring: importance of myofibroblasts. Wound Repair Regen 10:86–92

    Article  PubMed  Google Scholar 

  • Matsumura K, Campbell KP (1994) Dystrophin-glycoprotein complex: its role in the molecular pathogenesis of muscular dystrophies. Muscle Nerve 17:2–15

    Article  CAS  PubMed  Google Scholar 

  • Mauviel A (2005) Transforming growth factor-beta: a key mediator of fibrosis. Methods Mol Med 117:69–80

    CAS  PubMed  Google Scholar 

  • McPherron AC, Lee SJ (1997) Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA 94:12457–12461

    Article  CAS  PubMed  Google Scholar 

  • Milani S, Herbst H, Schuppan D, Kim KY, Riecken EO, Stein H (1990) Procollagen expression by nonparenchymal rat liver cells in experimental biliary fibrosis. Gastroenterology 98:175–184

    CAS  PubMed  Google Scholar 

  • Miura T, Kishioka Y, Wakamatsu J, Hattori A, Hennebry A, Berry CJ, Sharma M, Kambadur R, Nishimura T (2006) Decorin binds myostatin and modulates its activity to muscle cells. Biochem Biophys Res Commun 340:675–680

    Article  CAS  PubMed  Google Scholar 

  • Montesano R, Orci L (1988) Transforming growth factor beta stimulates collagen-matrix contraction by fibroblasts: implications for wound healing. Proc Natl Acad Sci USA 85:4894–4897

    Article  CAS  PubMed  Google Scholar 

  • Pannu J, Gardner H, Shearstone JR, Smith E, Trojanowska M (2006) Increased levels of transforming growth factor beta receptor type I and up-regulation of matrix gene program: a model of scleroderma. Arthritis Rheum 54:3011–3021

    Article  CAS  PubMed  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res 29:e45

    Article  CAS  PubMed  Google Scholar 

  • Raghu G, Chen YY, Rusch V, Rabinovitch PS (1988) Differential proliferation of fibroblasts cultured from normal and fibrotic human lungs. Am Rev Respir Dis 138:703–708

    CAS  PubMed  Google Scholar 

  • Ramos C, Montano M, Garcia-Alvarez J, Ruiz V, Uhal BD, Selman M, Pardo A (2001) Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ in growth rate, apoptosis, and tissue inhibitor of metalloproteinases expression. Am J Respir Cell Mol Biol 24:591–598

    CAS  PubMed  Google Scholar 

  • Roberts AB, Sporn MB (1996) Transforming growth factor-β. In: Clark RAF (ed) The molecular and cellular biology of wound repair. Plenum, New York, pp 275–308

    Google Scholar 

  • Ronnov-Jessen L, Petersen OW (1993) Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia. Lab Invest 68:696–707

    CAS  PubMed  Google Scholar 

  • Sappino AP, Masouyé I, Saurat JH, Gabbiani G (1990) Smooth muscle differentiation in scleroderma fibroblastic cells. Am J Pathol 137:585–591

    CAS  PubMed  Google Scholar 

  • Schmid P, Itin P, Cherry G, Bi C, Cox DA (1998) Enhanced expression of transforming growth factor-beta type I and type II receptors in wound granulation tissue and hypertrophic scar. Am J Pathol 152:485–493

    CAS  PubMed  Google Scholar 

  • Schnaper HW, Kopp JB, Poncelet AC, Hubchak SC, Stetler-Stevenson WG, Klotman PE, Kleinman HK (1996) Increased expression of extracellular matrix proteins and decreased expression of matrix proteases after serial passage of glomerular mesangial cells. J Cell Sci 109:2521–2528

    CAS  PubMed  Google Scholar 

  • Selman M, King TE, Pardo A (2001) Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 134:136–151

    CAS  PubMed  Google Scholar 

  • Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J (1997) Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Invest 100:768–776

    Article  CAS  PubMed  Google Scholar 

  • Surendran K, Simon TC, Liapis H, McGuire JK (2004) Matrilysin (MMP-7) expression in renal tubular damage: association with Wnt4. Kidney Int 65:2212–2222

    Article  CAS  PubMed  Google Scholar 

  • Takeda K, Hatamochi A, Ueki H, Nakata M, Oishi Y (1994) Decreased collagenase expression in cultured systemic sclerosis fibroblasts. J Invest Dermatol 103:359–363

    Article  CAS  PubMed  Google Scholar 

  • Thannickal VJ, Aldweib KD, Rajan T, Fanburg BL (1998) Upregulated expression of fibroblast growth factor (FGF) receptors by transforming growth factor-beta1 (TGF-beta1) mediates enhanced mitogenic responses to FGFs in cultured human lung fibroblasts. Biochem Biophys Res Commun 251:437–441

    Article  CAS  PubMed  Google Scholar 

  • Uria JA, Jimenez MG, Balbin M, Freije JMP, Lopez-Otin C (1998) Differential effects of transforming growth factor-beta on the expression of collagenase-1 and collagenase-3 in human fibroblasts. J Biol Chem 273:9769–9777

    Article  CAS  PubMed  Google Scholar 

  • Vogel KG, Paulsson M, Heinegard D (1984) Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem J 223:587–597

    CAS  PubMed  Google Scholar 

  • Wells RG (2000) Fibrogenesis V. TGF-β signaling pathways. Am J Physiol Gastrointest Liver Physiol 279:G845–G850

    CAS  PubMed  Google Scholar 

  • Westergren-Thorsson G, Hernnas J, Sarnstrand B, Oldberg A, Heinegard D, Malmstrom A (1993) Altered expression of small proteoglycans, collagen, and transforming growth factor-beta 1 in developing bleomycin-induced pulmonary fibrosis in rats. J Clin Invest 92:632–637

    Article  CAS  PubMed  Google Scholar 

  • Westergren-Thorsson G, Sime P, Jordana M, Gauldie J, Sarnstrand B, Malmstrom A (2004) Lung fibroblast clones from normal and fibrotic subjects differ in hyaluronan and decorin production and rate of proliferation. Int J Biochem Cell Biol 36:1573–1584

    Article  CAS  PubMed  Google Scholar 

  • Yamaguchi Y, Mann DM, Rouslahti E (1990) Negative regulation of transforming growth factor-beta by the proteoglycan decorin. Nature 19:281–284

    Article  Google Scholar 

  • Yamanouchi K, Soeta C, Naito K, Tojo H (2000) Expression of myostatin gene in regenerating skeletal muscle of the rat and its localization. Biochem Biophys Res Commun 270:510–516

    Article  CAS  PubMed  Google Scholar 

  • Yu WH, Yu S, Meng Q, Brew K, Woessner JF Jr (2000) TIMP-3 binds to sulfated glycosaminoglycans of the extracellular matrix. J Biol Chem 275:31226–31232

    Article  CAS  PubMed  Google Scholar 

  • Zanotti S, Negri T, Cappelletti C, Bernasconi P, Canioni E, Di Blasi C, Pegoraro E, Angelini C, Ciscato P, Prelle A, Mantegazza R, Morandi L, Mora M (2005) Decorin and biglycan expression is differentially altered in several muscular dystrophies. Brain 128:2546–2555

    Article  PubMed  Google Scholar 

  • Zanotti S, Saredi S, Ruggieri A, Fabbri M, Blasevich F, Romaggi S, Morandi L, Mora M (2007) Altered extracellular matrix transcript expression and protein modulation in primary Duchenne muscular dystrophy myotubes. Matrix Biol 26:615–624

    Article  CAS  PubMed  Google Scholar 

  • Zhang Z, Li XJ, Liu Y, Zhang X, Li YY, Xu WS (2007) Recombinant human decorin inhibits cell proliferation and downregulates TGF-β1 production in hypertrophic scar fibroblasts. Burns 33:634–641

    Article  PubMed  Google Scholar 

  • Zhu J, Li Y, Shen W, Qiao C, Ambrosio F, Lavasani M, Nozaki M, Branca MF, Huard J (2007) Relationships between transforming growth factor-beta1, myostatin, and decorin: implication for skeletal muscle fibrosis. J Biol Chem 282:25852–25863

    Article  CAS  PubMed  Google Scholar 

  • Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Pollini L, Morris D, Kim Y, DeLustro B, Sheppard D, Pardo A, Selman M, Heller RA (2002) Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci USA 99:6292–6297

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors are grateful to Professor Renato Iozzo of the Thomas Jefferson University of Philadelphia for helpful suggestions on the detection of the glycosylated form of decorin by Western blot and to Don Ward for help with the English.The EuroBioBank and Telethon Network of Genetic Biobanks (GTB07001F) are also gratefully acknowledged for providing biological samples.

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  1. Neuromuscular Diseases and Neuroimmunology Unit, Foundation Neurological Institute “C. Besta”, Milan, Italy

    Simona Zanotti, Sara Gibertini & Marina Mora

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  1. Simona Zanotti
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  2. Sara Gibertini
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  3. Marina Mora
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Correspondence to Marina Mora.

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The financial support of the Italian Ministry of Health is gratefully acknowledged.

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Zanotti, S., Gibertini, S. & Mora, M. Altered production of extra-cellular matrix components by muscle-derived Duchenne muscular dystrophy fibroblasts before and after TGF-β1 treatment. Cell Tissue Res 339, 397–410 (2010). https://doi.org/10.1007/s00441-009-0889-4

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