Autocrine overexpression of CTGF maintains fibrosis: RDA analysis of fibrosis genes in systemic sclerosis.

We have used representational difference analysis (RDA) to identify up-regulated genes in skin fibroblasts from fibrotic lesions obtained from patients with systemic sclerosis (scleroderma). RDA of cDNA libraries derived from fibroblasts from involved and uninvolved skin detected several differentially expressed genes. One such gene consistently up-regulated in scleroderma cells coded for human connective tissue growth factor (CTGF). Other studies described here show that the CTGF protein is readily detected in cultures of systemic sclerosis fibroblasts but was not detected in comparable normal cells. High levels of CTGF are also evident in biological fluids from patients with systemic sclerosis. TGFbeta stimulates CTGF production in both normal and systemic sclerosis fibroblasts with the latter found to be higher producers. Moreover, an analysis of constitutive and TGFbeta-induced CTGF gene activation showed altered and elevated transcriptional responses in systemic sclerosis cells compared with controls. CTGF stimulated a two- to threefold increase in proalpha1(I) collagen and fibronectin synthesis by both dermal and lung fibroblasts in culture and promoted significant matrix remodeling of fibroblast-populated three-dimensional collagen lattices. A direct relation between the overexpression of CTGF and elevated collagen synthesis was suggested by the observation that transfection of a CMV-CTGF cDNA construct and protein expression in fibroblasts increased the transcription of a Col 1alpha2 promoter-reporter construct to levels seen in systemic sclerosis fibroblasts. Using Col 1alpha2 promoter deletion constructs the CTGF responsive element was localized to the first 379 bp upstream of the transcriptional start site. These data indicate that there is an overexpression of CTGF in the systemic sclerosis cells, probably due to increased gene transcription, and suggest that the dysregulation of CTGF production is an important factor in fibroblast activation and the excessive deposition of collagen in systemic sclerosis.

[1]  N. Khalil TGF-β: from latent to active , 1999 .

[2]  V. Petkov,et al.  A preliminary study of long-term treatment with interferon gamma-1b and low-dose prednisolone in patients with idiopathic pulmonary fibrosis. , 1999, The New England journal of medicine.

[3]  M. Takigawa,et al.  Role and interaction of connective tissue growth factor with transforming growth factor‐β in persistent fibrosis: A mouse fibrosis model , 1999, Journal of cellular physiology.

[4]  H. Kleinman,et al.  Thymosin β4 Accelerates Wound Healing , 1999 .

[5]  C. Castagnoli,et al.  Expression and role of IL-15 in post-burn hypertrophic scars. , 1999, The Journal of investigative dermatology.

[6]  T. Wright,et al.  Identification of multiple, differentially expressed messenger RNAs in dermal fibroblasts from patients with systemic sclerosis. , 1999, Arthritis and rheumatism.

[7]  L. Lau,et al.  The CCN family of angiogenic regulators: the integrin connection. , 1999, Experimental cell research.

[8]  M. Boes,et al.  Connective tissue growth factor (IGFBP-rP2) expression and regulation in cultured bovine endothelial cells. , 1999, Endocrinology.

[9]  D. Brigstock The Connective Tissue Growth Factor/cysteine- Rich 61/nephroblastoma Overexpressed (ccn) Family* , 2022 .

[10]  A. K. Wilson,et al.  Localization of connective tissue growth factor during the period of embryo implantation in the mouse. , 1998, Biology of reproduction.

[11]  M. Takigawa,et al.  Establishment of the enzyme-linked immunosorbent assay for connective tissue growth factor (CTGF) and its detection in the sera of biliary atresia. , 1998, Biochemical and biophysical research communications.

[12]  Gary R. Grotendorst,et al.  Inhibition of TGF‐β‐stimulated CTGF gene expression and anchorage‐independent growth by cAMP identifies a CTGF‐dependent restriction point in the cell cycle , 1998, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[13]  E. Wilson,et al.  Identification of glycosylated 38-kDa connective tissue growth factor (IGFBP-related protein 2) and proteolytic fragments in human biological fluids, and up-regulation of IGFBP-rP2 expression by TGF-beta in Hs578T human breast cancer cells. , 1998, The Journal of clinical endocrinology and metabolism.

[14]  T. Lüscher,et al.  Connective tissue growth factor. Friend or foe? , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[15]  R. D. du Bois,et al.  Scleroderma lung fibroblasts exhibit elevated and dysregulated type I collagen biosynthesis. , 1997, Arthritis and rheumatism.

[16]  Richard A Flavell,et al.  The Transcription Factor GATA-3 Is Necessary and Sufficient for Th2 Cytokine Gene Expression in CD4 T Cells , 1997, Cell.

[17]  T. Sugimoto,et al.  Cloning of a mRNA preferentially expressed in chondrocytes by differential display-PCR from a human chondrocytic cell line that is identical with connective tissue growth factor (CTGF) mRNA. , 1997, Biochemical and biophysical research communications.

[18]  S. Jimenez,et al.  Pathogenesis of scleroderma. Collagen. , 1996, Rheumatic diseases clinics of North America.

[19]  Gary R. Grotendorst,et al.  Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. , 1996, The Journal of investigative dermatology.

[20]  Gary R. Grotendorst,et al.  A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor gene. , 1996, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[21]  Gary R. Grotendorst,et al.  Connective tissue growth factor gene expression in tissue sections from localized scleroderma, keloid, and other fibrotic skin disorders. , 1996, The Journal of investigative dermatology.

[22]  Gary R. Grotendorst,et al.  Significant correlation between connective tissue growth factor gene expression and skin sclerosis in tissue sections from patients with systemic sclerosis. , 1995, The Journal of investigative dermatology.

[23]  H. Ihn,et al.  Growth regulation in scleroderma fibroblasts: increased response to transforming growth factor-beta 1. , 1995, The Journal of investigative dermatology.

[24]  K. Miyazono,et al.  Collagen type I is not under autocrine control by transforming growth factor-beta 1 in normal and scleroderma fibroblasts. , 1994, Laboratory investigation; a journal of technical methods and pathology.

[25]  R. D. du Bois,et al.  Increased levels of endothelin-1 in bronchoalveolar lavage fluid from patients with systemic sclerosis contribute to fibroblast mitogenic activity in vitro. , 1994, American journal of respiratory cell and molecular biology.

[26]  C. Black,et al.  Immunocytochemical Localization and Serologic Detection of Transforming Growth Factor β1 , 1994 .

[27]  Gary R. Grotendorst,et al.  Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. , 1993, Molecular biology of the cell.

[28]  S. Jimenez,et al.  Regulation of human lung fibroblast alpha 1(I) procollagen gene expression by tumor necrosis factor alpha, interleukin-1 beta, and prostaglandin E2. , 1993, The Journal of biological chemistry.

[29]  M. Wigler,et al.  Cloning the differences between two complex genomes , 1993, Science.

[30]  Y. Deguchi Spontaneous increase of transforming growth factor beta production by bronchoalveolar mononuclear cells of patients with systemic autoimmune diseases affecting the lung. , 1992, Annals of the rheumatic diseases.

[31]  Gary R. Grotendorst,et al.  Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10 , 1991, The Journal of cell biology.

[32]  E. Leroy,et al.  Increased collagen synthesis by scleroderma skin fibroblasts in vitro: a possible defect in the regulation or activation of the scleroderma fibroblast. , 1974, The Journal of clinical investigation.

[33]  T. Nakanishi,et al.  Serum levels of connective tissue growth factor are elevated in patients with systemic sclerosis: association with extent of skin sclerosis and severity of pulmonary fibrosis. , 2000, The Journal of rheumatology.

[34]  C. Denton,et al.  Systemic sclerosis and related disorders , 2000 .

[35]  H. Ihn,et al.  Increased Expression of TGF-β Receptors by Scleroderma Fibroblasts: Evidence for Contribution of Autocrine TGF-β Signaling to Scleroderma Phenotype , 1998 .

[36]  Gary R. Grotendorst,et al.  Transforming growth factor beta induces anchorage-independent growth of NRK fibroblasts via a connective tissue growth factor-dependent signaling pathway. , 1997, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[37]  M Hubank,et al.  Identifying differences in mRNA expression by representational difference analysis of cDNA. , 1994, Nucleic acids research.