CCN2 Is Necessary for Adhesive Responses to Transforming Growth Factor-β1 in Embryonic Fibroblasts*

CCN2 is induced by transforming growth factor-β (TGFβ) in fibroblasts and is overexpressed in connective tissue disease. CCN2 has been proposed to be a downstream mediator of TGFβ action in fibroblasts; however, the role of CCN2 in regulating this process unclear. By using embryonic fibroblasts isolated from ccn2–/–mice, we showed that CCN2 is required for a subset of responses to TGFβ. Affymetrix genome-wide expression profiling revealed that 942 transcripts were induced by TGFβ greater than 2-fold in ccn2+/+ fibroblasts, of which 345 were not induced in ccn2–/–fibroblasts, including pro-adhesive and matrix remodeling genes. Whereas TGFβ properly induced a generic Smad3-responsive promoter in ccn2–/–fibroblasts, TGFβ-induced activation of focal adhesion kinase (FAK) and Akt was reduced in ccn2–/–fibroblasts. Emphasizing the importance of FAK and Akt activation in CCN2-dependent transcriptional responses to TGFβ in fibroblasts, CCN2-dependent transcripts were not induced by TGFβ in fak–/–fibroblasts and were reduced by wortmannin in wild-type fibroblasts. Akt1 overexpression in ccn2–/–fibroblasts rescued the TGFβ-induced transcription of CCN2-dependent mRNA. Finally, induction of TGFβ-induced fibroblast adhesion to fibronectin and type I collagen was significantly diminished in ccn2–/–fibroblasts. Thus in embryonic fibroblasts, CCN2 is a necessary cofactor required for TGFβ to activate the adhesive FAK/Akt/phosphatidylinositol 3-kinase cascade, FAK/Akt-dependent genes, and adhesion to matrix.

[1]  Yunliang Chen,et al.  Matrix contraction by dermal fibroblasts requires transforming growth factor-beta/activin-linked kinase 5, heparan sulfate-containing proteoglycans, and MEK/ERK: insights into pathological scarring in chronic fibrotic disease. , 2005, The American journal of pathology.

[2]  D. Brigstock,et al.  Cell Communication and Signaling Activation of Nuclear Factor Kappa B (nf-κb) by Connective Tissue Growth Factor (ccn2) Is Involved in Sustaining the Survival of Primary Rat Hepatic Stellate Cells , 2022 .

[3]  E. Greer,et al.  FOXO transcription factors at the interface between longevity and tumor suppression , 2005, Oncogene.

[4]  Feng Yang,et al.  Stromal expression of connective tissue growth factor promotes angiogenesis and prostate cancer tumorigenesis. , 2005, Cancer research.

[5]  D. Brigstock,et al.  Connective Tissue Growth Factor (CCN2) in Rat Pancreatic Stellate Cell Function: Integrin α5β1 as a Novel CCN2 Receptor , 2005 .

[6]  R. Nho,et al.  Role of Integrin-linked Kinase in Regulating Phosphorylation of Akt and Fibroblast Survival in Type I Collagen Matrices through a β1 Integrin Viability Signaling Pathway* , 2005, Journal of Biological Chemistry.

[7]  D. Abraham,et al.  N-terminal connective tissue growth factor is a marker of the fibrotic phenotype in scleroderma. , 2005, QJM : monthly journal of the Association of Physicians.

[8]  M. Jinnin,et al.  Constitutive phosphorylation of focal adhesion kinase is involved in the myofibroblast differentiation of scleroderma fibroblasts. , 2005, The Journal of investigative dermatology.

[9]  K. Elkon,et al.  Scleroderma fibroblasts demonstrate enhanced activation of Akt (protein kinase B) in situ. , 2005, The Journal of investigative dermatology.

[10]  Yunliang Chen,et al.  CCN2 (connective tissue growth factor) promotes fibroblast adhesion to fibronectin. , 2004, Molecular biology of the cell.

[11]  A. Nicholson,et al.  Gene expression profiling reveals novel TGFβ targets in adult lung fibroblasts , 2004, Respiratory research.

[12]  R. Fässler,et al.  Integrin-linked kinase: integrin's mysterious partner. , 2004, Current opinion in cell biology.

[13]  R. Nho,et al.  Focal Adhesion Kinase Is Upstream of Phosphatidylinositol 3-Kinase/Akt in Regulating Fibroblast Survival in Response to Contraction of Type I Collagen Matrices via a β1 Integrin Viability Signaling Pathway* , 2004, Journal of Biological Chemistry.

[14]  Yunliang Chen,et al.  Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts. , 2004, Molecular biology of the cell.

[15]  R. D. du Bois,et al.  Endothelin-1 Induces Expression of Matrix-associated Genes in Lung Fibroblasts through MEK/ERK* , 2004, Journal of Biological Chemistry.

[16]  Andrew Leask,et al.  TGF‐β signaling and the fibrotic response , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[17]  D. Brigstock,et al.  Connective Tissue Growth Factor (CCN2) Induces Adhesion of Rat Activated Hepatic Stellate Cells by Binding of Its C-terminal Domain to Integrin αvβ3 and Heparan Sulfate Proteoglycan* , 2004, Journal of Biological Chemistry.

[18]  Gary R. Grotendorst,et al.  Combinatorial signaling pathways determine fibroblast proliferation and myofibroblast differentiation , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[19]  T. Underhill,et al.  p38 MAP kinase signalling is required for hypertrophic chondrocyte differentiation. , 2004, The Biochemical journal.

[20]  B. Perbal.  CCN proteins: multifunctional signalling regulators , 2004, The Lancet.

[21]  D. Abraham,et al.  The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology. , 2003, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[22]  Giulio Gabbiani,et al.  Mechanisms of force generation and transmission by myofibroblasts. , 2003, Current opinion in biotechnology.

[23]  G. Gabbiani,et al.  The myofibroblast in wound healing and fibrocontractive diseases , 2003, The Journal of pathology.

[24]  B. Yoon,et al.  Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development , 2003, Development.

[25]  D. Abraham,et al.  Connective Tissue Growth Factor Gene Regulation , 2003, The Journal of Biological Chemistry.

[26]  R. Day,et al.  Myofibroblast Differentiation by Transforming Growth Factor-β1 Is Dependent on Cell Adhesion and Integrin Signaling via Focal Adhesion Kinase* , 2003, The Journal of Biological Chemistry.

[27]  J. Myllyharju.  Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis. , 2003, Matrix biology : journal of the International Society for Matrix Biology.

[28]  A. Colombatti,et al.  β1 Integrin-dependent Cell Adhesion to EMILIN-1 Is Mediated by the gC1q Domain* , 2003, The Journal of Biological Chemistry.

[29]  F. Martin,et al.  The Role of p42/44 MAPK and Protein Kinase B in Connective Tissue Growth Factor Induced Extracellular Matrix Protein Production, Cell Migration, and Actin Cytoskeletal Rearrangement in Human Mesangial Cells* , 2002, The Journal of Biological Chemistry.

[30]  D. Abraham,et al.  Prostacyclin derivatives prevent the fibrotic response to TGFβ2 by inhibiting the Ras/MEK/ERK pathway , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[31]  R. Goldschmeding,et al.  Gene regulation of connective tissue growth factor: new targets for antifibrotic therapy? , 2002, Matrix biology : journal of the International Society for Matrix Biology.

[32]  S. Sa,et al.  CTGF expression in mesangial cells: involvement of SMADs, MAP kinase, and PKC. , 2002, Kidney international.

[33]  E. Robertis,et al.  Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-β , 2002, Nature Cell Biology.

[34]  R. Kucherlapati,et al.  Functional Characterization of Transforming Growth Factor β Signaling in Smad2- and Smad3-deficient Fibroblasts* , 2001, The Journal of Biological Chemistry.

[35]  S. Sa,et al.  CTGF and SMADs, Maintenance of Scleroderma Phenotype Is Independent of SMAD Signaling* , 2001, The Journal of Biological Chemistry.

[36]  L. Lau,et al.  The Angiogenic Factors Cyr61 and Connective Tissue Growth Factor Induce Adhesive Signaling in Primary Human Skin Fibroblasts* , 2001, The Journal of Biological Chemistry.

[37]  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.

[38]  J. Romashkova,et al.  NF-κB is a target of AKT in anti-apoptotic PDGF signalling , 1999, Nature.

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

[40]  L. Lau,et al.  Fisp12/Mouse Connective Tissue Growth Factor Mediates Endothelial Cell Adhesion and Migration through Integrin αvβ3, Promotes Endothelial Cell Survival, and Induces Angiogenesis In Vivo , 1999, Molecular and Cellular Biology.

[41]  Gary R. Grotendorst.  Connective tissue growth factor: a mediator of TGF-β action on fibroblasts , 1997 .

[42]  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.

[43]  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.

[44]  Sharon M. Wahl.  Transforming growth factor beta: the good, the bad, and the ugly , 1994, The Journal of experimental medicine.

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

[46]  P. Bork.  The modular architecture of a new family of growth regulators related to connective tissue growth factor , 1993, FEBS letters.

[47]  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.

[48]  D. Abraham,et al.  Insights into the molecular mechanism of chronic fibrosis: the role of connective tissue growth factor in scleroderma. , 2004, The Journal of investigative dermatology.

[49]  A. Leask,et al.  Tumor necrosis factor alpha suppresses the induction of connective tissue growth factor by transforming growth factor-beta in normal and scleroderma fibroblasts. , 2000, The Journal of biological chemistry.

[50]  R. Golla,et al.  Vinculin in relation to stress fibers in spread platelets. , 1991, Cell motility and the cytoskeleton.

[51]  B. Weston,et al.  J Am Soc Nephrol 14: 601–610, 2003 CTGF Mediates TGF-�–Induced Fibronectin Matrix Deposition by Upregulating Active �5�1 Integrin in Human , 2022 .