Connective tissue growth factor and integrin αvβ6: A new pair of regulators critical for ductular reaction and biliary fibrosis in mice

Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell‐matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvβ6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvβ6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter‐driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvβ6 during liver injury induced by 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen‐inducible Cre‐loxP system down‐regulated integrin αvβ6 in DDC‐damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvβ6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis‐related genes, smaller areas of alpha‐smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvβ6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)‐β1 activation in vitro. Conclusions: CTGF and integrin αvβ6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF‐β1. CTGF and integrin αvβ6 are potential therapeutic targets to control DRs and fibrosis in related liver disease. (Hepatology 2015;61:678‐691)

[1]  E. Scott,et al.  Conditional knockout of CTGF affects corneal wound healing. , 2014, Investigative ophthalmology & visual science.

[2]  S. Forbes,et al.  Links between hepatic fibrosis, ductular reaction, and progenitor cell expansion. , 2014, Gastroenterology.

[3]  D. Schuppan,et al.  Evolving therapies for liver fibrosis. , 2013, The Journal of clinical investigation.

[4]  P. Weinreb,et al.  Lysophosphatidic acid increases proximal tubule cell secretion of profibrotic cytokines PDGF-B and CTGF through LPA2- and Gαq-mediated Rho and αvβ6 integrin-dependent activation of TGF-β. , 2012, The American journal of pathology.

[5]  W. Hauswirth,et al.  CCN2/CTGF regulates neovessel formation via targeting structurally conserved cystine knot motifs in multiple angiogenic regulators , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[6]  A. Gouw,et al.  Ductular reactions in human liver: Diversity at the interface , 2011, Hepatology.

[7]  Shangxin Liu,et al.  CCN2 is not required for skin development , 2011, Journal of Cell Communication and Signaling.

[8]  C. Sempoux,et al.  Cholangiolocellular Carcinoma: An Innocent-Looking Malignant Liver Tumor Mimicking Ductular Reaction , 2011, Seminars in liver disease.

[9]  D. Brigstock,et al.  Integrin expression and function in the response of primary culture hepatic stellate cells to connective tissue growth factor (CCN2) , 2010, Journal of cellular and molecular medicine.

[10]  R. Weimann,et al.  The alphavbeta6 integrin is a highly specific immunohistochemical marker for cholangiocarcinoma. , 2010, Journal of hepatology.

[11]  B. Perbal,et al.  Susceptibility to liver fibrosis in mice expressing a connective tissue growth factor transgene in hepatocytes , 2009, Hepatology.

[12]  A. Gressner,et al.  Connective tissue growth factor: a fibrogenic master switch in fibrotic liver diseases , 2008, Liver international (Print).

[13]  D. Schuppan,et al.  Inhibition of integrin alphavbeta6 on cholangiocytes blocks transforming growth factor-beta activation and retards biliary fibrosis progression. , 2008, Gastroenterology.

[14]  Jen-Jung Pan,et al.  Connective tissue growth factor with a novel fibronectin binding site promotes cell adhesion and migration during rat oval cell activation , 2008, Hepatology.

[15]  H. Friess,et al.  Integrin alphavbeta6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies. , 2008, Journal of hepatology.

[16]  A. Gown,et al.  Antibody-mediated blockade of integrin alpha v beta 6 inhibits tumor progression in vivo by a transforming growth factor-beta-regulated mechanism. , 2008, Cancer research.

[17]  P. Weinreb,et al.  Role of αvβ6 integrin in acute biliary fibrosis , 2007 .

[18]  K. Zatloukal,et al.  A new xenobiotic-induced mouse model of sclerosing cholangitis and biliary fibrosis. , 2007, The American journal of pathology.

[19]  W. Parks,et al.  Transforming growth factor‐beta differentially regulates oval cell and hepatocyte proliferation , 2007, Hepatology.

[20]  Z. Tong,et al.  Intrinsic biological activity of the thrombospondin structural homology repeat in connective tissue growth factor. , 2006, The Journal of endocrinology.

[21]  D. Rifkin,et al.  Fibronectin is required for integrin o:vβ6‐mediated activation of latent TGF‐β complexes containing LTBP‐1 , 2005 .

[22]  C. Ambrose,et al.  TWEAK induces liver progenitor cell proliferation. , 2005, The Journal of clinical investigation.

[23]  B. Petersen,et al.  Role of connective tissue growth factor in oval cell response during liver regeneration after 2-AAF/PHx in rats. , 2005, Gastroenterology.

[24]  M. Richardson,et al.  Fibrosis correlates with a ductular reaction in hepatitis C: Roles of impaired replication, progenitor cells and steatosis , 2005, Hepatology.

[25]  K. Simon,et al.  Function-blocking Integrin αvβ6 Monoclonal Antibodies , 2004, Journal of Biological Chemistry.

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

[27]  N. Kaminski,et al.  The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. , 1999, Cell.

[28]  D. Abraham,et al.  CCN2 is required for bleomycin-induced skin fibrosis in mice. , 2011, Arthritis and rheumatism.

[29]  P. Weinreb,et al.  Role of alphavbeta6 integrin in acute biliary fibrosis. , 2007, Hepatology.

[30]  D. Rifkin,et al.  Fibronectin is required for integrin alphavbeta6-mediated activation of latent TGF-beta complexes containing LTBP-1. , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[31]  K. Simon,et al.  Function-blocking integrin alphavbeta6 monoclonal antibodies: distinct ligand-mimetic and nonligand-mimetic classes. , 2004, The Journal of biological chemistry.