Wound Healing/Plastic Surgery| Volume 185, ISSUE 2, e109-e119, December 2013

CCN2 is transiently expressed by keratinocytes during re-epithelialization and regulates keratinocyte migration in vitro by the ras-MEK-ERK signaling pathway



      CCN2 (previously known as connective tissue growth factor) is a multifunctional matricellular protein that has numerous effects on cell life and cell interactions with the connective tissue. Although the importance of CCN2 for the fibrotic process in wound healing has been well studied, the involvement of CCN2 in keratinocyte function has not yet been explored. Therefore, the aim of the present study was to investigate the role of CCN2 in the epidermis during wound healing.

      Materials and methods

      Immunohistochemistry was done on sections from full-thickness porcine wounds. The effect of CCN2 on the migration of cultured human keratinocytes exposed to scratch wounds, the effect on phosphorylation of extracellular signal-related kinases (ERK), and the effect of adding inhibitors to the ERK/mitogen-activated protein kinase pathway to human keratinocytes were studied.


      The CCN2 protein was transiently expressed in vivo at the leading keratinocyte edge during re-epithelialization of full-thickness porcine wounds. In vitro, exogenous addition of CCN2 to human keratinocyte cultures regulated keratinocyte migration and resulted in phosphorylation of ERK. The addition of inhibitors of ERK/mitogen-activated protein kinase counteracted the effect of CCN2 on migration.


      CCN2 was transiently expressed at the leading keratinocyte edge in vivo. The biologic importance of this was supported in vitro, because CCN2 regulated human keratinocyte migration through activation of the Ras-mitogen-activated protein kinase kinase-ERK signal transduction pathway.


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        • Gurtner G.C.
        • Werner S.
        • Barrandon Y.
        • Longaker M.T.
        Wound repair and regeneration.
        Nature. 2008; 453: 314
        • Frazier K.
        • Williams S.
        • Kothapalli D.
        • Klapper H.
        • Grotendorst G.R.
        Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor.
        J Invest Dermatol. 1996; 107: 404
        • Raja
        • Sivamani K.
        • Garcia M.S.
        • Isseroff R.R.
        Wound re-epithelialization: modulating keratinocyte migration in wound healing.
        Front Biosci. 2007; 12: 2849
        • Santoro M.M.
        • Gaudino G.
        Cellular and molecular facets of keratinocyte reepithelization during wound healing.
        Exp Cell Res. 2005; 304: 274
        • Brigstock D.R.
        The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family.
        Endocr Rev. 1999; 20: 189
        • Lau L.F.
        • Lam S.C.
        The CCN family of angiogenic regulators: the integrin connection.
        Exp Cell Res. 1999; 248: 44
        • Perbal B.
        CCN proteins: multifunctional signalling regulators.
        Lancet. 2004; 363: 62
        • Leask A.
        Signaling in fibrosis: targeting the TGF beta, endothelin-1 and CCN2 axis in scleroderma.
        Front Biosci (Elite Ed). 2009; 1: 115
        • Igarashi A.
        • Okochi H.
        • Bradham D.M.
        • Grotendorst G.R.
        Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair.
        Mol Biol Cell. 1993; 4: 637
        • Grotendorst G.R.
        Connective tissue growth factor: a mediator of TGF-beta action on fibroblasts.
        Cytokine Growth Factor Rev. 1997; 8: 171
        • Khoo Y.T.
        • Ong C.T.
        • Mukhopadhyay A.
        • et al.
        Upregulation of secretory connective tissue growth factor (CTGF) in keratinocyte-fibroblast coculture contributes to keloid pathogenesis.
        J Cell Physiol. 2006; 208: 336
        • Amjad S.B.
        • Carachi R.
        • Edward M.
        Keratinocyte regulation of TGF-beta and connective tissue growth factor expression: a role in suppression of scar tissue formation.
        Wound Repair Regen. 2007; 15: 748
        • Phanish M.K.
        • Wahab N.A.
        • Hendry B.M.
        • Dockrell M.E.
        TGF-beta1-induced connective tissue growth factor (CCN2) expression in human renal proximal tubule epithelial cells requires Ras/MEK/ERK and Smad signalling.
        Nephron Exp Nephrol. 2005; 100: e156
        • Li B.X.
        • Tang Y.T.
        • Wang W.
        • et al.
        Fluorofenidone attenuates renal interstitial fibrosis in the rat model of obstructive nephropathy.
        Mol Cell Biochem. 2011; 354: 263
        • Kantarci A.
        • Black S.A.
        • Xydas C.E.
        • et al.
        Epithelial and connective tissue cell CTGF/CCN2 expression in gingival fibrosis.
        J Pathol. 2006; 210: 59
        • Zuehlke J.
        • Ebenau A.
        • Krueger B.
        • Goppelt-Struebe M.
        Vectorial secretion of CTGF as a cell-type specific response to LPA and TGF-beta in human tubular epithelial cells.
        Cell Commun Signal. 2012; 10: 25
        • Huang C.
        • Jacobson K.
        • Schaller M.D.
        MAP kinases and cell migration.
        J Cell Sci. 2004; 117: 4619
        • Hood J.D.
        • Cheresh D.A.
        Role of integrins in cell invasion and migration.
        Nat Rev Cancer. 2002; 2: 91
        • Li J.
        • Zhao Z.
        • Liu J.
        • et al.
        MEK/ERK and p38 MAPK regulate chondrogenesis of rat bone marrow mesenchymal stem cells through delicate interaction with TGF-beta1/Smads pathway.
        Cell Prolif. 2010; 43: 333
        • Park S.
        • Jung H.H.
        • Park Y.H.
        • Ahn J.S.
        • Im Y.H.
        ERK/MAPK pathways play critical roles in EGFR ligands-induced MMP1 expression.
        Biochem Biophys Res Commun. 2011; 407: 680
        • Walshe T.E.
        • Leach L.L.
        • D'Amore P.A.
        TGF-beta signaling is required for maintenance of retinal ganglion cell differentiation and survival.
        Neuroscience. 2011; 189: 123
        • Secker G.A.
        • Shortt A.J.
        • Sampson E.
        • Schwarz Q.P.
        • Schultz G.S.
        • Daniels J.T.
        TGFbeta stimulated re-epithelialisation is regulated by CTGF and Ras/MEK/ERK signalling.
        Exp Cell Res. 2008; 314: 131
        • Kennedy L.
        • Liu S.
        • Shi-Wen X.
        • et al.
        CCN2 is necessary for the function of mouse embryonic fibroblasts.
        Exp Cell Res. 2007; 313: 952
        • Kiwanuka E.
        • Hackl F.
        • Philip J.
        • Caterson E.J.
        • Junker J.P.
        • Eriksson E.
        Comparison of healing parameters in porcine full-thickness wounds transplanted with skin micrografts, split-thickness skin grafts, and cultured keratinocytes.
        J Am Coll Surg. 2011; 213: 728
        • Hackl F.
        • Bergmann J.
        • Granter S.R.
        • et al.
        Epidermal regeneration by micrograft transplantation with immediate 100-fold expansion.
        Plast Reconstr Surg. 2012; 129: 443e
        • Svensjo T.
        • Yao F.
        • Pomahac B.
        • Eriksson E.
        Autologous keratinocyte suspensions accelerate epidermal wound healing in pigs.
        J Surg Res. 2001; 99: 211
        • Svensjo T.
        • Yao F.
        • Pomahac B.
        • Winkler T.
        • Eriksson E.
        Cultured autologous fibroblasts augment epidermal repair.
        Transplantation. 2002; 73: 1033
        • Cory G.
        Scratch-wound assay.
        Methods Mol Biol. 2011; 769: 25
        • Samarin J.
        • Rehm M.
        • Krueger B.
        • Waschke J.
        • Goppelt-Struebe M.
        Up-regulation of connective tissue growth factor in endothelial cells by the microtubule-destabilizing agent combretastatin A-4.
        Mol Cancer Res. 2009; 7: 180
        • Samarin J.
        • Cicha I.
        • Goppelt-Struebe M.
        Cell type-specific regulation of CCN2 protein expression by PI3K-AKT-FoxO signaling.
        J Cell Commun Signal. 2009; 3: 79
        • Tall E.G.
        • Bernstein A.M.
        • Oliver N.
        • Gray J.L.
        • Masur S.K.
        TGF-beta-stimulated CTGF production enhanced by collagen and associated with biogenesis of a novel 31-kDa CTGF form in human corneal fibroblasts.
        Invest Ophthalmol Vis Sci. 2010; 51: 5002
        • Tomic-Canic M.
        • Komine M.
        • Freedberg I.M.
        • Blumenberg M.
        Epidermal signal transduction and transcription factor activation in activated keratinocytes.
        J Dermatol Sci. 1998; 17: 167
        • Grotendorst G.R.
        • Okochi H.
        • Hayashi N.
        A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor gene.
        Cell Growth Differ. 1996; 7: 469
        • Cowley S.
        • Paterson H.
        • Kemp P.
        • Marshall C.J.
        Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells.
        Cell. 1994; 77: 841
        • Dudley D.T.
        • Pang L.
        • Decker S.J.
        • Bridges A.J.
        • Saltiel A.R.
        A synthetic inhibitor of the mitogen-activated protein kinase cascade.
        Proc Natl Acad Sci USA. 1995; 92: 7686
        • Leask A.
        • Sa S.
        • Holmes A.
        • Shiwen X.
        • Black C.M.
        • Abraham D.J.
        The control of CCN2 (CTGF) gene expression in normal and scleroderma fibroblasts.
        Mol Pathol. 2001; 54: 180
        • Shi-Wen X.
        • Leask A.
        • Abraham D.
        Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis.
        Cytokine Growth Factor Rev. 2008; 19: 133
        • Wang J.F.
        • Olson M.E.
        • Ball D.K.
        • Brigstock D.R.
        • Hart D.A.
        Recombinant connective tissue growth factor modulates porcine skin fibroblast gene expression.
        Wound Repair Regen. 2003; 11: 220
        • Wang J.F.
        • Olson M.E.
        • Ma L.
        • Brigstock D.R.
        • Hart D.A.
        Connective tissue growth factor siRNA modulates mRNA levels for a subset of molecules in normal and TGF-beta 1-stimulated porcine skin fibroblasts.
        Wound Repair Regen. 2004; 12: 205
        • Nanney L.B.
        • Woodrell C.D.
        • Greives M.R.
        • et al.
        Calreticulin enhances porcine wound repair by diverse biological effects.
        Am J Pathol. 2008; 173: 610
        • Sullivan T.P.
        • Eaglstein W.H.
        • Davis S.C.
        • Mertz P.
        The pig as a model for human wound healing.
        Wound Repair Regen. 2001; 9: 66
        • Gallant-Behm C.L.
        • Tsao H.
        • Reno C.
        • Olson M.E.
        • Hart D.A.
        Skin wound healing in the first generation (F1) offspring of Yorkshire and red Duroc pigs: evidence for genetic inheritance of wound phenotype.
        Burns. 2006; 32: 180
        • Zhu K.Q.
        • Engrav L.H.
        • Tamura R.N.
        • et al.
        Further similarities between cutaneous scarring in the female, red Duroc pig and human hypertrophic scarring.
        Burns. 2004; 30: 518
        • Sugioka K.
        • Yoshida K.
        • Kodama A.
        • et al.
        Connective tissue growth factor cooperates with fibronectin in enhancing attachment and migration of corneal epithelial cells.
        Tohoku J Exp Med. 2010; 222: 45
        • Dhar A.
        • Ray A.
        The CCN family proteins in carcinogenesis.
        Exp Oncol. 2010; 32: 2
        • Quan T.
        • He T.
        • Kang S.
        • Voorhees J.J.
        • Fisher G.J.
        Connective tissue growth factor: expression in human skin in vivo and inhibition by ultraviolet irradiation.
        J Invest Dermatol. 2002; 118: 402
        • Quan T.
        • Shin S.
        • Qin Z.
        • Fisher G.J.
        Expression of CCN family of genes in human skin in vivo and alterations by solar-simulated ultraviolet irradiation.
        J Cell Commun Signal. 2009; 3: 19
        • Rittie L.
        • Perbal B.
        • Castellot Jr., J.J.
        • Orringer J.S.
        • Voorhees J.J.
        • Fisher G.J.
        Spatial-temporal modulation of CCN proteins during wound healing in human skin in vivo.
        J Cell Commun Signal. 2011; 5: 69
        • Kapoor M.
        • Liu S.
        • Huh K.
        • Parapuram S.
        • Kennedy L.
        • Leask A.
        Connective tissue growth factor promoter activity in normal and wounded skin.
        Fibrogenesis Tissue Repair. 2008; 1: 3
        • Krueger J.S.
        • Keshamouni V.G.
        • Atanaskova N.
        • Reddy K.B.
        Temporal and quantitative regulation of mitogen-activated protein kinase (MAPK) modulates cell motility and invasion.
        Oncogene. 2001; 20: 4209
        • Pickles M.
        • Leask A.
        Analysis of CCN2 promoter activity in PANC-1 cells: regulation by ras/MEK/ERK.
        J Cell Commun Signal. 2007; 1: 85
        • Ponticos M.
        • Holmes A.M.
        • Shi-wen X.
        • et al.
        Pivotal role of connective tissue growth factor in lung fibrosis: MAPK-dependent transcriptional activation of type I collagen.
        Arthritis Rheum. 2009; 60: 2142
        • Tan T.W.
        • Lai C.H.
        • Huang C.Y.
        • et al.
        CTGF enhances migration and MMP-13 up-regulation via alphavbeta3 integrin, FAK, ERK, and NF-kappaB-dependent pathway in human chondrosarcoma cells.
        J Cell Biochem. 2009; 107: 345
        • Leask A.
        CCN6 (WISP3): a new anti-cancer therapy?.
        J Cell Commun Signal. 2010; 4: 199
        • Dornhofer N.
        • Spong S.
        • Bennewith K.
        • et al.
        Connective tissue growth factor-specific monoclonal antibody therapy inhibits pancreatic tumor growth and metastasis.
        Cancer Res. 2006; 66: 5816
      1. Finger EC, Cheng CF, Williams TR, et al. CTGF is a therapeutic target for metastatic melanoma. Oncogene [Epub ahead of print].