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G.A. Secker, A.J. Shortt, E.M. Sampson, G.R. Grotendorst, G.S. Schultz, J.T. Daniels; CTGF Stimulates Human Corneal Epithelial Cell Re–Epithelialisation and Migration Through the Ras/MEK/ERK Pathway and May Act Downstream of TGFß1 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5026.
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© ARVO (1962-2015); The Authors (2016-present)
Connective tissue growth factor (CTGF) is a cysteine–rich, heparin–binding protein previously shown to have no effect on epithelial cells. However, we have previously found that CTGF stimulates re–epithelialisation of the human corneal epithelial cell line, HCE–T (ARVO abstract 2139). The current study investigated the cellular mechanisms and potential signalling pathways involved in CTGF–mediated re–epithelialisation. As CTGF is a downstream mediator of TGFß1 profibrotic action in fibroblasts, this study also investigated the hypothesis that TGFß1 stimulates up–regulation of CTGF in epithelial cells.
Confluent monolayers of HCE–T and a keratinocyte (HaCat) cell line were scratch wounded using a pipette tip. Digital photographs were taken immediately and at 8 hours post wounding. Image Tool software was used to determine wound width. Migration and proliferation was assessed using a colony dispersion assay and WST–1 reagent respectively. Serum free conditions, concentrations of CTGF, TGF ß1 and Ras/MEK/ERK signalling pathway inhibitors were compared in the above assays. CTGF protein levels were analysed by ELISA, phosphorlyated ERK was detected by western blotting. Data was analysed by one–way analysis of variance.
Percentage re–epithelialisation and migration was increased by TGFß1 and CTGF in HCE–T and HaCat cells (p<0.05). Proliferation was decreased or unaffected. TGFß1 stimulated CTGF protein production in HCE–T cells. Antisense oligonucleotides to CTGF blocked the functional effects of TGF ß1 (p<0.05). CTGF stimulated phosphorylation of ERK1/2 in HCE–T cells. Ras/MEK/ERK pathway inhibitors functionally blocked CTGF stimulated re–epithelialisation and migration (p<0.05).
CTGF promotes human corneal epithelial cell re–epithelialisation by stimulating migration through the Ras/MEK/ERK pathway. CTGF may act downstream of TGF ß1 since TGFß1 up–regulates CTGF protein production in HCE–T and CTGF antisense oligonucleotides functionally blocks TGFß1 activity. These novel data suggest a role for CTGF in corneal epithelial wound healing and were corroborated by the skin epithelial cell line HaCat indicating that these finding may be relevant to other stratified epithelia.
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