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Epidermal Growth Factor Synergism with TGF-β1 via PI-3 Kinase Activity in Corneal Keratocyte Differentiation

From the Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, School of Medicine, New Orleans, Louisiana.

PURPOSE. To investigate the action of epidermal growth factor (EGF) on corneal keratocyte differentiation and its effects in conjunction with transforming growth factor (TGF)-β1.

METHODS. Rabbit corneal keratocytes (RCKs) were treated with EGF, TGF-β1, or EGF plus TGF-β1 in the presence or absence of inhibitors of EGF-receptor (EGF-R), neutralizing concentrations of EGF antibody and of signaling kinases for 2 days to 1 week. RCK differentiation to myofibroblasts was identified with anti-aldehyde dehydrogenase (ALDH)-1 and -smooth muscle actin (-SMA) antibodies. Cell proliferation was evaluated with anti-Ki-67 antibody. Extracellular matrix (ECM) components were assayed by immunochemistry and Western blot. Cell migration images were captured with a camera attached to the microscope, and the area of the wound was calculated using imaging software.

RESULTS. RCKs cultured in serum-free DMEM/F12 without frequent changes of medium maintained the phenotype for more than 1 month. EGF stimulated differentiation into a proto-myofibroblast phenotype with the loss of dendritic shape and the expression of -SMA. Treatment with TGF-β1 stimulated 12% of the cells to differentiate to defined myofibroblasts, but in the presence of EGF, TGF-β1 induced 90% of RCKs to transform into myofibroblasts. Inhibition of EGF-R activation and of the phosphatidylinositol-3 kinase (PI-3K)/Akt-1 pathway prevented the action of EGF on TGF-β1 cell differentiation. TGF-β1 in the presence of EGF also increased cell migration, which is inhibited by blocking EGF-R activation.

CONCLUSIONS. These data show that EGF contributes to the differentiation and migration of myofibroblasts induced by TGF-β1 through EGF-R activation and that it is an important modulator of wound healing and scar tissue formation.