Epidermal growth factor synergism with TGF-β1 via PI-3 kinase activity in corneal keratocyte differentiation

PURPOSE. To investigate the action of epidermal growth factor (EGF) on corneal keratocyte differentiation and its effects in conjunction with transforming growth factor (TGF)-beta 1. METHODS. Rabbit corneal keratocytes (RCKs) were treated with EGF, TGF-beta 1, or EGF plus TGF-beta 1 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 alpha-smooth muscle actin (alpha-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 protomyofibroblast phenotype with the loss of dendritic shape and the expression of alpha-SMA. Treatment with TGF-beta 1 stimulated 12% of the cells to differentiate to defined myofibroblasts, but in the presence of EGF, TGF-beta 1 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-beta 1 cell differentiation. TGF-beta 1 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-beta 1 through EGF-R activation and that it is an important modulator of wound healing and scar tissue formation.