Epidermal growth factor synergism with TGF-beta1 via PI-3 kinase activity in corneal keratocyte differentiation.
OBJECTIVE
To investigate the action of epidermal growth factor (EGF) on corneal keratocyte differentiation and its effects in conjunction with transforming growth factor (TGF)-beta1.


METHODS
Rabbit corneal keratocytes (RCKs) were treated with EGF, TGF-beta1, or EGF plus TGF-beta1 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 proto-myofibroblast phenotype with the loss of dendritic shape and the expression of alpha-SMA. Treatment with TGF-beta1 stimulated 12% of the cells to differentiate to defined myofibroblasts, but in the presence of EGF, TGF-beta1 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-beta1 cell differentiation. TGF-beta1 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-beta1 through EGF-R activation and that it is an important modulator of wound healing and scar tissue formation.
He
Jiucheng
J
Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, School of Medicine, New Orleans, Louisiana 70112, USA.
Bazan
Haydee E P
HE
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D004357:Drug Synergism; D004815:Epidermal Growth Factor / Q000494:pharmacology / Q000502:physiology*; D066246:ErbB Receptors / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology / Q000502:physiology*
D011994:Recombinant Proteins; D053773:Transforming Growth Factor beta1; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors