Activation of epidermal growth factor receptor during corneal epithelial migration.
OBJECTIVE
Epidermal growth factor (EGF) and related growth factors: transforming growth factor (TGF)-alpha, heprin-ding (HB)-EGF, and amphiregulin (AR), have been shown to stimulate events associated with epithelial wound repair. These growth factors function by binding to a common EGF receptor (EGFR), tyrosine kinase. We have used in vivo and organ culture wound-healing models to examine the kinetics and extent of EGFR activation during corneal epithelial wound repair and whether the epithelium itself produces EGFR ligands capable of stimulating the healing process.


METHODS
In the in vivo model, 3-mm debridement wounds were made in rat corneas and allowed to heal in situ. Activation of EGFR was analyzed by 1) indirect immunofluorescence microscopy, 2) immunoprecipitation using anti-EGFR and anti-phosphotyrosine (anti-PT), and 3) binding-site localization using EGF-fluorescein isothiocyanate (FITC). Relative levels of mRNA for EGF, TGF-alpha, HB-EGF, and AR were determined using reverse transcription-polymerase chain reaction. To determine whether inhibiting EGFR activation slows epithelial migration, wounded corneas were allowed to heal in organ culture in the presence of tyrphostin AG1478 (0-50 microM), a specific inhibitor of EGFR kinase activity.


RESULTS
In unwounded corneas, EGFR was localized in basal cells and appeared to be membranous. Within 1 hour after wounding, EGFR was no longer immunolocalized in the membranes of cells migrating into the wound area. EGF-FITC-binding assays indicated that EGFR ligands could penetrate all the way to the limbus. Immunoprecipitation showed that EGFR was phosphorylated on tyrosine residues within 30 minutes after wounding and that phosphorylation levels increased after wounding. Levels of mRNA for TGF-alpha, HBEGF, and AR all appeared to increase after wounding. In organ culture experiments, tyrphostin AG1478 inhibited migration rates in a dose-dependent manner.


CONCLUSIONS
These data indicate that EGFR was activated during corneal epithelial wound healing in vivo. Furthermore, this activation appears to be a necessary component of the process, because inhibition of the EGFR signaling cascade significantly slowed migration rates.
Zieske
J D
JD
Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA. zieske@vision.eri.harvard.edu
Takahashi
H
H
Hutcheon
A E
AE
Dalbone
A C
AC
D066258:Amphiregulin; D000818:Animals; D001665:Binding Sites; D002465:Cell Movement / Q000187:drug effects; D003646:Debridement; D066255:EGF Family of Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology / Q000293:injuries / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D009924:Organ Culture Techniques; D010766:Phosphorylation; D011233:Precipitin Tests; D011799:Quinazolines; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020032:Tyrphostins / Q000494:pharmacology; D014945:Wound Healing / Q000502:physiology
D066258:Amphiregulin; C587850:Areg protein, rat; D066255:EGF Family of Proteins; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D006133:Growth Substances; C587885:Hbegf protein, rat; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011799:Quinazolines; D012333:RNA, Messenger; D020032:Tyrphostins; C101044:RTKI cpd; D004815:Epidermal Growth Factor; D066246:ErbB Receptors