Human corneal epithelial cell proliferation by epiregulin and its cross-induction by other EGF family members.
OBJECTIVE
To investigate the effects of epiregulin, a newly identified member of the epidermal growth factor (EGF) family, on the proliferation of human corneal epithelial cells (HCECs).


METHODS
The proliferation of HCECs was determined by cell counting and BrdU incorporation assays at specific times after exposure to different concentrations of human recombinant epiregulin (0 to 20 ng/ml). Immunohistochemical staining was used to localize epiregulin in cadaveric corneas. RT-PCR and real-time PCR were used to determine the expression levels of epiregulin in cultured and cadaveric HCECs. To examine the interaction between epiregulin and epidermal growth factor receptors (EGFRs), the phosphorylation of ErbB1 and ERK1/ERK2 (ERK1/2) was estimated by western blot analysis in the presence or absence of AG1478, a specific inhibitor of EGFR kinase activity. To search for cross-induction of epiregulin by other EGF family members, the expressions of EGF, heparin-binding epidermal growth factor-like growth factor (HB-EGF), amphiregulin (AR), and transforming growth factor-alpha (TGF-alpha) mRNA were determined by real-time PCR in the presence of 10 ng/ml of epiregulin. Conversely, the expression of epiregulin was also determined following the incubation of HCECs with 10 nM of either of EGF, HB-EGF, TGF-alpha, or AR.


RESULTS
The mRNA of epiregulin was expressed in cultured HCECs and HCECs obtained from cadaveric eyes. Epiregulin was strongly detected in the limbal epithelium and basal epithelium of the peripheral cornea, but it was weakly detected in the central corneal epithelium. HCECs proliferated in the presence of epiregulin in a dose-dependent manner as detected by an increase in cell numbers or in BrdU incorporation. When HCECs were incubated with exogenous epiregulin, the expression of the mRNA of epiregulin was upregulated as detected by real-time PCR, and the phosphorylation of ErbB1 and ERK1/2 was upregulated in a dose-dependent manner as shown by western blot analysis. These upregulations were inhibited by AG1478, a specific inhibitor of EGFR kinase activity. Epiregulin increased the expression of HB-EGF and AR, while TGF-alpha, HB-EGF, AR, and EGF increased the expression of epiregulin in HCECs.


CONCLUSIONS
These findings indicate that epiregulin played an autocrine role in the proliferation of HCECs presumably through cross-induction with other EGF family members.
Morita
Shin-ichi
S
Department of Ophthalmology, Ehime University School of Medicine, Ehime, Japan. morita21@m.ehime-u.ac.jp
Shirakata
Yuji
Y
Shiraishi
Atsushi
A
Kadota
Yuko
Y
Hashimoto
Koji
K
Higashiyama
Shigeki
S
Ohashi
Yuichi
Y
D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000008:administration & dosage / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D066260:Epiregulin; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D016850:Limbus Corneae / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000494:pharmacology; D014018:Tissue Distribution; D015854:Up-Regulation
C587880:EREG protein, human; D066260:Epiregulin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases