Corneal epithelial wound healing impaired in keratinocyte-specific HB-EGF-deficient mice in vivo and in vitro.
OBJECTIVE
To study the role played by HB-EGF in corneal epithelial wound healing.


METHODS
A 2-mm corneal epithelial wound was created in keratinocyte-specific, HB-EGF-deficient mice--HB(lox/lox):K-5Cre (HB(-/-))--and the speed of wound healing was compared with that in wild-type (WT) mice. Cultured confluent mouse corneal epithelial cells (MCECs) from WT and HB(-/-) mice were scraped, and the bare area was measured. The proliferation of MCECs was determined by BrdU incorporation. The degree of attachment of WT and HB(-/-) MCECs was also determined. The mRNA expression of EGF family and cell adhesion molecules was determined by real-time PCR.


RESULTS
Corneal epithelial wound healing was significantly delayed in HB(-/-) mice, and the expression of HB-EGF was detected at the leading edge of the wound in HB(lox/+):K5-Cre (HB(+/-)) mice by the presence of lacZ staining. The wound closure was significantly impaired in HB(-/-) MCECs and was improved by adding HB-EGF. The number of BrdU-positive MCECs of WT and HB(-/-) mice was not significantly different, and both increased to different degrees when HB-EGF was added. The adhesion of isolated HB(-/-) MCECs was lower than that of WT MCECs, but the degree of adhesion was restored by adding HB-EGF. The expression of epiregulin was upregulated in HB(-/-) MCECs, and α6- and β1-integrin were upregulated by adding HB-EGF.


CONCLUSIONS
HB-EGF plays an important role in corneal epithelial cell healing by enhancing cellular attachment and part of cell proliferation.
Yoshioka
Ryuji
R
Department of Ophthalmology, Ehime University Graduate School of Medicine, Ehime, Japan. ryoshiok@m.ehime-u.ac.jp
Shiraishi
Atsushi
A
Kobayashi
Takeshi
T
Morita
Shin-Ichi
S
Hayashi
Yasuhito
Y
Higashiyama
Shigeki
S
Ohashi
Yuichi
Y
D000818:Animals; D015815:Cell Adhesion Molecules / Q000235:genetics; D049109:Cell Proliferation; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000235:genetics; D019573:Epithelium, Corneal / Q000166:cytology*; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins / Q000502:physiology*; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014945:Wound Healing / Q000502:physiology*
D015815:Cell Adhesion Molecules; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D004815:Epidermal Growth Factor