Up-regulation of urokinase-type plasminogen activator in corneal epithelial cells induced by wounding.
OBJECTIVE
To investigate the possible role of urokinase-type plasminogen activator (uPA) in corneal epithelial wound healing by examining its expression both in the rabbit corneal epithelium in situ and in rabbit corneal epithelial (RCE) cells in vitro.


METHODS
The rabbit cornea was subjected to mechanical wounding, and frozen sections of the tissue were subsequently prepared and subjected both to immunostaining with antibodies to uPA and to in situ zymography for the detection of PA activity. RCE cell monolayers were also subjected to scrape wounding, after which they were immunostained for uPA. The amounts of uPA protein in the culture medium and of uPA mRNA in cell lysates were also determined by enzyme-linked immunosorbent assay and by reverse transcription and real-time quantitative polymerase chain reaction analysis, respectively.


RESULTS
Immunostaining and in situ zymography of the wounded cornea revealed that uPA was restricted to the leading edge of the migrating corneal epithelium. In contrast, tissue-type PA was expressed throughout the corneal epithelium. Scraping of RCE cell monolayers induced the expression of uPA in the migrating cells at the wound edge. The amount of uPA in the culture medium of RCE cells increased with the number of scrape wounds applied. Wounding also induced a time-dependent increase in the abundance of uPA mRNA in the cell monolayers. The migration of RCE cells was inhibited by antibodies to uPA.


CONCLUSIONS
Mechanical wounding induces up-regulation of uPA at both the protein and mRNA levels in corneal epithelial cells. uPA may thus contribute to epithelial cell migration during corneal epithelial wound healing.
Watanabe
Masanao
M
Tokyo New Drug Research Laboratories, Kowa Company Ltd., Tokyo, Japan. mwatanb@kowa.co.jp
Yano
Wataru
W
Kondo
Shoichi
S
Hattori
Yukio
Y
Yamada
Naoyuki
N
Yanai
Ryoji
R
Nishida
Teruo
T
D000818:Animals; D000911:Antibodies, Monoclonal; D002465:Cell Movement; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000201:enzymology; D019573:Epithelium, Corneal / Q000201:enzymology* / Q000293:injuries / Q000473:pathology; D015807:Eye Injuries, Penetrating / Q000201:enzymology* / Q000473:pathology; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic; D008297:Male; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D014945:Wound Healing
D000911:Antibodies, Monoclonal; D012333:RNA, Messenger; D014568:Urokinase-Type Plasminogen Activator