ERK1/2 activation regulates the wound healing process of rabbit corneal endothelial cells.
OBJECTIVE
To evaluate the activation of ERK, a MAP kinase, in the regulation of proliferation and migration of rabbit corneal endothelial cells during the healing process after scrape wound injury.


METHODS
Cultured rabbit corneal endothelial cells were used in this study. Immunocytochemistry and Western blot analysis were performed to demonstrate the phosphorylation of ERK after scrape injury. The injured cell sheets were treated with different concentrations of ERK inhibitor, PD98059. The wound closure at 24, 48, and 72 hr following injury was evaluated. In order to evaluate the role of proliferation during wound healing, immunocytochemistry with Ki67 and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay were performed. The role of cell migration in wound healing was evaluated by quantification with the time lapse microscopic video imaging system.


RESULTS
Immunocytochemical and Western blot analysis revealed phosphorylation of ERK in the rabbit corneal endothelial cells after wounding. Inhibition of ERK phosphorylation by PD58059 resulted in a dose-dependent delay of wound closure. The inhibition of wound healing became significant from 48 to 72 hr in cells treated with 10 and 50 micro M of PD58059 (p < 0.05). ERK inhibition suppressed cell proliferation as indicated by Ki67 immunostaining and MTS assay. The time lapse microscopic video imaging analysis indicated delayed cell migration caused by PD98059.


CONCLUSIONS
ERK activation regulates wound repair of rabbit corneal endothelial cells. Both cell proliferation and migration are involved in this process.
Chen
Wei-Li
WL
Department of Ophthalmology and Center of Corneal Tissue Engineering and Stem Cell Biology, National Taiwan University Hospital, Taipei, Taiwan.
Lin
Chung-Tien
CT
Li
Jing-Wen
JW
Hu
Fung-Rong
FR
Chen
Ching-Chow
CC
D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004728:Endothelium, Corneal / Q000201:enzymology* / Q000293:injuries / Q000473:pathology; D004789:Enzyme Activation; D015807:Eye Injuries, Penetrating / Q000201:enzymology* / Q000473:pathology; D007150:Immunohistochemistry; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D011817:Rabbits; D014945:Wound Healing / Q000502:physiology*
D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3