Abstract
Purpose: :
To evaluate the role of ERK MAP kinase (ERK1/2) in the proliferation and migration of rabbit corneal endothelial cells.
Methods: :
Primary culture of rabbit corneal endothelial cells were used in this study. Cells were divided into following groups: (1) control group (2) medium adding with 10uM of ERK inhibitor PD58059 (3) medium adding with 50uM of ERK inhibitor PD58059. For cell migration cell assay, confluent cells were treated with straight line scrape injury. The wound healing processes were evaluating 12 hours, 24, 36, 48 and 72 hours later. In addition, time lapse microscopy was also used to evaluating the lamellipodia formation in different groups. For cell proliferation assay, Ki67 positive at wound edge and MTS assay were used. Western blot analysis and Immunocytochemical study were also used to demonstrate the phosphorylation of ERK after injury in different groups.
Results: :
ERK inhibitors PD58059 can dose–dependently inhibit the wound healing process in the wounding assays. The difference of wound healing rate becomes significant from 24 hours to 48 hours in cells treated with 50uM of ERK inhibitor PD58059. However, there was no significant differences between control groups and cells treated with 10uM of ERK inhibitor PD58059. ERK inhibition also inhibits cell proliferation in Ki67 positive cells and MTS assay in group 3 instead of group 2. Immunocytochemical study and Western blot analysis demonstrated the phosphorylation ERK after making wound, and the phosphoryling process was most significant from 5 minutes to 30 minutes after injury.
Conclusions: :
This study demonstrated that ERK plays an important role in coordinating the proliferation and migration of rabbit corneal endothelial cells in wound healing process.
Keywords: cornea: endothelium • signal transduction • wound healing