[Effect of epidermal growth factor on expression of cell cycle-regulatory proteins and proliferation of rabbit corneal epithelial cells].
OBJECTIVE
To investigate the effects of epidermal growth factor (EGF) on the proliferation of cultured rabbit corneal epithelial (RCE) cells and the expression of cell cycle-regulatory proteins in these cells.


METHODS
It was an experimental study. Cultured RCE cells were exposed to EGF at different concentrations (0, 1, 5, 10, 25 and 50 microg/L) and different time (0, 2, 4, 6, 8 and 10 days). Cell morphologic changes were observed under a phase contrast microscope. Cell proliferation was measured using the WST-1 cell proliferation assay. The expression of cell cycle-regulatory proteins, cyclinD1, CDK4, p27, p21 and p18, was examined using Western blot analysis. The data was statistically analyzed with SPSS 11.5 software, difference between two groups and multiple groups was compared by t test and ANOVA, respectively.


RESULTS
Treatment with EGF did not obviously change the cell morphology. EGF significantly induced the proliferation of RCE cells in a dose- and time-dependent manner. In cells treated with different concentrations of EGF, the proliferation rate of 5, 10, 25 and 50 microg/L EGF group at 72 h were 9.0%, 23.5%, 20.8% and 17.7%, respectively. The difference was statistically significant as compared with the control group (F = 45.48, P < 0.01). EGF at 10 microg/L showed maximally stimulating effect. After EGF treatment for 0, 24, 48 and 72 h, the expression of cyclinD1 and CDK4 proteins was markedly increased (cyclinD1/beta-actin ratio was 0.253, 0.591, 0.885 and 1.043; CDK4/beta-actin ratio was 0.422, 0.588, 0.804 and 1.241 at different periods, respectively). Furthermore, p27 protein expression was significantly inhibited by EGF (p27/beta-actin ratio was 0.225, 0.163, 0.107 and 0.082 after EGF treatment for 0, 24, 48 and 72 h, respectively). However, there was no detectable difference in p21 and p18 protein expression between EGF treatment and control groups (p21/beta-actin ratio was 0.432, 0.391, 0.407 and 0.329 and p18/beta-actin ratio was 0.268, 0.274, 0.231 and 0.309, respectively).


CONCLUSIONS
This study suggests that EGF could induce RCE cell proliferation. EGF up-regulates cyclinD1 and CDK4 and down-regulates p27 in RCE cells. These changes may be the causes for EGF-induced proliferation of RCE.
Wang
You-dong
YD
Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University, Shenyang 110005, China. wyoudong@mail.cmu.edu.cn
Zhang
Jin-song
JS
D000199:Actins; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology*; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects*; D011817:Rabbits; D015854:Up-Regulation
D000199:Actins; D018797:Cell Cycle Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004815:Epidermal Growth Factor; D051358:Cyclin-Dependent Kinase 4