ANALYSIS OF EXPRESSION KINETICS OF CELL CYCLE MACHINERY DURING CORNEAL EPITHELIAL WOUND HEALING AND EPITHELIAL GROWTH INFLUENCED BY GROWTH FACTORS AND EXTRACELLULAR MATRIX

Abstract

Purpose: Corneal injuries induce an increase in receptor tyrosine kinase ligands and extracellular matrix proteins (ECMs) in lacrimal and stromal secretions. These factors play very important roles in promoting wound healing. This study compared the expression pattern of various cell cycle proteins during corneal epithelial wound healing and epithelial growth promoted by hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and ECMs.

Methods: Wounds were created on live rabbit corneas by complete epithelial debridement. Regenerated epithelium was collected from 1-, 2-, 4-, and 8-day post injury corneas. The protein expression was analyzed by immunoblotting and immunofluorescence. The effect of HGF, KGF and ECMs (laminin, collagens I and IV and fibronectin) on cell cycle proteins expression was evaluated in rabbit corneal epithelial primary cells cultured in DMEM-F12/FCS.

Results: While the expression of cell cycle progressing cyclin E, cyclin A and cyclin-dependent kinases (CDK-2 and CDK4) increased gradually between 1-4 days and declined by day 8, cell cycle inhibitor p27^kip exhibited biphasic expression. Its level was higher one day after the injury but decreased by day 2. At later time points its level increased and peaked at day 8. The kinetics of another inhibitor p21^cip expression was similar to cyclins and CDKs. DAPI-staining of cornea cross-sections indicated restoration of multilayer epithelium by day 8. Fluorescein staining revealed near complete wound closure within 24 h following debridement indicating migration of epithelium from the limbus. Treatment of cells with HGF and KGF (20 ng/ml) for 24 h upregulated cyclins, CDKs and p21^cip but suppressed p27^kip expression. Interestingly, all ECMs (10 μg/ml) increased p21^cip and p27^kip levels in 24 h.

Conclusions: Increase in cyclins and CDKs levels between 2-4 days after the injury indicates robust proliferative activity which is required to restore multilayer epithelial architecture. Elevated levels of p27^kip in 8-day post-wound cornea could lead to suppression of cell division and prevention of epithelial hyperplasia. Further, the different patterns of expression of various cell cycle proteins as effected by HGF, KGF and ECMs could lead to the regulation of cell adhesion, migration and proliferation phases during wound healing.

Keywords: 482 cornea: epithelium • 765 wound healing • 480 cornea: basic science