Role of Epidermal Growth Factor Receptor and Src-Family Kinase Activation in Human Corneal Epithelial Wound Healing

Abstract

Purpose : Corneal epithelial wounds are a serious global concern that can lead to deeper corneal scarring and blindness. Our previous study suggests activation of epidermal growth factor receptor (EGFR) and Src-family kinases (SFK) may play a role in corneal epithelial wound healing in murine derived corneal epithelial cells. To further understand the role of the EGFR/SFK pathway in wound healing, wound assays using immortalized human corneal epithelial cells and primary human corneal epithelial cells were examined in the presence and absence of tyrosine kinase inhibitors. Using live cell imaging and Western blot analysis, cellular events were correlated to cell migration rates.

Methods : Monolayers derived from 2.040 pRSV-T ATCC (2.040) immortalized cells and primary human corneal epithelial cells (HCEC) were established and subsequently wounded. Cells were incubated with small molecule tyrosine kinase inhibitors or vehicle at various concentrations in pairs. Wound assays were either imaged with a live cell imaging system and analyzed with Image J software, or collected as protein lysates 24 hours after wounding for Western blot analysis.

Results : Cells incubated with tyrosine kinase inhibitors showed marked inhibition of cell migration into the wound gaps 24 hours post wounding at all concentrations tested, while cells with vehicle only demonstrated near complete resolution of the wound area. There was a strong correlation between cell migration and activation of EGFR and SFKs. Treatment with tyrosine kinase inhibitors inhibited cell migration and this correlated with decreased levels of activated EGFR and SFK in wounded 2.040 and HCEC.

Conclusions : Enhanced corneal epithelial wound healing was previously observed in murine derived corneal epithelial cells with increased levels of activated EGFR and SFK. The introduction of small molecule tyrosine kinase inhibitors inhibited the capacity of wounded human corneal epithelial cells to migrate and close wounds, paralleling decreased activation of EGFR and SFK on Western blot. Results from this study suggest a critical role for EGFR and SFK activation in human corneal epithelial wound healing with small molecule tyrosine kinase inhibitors potentiating EGFR/SFK dependent signaling. Further studies to enhance human corneal epithelial wound healing through increased activation of EGFR and SFK should be conducted.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.