ABSTRACT
PURPOSE: In many cell types, the E3 ubiquitin ligase, c-Cbl, induces ligand-dependent ubiquitylation of the epidermal growth factor receptor (EGFR) and targets the receptor for lysosomal degradation. The goal of this study was to determine whether c-Cbl is a negative regulator of EGFR in the corneal epithelium and if it can be inhibited to promote corneal epithelial homeostasis. METHODS: Expression and activity of c-Cbl were blocked in immortalized human corneal epithelial cells (hTCEpi) using RNAi and pharmacological agents ([4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine] or PP1). Following c-Cbl inhibition, cells were assessed for ligand-dependent receptor ubiquitylation, receptor phosphorylation, and in vitro wound healing. Subsequent experiments used PP1 in hTCEpi cells and monitored in vivo murine corneal epithelial wound healing. RESULTS: Knockdown and inhibition of c-Cbl decreased ligand-dependent ubiquitylation of the EGFR and prolonged receptor activity as measured by tyrosine phosphorylation. Further, these treatments also increased the extent of ligand-dependent corneal epithelial wound healing in vitro and in vivo. CONCLUSION: Manipulating the duration of EGFR activity can enhance the rate of restoration of the corneal epithelial layer. Based on our findings, c-Cbl is a new therapeutic target to enhance EGFR-mediated corneal epithelial homeostasis that bypasses the limitations of previous approaches.
