Abstract
Purpose :
Epidermal growth factor receptor (EGFR) signaling is required for homeostasis and full differentiation of the corneal epithelium, but stimulation with exogenous EGF is of limited therapeutic use due to receptor downregulation. The E3 ubiquitin ligase, c-Cbl, is a key regulator of EGFR downregulation and indirect inhibition of c-Cbl enhances EGFR-mediated corneal epithelial regeneration. This study was designed to assess how knockout of c-Cbl affects EGFR endocytic trafficking and EGFR-mediated responses (e.g. proliferation, migration, differentiation).
Methods :
Human telomerase immortalized corneal epithelial (hTCEpi) cells were bioengineered with CRISPR/CAS9 to knockout c-Cbl. C-Cbl knockout and control (CAS9 expressing) hTCEpi cells were assayed for ligand-mediated responses. EGFR phosphorylation and degradation were measured by immunoblot analysis. Cell growth was monitored using microscopy and alamar blue assay. Cell migration was measured with microscopy and transwell migration assays. The ligand-dependent EGFR endocytic trafficking was monitored with indirect immunofluorescence.
Results :
Knockout of c-Cbl enhances the ligand-dependent EGFR phosphorylation by 2.5 fold (p <0.001) and doubles the receptor’s half-life (p < 0.05). This is a consistent with the c-Cbl knockout cells having prolonged localization of the EGFR at the plasma membrane following EGF treatment and retention of the receptor in the early endosome. Coincident with these changes in receptor signaling and trafficking, ligand-stimulated cell migration is enhanced in c-Cbl knockout cells as compared to control cells. Ligand-dependent cell proliferation is slightly elevated in these cell.
Conclusions :
These studies demonstrate that c-Cbl negatively regulates signaling by slowing the endocytic trafficking of the EGF:EGFR complex. These slowed kinetics increase receptor activity and preferentially drives cell migration over cell proliferation.
This is a 2020 ARVO Annual Meeting abstract.