Abstract
Purpose :
The cornea is the most anterior portion of the eye and serves to refract light onto the retina and protect the ocular surface. The corneal epithelium (CE) often sustains damage from trauma due to its position as the interface with the external environment. A properly formed cornea is required for clear vision, so ensuring the CE heals correctly is crucial. Growth factor receptors, like c-Met, mediate CE homeostasis and re-epithelialization when activated by their cognate ligand. c-Met is activated by hepatocyte growth factor (HGF) to enhance corneal epithelial biology. Despite promising responses to HGF in murine models, clinical studies have limited efficacy. This may be due to desensitization, where the c-Met receptor is ubiquitylated after stimulation and the c-Met:HGF complex is endocytosed and degraded in the lysosome. We hypothesize that preventing ubiquitylation knocking out c-Cbl/Cbl-b in CE cells will extend c-Met signaling, slow trafficking and degradation, and accelerate corneal wound healing.
Methods :
These studies use immortalized human corneal epithelial (hTCEpi) cells transfected with Cas9 and c-Cbl/Cbl-b guide RNA to knockout both proteins (DKO). Parental control cells expressed Cas9 only (Cas9). Following HGF treatment, ligand-dependent ubiquitylation was measured by immunoprecipitating ubiquitin and immunoblotting for the presence of c-Met. Cells were monitored for the magnitude and duration of c-Met receptor signaling via immunoblot analysis. The route of c-Met endocytic trafficking was assessed by immunofluorescence. The effect on in vitro wound healing was observed via live cell video microscopy that followed the rate of cells into an acellular area. Western blots were subject to densitometry. Blots and wound healing assay were subject to two-way ANOVA with multiple comparisons and area under the curve analysis.
Results :
In DKO cells, c-Met does not have detectable levels of ubiquitylation. Time course blots show 180% more area under the curve in DKO cells compared to Cas9. DKO cells exhibited slowed receptor internalization. Untreated cells showed similar rates of closure in the wound healing assay while treated DKO cells were 2x faster.
Conclusions :
c-Cbl and Cbl-b are E3 ubiquitin ligases that negatively regulate c-Met signaling. As such, they are two promising pharmacologic targets for enhancing c-Met-mediated CE re-epithelialization.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.