Knockout of c-Cbl and Cbl-b enhances corneal re-epithelialization in male, but not female, mice.

Abstract

Tissue culture studies indicate c-Cbl and Cbl-b are negative regulators of growth factor receptor signaling. Inhibiting c-Cbl and Cbl-b is a potential therapeutic strategy for enhancing growth factor-mediated corneal re-epithelialization. An inducible, tissue-specific mouse model was used to assess the role of c-Cbl and Cbl-b in the corneal epithelium to determine the therapeutic potential of c-Cbl/Cbl-b inhibitors. Mice were generated by breeding c-Cbl and Cbl-b floxed mice with Keratin 14 (Krt14) ERTCre expressing mice. Knockdown was assessed by PCR and immunofluorescence. Corneal morphology was assessed using H&E staining. Ki67 immunostaining was used to measure cell proliferation. The functional consequence of c-Cbl/Cbl-b knockout was determined by measuring HGF-dependent corneal re-epithelialization following a mechanical debridement wound. Knockout of c-Cbl and Cbl-b was tamoxifen-dependent and did not affect the overall structure of the cornea. In male c-Cbl/Cbl-b knockout mice, corneal epithelial thickness and cell density were unchanged from controls, and there was enhanced HGF-dependent corneal re-epithelialization. Female knockout mice had reduced corneal epithelial thickness and cell density and increases in basal epithelial cell proliferation. However, there was no increase in HGF-dependent corneal re-epithelialization. Despite experimental considerations due to the Cre and tamoxifen toxicities, c-Cbl/Cbl-b/Krt14 ERTCremice are a viable model for studying c-Cbl and Cbl-b in corneal epithelial biology. Knockout of c-Cbl and Cbl-b affects corneal epithelial homeostasis and regeneration in both sexes of mice. However, the role of c-Cbl/Cbl-b in male knockout mice is stronger in re-epithelialization, whereas in female mice it regulates homeostasis.