Abstract
Purpose :
Protein ubiquitination status is fundamental to the regulation of many different cellular processes. However, its role in controlling corneal epithelial renewal, remains largely unknown. In the present study, we profile its involvement in corneal epithelial wound healing and thereby gain insight into how it affects renewal.
Methods :
Western blot was performed to detect the level of ubiquitination, as well as E3 Ubiquitin Ligase UBR5 in murine corneal epithelium during the wound healing process. Human corneal epithelial cells (HCEC) were transfected with siRNA targeted to UBR5 using Lipofectamine RNAiMAX reagent. MTS and a wound-healing assay evaluated the effects of UBR5 on HCEC proliferation and migration. Flow cytometry determined cell cycle progression.
Results :
Both global protein ubiquitination and UBR5 expression levels were dramatically upregulated during murine corneal epithelial wound healing. Knockdown of UBR5 in HCEC led to a significant decrease (60±3%) in cell proliferation, induced cell cycle G2 arrest and inhibited cell migration (by 67%). Furthermore, UBR5 downregulation inhibited the PI3K/Akt signaling pathway.
Conclusions :
Our results demonstrate that UBR5 upregulation promotes through stimulation of the PI3K/AKT signaling pathway HCEC proliferation and migration. This association indicates that increases in protein ubiquitination contribute to hastening these responses during corneal epithelial wound healing.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.