Effect of Vitamin D Receptor Knockout on Cornea Epithelium Wound Healing and Tight Junctions

Abstract

Purpose.: Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1α hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction–associated proteins occludin and ZO-1, and tight junction numbers in mouse corneas. Methods.: Epithelial wounds (2-mm) were made with an agar brush on 4-week-old and 10-week-old wild-type, heterozygous, and VDR knockout mouse corneas. Mice were on a normal or high lactose, Ca²⁺, and PO₄ ⁻ diet. Wound-healing area was measured over time. Real-time PCR was used to quantify occludin and ZO-1 message expression. Western blot was used for protein expression. Transmission electron microscopy was used to examine corneal epithelium and endothelium tight junctions. Immunofluorescence was used to examine epithelial ZO-1 distribution. Results.: Results showed a decreased healing rate in 10-week-old VDR knockout mice compared with wild-types. Vitamin D receptor knockout mice on the special diet had no difference in healing rate compared with wild-types. Real-time PCR showed decreased expression of occludin and ZO-1 in 10-week-old VDR knockout mice compared with wild-types. Western blot of 10-week-old knockout mouse corneas showed decreased occludin expression compared with wild-types. Transmission electron microscopy showed a significant difference in tight junction numbers in VDR knockouts versus wild-types. Immunofluorescence showed a change in ZO-1 distribution among genotypes. Conclusions.: Vitamin D receptor knockout affects mouse corneal epithelium wound healing and tight junction integrity.