Abstract
Purpose :
Vitamin D (Vit D) acts primarilly on cells via the nuclear-associated Vit D receptor (VDR). Our previous work has identified a number of VDR-independent actions of Vit D on corneal epithelial cells. The protein disulfide isomerase family member 3 (Pdia3) has been identified as a potential membrane-associated Vit D receptor (Rohe et al, Steroids, 2012) and we hypothesize Pdia3 is responsible for VDR-independent effects of Vit D in the cornea.
Methods :
Effects of 1,25-Vit D3 and 24,25-Vit D3 Vit D on stromal cell migration were tested using a scratch-wounded assay in primary stromal fibroblasts cultured from wild-type (VDR WT) and VDR knockout (VDR KO) mice. The Pdia3 inhibitors LOC14 and punicalagin were used to explore the role of Pdia3 in Vit D-stimulated stromal cell migration. Western blot was used to determine how Vit D affects Pdia3 protein levels in human and mouse primary stromal cells HPCSC and MPCSC), and VDR WT and VDR KO cells. Pdia3 expression was detected using immunostaining in mouse corneas.
Results :
1,25-Vit D3 (20 nM) significantly decreased HPCSC and VDR WT MPCSC migration, and increased VDR KO MPCSC migration. Both Pdia3 inhibitors (LOC14 (20 μM) and punicalagin (6 μM)) blocked the increase in migration observed in 1,25-Vit D3–treated VDR KO MPCSC. 24,25-Vit D3 (100 nM) had no effect on stromal cell migration. 1,25-Vit D3 (20 nM) and 24,25-Vit D3 (100 nM) significantly increased Pdia3 protein expression levels in HPCSC, VDR WT MPCSC and VDR KO MPCSC. Pdia3 protein expression was observed in mouse corneal epithelium, keratocytes, and endothelium.
Conclusions :
1,25-Vit D3 affects corneal stromal cell migration through both the VDR and Pdia3 pathways. VDR KO results in increased migration, and blocking Pdia3 in VDR KO cells effectively negates the 1,25-Vit D3 response. We conclude that Pdia3 is at least partly responsible for the 1,25-Vit D-induced increase in stromal cell migration. 1,25-Vit D3 and 24,25-Vit D3 increased Pdia3 expression in corneal stromal cells with or without active VDR signaling, indicating that Vit D has regulatory feedback control over Pdia3 expression, and this control is at least partialy the result of a VDR-independent signaling pathway.
This is a 2020 ARVO Annual Meeting abstract.