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Mitchell A Watsky, Xiaowen Lu, Zhong Chen; Vitamin D Receptor Knockout Effects on Mouse Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2625.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the effects on Vitamin D Receptor (VDR) knockout and 1,25- and 24,25-dihyroxyvitamin D3 (Vit D3) on cultured mouse corneal epithelial cell proliferation and on the Vit D activating enzyme CYP27B1 and inactivating enzyme CYP24A1. We also examined effects of vitamin D on mouse corneal epithelial intracellular calcium levels.
Cultured mouse corneal epithelial cell (MCEC) proliferation was measured by reduction of MTT, which corresponds to the living cell number and metabolic activity. Total RNA and protein were isolated from mouse corneas and cultured mouse primary corneal epithelial cells. Transcript levels of CYP24A1 and CYP27B1 were assessed by qPCR, and Western blotting was used to detect CYP24A1 and CYP27B1 protein levels. Corneal epithelial calcium levels were recorded in excised, ex vivo mouse corneas using the calcium-sensitive dye Cal-520® and multi-photon microscopy.
24,25-Vit D3 (50 nM) significantly increased proliferation in corneal epithelial cells cultured from both VDR wild type (WT) and knockout (KO) mouse corneas. 1,25-Vit D3 (10 nM ) had no effect on VDR WT mouse corneal epithelial cell proliferation but significantly increased VDR KO epithelial cell proliferation. Examining whole cornea mRNA levels, CYP24A1 and CYP27B1 mRNA levels were significantly increased in VDR KO mouse corneas while protein expression of CYP24A1 and CYP27B1 was significantly decreased. CYP24A1 and CYP27B1 mRNA and protein levels were significantly increased in VDR WT and VDR KO mouse corneal epithelial cells cultured with 1,25-Vit D3 and 24,25-Vit D3. Neither 1,25-Vit D3 nor 24,25-Vit D3 affected intracellular calcium levels in ex vivo mouse corneal epithelium, while the positive control ATP (5 µmol) rapidly increased cytosolic calcium levels.
1,25-Vit D3 stimulates VDR KO mouse corneal epithelial cell proliferation, and 24,25-Vit D3 stimulates VDR WT and VDR KO mouse corneal epithelial cell proliferation. VDR KO leads to reduced corneal CYP24A1 and CYP27B1 expression, while 1,25-Vit D3 and 24,25-Vit D3 increase CYP24A1 and CYP27B1 expression independent of VDR. It appears that VDR contributes to Vit D3 signaling in the corneal epithelium but a secondary signaling pathway is also involved. Corneal epithelial Vit D3 signaling does not appear to involve acute changes in intracellular calcium concentration.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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