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Rose Y Reins, Alison M McDermott; Vitamin D Activation and Function in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1190.
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© ARVO (1962-2015); The Authors (2016-present)
Vitamin D has many functions, including an immunomodulatory role during infection and a role in wound healing. Our previous studies have shown that human corneal epithelial cells (HCEC) respond to vitamin D and express the enzymes necessary for activation. In the current study, we examined vitamin D receptor (VDR) expression and activation of vitamin D metabolites in HCEC, as well as involvement of vitamin D-related genes during wound healing.
Telomerase-immortalized human corneal epithelial cells (hTCEpi) were grown on 8-well chamber slides and stimulated with 1,25D3 (10-7M) for 24 hours. Cells were fixed in methanol, blocked, and incubated with a rat anti-VDR antibody for immunofluorescence analysis. hTCEpi were also treated with D3 or 25D3 (10-6 M-10-9 M) for 24 hours and 1,25D3 was quantitated in cell supernatants with a 1,25D3 immunoassay kit. For organ culture wounding studies, epithelium was removed from human donor corneas (original sample), allowed to regrow, and collected again after 48 hours (regrown sample). RNA was isolated and analyzed by RT-PCR for expression of CYP27B1, the hydroxylase that converts vitamin D to its active form, 1,25D3.
hTCEpi expressed the VDR as shown by immunofluroescent staining. Stimulating these cells with 1,25D3 resulted in an increase in the nuclear localization of the VDR (n=3). In addition, when stimulated with 25D3, hTCEpi were able to produce active 1,25D3, with 10-7 M 25D3 treatment yielding the highest concentration (318 pmol/L). When cells were treated with the unhydroxylated D3, there was also a significant increase in 1,25D3 above unstimulated control supernatants, although at very low levels (< 6.4 pmol/L; n=4). In the wounded corneas, CYP27B1 expression was increased (1.65-fold) following wound closure (n=4).
In addition to expressing activating vitamin D enzymes, hTCEpi also express a functional VDR that is responsive to 1,25D3 treatment, as seen by increased nuclear localization. Importantly, hTCEpi are able to convert inactive D3 and 25D3 to the biologically functional 1,25D3 metabolite, which can then in turn activate the VDR in these cells. During corneal wounding, the expression of CYP27B1 increases, which could result in enhanced activation of vitamin D, aiding in the wound healing process.
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