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Rose Y Reins, Fahmi Mesmar, Cecilia Williams, Alison M McDermott; Microarray Analysis of Vitamin D Treated Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4900.
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© ARVO (1962-2015); The Authors (2016-present)
Vitamin D is an important biological mediator in a variety of tissues. Our previous studies show that human corneal epithelial cells (HCEC) have a functional vitamin D receptor (VDR) and respond to vitamin D by dampening TLR-induced inflammation. Here, we used genome-wide microarray analysis to examine vitamin D’s effect on corneal gene expression, further characterizing its influence on immune-related genes in HCEC.
Telomerase-immortalized (hTCEpi) and SV40-HCEC were treated with 1,25D3 (10-7M) or vehicle (0.01% ethanol/PBS) for 6h and RNA collected. Biological replicates and dye-swapped samples were used in genome-wide microarray analysis using Human Op-Arrays (Microarrays Inc) with analysis in R software. Functional groups and gene annotation clusters were identified using Pathway Studio (Elsevier Inc), DAVID, and PANTHER software. Gene expression was validated using qPCR in both cell lines as well as in primary HCEC, isolated from human donor corneas. For confirmation of IκBα protein, hTCEpi were treated with 1,25D3 (10-7M) for 24h and cell lysates used in an ELISA.
In hTCEpi, 308 genes were differentially expressed with vitamin D treatment, while in SV40s, 69 genes were changed. The expression of 24 genes was influenced by vitamin D in both cell lines. Regulated genes were identified that are involved in MAPK and TLR signaling pathways. qPCR confirmed the vitamin D-mediated upregulation (expressed as relative fold-change) of CYP24A1 (11,528+3215), SERPINB1 (4.6+0.7), IκBα (1.9+0.03), DUSP10 (5.0+1.5), IGFBP3 (9.9+1.6), and IL1RL (46.6+11.5) in hTCEpi, which exhibited a larger response to vitamin D than SV40 or primary HCEC. In addition to increased transcript levels, IκBα protein was also increased by 28% following 24h of vitamin D treatment.
This study is the first to report vitamin D’s influence on corneal gene transcription using microarray analysis, and shows that vitamin D regulates multiple genes in HCEC, including genes important in the inflammatory response. Interestingly, two cell lines commonly used in corneal research showed different levels of responsiveness to vitamin D, with hTCEpi more closely resembling primary cells. Vitamin D treatment increased the expression of IκBα, thereby increasing the cell’s capacity for inhibiting NF-κB activity, providing a possible mechanism for vitamin D’s ability to dampen TLR-induced inflammation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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