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Y. Hori, S. Spurr-Michaud, P. Argüeso, I.K. Gipson; Effect of Retinoic Acid on Gene Expression in Human Conjunctival Epithelial Cells as Determined by Microarray Analysis . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3792.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Vitamin A deficiency causes abnormal differentiation and keratinization of the ocular surface epithelia. The mechanism through which vitamin A acts to maintain the wet-surfaced phenotype of the epithelia is not well understood. To investigate vitamin A responsive genes at the ocular surface, we used microarray analysis on cultures of a TERT-immortalized human conjunctival epithelial cell line (HCjE) grown with and without retinoic acid (RA). Methods: HCjE cells were cultured in DMEM/F12 and 100nM RA for 0 (control), 3, 6, 12, 24 and 48 h. Total RNA was isolated and used as a template to synthesize a double strand cDNA with T7-(dT)24 oligomer primers. Biotin-labeled complementary RNA (cRNA) was produced by in vitro transcription and hybridized to an Affymetrix human microarray chip (HG-U133A). Gene expression in cultures treated with RA over the course of 3-48 h was compared to the no RA control. The results were analyzed using Rosetta Resolver software. Results: Forty-nine percent of the genes represented on the U133A array (10889 out of 22383) were detected in control samples. Compared with the control, the expression of 70, 38, 20, 75, and 79 genes were upregulated more than 2-fold at 3, 6, 12, 24, and 48 h, respectively. In contrast, 54, 30, 29, 63, and 155 genes were downregulated at each time point, respectively. Maximum change of gene expression occurred at the early 3 h time point and the late 24 and 48 h time points. In the early phase of the response, 3 and 6 h, short-chain dehydrogenasereductase 1 (SDR1), an enzyme associated with RA metabolism, was the most upregulated gene (19 to 26 fold). Mitogen-activated protein kinase kinase 2 (MAP2K2) was the most downregulated gene at 3 h (13 fold). During the late phase of the response, matrix metalloproteinases (MMPs) 3 and 7 were the most upregulated genes (16 and 8 fold, respectively). Rabkinesin 6, a kinesin-like protein, was the most downregulated gene, 65 fold at 48 h. Keratin 13, which is known to be present in non-keratinized epithelia, was upregulated at 6 to 48 h (9 to 11 fold), but curiously, keratin 1, which is expressed in keratinized epithelia, was also upregulated at 48 h (5 fold). Conclusions: There are two peaks in gene expression in conjunctival epithelium in response to RA. They occur early (3 h) and late (24 and 48 h). These early and late peaks may represent the genes regulated directly and indirectly, respectively. Further investigation of the genes expressed in response to RA may provide new insight into the role of the retinoid in conferring phenotypic characteristics to wet-surfaced epithelia.
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