Project description:Purpose. How vitamin A contributes to the maintenance of the wet-surfaced phenotype at the ocular surface is not well understood. We sought to identify vitamin A responsive genes in ocular surface epithelia using gene microarray analysis of cultures of a human conjunctival epithelial cell line (HCjE) grown with all-trans-retinoic acid (RA). The analysis showed that the membrane-associated mucin MUC16 was induced by RA and that secretory phospholipase A2 Group IIA (sPLA2-IIA), the gene most upregulated by RA, was induced earlier. Since eicosanoids, metabolites of arachidonic acid, which is produced by sPLA2 catalysis of membrane phospholipids, have been demonstrated to affect mucin production, we sought to determine if the sPLA2 induction in HCjE cells was associated with RA induction of MUC16. Methods. HCjE cells were cultured with or without RA for 3, 6, 24 and 48 hours. Complementary RNA prepared from RNA of the HCjE cells was hybridized to human gene chips (HG-U133A; Affymetrix) and analyzed using Rosetta Resolver software. Microarray data on mucin expression were validated by real-time PCR. To investigate whether sPLA2 is associated with RA-induced MUC16 upregulation, HCjE cells were incubated with RA and the broad spectrum PLA2 inhibitor, aristolochic acid (ArA) or the specific sPLA2-IIA inhibitor LY315920, followed by analysis of MUC16 mRNA and protein by real-time PCR and Western blot analysis. Results. After RA addition, 28 transcripts were upregulated and 6 downregulated by over 2.0-fold (p < 0.01) at both 3 and 6 hours (early phase). Eighty gene transcripts were upregulated and 45 downregulated at both 24 and 48 hours (late phase). Group IIA sPLA2, significantly upregulated by 24 hours, and MUC16 were the most upregulated RNAs by RA at 48 hours. sPLA2 upregulation by RA was confirmed by Western blot analysis. When HCjE cells were incubated with RA plus ArA or specific inhibitor of sPLA2-IIA, LY315920, the RA-induced MUC16 mRNA was significantly reduced (p < 0.01). Conclusion. The retinoic acid-associated upregulation of membrane-associated mucin MUC16 at late phase appears to be through sPLA2-IIA. Upregulation of this hydrophilic membrane-associated mucin may be one of the important mechanisms by which vitamin A facilitates maintenance of the wet-surfaced phenotype on the ocular surface. Experiment Overall Design: Time course of retinoic acid treatment of human conjunctival epithelial cells: 0 (control), 3, 6, 12, 24, and 48 hours. 2 samples per time point.

Project description:Human conjunctival cell lines are useful tools for modeling ocular surface disease and evaluation of ocular drugs. Here we demonstrate that the IOBA-NHC and the ChWK conjunctival epithelial cell lines show, using an unbiased gene microarray approach, unique gene expression signatures that differ from primary conjunctival epithelial cells (PCEC) and conjunctival tissue. Globally, the expression profile obtained with the Affymetrix U133A chip (>22000 genes) from PCEC was clustered more closely to conjunctival tissue than either of the 2 cell lines. However, when restricted to Gene Ontology sub-categories: cellular defense, viral replication/cycling, antigen presentation, anti-oxidant pathways and ubiquitin ligase complex, the cell lines correlated reasonably well to PCEC (r > 0.70). In the category response to inflammation, correlation of cell lines to PCEC was poor (r = -0.012 and â0.041 for IOBA-NHC and ChWK respectively). In general, the expression profile in IOBA-NHC cells was better correlated to PCEC than the ChWK cells. This was statistically significant (p<0.05) when one considers all the genes on the chip, or for proteins in the extracellular region, response to wounding, stress, lipid, protein and organic acid metabolism, development and differentiation. Our results are useful for the choice of conjunctival cell lines, if necessary, in future experiments, to increase validity of extrapolation to clinical scenarios. Experiment Overall Design: Affymetrix U133A Genechip Experiment Overall Design: Experimental samples: Experiment Overall Design: IOBA-NHC cells (5 samples) Experiment Overall Design: Chang conjunctival epithelial cells WK derivative (4 samples) Experiment Overall Design: Primary conjunctival epithelial cells from explants (3 samples, obtained from cadaveric human explants) Experiment Overall Design: Conjunctival tissue from pterygium study where small piece uninvolved conjunctiva harvested (4 patients' RNA pooled to form one sample, total number of samples: 4) Experiment Overall Design: RT and hybridisation 16 hr according to Affymetrix protocol Experiment Overall Design: Labeling with biotin Experiment Overall Design: Washing microfluidics station 450 Experiment Overall Design: Analysis with Genespring GX 7.3.1 Experiment Overall Design: RMA normalisation following by normalisation to chip level median signal Experiment Overall Design: These processed data used for correlation analysis Experiment Overall Design: Further gene level normalisation to primary conjunctival epithelial cells samples for the purpose of fold change analysis to compare expression in IOBA or ChWK cells vs primary conjunctival epithelial cells.

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