Microarray Analysis of Dexamethasone–induced Changes in Human Trabecular Meshwork Cells

Abstract

Abstract: : Purpose: To identify corticosteroid responses in human trabecular meshwork (HTM) cells using microarray technology. Previous studies have shown that long–term dexamethasone (DEX) treatment of human trabecular meshwork cells result in a dramatic increase in the expression of myocilin (MYOC). This study aims to identify genes that respond to long–term DEX treatment that fall within known GLC loci. Methods: Cultured fifth passage HTM cell lines from three donors with no history of glaucoma were untreated or treated with 100 micromolar DEX for 21 days, after which total RNA was extracted. Gene expression was determined using a minimum of three Affymetrix U133A gene chips per assay condition per donor. Genechip data were scaled to the same target intensity of 1500 and compared using MAS 5.0 software. To reduce random fluctuations in the data, strict filtering for inclusion required that the average fold–change difference for all three cell lines be greater than 4.0 and call change be the same for all three cell lines. Certain findings were confirmed by realtime PCR using intron–spanning primers to calculate a relative fold change between DEX and untreated cell lines. Results: Using the strict filtering described above, DEX treatment increased expression of 40 genes greater than 4–fold (range 227 – 4) while 26 genes had a decrease in expression (range: –19 to –4). The average increase in expression for MYOC was 13–fold. Three of the genes identified are located within inclusion intervals for the GLC1B (2cen–q13) and GLC1C (3q21–q24) loci. Realtime PCR confirmed the genechip data findings within a cell line although the relative fold change values were often higher using realtime PCR. Conclusions: Prolonged Dex treatment alters the expression of many genes in the trabecular meshwork, including genes located within known glaucoma genetic inclusion intervals for which the genes have not been identified. Microarrays provide a powerful tool to initially identify genes of interest so they can be further studied using additional techniques such as proteomic and siRNA approaches to characterize pathways to elucidate the pathological responses that involve steroid–induced glaucoma.

Keywords: gene microarray • gene/expression • trabecular meshwork