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Sanghamitra Mishra, Katherine Peterson, Alan Berger, Graeme Wistow; Gene expression profile of Retinal Pigment Epithelium derived ARPE-19 cells under serum starvation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4586.
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© ARVO (1962-2015); The Authors (2016-present)
Age Related Macular Degeneration (AMD) is associated with formation of subretinal deposits (drusen) and damage to Bruch’s membrane (BM) basal to the retinal pigment epithelium (RPE). This could lead to serum deprivation of overlying RPE. Here we studied gene expression in the human RPE derived cell line, ARPE-19, during a seven day time course of serum starvation.
Gene expression levels in serum starved ARPE-19 cells were measured for 0, 1, 3, 5, and 7 days using cDNA microarrays. Standard statistical tools and pathway analysis software were used to identify regulated genes and altered pathways. Differentially expressed genes and pathways, identified from microarray analysis, and the effect of supplements were confirmed by qPCR, western blot analysis and confocal microscopy.
Differentially expressed genes at days 1, 3, 5, and 7 were identified. The most notable changes in gene expression were up-regulation of cholesterol and lipid gene pathways and down-regulation of methallothionein genes. This was confirmed by qPCR and western blotting. Supplementing LDL reversed the change in cholesterol pathway genes. Uptake of LDL in the serum starved cells was also tested. Cells at days 3, 5 and 7 of serum starvation showed increasing uptake of LDL-Dy549, whereas no uptake was seen at Day 0. The labeled LDL uptake was abolished in the presence of unlabeled LDL. Downregulation of metallothionein genes along with upregulation of zinc binding genes is consistent with a reduction in the intracellular levels of bio-available zinc. Zinc supplementation reversed expression changes of the metallothionein and zinc-transporter genes.
Serum starved ARPE-19 cells showed significant changes in expression patterns of cholesterol and zinc-related pathway genes. Cholesterol synthesis enzymes were upregulated and cells showed increased uptake of exogenous cholesterol. This suggests that serum deprivation initiates a cellular program requiring cholesterol in RPE cells. The cells also have an increased requirement of zinc. Zinc-binding proteins frequently function as regulators of transcription, which have a profound effect on cell function when altered. These observations are interesting, considering that both cholesterol and zinc have been implicated in AMD. This raises the possibility that the response of RPE-derived cells to serum-deprivation may model some processes related to AMD.
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