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  • Title: Changes in retinal pigment epithelial gene expression induced by rod outer segment uptake.
    Author: Chowers I, Kim Y, Farkas RH, Gunatilaka TL, Hackam AS, Campochiaro PA, Finnemann SC, Zack DJ.
    Journal: Invest Ophthalmol Vis Sci; 2004 Jul; 45(7):2098-106. PubMed ID: 15223782.
    Abstract:
    PURPOSE: Rod outer segment (ROS) uptake, a crucial function of the retinal pigment epithelium (RPE), probably involve multiple proteins, yet only a small number have so far been identified. The goal of this study was to find additional genes involved in ROS uptake and degradation by identifying ROS-induced gene expression changes. METHODS: Human RPE-derived ARPE-19 cells were harvested 3 and 12 hours after addition of bovine ROS. Gene expression profiles were compared with control cultures by using a custom human retina cDNA microarray and were validated by quantitative real-time RT-PCR (QPCR). ROS binding and internalization were quantitated with a fluorescence assay. RESULTS: Alterations in the expression levels of multiple genes (especially ones involved in transcriptional regulation, signal transduction, or protein modification) were detected 3 hours after ROS challenge, whereas by 12 hours most had returned to baseline. QPCR results corroborated the microarray results for seven of the eight genes tested. Time-course QPCR experiments on an independent sample set demonstrated characteristic temporal expression changes for each gene. Protein levels of one of these, plasminogen activator inhibitor-1 (PAI-1), were tested and found to parallel the mRNA changes. In addition, exogenous PAI-1 inhibited ROS uptake by RPE cells in vitro, consistent with its putative function in integrin receptor regulation. CONCLUSIONS: ROS uptake is associated with regulation of multiple RPE genes in a gene-specific temporal pattern. These genes are candidates for involvement in ROS uptake and degradation, particularly PAI-1, for which the study provided evidence suggesting that it may participate in the negative feedback of ROS uptake.
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