Changes in retinal pigment epithelial gene expression induced by rod outer segment uptake

Itay Chowers, Yoonhee Kim, Ronald H. Farkas, Tushara L. Gunatilaka, Abigail S. Hackam, Peter A. Campochiaro, Silvia C. Finnemann, Donald J. Zack

Research output: Contribution to journalArticle

19 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)2098-2106
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume45
Issue number7
DOIs
StatePublished - Jul 1 2004
Externally publishedYes

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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    Chowers, I., Kim, Y., Farkas, R. H., Gunatilaka, T. L., Hackam, A. S., Campochiaro, P. A., Finnemann, S. C., & Zack, D. J. (2004). Changes in retinal pigment epithelial gene expression induced by rod outer segment uptake. Investigative Ophthalmology and Visual Science, 45(7), 2098-2106. https://doi.org/10.1167/iovs.03-0863