Changes in retinal pigment epithelial gene expression induced by rod outer segment uptake.
OBJECTIVE
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.
Chowers
Itay
I
Guerrieri Center for Genetic Engineering and Molecular Ophthalmology, Wilmer Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
Kim
Yoonhee
Y
Farkas
Ronald H
RH
Gunatilaka
Tushara L
TL
Hackam
Abigail S
AS
Campochiaro
Peter A
PA
Finnemann
Silvia C
SC
Zack
Donald J
DJ
D000818:Animals; D002417:Cattle; D002460:Cell Line; D004705:Endocytosis; D005136:Eye Proteins / Q000235:genetics*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology*; D010857:Pigment Epithelium of Eye / Q000502:physiology*; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012374:Rod Cell Outer Segment / Q000502:physiology*
D005136:Eye Proteins; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger