Down-regulation of RPE65 protein expression and promoter activity by retinoic acid.
OBJECTIVE
RPE65 is critical for the normal formation of 11-cis retinal and thus photoreceptor function. Opsin expressed in HEK293 cells has been reported to form rhodopsin on the addition of all-trans retinol, indicating that the machinery for retinoid isomerization is present. RPE65 has been previously identified in HEK293 cells at both the RNA and protein levels. To further understand retinoid metabolism in these cells and the control of RPE65 expression, HEK293 cells were used as a model to determine if retinoic acid (RA) affects RPE65 promoter activity.


METHODS
RPE65 levels were determined by Western blots. RA regulation of RPE65 promoter activity was monitored using the luciferase reporter assay after transient transfection of HEK293 cells with the RPE65 promoter. Deletion and truncation promoter mutants were assessed for activity.


RESULTS
RA down-regulates RPE65 protein expression and promoter activity. The RA receptors (RARs), RARalpha, -beta, and -gamma, and the retinoid X receptors (RXRs), RXRalpha, -beta, and -gamma, were all identified in these cells and shown to mediate the regulation of RPE65 mRNA expression. After deletion of the AP1, AP4 or NF1 transcription factor binding sites, the RA down-regulation was decreased, but the decrease was not associated with a single transcription factor. The truncation promoter constructs P60, P153 and P257 showed increases in promoter activity, indicating an inhibitory element had been removed, and the down-regulatory effect of RA was decreased.


CONCLUSIONS
The down-regulation of RPE65 by RA is occurring at the transcription level. Multiple elements in the RPE65 promoter may contribute to this regulation.
Chen
Yumei
Y
Department of Ophthalmology, Medical University of South Carolina, Charleston, SC 29425, USA.
Ma
Jian-xing
JX
Crouch
Rosalie K
RK
D015139:Blotting, Southern; D015153:Blotting, Western; D002352:Carrier Proteins; D002453:Cell Cycle; D002460:Cell Line; D015536:Down-Regulation; D005136:Eye Proteins; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007668:Kidney / Q000166:cytology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D010857:Pigment Epithelium of Eye / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology*; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D002352:Carrier Proteins; D005136:Eye Proteins; D008156:Luciferases; D011506:Proteins; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase