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E. Chaum, J. Yin, H. Yang, X. Yang, J. C. Lang; Quantitative AP-1 Gene Regulation by Oxidative Stress in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5933.
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We have previously shown that the retinal pigment epithelium (RPE) demonstrates stereotypic changes in the expression of specific AP-1 family genes during the early cellular responses to oxidative stress (OS). The purpose of this study was to correlate AP-1 gene family transcription with quantitative changes in protein expression in response to OS.
Confluent ARPE-19 cells were cultured in defined medium for three days to stabilize gene expression, and then were treated to different levels of OS (0-500µM H2O2). RNA and protein were isolated from the cells for up to 8-hours following a one-hour OS treatment using a no-rinse method. Gene expression was quantified by real-time PCR and Western blotting methods. Total AP-1 protein expression levels, normalized to β-actin, were quantified. We examined the expression of FosB, c-Fos, Fra-1, JunB, c-Jun, ATF-2, and ATF3 genes.
The expression of FosB protein increased by up to 3.3-fold over controls 4 hours after OS and remained elevated 1.7-fold after 8 hours. Similarly, ATF-3 protein expression increased from 1.5-fold to 1.8-fold in response to increasing levels of OS (200-500µM H2O2) after 6 hours. Increased c-Fos, JunB, and Fra-1 protein expression was not seen despite increased levels of transcription. Finally, c-Jun, and ATF-2 expression levels were not increased either at the level of protein or mRNA.
These studies confirm that the expression of the AP-1 family genes FosB and ATF3 is regulated in a dose-dependent manner in response to OS, in the RPE, at the level of both transcription and translation.
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