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E. Chaum, H. Yang, X. Yang, J. C. Lang; Mechanisms of AP-1 Gene Regulation by Oxidative Stress in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3775.
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We have previously shown that the retinal pigment epithelium demonstrates stereotypic changes in the expression of specific genes during the early cellular responses to oxidative stress (OS), in particular, AP-1 family genes. The purpose of this study was to determine whether this OS-induced transcription is translated into increased AP-1 proteins levels and to identify the signaling pathways regulating the response.
Confluent ARPE-19 cells were cultured for three days in defined medium to stabilize gene expression. The cells were exposed to varying levels of OS (0 - 500µM H2O2) for 1- or 4-hours. RNA and protein were isolated from the cells using a no-rinse method and gene-specific expression was quantified by real-time PCR. Levels of AP-1 proteins FosB, c-Fos, JunB, and ATF3 were determined by Western blotting. Inhibition studies are being performed following incubation of the cells in the presence of the MEK/ERK pathway inhibitor U0126 (50µM) or the PI3-K/AKT pathway inhibitor LY294002 (10µM).
We quantified the translation of FosB, and other AP-1 proteins for 6-hours following OS and correlated the protein levels with the increases in transcription seen. The peak level of AP-1 protein synthesis occurred 2-hours after OS. Total FosB protein levels increased up to 2.5-fold for the highest levels of OS and were quantitatively correlated with increased transcription at higher levels of OS.
Our studies have shown that oxidative stress can quantitatively regulate AP-1 gene expression in the RPE. OS-induced AP-1 transcription is correlated with increased AP-1 protein levels in the cell, but at a quantitatively reduced level relative to transcription. The pathway inhibition studies are in progress and our results will be reported at the meeting.
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