Quantitative AP‐1 gene regulation by oxidative stress in the human retinal pigment epithelium

Abstract

The purpose of this study was to characterize the early molecular responses to quantified levels of oxidative stress (OS) in the human retinal pigment epithelium (RPE). Confluent ARPE-19 cells were cultured for 3 days in defined medium to stabilize gene expression. The cells were exposed to varying levels of OS (0-500 microM H(2)O(2)) for 1-8 h and gene expression was followed for up to 24-h after OS. Using real-time qPCR, we quantified the expression of immediate early genes from the AP-1 transcription factor family and other genes involved in regulating the redox status of the cells. Significant and quantitative changes were seen in the expression of six AP-1 transcription factor genes, FosB, c-Fos, Fra-1, c-Jun, JunB, and ATF3 from 1-8 h following OS. The peak level of induced transcription from OS varied from 2- to 128-fold over the first 4 h, depending on the gene and magnitude of OS. Increased transcription at higher levels of OS was also seen for up to 8-h for some of these genes. Protein translation was examined for 24-h following OS using Western blotting methods, and compared to the qPCR responses. We identified six AP-1 family genes that demonstrate quantitative upregulation of expression in response to OS. Two distinct types of quantifiable OS-specific responses were observed; dose-dependent responses, and threshold responses. Our studies show that different levels of OS can regulate the expression of AP-1 transcription factors quantitatively in the human RPE in vitro.