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E. Chaum, J. Yin, W. Huo, J. C. Lang; Quantitative Analysis of Gene Regulation in Response to Serial Oxidative Stress in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1829.
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Oxidative stress (OS) is implicated in the pathogenesis of many age-related diseases, including AMD. We have previously shown that OS to the retinal pigment epithelium (RPE) is characterized by stereotypic changes in the expression of specific AP-1 family genes. The purpose of this study was to evaluate the quantitative molecular responses to repetitive OS cycles in the human RPE.
Confluent ARPE-19 cells cultured in defined medium were treated to serial OS (350-500µM H2O2) over a period of 28 hours. RNA was isolated from parallel cultures at various time points following a one-hour OS using our no-rinse method. The cells were exposed to 3 cycles of OS over the period of analysis. Expression of the AP-1 family genes FosB, c-Fos, Fra-1, JunB, and ATF3, transcription factor (TF) genes EGR1 and EGR2, and OS-response genes SOD1, HMOX-1, TXNIP, CAT, TXN, and GSR were examined using qPCR methods.
Real-time qPCR confirmed transcriptional responses to 3 serial OS cycles with a delay time of four hours in the AP-1 family genes c-Fos (83-fold, 6.7-fold, and 1.4-fold) and ATF-3 (6.8-fold, 1.7-fold, and 1.4-fold). FosB did not demonstrate a significant secondary or tertiary response to OS (56-fold, 1.2-fold, and 1.4-fold). OS induced EGR2 transcription over all 3 cycles (16-fold, 2-fold, and 2-fold), whereas EGR1 appeared to have primarily an initial response (7.9-fold, 1.3-fold, and 1.9-fold). HMOX-1 showed a large single qualitative response to an initial OS, but none thereafter (150-fold, 0.7-fold, and 0.8 fold) Thioredoxin interacting protein was strongly induced for 2 OS cycles only (1000-fold, 24-fold, and 1.2-fold). Other OS-response genes did not respond to serial OS.
These studies show that the AP-1 family genes c-Fos and ATF-3, the TF gene EGR2, and OS-response gene TXNIP can be serially upregulated over a period of hours in response to strong OS. We hypothesize that these genes play a key role in protecting the RPE from strong OS and may regulate downstream networks of typical OS response genes.
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