Abstract
Purpose: :
Previous work in our lab has suggested that the retinal pigment epithelium demonstrates stereotypic changes in the expression of specific genes involved in the early cellular responses to oxidative stress. The purpose of this study was to analyze and characterize the molecular responses of a panel of OS–specific genes to quantified levels of oxidative stress.
Methods: :
Confluent ARPE–19 cells were cultured for three days in defined NR–1 medium to stabilize gene expression. The cells were exposed to varying levels of OS (0, 50, 100, 200, 350, or 500 µM H2O2). RNA was isolated from the cells using a no–rinse method after 1–hour or 4–hours and compared to no–OS controls. Total cellular RNA was isolated at each time point, DNase treated, and reverse transcribed. Gene–specific expression was quantified by real–time PCR on the ABI 7700 System.
Results: :
We quantified the expression of nine transcription factor genes, 5 chaperone protein genes and eight genes involved in regulating the redox status of the cells. Significant and quantitative changes were seen in the expression of five AP–1 transcription factor genes, FosB, c–Fos, FosL, JunB, and ATF3 within one hour of OS The peak level of induction at one hour of OS varied from 2–fold to 64–fold, depending on the gene. Sustained levels of transcription were also seen after 4 hours of OS. Transcription of the crystallin gene CRYBA and OS–response genes HO–1 and thioredoxin was also induced. The molecular responses seen were threshold, dose–dependent or mixed, depending on the gene. The other transcription factors and OS–response genes tested did not show any transcriptional response in the first 4 hours after stress.
Conclusions: :
We have identified eight genes in 3 different classes that demonstrate quantitative gene regulation in response to OS. These include AP–1 transcription factor genes, crystallin CRYBA and two OS–response genes. Three distinct types of quantifiable OS–specific responses are seen in the RPE. Our data shows that extracellular stressors such as OS can quantitatively regulate gene expression in the human RPE.
Keywords: gene/expression • retinal pigment epithelium • oxidation/oxidative or free radical damage