Molecular responses transduced by serial oxidative stress in the retinal pigment epithelium: feedback control modeling of gene expression.

The purpose of this study was to characterize the early molecular responses to quantified levels of serial 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 serially exposed to high levels of OS (500 μM H(2)O(2)) with up to 3 distinct stimuli presented 4 h apart. Gene expression was followed for at least 8 h following initiation of each OS. Using real time qPCR, we quantified the expression of immediate early genes from the AP-1 and EGR transcription factor families and other 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. The peak level of induced transcription from OS varied from two-fold to >64-fold over the first 4 h, depending on the gene and magnitude of OS. Serial responses were characterized and distinct types of quantifiable OS-specific responses were observed. The responses manifested controlled serial increases in expression of these genes in a manner dependent upon the rate of increase in transcription, the relative duration of the transcriptional stimulus, and the characteristic relaxation to the initial steady state. This complexity suggests a mechanism whereby the rate of increase in transcription directs alternative paths to effect downstream gene-specific responses to OS. The molecular mechanisms by which these signals are transduced and controlled in the RPE are speculative. However, the rapidity of the TF response, and the known autoregulation of TF promoters by their gene products suggests that the early control is likely mediated by phosphorylation and activation of existing AP-1 and EGR protein pools within the cell, modulated by the opposing activity of kinase and phosphatase enzymes. Because of the differences in both initial and serial responses to the input stresses, it appears that control theory may provide a useful characterization of the distinctions.