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H. Yang, X. Yang, J.C. Lang, E. Chaum; Antioxidant Modulation of Quantitative Gene Regulation in the Human Retinal Pigment Epithelium Under Oxidative Stress . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2965.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies of oxidative stress in the retinal pigment epithelium have demonstrated quantitative changes in the expression of various genes including transcription factors, chaperone proteins, and antioxidant genes. The purpose of this study was to determine whether the molecular responses to quantified levels of OS in the human RPE are modulated by antioxidant vitamin treatment.
Confluent ARPE–19 cells were cultured for three days in defined NR–1 medium in the presence of varying concentrations of vitamin C (0.01 mM to 0.2 mM) to stabilize gene expression. The vitamin C was removed 24 hours prior to treatment with 500 µM H2O2. RNA was isolated from the cells using a no–rinse method after 1–hour or 4–hours of OS and compared to no–OS controls. Gene–specific expression was quantified by real–time PCR on the ABI 7700 System.
We quantified the expression of four AP–1 transcription factors, the crystallin CRYBA heme oxygenase–1, and ß–actin. Quantitative changes in the level of transcription of FosB, c–Fos, JunB, ATF3, and CRYBA were seen within one hour of OS. The peak level of induction one hour after OS varied from 5–fold to 128–fold, depending on the gene. There was a statistically significant and dose–dependent reduction in the activation of AP–1 genes and CRYBA gene transcription (2– to 8–fold) after pretreatment with vitamin C. Heme oxygenase–1 transcription after OS did not demonstrate any response to pretreatment with vitamin C.
Vitamin C pretreatment modulates the expression of AP–1 transcription factors and crystallin CRYBA in RPE cells under oxidative stress. Modulation of the transcriptional response appears to be dose–dependent and quantitative in nature.
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