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N. Miyamoto, H. Izumi, R. Miyamoto, K. Kohno, A. Tawara; Transcriptional Regulation of Activating Transcription Factor 4 Under Oxidative Stress in Retinal ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4107.
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Oxidative stress plays an important role in pathogenesis of various ocular disease such as retinopathy, glaucoma and age-related macular degeneration. The eye is protected against oxidative stress by several mechanisms. We have previously shown that transcription factor Foxo3a functions to protect trabecular meshwork cells from oxidative stress. We have also reported that Activating transcription factor 4 (ATF4) is upregulated in drug-resistant cells and protects from oxidative stress through the gluthathione biosynthesis. Thus, in addition to the transcription factor Foxo3a, ATF4 gene is thought to be one of the critical transcriptional factor for cellular response against oxidative stress.ATF4 is induced by various stresses including endoplasmic reticulum (ER) stress and oxidative stress, and ATF4 expression is regulated translationally through the PERK pathway of eIF2α phosphorylation. Here, we investigated the transcriptional regulation of ATF4 gene under oxidative stress using SV-40 transformed retinal pigmentosal ARPE-19 cells
ARPE-19 cells were treated with ER stress and oxidative stress. The gene expression of transcription factors were investigated by western blot, reporter assays and siRNA strategy. Cellular sensitivity against oxidative stress was also determined with the WST8-assays.
The expression of two transcriptional factors, ATF4 and Nrf2, was significantly induced by hypoxia and thapsigargin (TG). Nrf2 regulator Keap1 was down-regulated by hypoxia. Down-regulation of Nrf2 abolished the ATF4 expression. On the other hand, down-regulation of Keap1 enhanced the expression both Nrf2 and ATF4. The promoter activity of ATF4 was transactivated by the co-transfection of Nrf2 expression plasmids and reduced by the transfection of Nrf2-specific siRNA.Nrf2 down-regulation almost abolished the ATF4 induction by hypoxia and TG. Consistent with these findings, the promoter activity of ATF4 was augmented by the treatment with TG, HCA, H2O2, and hypoxia. However, stress induction of ATF4 promoter activity was observed ever when the mutation was introduced into ARE site. Further, stress induction of ATF4 promoter was completely abolished when 5`UTR of ATF4 gene was deleted. Down-regulation of ATF4 render ARPE-19 cells sensitive to oxidative stress.
These results suggest that the stress induction of ATF4 is significantly regulated transcriptinally through Nrf2-dependent manner.
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