Unknown

The NRF2 is a transcription factor that protects against oxidative stress by binding to the antioxidant response element (ARE) in the promoter of its target genes, including NQO1 and HO-1. It was studied whether NRF2 activity is regulated by 4-Acetoxyphenol (4-AC) in human retina pigment epithelium cell lines (ARPE-19) by examining its effect on ARE activity using luciferase reporter assay. Pretreatment with 5 μM 4-AC for 24 hours induced a significant increase in luciferase activity by 2.7 ± 0.6-fold, indicating activation of NRF2 transcription factor. NRF2 mRNA level was not affected by 4-AC, measured by (q)RT-PCR, suggesting 4-AC functions at the NRF2 protein level. Normally, NRF2 is localized in the cytoplasm of ARPE-19 cells. Treatment with 200 μM tBHP for 30 minutes only occasionally induced the translocation of NRF2 to the nucleus. Pretreatment with 5 μM 4-AC for 24 hours resulted in robust translocation of NRF2 to the nucleus with or without induction of oxidative stress. There was also examined the NRF2 stability in response to 4-AC treatment. The half-life of the NRF2 in ARPE-19 cells in the presence of 4-AC was evaluated by Western blot. The amount of NRF2 was decreased to approximately 25% after treatment with cycloheximide for 30 minutes. However, pretreatment with 4-AC significantly extended NRF2 protein half-life, as approximately 96% of NRF2 protein was detected at 30 minutes and approximately 83% of NRF2 protein was detected at 45 minutes after cycloheximide treatment