Abstract
Antioxidants cause stabilization and nuclear translocation of NF-E2-related factor 2 (Nrf2), where it binds to the antioxidant response element (ARE) and induces up-regulation of defensive genes that protect cells against oxidative and electrophilic stress. Bach1, the negative regulator of Nrf2, competes with Nrf2 for binding to the ARE in the human NQO1 promoter. In this study, we demonstrate that Bach1 exits the nucleus within 1-2 h upon antioxidant treatment. Genistein, an inhibitor of tyrosine kinases, blocked nuclear export of Bach1. Site-directed mutagenesis and immunoprecipitation assays identified tyrosine 486 that was phosphorylated in response to the antioxidant and was essential for nuclear export of Bach1. Chromatin immunoprecipitation assays revealed a competitive interplay between Bach1 and Nrf2 at 1-2 and 4 h for binding to the human NQO1 ARE. Luciferase and real time PCR assays showed a significant decrease in antioxidant induction of reporter activity and mRNA levels in cells transfected with mutant Bach1 compared with wild type. This decrease was due to the absence of nuclear export of the mutant protein. Bach1 levels inside the nucleus returned to normal at 4 h after antioxidant treatment in the absence but not in the presence of protein synthesis inhibitor cycloheximide. In addition, antioxidant treatment increased the transcription of Bach1 as shown by pulse chase and real time PCR experiments. Taken together, these results indicate that increased synthesis of Bach1 restored its nuclear levels to normal at 4 h. In conclusion, antioxidant-induced tyrosine 486 phosphorylation leads to nuclear exit of Bach1, thus allowing Nrf2 access to the ARE.
Highlights
Grant R01 GM047466. 1 To whom correspondence should be addressed: Dept. of Pharmacology and repress defensive gene expression [1, 2, 5]
The cellular stress causes NF-E2-related factor 2 (Nrf2) to be released from its cytosolic inhibitor, INrf2, and translocation into the nucleus leading to the activation of antioxidant response element (ARE)-mediated gene expression [9, 10]
Nrf2 cannot bind to the ARE as a monomer, but requires dimerization with one of the small Maf or Jun proteins to bring about transactivation [11, 12]
Summary
Cell Cultures—Human hepatoblastoma (HepG2) and mouse hepatoma (Hepa-1) cells were obtained from the American Type Culture Collection. The cDNA was subcloned into pcDNA 3.1/V5-His Topo vector by TA cloning (Invitrogen) This plasmid encodes the V5-tagged Bach wild type (WT) protein designated Bach1V5. Subcellular Fractionation and Western Blotting—Hepa-1 and HepG2 cells, seeded in 100-mm plates and treated/transfected as displayed in the figures, were washed twice with icecold phosphate-buffered saline, trypsinized, and centrifuged at 1500 rpm for 5 min. Immunoprecipitation—For immunoprecipitation, 1 mg of whole cell lysates was equilibrated in RIPA buffer, precleared by protein AG-agarose (Santa Cruz Biotechnology), and extracts were incubated with respective antibodies (1 g) at 4 °C overnight. After 24 h of transfection, the cells were washed with 1ϫ phosphate-buffered saline and lysed in 1ϫ passive lysis buffer from the Dual Luciferase reporter assay system kit (Promega, Madison, WI). Immunoprecipitates were boiled in 1ϫ SDS buffer and resolved on 10% SDS gel
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