Abstract
The ubiquitous toxic metalloid arsenic elicits pleiotropic adverse and adaptive responses in mammalian species. The biological targets of arsenic are largely unknown at present. We analyzed the signaling pathway for induction of detoxification gene NAD(P)H-quinone oxidoreductase (Nqo1) by arsenic. Genetic and biochemical evidence revealed that induction required cap 'n' collar basic leucine zipper transcription factor Nrf2 and the antioxidant response element (ARE) of Nqo1. Arsenic stabilized Nrf2 protein, extending the t(1/2) of Nrf2 from 21 to 200 min by inhibiting the Keap1 x Cul3-dependent ubiquitination and proteasomal turnover of Nrf2. Arsenic markedly inhibited the ubiquitination of Nrf2 but did not disrupt the Nrf2 x Keap1 x Cul3 association in the cytoplasm. In the nucleus, arsenic, but not phenolic antioxidant tert-butylhydroquinone, dissociated Nrf2 from Keap1 and Cul3 followed by dimerization of Nrf2 with a Maf protein (Maf G/Maf K). Chromatin immunoprecipitation demonstrated that Nrf2 and Maf associated with the endogenous Nqo1 ARE enhancer constitutively. Arsenic substantially increased the ARE occupancy by Nrf2 and Maf. In addition, Keap1 was shown to be ubiquitinated in the cytoplasm and deubiquitinated in the nucleus in the presence of arsenic without changing the protein level, implicating nuclear-cytoplasmic recycling of Keap1. Our data reveal that arsenic activates the Nrf2/Keap1 signaling pathway through a distinct mechanism from that by antioxidants and suggest an "on-switch" model of Nqo1 transcription in which the binding of Nrf2 x Maf to ARE controls both the basal and inducible expression of Nqo1.
Highlights
The ubiquitous toxic metalloid arsenic elicits pleiotropic adverse and adaptive responses in mammalian species
Chromatin immunoprecipitation demonstrated that Nrf2 and Maf associated with the endogenous Nqo1 antioxidant response element (ARE) enhancer constitutively
The results indicated that Nrf2 but not Keap1, Cullin 3 (Cul3), or ubiquitin was recruited to ARE in the presence of arsenic or tBHQ
Summary
Cell Culture and Treatment—Mouse hepa1c1c7 cells were provided by Dr J. Cell lysate was Nrf prepared, and luciferase activity was measured using the dual luciferase assay dependence of Nqo induction. HCl, pH 7.4, 10 mg of bovine serum albumin, 0.1 ml of 1.5% Tween 20, 10 l of 7.5 mM FADϩ (Sigma), 0.1 ml of 150 mM RESULTS glucose 6-phosphate (Sigma), 9 l of 50 mM NADPϩ (sigma), 30 Arsenic Potently Activates the Nrf2/ARE Pathway by Stabilizunits of yeast glucose-6-phosphate dehydrogenase (Sigma), 4.4 ing Nrf2—Examining induction of a panel of cellular defense mg of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium genes by arsenic revealed that arsenic robustly induced the mRNA expression of xenobiotic-metabolizing enzyme NQO1 in hepa1c1c7, a mouse hepatoma cell line with minimal deviation from hepatocytes and highly responsive to arsenic (Fig. 1A).
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