Search

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 t1/2 of Nrf2 from 21 to 200 mm by inhibiting the Keap1·Cul3-dependent ubiquitination and proteasomal turnover of Nrf2. Arsenic markedly inhibited the ubiquitination of Nrf2 but did not disrupt the Nrf2·Keap1·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·Maf to ARE controls both the basal and inducible expression of Nqo1. [ABSTRACT FROM AUTHOR]

Chromium(VI) (Cr) is a major environmental toxic metal and human carcinogen. The molecular events mediating cellular responses to Cr(VI) are not clear at present. We show that Cr(VI) potently induced apoptosis and production of reactive oxygen species (ROS) in a concentration-dependent manner. Mouse embryonic fibroblast cells lacking Nrf2 exhibited elevated ROS production and apoptosis, which were markedly further increased by Cr(VI). Protection by Nrf2 correlated with induction of cytoprotective genes Ho-1 and Nqo1. Induction of the genes by Cr(VI) involved inhibition of ubiquitination of Nrf2 and accumulation of Nrf2 into the nucleus. In the nucleus, treatment with Cr(VI), but not phenolic antioxidant tBHQ, librates Nrf2 from Nrf2/Keap1 association and recruits Nrf2 to the antioxidant response elements (ARE) located in the enhancers of Ho-1 and Nqo1. Activation of Nrf2 by Cr(VI) was accompanied by the nuclear translocation and deubiquitination of Keap1 implicating recycling of Keap1 in Nrf2 signaling. Thus, protection against Cr(VI) toxicity involves a transcriptional signaling loop that includes activation of Nrf2 signaling by toxic metal, transcription of ARE-driven genes, and reduction of ROS production. [ABSTRACT FROM AUTHOR]

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.