Quinone oxidoreductases (NQO1 and NQO2) catalyze the detoxification of quinines, thus protecting cells from oxidative stress and neoplasia. The NQO1 gene is coordinately induced with other detoxifying enzyme genes in response to xenobiotics (Beta-naphthoflavone (Beta-NF)) and antioxidants (tert-butyl hydroquinone 9t-BHQ)). Deletion mutagenesis of the NQO1 gene promoter identified and antioxidant response element (ARE) that is required for NQO1 gene expression and induction in response to Beta-NF and t-BHQ. The nuclear factors Nrf2 and Nrf1 heterodimerize with Jun proteins and bind to the ARE. This complex upregulates basal expression and coordinated induction of NQO1 and other genes. The Nrf-Jun dimerization requires unknown cytosolic factors. A cytosolic inhibitor of Nrf2 that retains it in the cytoplasm, Inrf2, was cloned and sequenced. Treatment of cells with t-BHQ caused the release of Nrf2 which moved into nucleus, inducing NQO1 gene expression. The mechanism of the release of Nrf2, from Inrf2, remains unknown. The role of the newly identified Nrf3 and large Maf proteins, in ARE-mediated regulation of NQO1 and other genes, also remains unknown. Analysis of NQO1 gene expression showed large variations among human tissues. The major goals of this proposal are to elucidate the mechanisms that regulate the ARE-mediated expression and coordinated induction of NQO1 and other detoxifying enzyme genes in response to xenobiotics and antioxidants. We will perform transfection, band/super shift and immunoprecipitation assays to investigate the role of Nrf3 and large Maf proteins in this process. PCR generated deletions of INrf2 will be used in nuclear translocation, transfection and immunoprecipitation assays to precisely map the domain of INrf that interacts with Nrf2. Similar assays will be used to determine xenobiotic and antioxidant induced phosphorylation/dephosphorylation and/or redox regulation of INrf2, Nrf2 and/or c-Jun leading to the release of Nrf2 form INrf2 and stimulation of heterodimerization/binding of Nrf2/c-Jun to the ARE. Mass spectrophotometry will be used to detect the phosphorylation/dephosphorylation sites of INrf2, Nrf2 and/or c-Jun. Deletion mapping and transfection studies are also planned to identify cis-elements and trans-acting factors that regulate expression and induction of the NQO2 gene. We will use Northern, Western, DNaseI hypersensitivity, footprinting, band/super shift assays and transgenic NQO1-/- mice that express the human NQO1 gene to study the mechanism of tissue specific expression and induction NQO1.
Showing the most recent 10 out of 19 publications
