Quinone Oxidoreductases (NQO1 and NQ02) are cytosolic proteins that catalyze detoxification of quinones and protect against oxidative stress and neoplasia. Antioxidant response element (ARE) and nuclear factor Nrf2 regulate coordinated induction of detoxifying genes including NQO1 and NQO2 in response to antioxidants. Nrf2 is retained in the cytoplasm by inhibitor INrf2. PKC phosphorylation of Nrf2S40 results in the release of Nrf2 from INrf2. Nrf2 translocates to the nucleus, binds to ARE, and activate gene expression. Once this is done, Nrf2 is exported out, binds to INrf2 and degrade. The PKC isoform(s) that phosphorylates Nrf2S40 and the mechanism of Nrf2 export remains unknown. Antioxidant- induced nuclear accumulation of Bachl leads to repression of Nrf2-induced genes by unknown mechanism. Nrf2 expression varies significantly among tissues, raising questions regarding tissue specific Nrf2 protection against carcinogenesis. NQO1 gene expression varies among human tissues by unknown mechanism. We have generated humanized mice that express human NQO1 gene in NQO1-/- mice. The major goals of this proposal are to elucidate the mechanism of Nrf2 regulation of detoxifying genes in response to antioxidants, determine in vivo role of Nrf2 in carcinogenesis and investigate the mechanism of tissue specific expression of NQO1 gene.
Four aims are planned.
Aim 1 will determine the mechanism of signal transduction from antioxidants to Nrf2/ARE. The chemical inhibitors and Si RNA will be used in transfection, reporter assays, immunohistochemistry and immunoprecipitation to identify the PKC isoform(s) that phosphorylate Nrf2S40 for release of Nrf2. Similar strategies will be used to investigate the hypothesis that tyrosine kinase-mediated phosphorylation of Nrf2Y568 regulates nuclear export of Nrf2. The Nrf2 phosphorylation at sites other than S40/Y568 and its role in ARE-mediated gene expression will also be investigated.
Aim 2 will use kidney carcinoma cells stably expressing tetracycline- inducible Flag-Nrf2 to identify the heterodimeric partner of Nrf2 in upregulation of ARE-mediated gene expression. In addition, the experiments will investigate the hypothesis that antioxidant induced modification(s) of Bachl leads to delayed nuclear accumulation of Bachl and negative regulation of ARE- mediated gene expression.
Aim 3 will use Cre-Lox system to generate mice that demonstrate deletion of exon 2-4 of Nrf2 gene in liver and skin. The sensitivity of conditional Nrf2 knockout mice expressing the null genotype will be compared with that of mice expressing Nrf2, following carcinogen and ultraviolet exposure, to develop liver and skin tumors.
Aim 4 will use humanized mice that express human NQO1 in NQO1-/- mice in DNasel hypersensitivity and in vivo.and in vitro footprinting assays to identify the cis- element(s) and trans-acting factor(s) that regulate variations in NQO1 gene expression among the tissues. ? ? ? ? ? ? ?
Showing the most recent 10 out of 19 publications
