Nuclear transcription factor Nrf2 regulates ARE-mediated expression and coordinated induction of a battery of antioxidant genes. The products of these genes are known to play critical roles in cellular protection against oxidative stress and neoplasia. Recently, we cloned and sequenced a cytosolic inhibitor of Nrf2 designated as INrf2. INrf2 retains Nrf2 in the cytosol. Antioxidants antagonize INrf2 retention of Nrf2. Nrf2 releases from INrf2. Nrf2 translocates in the nucleus, binds to ARE and activate ARE-mediated expression of antioxidant genes. Preliminary studies have shown that INrf2 is phosphorylated at S104 and that mutant INrf2S104A failed to significantly repress ARE-mediated gene expression as observed with wild type INrf2. The studies have also shown that INrf2 gene is ubiquitously expressed, down regulated in hepatoma cells and induced in response to antioxidants. The goals of this proposal are: 1) elucidate the role of phosphorylation/dephosphorylation and/or redox modification of INrf2 in homodimerization of INrf2, Nrf2 degradation in basic conditions and antioxidant-induced release of Nrf2;2) generate conditional knockout mice that do not express INrf2 in liver and epidermis of skin and investigate in vivo roles of INrf2 in Nrf2 signaling and susceptibility to carcinogens;and 3) characterize the cis-elements and trans-acting factors that regulate basal expression, down regulation of INrf2 in hepatoma cells and induction in response to antioxidants. To this end, we will use transfection, orthophosphate labeling, immunoprecipitation and Western analysis along with inhibitors of phosphorylation and phospho-specific antibodies and redox modulators. This will determine if INrf2 is phosphorylated/dephosphorylated and/or redox modulated in basic conditions and in response to antioxidants that leads to homodimerization and/or Nrf2 degradation and/or release of Nrf2 from INrf2. Mass spectrophotometry will be used to precisely map the modified sites in INrf2. We will use Cre-Lox system to delete exon 2-4 from endogenous INrf2 gene in mouse models. This will generate conditional knockout mice that do not express the INrf2 gene in liver and epidermis of skin. These will be analyzed for the role of INrf2 in Nrf2 signaling and their susceptibility to benzo(a)pyrene induced skin and liver tumors as compared to wild type mice. PCR will be used to construct INrf2 deletions, internal deletions and mutations, including Oct-1, c/EBP and ARE elements. The transfection and band/super shift assays will be used to identify the cis-elements and trans-acting factors that control the down regulation of INrf2 gene in hepatic tumor cells and antioxidant induction.
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