Nrf2/ARE-mediated coordinated activation of quinone oxidoreductases (NQO1 and NQO2), and other detoxifying enzyme gene expression, is one mechanism of critical importance in prevention of chemicals and radiation induced oxidative/electrophilic stress and cancer. Under normal conditions, INrf2 (Keap1)/Cul3-Rbx1 complex is constantly degrading Nrf2. In response to stress, Nrf2 is switched 'ON'and then 'OFF'by early and delayed mechanisms. Modification of INrf2Cys151 and PKC phosphorylation of Nrf2Ser40 results in the release of Nrf2 from INrf2. Nrf2 translocates to the nucleus, binds ARE and leads to activation of gene expression. Recent studies demonstrate that negative regulators of Nrf2, including Bach1 and Fyn, are exported out of nucleus during pre-induction response to antioxidant through an unknown mechanism to allow unimpeded Nrf2 activation. This is followed by activation of a delayed mechanism to switch 'OFF'Nrf2 activation. Activated GSK3 phosphorylates Fyn that translocates to nucleus. Fyn phosphorylates Nrf2Tyr568 resulting in nuclear export/degradation of Nrf2. However, the mechanism of antioxidant regulation of GSK3 remains unknown. The switching 'ON'and 'OFF'of Nrf2 presumably prevents apoptosis and promotes cell survival. Large variations in Nrf2 expression among tissues warrant studies on tissue specific response to chemical/UVB carcinogenesis. Furthermore, mechanism of Nrf2 control of apoptosis/cell survival is unknown. The goals of this proposal are to elucidate the oxidative/electrophilic signaling and mechanism of Nrf2 regulation of coordinated induction of the NQO1, NQO2 and other detoxifying gene expression in response to antioxidants and its significance in apoptotic cell death and cell survival and carcinogenesis. We plan four aims:
Aim 1 will elucidate the mechanisms that regulate antioxidant-induced nuclear export/degradation followed by new synthesis/nuclear import of negative factors Bach1 and Src subfamily members Fyn, Src, Yes, and Fgr, and determine its physiological significance in Nrf2/ARE-mediated gene expression apoptosis and cell survival.
Aim 2 will investigate the hypothesis that antioxidants modulate PI3K/AKT/PP2A to control GSK3 which in turn controls Src subfamily members, leading to phosphorylation of Nrf2Tyr568, nuclear export and degradation of Nrf2, and control of ARE-gene expression. In addition, determine the physiological significance of PI3K/Akt/GSK3 /Fyn regulated nuclear export/degradation of Nrf2 in apoptosis/cell survival.
Aim 3 will determine in vivo role of Nrf2 in benzo(a)pyrene (BP)-induced hepatic and UVB-induced skin carcinogenesis. We will also test the hypothesis that lower doses of BP and shorter exposure to UVB leads to Nrf2 activation and protection while higher doses BP and longer UVB exposure leads to Nrf2 degradation and carcinogenesis.
Aim 4 will investigate the molecular mechanism of the role of antioxidant activation of Nrf2 in prevention of apoptosis and promotion of cell survival. When completed, studies will provide significant novel information on Nrf2/associated factors as possible targets for chemoprotective drug development.
Nrf2 study is of considerable importance in prevention of xenobiotic, nutrient and radiation-induced oxidative/electrophilic stress. Nrf2 serves as a sensor of oxidative/electrophilic stress and initiates a signal cascade that leads to the coordinated activation of a battery of antioxidant/defensive genes including quinone oxidoreductase genes. The products of these genes play critical roles in cellular protection against chemical induced cytotoxicity and neoplasia. In the competing continuation proposal, we plan to investigate the mechanism of xenobiotic and nutrient modification of positive and negative factors that regulate Nrf2 activation and repression, investigate in vivo roles of Nrf2 in coordinated activation of antioxidant genes and susceptibility to benzo(a)pyrene and UVB-induced carcinogenesis, and test the hypothesis that Nrf2 activation of anti- apoptotic factors prevents apoptosis and promotes cell survival. These studies upon completion will provide a better understanding of the mechanisms of the cellular protection against chemical/radiation stress and chemoprevention. The studies may also provide the basis for further analysis of identified targets (cis- and trans-elements) that could ultimately allow us to increase the chemoprotective capacity of cells.
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