Multidrug resistance-associated proteins (Mrps) play a key role in hepatic detoxication by transporting Phase-ll conjugates and other organic compounds out of hepatocytes. Similarly, upregulation of Mrps in tumor cells confers resistance to chemotherapeutic drugs by transporting these cytotoxic compounds out of cells. The regulatory mechanisms governing Mrp expression in normal and diseased liver, and in tumor cells, are not understood. Thus, the overall goal of this application is to study the molecular mechanisms of transcriptional regulation of Mrps. Nuclear factor E2 related-factor 2 (Nrf2) is emerging as a critical transcription factor in regulation of both constitutive and inducible expression of Phase-ll enzymes. Because Mrps play a key role in the efflux of Phase-ll conjugates, we hypothesize that Mrps are coordinately regulated with Phase-ll enzymes by Nrf2. We recently examined Mrp expression in three different models: 1) mice treated with monofunctional inducers that selectively upregulate Phase-ll enzymes, 2) bile-duct ligation, a surgical model of extrahepatic cholestasis, and 3) mice with targeted disruption of glutathione synthesis. In each model, induction of Mrps and classical Nrf2 target genes was observed. Moreover, using in-silico analysis, we identified putative Nrf2-responsive sequences, known as electrophile response elements (EpREs), in the 5'flanking regions of the Mrp2, 3, and 4 genes. We hypothesize that activation and subsequent binding of Nrf2 to these EpREs results in increased expression of Mrp2, 3, and 4. Thus we propose Mrps belong to the battery of Nrf2-regulated detoxication genes. To test this hypothesis, we will determine: 1) the role of Nrf2 in Mrp induction in mice, taking advantage of Nrf2-null mice, 2) Nrf2 activation and subsequent translocation to the nucleus, 3) critical response elements by in vitro and in vivo reporter gene assay in combination with promoter deletion analysis, 4) Nrf2 binding to EpREs identified in Mrp 5'flanking regions, and 5) specificity of Nrf2 binding to Mrp promoter regions. Data from the experiments in this proposal will provide novel insight into the transcriptional regulation of Mrps. Elucidation of the role of Nrf2 in the regulation of the efflux transport process will have significant ramifications in toxicology, xenobiotics disposition, drug-drug interaction, and cancer chemoprevention.

R01ES009716

2000-08-01

2011-07-31