Regulation of Hepatic Excretion of Xenobiotics by Mrps

描述（由申请人提供）：多药耐药相关蛋白（Mrps）在肝脏解毒中发挥关键作用，通过将ii相偶联物和其他有机化合物运输出肝细胞。同样，肿瘤细胞中Mrps的上调通过将这些细胞毒性化合物运输出细胞而赋予化疗药物耐药性。在正常、病变肝脏和肿瘤细胞中，Mrp表达的调控机制尚不清楚。因此，本应用的总体目标是研究Mrps转录调控的分子机制。核因子E2相关因子2 （Nrf2）正逐渐成为调控ii相酶组成型和诱导型表达的关键转录因子。由于Mrps在phase - ii偶联物的外排中起关键作用，我们假设Mrps与phase - ii酶通过Nrf2协调调节。我们最近在三种不同的模型中检测了Mrp的表达：1)用单功能诱导剂治疗的小鼠选择性上调ii相酶，2)胆管结扎，肝外胆汁淤积的手术模型，以及3)靶向破坏谷胱甘肽合成的小鼠。在每个模型中，观察到Mrps和经典Nrf2靶基因的诱导。此外，通过计算机分析，我们在Mrp2、3和4基因的5'侧区确定了假定的nrf2响应序列，称为亲电响应元件（EpREs）。我们假设Nrf2的激活和随后与这些EpREs的结合导致Mrp2、3和4的表达增加。因此，我们认为Mrps属于nrf2调节的解毒基因群。为了验证这一假设，我们将确定：1)Nrf2在小鼠Mrp诱导中的作用，利用Nrf2缺失的小鼠，2)Nrf2激活并随后易位到细胞核，3)通过体外和体内报告基因测定结合启动子缺失分析的关键反应元件，4)Nrf2结合Mrp 5'侧翼区域鉴定的EpREs， 5) Nrf2结合Mrp启动子区域的特异性。本提案中的实验数据将为Mrps的转录调控提供新的见解。阐明Nrf2在外排转运过程中的调节作用将在毒理学、外源药物处置、药物-药物相互作用和癌症化学预防方面产生重大影响。

DESCRIPTION (provided by applicant): 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.