Regulation of Hepatic Excretion of Xenobiotics by Mrps

Mrps 对异生物质肝脏排泄的调节

• 批准号: 7093310
• 负责人: CURTIS D KLAASSEN
• 金额: $ 34.91万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-10 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7093310
• 关键词: biliary tract; cholanate compound; cholestasis; confocal scanning microscopy; detoxification; disease /disorder model; excretion; gel mobility shift assay; gene deletion mutation; gene expression; gene induction /repression; genetic regulatory element; genetically modified animals; glutathione; immunocytochemistry; laboratory mouse; liver disorder; multidrug resistance; protein structure function; tissue /cell culture; transcription factor; western blottings

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.

描述（由申请人提供）：多药耐药相关蛋白（Mrps）通过将II相结合物和其他有机化合物转运出肝细胞，在肝脏解毒中发挥关键作用。类似地，肿瘤细胞中Mrps的上调通过将这些细胞毒性化合物转运出细胞而赋予对化疗药物的抗性。在正常和病变的肝脏，以及在肿瘤细胞中，Mrp表达的调控机制还不清楚。因此，本申请的总体目标是研究Mrps转录调控的分子机制。核因子E2相关因子2（Nrf 2）正在成为调节II期酶的组成型和诱导型表达的关键转录因子。因为Mrps在II期缀合物的流出中起关键作用，所以我们假设Mrps与II期酶通过Nrf 2协同调节。我们最近在三种不同的模型中检测了Mrp的表达：1）用选择性上调II期酶的单功能诱导剂治疗的小鼠，2）胆管结扎，肝外胆汁淤积的手术模型，和3）谷胱甘肽合成靶向中断的小鼠。在每个模型中，观察到Mrps和经典Nrf 2靶基因的诱导。此外，使用计算机模拟分析，我们确定了推定的Nrf 2响应序列，称为亲电响应元件（EpRE），在5'侧翼区的Mrp 2，3和4基因。我们假设Nrf 2的激活和随后与这些EpRE的结合导致Mrp 2、3和4的表达增加。因此，我们建议Mrps属于电池的Nrf 2调节解毒基因。为了验证这个假设，我们将确定：1）Nrf 2在小鼠中Mrp诱导中的作用，利用Nrf 2缺失小鼠，2）Nrf 2活化和随后易位至细胞核，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应答元件，4）Nrf 2与在Mrp 5'侧翼区鉴定的EpRE的结合，和5）Nrf 2与Mrp启动子区结合的特异性。从实验中的数据在这个建议将提供新的见解Mrps的转录调控。阐明Nrf 2在调节外排转运过程中的作用将在毒理学、外源性物质处置、药物-药物相互作用和癌症化学预防方面产生重大影响。