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Xenobiotic sensors PXR and CAR and regulation of the UGT1 locus

异生素传感器 PXR 和 CAR 以及 UGT1 基因座的调节

基本信息

批准号：
8117524
负责人：
Robert H Tukey
金额：
$ 31.26万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8117524
关键词：
Adult Agonist Alleles Androstanes Animal Model Animals Bile Acids Bilirubin Binding Biochemical Breeding Cells Cessation of life Chemicals Clinical Complement Coupled Depressed mood Development Disease Drug Interactions Environmental Carcinogens Fatty Acids Gene Cluster Gene Expression Gene Targeting Generations Genes Genetic Glucuronic Acids Glucuronosyltransferase Health Hepatic Hepatocyte Hormonal Hormones Human Hyperbilirubinemia In Vitro Individual Inherited Knockout Mice LXRalpha protein Ligands Link Liver Mediating Mediator of activation protein Metabolic syndrome Modeling Molecular Mus Neonatal Nuclear Receptors Organism PPAR alpha Pattern Peroxisome Proliferator-Activated Receptors Pharmaceutical Preparations Play Pregnancy Process Proteins Receptor Activation Regulation Role Serum Steroids Stress Syndrome Technology Testing Tissues Toxic Environmental Substances Transcriptional Regulation Transgenic Mice UGT1A1 gene Work Xenobiotic Metabolism Xenobiotics constitutive androstane receptor dietary constituent drug metabolism human tissue in vivo positional cloning pregnane X receptor pregnant protein expression receptor receptor expression research study sensor steroid hormone tool

项目摘要

DESCRIPTION (provided by applicant): In humans, the UGT1 locus encodes 9 functional UGT1A genes. These genes are expressed in a strict tissue specific fashion, with hepatic UGT1A1 expressed at low levels in individuals that inherit the UGT1A1*28 Gilbert's allele. Experiments conducted in transgenic mice expressing the UGT1A1*28 allele as part of the UGT1 locus (Tg-UGT1*28) have demonstrated that expression of all 9 UGT1A proteins closely mimics the differential control that is observed in human tissues. Remarkably, hepatic UGT1A1 expression is depressed as a result of altered expression of the UGT1A1*28 gene in Tg-UGT1*28 mice. Using Tg-UGT1*28 as a model to study expression of the UGT1 locus, the treatment of mice with compounds that active the Ah receptor (AhR), the pregnenalone X-receptor (PXR), the constitutive androstane receptor (CAR), the peroxisome proliferator- activated receptor alpha (PPAR1) and the liver X-receptor (LXR) all profoundly induce various UGT1A genes. PXR and CAR play key roles in many aspects of drug metabolism since they have been described as xenobiotic sensors and are believed to be regulated by a host of steroid and other hormonal activators. We propose that PXR and CAR are regulated in part by the steady-state levels of steroids and hormones. Thus, to fully understand the role of these xenobiotic receptors towards human UGT1A gene expression and human glucuronidation, mouse genetics will be exploited to examine their contribution towards tissue specific and inducible expression of the UGT1 locus. Experiments will be described demonstrating that regulation of the UGT1 locus in Tg-UGT1*28 mice that are Pxr-null and Car-null participate in the constitutive and inducible expression of the UGT1A genes. Since PXR and CAR have been shown to act in concert with many of the same target genes, this application will focus its efforts on defining the role of PXR and CAR in controlling the inducible and tissue specific expression of the UGT1 locus. Experiments conducted in vitro in primary hepatocytes as well as in vivo in UGT1 mice that lack Pxr and/or Car will examine the molecular mechanisms associated with and further link the relationship between xenobiotic receptor expression and the inducible and humoral control of the UGT1 locus. These studies will be coupled with recent technologies employing a combination of reverse genetics and biochemical analysis that have resulted in the functional deletion of the entire Ugt1 locus in mice and the generation of humanized UGT1 (hUGT1*28) mice. Since hUGT1*28 mice are hyperbilirubinemic as a result of the diminished hepatic UGT1A1 gene expression, these mice will be exploited to study the role of PXR and CAR towrds controlling UGT1A1 gene expression and hyperbilirubinemia. To this end, the specific aims of this competitive renewal are 1. Determine the role of PXR and CAR towards regulation of the human UGT1A1 gene, 2. Determine the role of PXR and CAR towards regulation of the UGT1 locus in vitro, 3. Evaluate the role of human PXR and regulation of the human UGT1 locus. 4. Examine the hormonal implications of PXR and CAR directed activation of the UGT1 locus in maternal liver during pregnancy. PUBLIC HEALTH RELEVANCE: The ability to adequately eliminate steroids, bile acids, drugs and environmental toxicants from our cells and tissues by the enzymatic process that leads to the attachment of glucuronic acid to these agents is a crucial step in protecting the organism from a toxic or carcinogenic episode. How human UDP- glucuronosyltransferases (UGTs) are regulated in vivo (in the tissues) is very poorly understood, primarily because the sophisticated tools necessary to develop animal models carrying these human genes have not been developed. This application is the first attempt to investigate how human UGTs are regulated in mice, and to apply this animal to understand the impact of glucuronidation towards drug-drug interactions, drug metabolism and eventually disease.

描述（由申请人提供）：在人体中，UGT 1基因座编码9个功能性UGT 1A基因。这些基因以严格的组织特异性方式表达，在遗传UGT 1A 1 *28吉尔伯特等位基因的个体中，肝脏UGT 1A 1表达水平较低。在表达UGT 1基因座（Tg-UGT 1 *28）一部分的UGT 1A 1 *28等位基因的转基因小鼠中进行的实验证明，所有9种UGT 1A蛋白的表达与在人体组织中观察到的差异对照非常相似。值得注意的是，由于Tg-UGT 1 *28小鼠中UGT 1A 1 *28基因的表达改变，肝脏UGT 1A 1表达受到抑制。使用Tg-UGT 1 *28作为研究UGT 1基因座表达的模型，用激活Ah受体（AhR）、依那酮X-受体（PXR）、组成型雄烷受体（CAR）、过氧化物酶体增殖物激活受体α（PPAR 1）和肝X-受体（LXR）的化合物处理小鼠均显著诱导各种UGT 1A基因。PXR和CAR在药物代谢的许多方面发挥关键作用，因为它们被描述为异生物质传感器，并且被认为是由类固醇和其他激素激活剂调节的。我们认为PXR和CAR部分受类固醇和激素稳态水平的调节。因此，为了充分了解这些外源性受体对人UGT 1A基因表达和人葡萄糖醛酸化的作用，将利用小鼠遗传学来检查它们对UGT 1基因座的组织特异性和诱导性表达的贡献。将描述实验，证明Pxr缺失和Car缺失的Tg-UGT 1 *28小鼠中UGT 1基因座的调节参与UGT 1A基因的组成型和诱导型表达。由于PXR和CAR已被证明与许多相同的靶基因协同作用，因此本申请将集中精力定义PXR和CAR在控制UGT 1基因座的诱导型和组织特异性表达中的作用。在体外原代肝细胞中以及在缺乏Pxr和/或Car的UGT 1小鼠体内进行的实验将检查与异生素受体表达和UGT 1基因座的诱导型和体液控制之间的关系相关的分子机制，并进一步将其联系起来。这些研究将与最近的技术相结合，采用反向遗传学和生化分析的组合，导致小鼠中整个Ugt 1基因座的功能缺失和人源化UGT 1（hUGT 1 *28）小鼠的产生。由于hUGT 1 *28小鼠由于肝脏UGT 1A 1基因表达减少而导致高胆红素血症，因此将利用这些小鼠研究PXR和CAR towards控制UGT 1A 1基因表达和高胆红素血症的作用。为此，本次竞争性更新的具体目标是1。确定PXR和CAR对人UGT 1A 1基因调节的作用，2.确定PXR和CAR对体外UGT 1基因座调节的作用，3.评价人PXR的作用和人UGT 1基因座的调节。4.检查怀孕期间母体肝脏中PXR和CAR定向激活UGT 1基因座的激素影响。公共卫生关系：通过导致葡萄糖醛酸附着于这些物质的酶促过程，从我们的细胞和组织中充分消除类固醇、胆汁酸、药物和环境毒物的能力是保护生物体免受有毒或致癌事件的关键步骤。人UDP-葡萄糖醛酸基转移酶（UGT）如何在体内（在组织中）被调节的了解非常少，主要是因为还没有开发出开发携带这些人基因的动物模型所必需的复杂工具.该应用是首次尝试研究人类UGT如何在小鼠中调节，并应用该动物了解葡萄糖醛酸化对药物相互作用，药物代谢和最终疾病的影响。