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

• 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
• 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

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）：在人类中，UGT1基因座编码9个功能性UGT1A基因。这些基因以严格的组织特异性表达，肝UGT1A1在继承UGT1A1*28 Gilbert等位基因的个体中以低水平表示。在表达UGT1A1*28等位基因的转基因小鼠中进行的实验作为UGT1基因座的一部分（TG-ugt1*28）表明，所有9种UGT1A蛋白的表达都紧密模仿了人类组织中观察到的差异对照。值得注意的是，由于Tg-ugt1*28小鼠的UGT1A1*28基因的表达改变，肝UGT1A1的表达降低。使用TG-ugt1*28作为研究UGT1基因座表达的模型，用活跃AH受体（AHR）的化合物处理小鼠，妊娠烯酮X受体（PXR），本构型雄激素受体（CAR），过氧化物组扩散的受体Alpha（PPAR1）和lxR X-RECRILY X-RECR（LXR） UGT1A基因。 PXR和CAR在药物代谢的许多方面都起着关键作用，因为它们被描述为异生物传感器，并且被认为受到许多类固醇和其他激素活化剂的调节。我们建议PXR和CAR部分由类固醇和激素的稳态水平调节。因此，为了充分了解这些异种生物受体对人UGT1A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UGT1基因座组织特异性和诱导表达的贡献。将描述实验表明，在Tg-ugt1*28小鼠中对UGT1基因座的调节为PXR-NULL和CAR-NULL参与UGT1A基因的组成型和诱导表达。由于已显示PXR和CAR与许多相同的靶基因一起起作用，因此该应用将重点放在定义PXR和CAR在控制UGT1基因座的诱导型和组织特定表达方面的作用上。缺乏PXR和/或CAR的UGT1小鼠的原发性肝细胞和体内进行体外进行的实验将检查与异生物生物受体表达与UGT1轨迹的诱导型和体液控制之间的关系。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，从而导致小鼠中整个UGT1基因座的功能缺失以及人源化的UGT1（HUGT1*28）小鼠的产生。由于Hugt1*28小鼠是由于肝UGT1A1基因表达降低而导致的高胆红素，因此这些小鼠将被利用以研究控制UGT1A1基因表达和高胆红素血症的PXR和CAR TOWRDS的作用。为此，这种竞争性更新的具体目的是1。确定PXR和CAR在调节人UGT1A1基因的调节中的作用，2。确定PXR和CAR在体外调节UGT1基因座的作用，3。评估人类PXR的作用以及人类UGT1 locus的作用。 4。检查怀孕期间孕产妇肝脏中PXR和CAR的激素指示的激素。公共卫生相关性：通过酶促过程从我们的细胞和组织中充分消除类固醇，胆汁酸，药物和环境有毒物质的能力，从而导致葡萄糖酸与这些药物的附着是保护生物体免受有毒或癌发作的保护是至关重要的一步。人们如何在体内（在组织中）调节人UDP-葡萄糖醛酸糖基转移酶（UGT），这主要是因为尚未开发出携带这些人类基因所需的复杂工具。该应用是研究小鼠中人类UGT的首次尝试，并应用该动物以了解葡萄糖醛酸化对药物 - 药物相互作用，药物代谢和最终疾病的影响。