Xenobiotic sensors PXR and CAR and regulation of the UGT1 locus

描述(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方式表达，在遗传UGT1A1*28吉尔伯特等位基因的个体中，肝脏UGT1A1的表达水平较低。在表达UGT1A1*28等位基因作为UGT1基因座(TG-UGT1*28)一部分的转基因小鼠中进行的实验表明，所有9种UGT1A蛋白的表达都接近于在人类组织中观察到的差异控制。值得注意的是，由于UGT1A1*28基因在TG-UGT1*28小鼠中表达的改变，肝脏UGT1A1的表达受到抑制。以Tg-UGT1*28为模型研究UGT1基因座的表达，小鼠用激活AH受体(AhR)、孕酮X受体(PXR)、构成雄烷受体(CAR)、过氧化物酶体增殖物激活受体α(PPAR1)和肝X受体(LXR)的化合物处理后，都能深刻地诱导各种UGT1A基因的表达。PXR和CAR在药物代谢的许多方面发挥着关键作用，因为它们被描述为外来生物传感器，并被认为受到一系列类固醇和其他激素激活剂的调节。我们认为PXR和CAR在一定程度上受到类固醇和激素稳态水平的调节。因此，为了充分了解这些异种受体对人类UGT1A基因表达和人类葡萄糖醛酸化的作用，我们将利用小鼠遗传学来检测它们对UGT1基因座组织特异性和诱导性表达的贡献。将描述的实验表明，在PXR缺失和CAR缺失的TG-UGT1*28小鼠中，UGT1基因的调节参与了UGT1A基因的结构性和诱导性表达。由于PXR和CAR已被证明与许多相同的靶基因协同作用，本申请将致力于确定PXR和CAR在控制UGT1基因的诱导性和组织特异性表达中的作用。在原代肝细胞以及在体内缺乏PXR和/或CAR的UGT1小鼠身上进行的实验将检验与异种受体表达相关的分子机制，并进一步联系UGT1基因座的诱导和体液控制之间的关系。这些研究将与采用反向遗传学和生化分析相结合的最新技术相结合，这些技术已导致小鼠完整的UGT1基因座功能缺失，并产生人源化的UGT1(hUGT1*28)小鼠。由于hUGT1*28小鼠由于肝脏UGT1A1基因表达降低而导致高胆红素血症，因此将利用这些小鼠来研究PXR和CARDS对UGT1A1基因表达和高胆红素血症的控制作用。为此，本次竞争性更新的具体目的是：1.确定PXR和CAR对人类UGT1A1基因的调控作用；2.确定PXR和CAR在体外对UGT1基因座的调控作用；3.评估PXR和CAR对人UGT1基因座的调控作用。4.研究孕期PXR和CAR诱导的母体肝脏UGT1基因的激活对激素的影响。与公共健康相关：通过酶促过程充分清除细胞和组织中的类固醇、胆汁酸、药物和环境毒物的能力，导致葡萄糖醛酸与这些物质结合，是保护生物体免受有毒或致癌事件影响的关键一步。人类UDP-葡萄糖醛酸基转移酶(UGT)在体内(在组织中)是如何调节的，人们对此知之甚少，主要是因为开发携带这些人类基因的动物模型所需的复杂工具尚未开发出来。这项应用是第一次尝试研究人类UGT在小鼠中是如何调节的，并应用这种动物来了解葡萄糖醛酸化作用对药物相互作用、药物代谢和最终疾病的影响。

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.