Xenobiotic sensors PXR and CAR and regulation of the UGT1 locus

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方式表达，在继承 UGT1A1*28 吉尔伯特等位基因的个体中，肝脏 UGT1A1 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-UGT1*28 小鼠中 UGT1A1*28 基因表达的改变，肝脏 UGT1A1 表达受到抑制。使用 Tg-UGT1*28 作为研究 UGT1 基因座表达的模型，用激活 Ah 受体 (AhR)、孕烯酮 X 受体 (PXR)、组成型雄甾烷受体 (CAR)、过氧化物酶体增殖物激活受体 α (PPAR1) 和肝脏 X 受体 (LXR) 的化合物治疗小鼠，均可显着诱导各种 UGT1A 基因。 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源传感器，并被认为受到许多类固醇和其他激素激活剂的调节。我们认为 PXR 和 CAR 部分受类固醇和激素的稳态水平调节。因此，为了充分了解这些外源受体对人类 UGT1A 基因表达和人类葡萄糖醛酸化的作用，将利用小鼠遗传学来检查它们对 UGT1 基因座的组织特异性和诱导性表达的贡献。将描述实验，证明 Pxr 无效和 Car 无效的 Tg-UGT1 * 28 小鼠中 UGT1 基因座的调节参与 UGT1A 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确定 PXR 和 CAR 在控制 UGT1 基因座的诱导型和组织特异性表达中的作用。在原代肝细胞中进行的体外实验以及在缺乏 Pxr 和/或 Car 的 UGT1 小鼠体内进行的实验将检查与外源性受体表达和 UGT1 基因座的诱导性和体液控制相关的分子机制，并进一步联系它们之间的关系。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，这些技术导致小鼠中整个 Ugt1 基因座的功能性缺失，并产生人源化 UGT1 (hUGT1*28) 小鼠。由于 hUGT1*28 小鼠因肝脏 UGT1A1 基因表达减少而出现高胆红素血症，因此将利用这些小鼠来研究 PXR 和 CAR 控制 UGT1A1 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UGT1A1基因调节的作用，2.确定PXR和CAR在体外对UGT1基因座调节的作用，3.评估人PXR和对人UGT1基因座调节的作用。 4. 检查怀孕期间 PXR 和 CAR 定向激活母体肝脏中 UGT1 位点的激素影响。公共健康相关性：通过酶促过程将葡萄糖醛酸附着到这些物质上，从而充分消除我们细胞和组织中的类固醇、胆汁酸、药物和环境毒物，这是保护生物体免受有毒或致癌事件影响的关键一步。人们对人 UDP-葡萄糖醛酸基转移酶 (UGT) 在体内（组织中）如何受到调节知之甚少，主要是因为尚未开发出开发携带这些人类基因的动物模型所需的复杂工具。该应用是首次尝试研究人类 UGT 如何在小鼠体内受到调节，并应用该动物来了解葡萄糖醛酸化对药物-药物相互作用、药物代谢以及最终疾病的影响。

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.