Xenobiotic sensors PXR and CAR and regulation of the UGT1 locus

描述（由申请人提供）：在人类中，UGT1位点编码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表达水平较低。在UGT1基因座（Tg-UGT1*28）中表达UGT1A1*28等位基因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控制非常相似。值得注意的是，在Tg-UGT1*28小鼠中，由于UGT1A1*28基因表达的改变，肝脏UGT1A1表达被抑制。以Tg-UGT1*28为模型研究UGT1位点的表达，激活Ah受体（AhR）、孕烯素x受体（PXR）、组成型雄甾烷受体（CAR）、过氧化物酶体增殖体激活受体α (PPAR1)和肝脏x受体（LXR）的化合物处理小鼠均能深刻诱导各种UGT1A基因。PXR和CAR在药物代谢的许多方面起着关键作用，因为它们被描述为外源传感器，并被认为是由许多类固醇和其他激素激活剂调节的。我们认为PXR和CAR在一定程度上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受体对人类UGT1A基因表达和人类糖醛酸化的作用，我们将利用小鼠遗传学来研究它们对UGT1基因座组织特异性和诱导表达的贡献。实验将证明，在Pxr-null和Car-null的Tg-UGT1*28小鼠中，UGT1位点的调控参与了UGT1A基因的组成性和诱导性表达。由于PXR和CAR已被证明与许多相同的靶基因协同作用，本应用将集中在确定PXR和CAR在控制UGT1基因座的诱导和组织特异性表达中的作用。在体外原代肝细胞和体内缺乏Pxr和/或Car的UGT1小鼠中进行的实验将研究与外源受体表达与UGT1位点诱导和体液控制之间的关系相关的分子机制。这些研究将结合最近的技术，采用反向遗传学和生化分析的组合，导致小鼠Ugt1位点的整个功能缺失，并产生人源化Ugt1 （hUGT1*28）小鼠。由于hUGT1*28小鼠由于肝脏UGT1A1基因表达减少而出现高胆红素血症，这些小鼠将被用于研究PXR和CAR在控制UGT1A1基因表达和高胆红素血症中的作用。为此，这一竞争性更新的具体目标是：1。确定PXR和CAR在人类UGT1A1基因调控中的作用，1。2 .确定PXR和CAR在体外调控UGT1位点中的作用。评估人类PXR的作用和对人类UGT1位点的调控。4. 研究PXR和CAR在妊娠期间对母体肝脏UGT1基因座激活的激素影响。公共卫生相关性：通过葡萄糖醛酸与细胞和组织的酶促过程，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一步。人类UDP-葡萄糖醛酸转移酶（UGTs）是如何在体内（组织中）调节的，人们知之甚少，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。该应用程序是首次尝试研究人类ugt如何在小鼠中调节，并应用这种动物来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。

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.