SIGNALING OF PREGNANE X RECEPTOR

妊娠 X 受体的信号传导

• 批准号: 6387275
• 负责人: Bingfang Yan
• 金额: $ 23.04万
• 依托单位: UNIVERSITY OF RHODE ISLAND
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387275
• 关键词: antisense nucleic acid; biological signal transduction; cytochrome P450; drug metabolism; gene induction /repression; genetic polymorphism; laboratory rabbit; laboratory rat; pharmacology; pregnenolone; receptor expression; rifamycins; site directed mutagenesis; steroid hormone receptor; transfection

DESCRIPTION (Adapted from the applicant's abstract): Cytochrome P4503A (CYP3A) enzymes involve the metabolism of two thirds of drugs and other xenobiotics. Induction of CYP3A enzymes by many compounds is known as an important contributing factor to many failures of therapy or severe toxicity. CYP3A induction is featured by marked species difference, structural diversity of the inducers, and inter-individual variation. Analyses of CYP3A promoters locate three cis-response elements likely involved in CYP3A induction. A reporter gene construct containing one of the elements can be transactivated by an orphan receptor designated the pregnane X receptor (PXR), and the differential activation of mouse and human PXRs by several compounds largely reflects the species difference observed in vivo. The central hypothesis of the proposed studies is that PXR plays a determinant role in CYP3A induction and multiplicity/polymorphism, along with inducibility of PXR, are responsible for the species difference, inducer diversity and individual variation. The specific aims of this project are: (1) to determine the multiplicity/polymorphism of PXR in humans; (2) to determine the essentiality of PXR in the CYP3A induction; (3) to determine the synergistic effects of PXR inducers on PXR activator-mediated CYP3A induction; and (4) to determine important residues of PXRs in conferring CYP3A induction by rifampicin (RIF) and pregnenolone 16alpha-carbonitrile (PCN). As part of the studies to determine the molecular basis for the existence of multiple forms and polymorphic variants of PXRs in humans, a cDNA-trapping method will be used to screen cDNA libraries from hepatic and extrahepatic tissues. Transient cotransfection experiments with a CYP3A reporter will be conducted to determine the activation profile of each PXR. To determine the essentiality of PXR in CYP3A induction, PXR antisense constructs will be tested for their ability to block CYP3A induction; and PXR chimeras with a ligand binding domain from another species will be tested for their ability to modulate CYP3A expression in response to species-selective activators. To determine the synergistic effects of PXR inducers on PXR activator-mediated CYP3A induction, rats and hepatocytes will be treated with PXR inducers, PXR activators, or in combination; induction of CYP3A will be determined by Northern and Western blot analyses. Site-directed mutagenesis experiments will be conducted to determine functionally important residues of PXRs in conferring CYP3A induction by RIF and PCN. Significant progress has been made toward the proposed objective. Full-length cDNAs encoding multiple forms of rodent and human PXRs have been isolated. Several compounds are found to drastically increase rPXR-1 mRNA levels. These results support our hypothesis that multiplicity and polymorphism along with inducibility of PXR are responsible for species difference, inducer diversity and individual variation featured by CYP3A induction.

描述（改编自申请人的摘要）：细胞色素P4503A（CYP3A） 酶涉及三分之二的药物和其他异种生物的代谢。 许多化合物诱导CYP3A酶被称为重要 导致许多治疗或严重毒性失败的因素。 CYP3A 诱导由明显的物种差异，结构多样性的特征 诱导者和个体间差异。 CYP3A启动子的分析定位 CIP3A诱导可能涉及的三个顺式反应元件。记者基因 包含其中一个元素之一的构造可以通过孤儿进行反式激活 受体指定妊娠X受体（PXR），差异 几种化合物对小鼠和人PXR的激活主要反映了 体内观察到的物种差异。提议的中心假设 研究是，PXR在CYP3A诱导和 多样性/多态性以及PXR的诱导性，负责 物种差异，诱导剂多样性和个体变异。这 该项目的具体目的是：（1）确定 PXR在人类中的多样性/多态性； （2）确定重要性 CYP3A诱导中的PXR； （3）确定PXR的协同作用 PXR激活剂介导的CYP3A诱导诱导； （4）确定 PXR的重要残基在利福平（RIF）授予CYP3A诱导中 和妊娠16Alpha-碳硝基（PCN）。作为研究的一部分 确定存在多种形式的分子基础和 PXR的多态性变体在人类中，将使用一种cDNA捕获方法来 从肝和肝外组织中筛选cDNA文库。瞬态 将对CYP3A报告基因进行共转染实验，以确定 每个PXR的激活曲线。确定pxr的本质 CYP3A诱导，PXR反义构建体的能力将测试 块CYP3A诱导；和PXR嵌合体，具有配体结合域的 将测试另一个物种的调节CYP3A表达的能力 响应物种选择性激活剂。确定协同作用 PXR诱导剂对PXR激活剂介导的CYP3A诱导，大鼠和大鼠的影响 肝细胞将用PXR诱导剂，PXR激活剂或IN治疗 组合; CYP3A的诱导将由Northern和Western印迹确定 分析。将进行定向诱变实验以确定 PXR在功能上重要的残基在RIF授予CYP3A诱导中 和PCN。对拟议的目标取得了重大进展。 编码多种形式的啮齿动物和人PXR的全长cDNA已 孤立。发现几种化合物大大增加了RPXR-1 mRNA 水平。这些结果支持我们的假设，即多样性和多态性 除PXR的诱导性外，还导致物种差异，诱导剂 CYP3A诱导的多样性和个体变异。