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(摘自申请者摘要)：细胞色素P4503A(CyP3A) 酶涉及三分之二的药物和其他外来物质的新陈代谢。 许多化合物对CYP3A酶的诱导是已知的一种重要的 导致许多治疗失败或严重毒性的因素。细胞色素P3A 诱导的特点是物种差异显著，结构多样性 诱因和个体间变异。细胞色素P3A基因启动子定位分析 三个顺式反应元件可能参与了细胞色素P3A的诱导。一种报道基因 包含其中一个元素的构造可以由孤儿事务激活 受体命名为孕烷X受体(PXR)，并区分 几种化合物对小鼠和人PXR的激活在很大程度上反映了 在活体内观察到物种差异。建议中的中心假设 研究表明，PXR在细胞色素P3A的诱导和释放中起决定性作用 多发性/多态，以及PXR的诱导性，是导致 物种差异、诱导子多样性和个体变异。这个 本项目的具体目标是：(1)确定 PXR在人类中的多样性/多态性；(2)确定其重要性 PXR在细胞色素P3A诱导中的作用；(3)确定PXR的协同作用 诱导剂对PXR激活剂介导的细胞色素P3A的诱导；以及(4)确定 PXRs在利福平诱导细胞色素P3A中的重要残基 孕烯醇酮16α-碳腈(PCN)。作为研究的一部分， 确定多种形式存在的分子基础和 在人类中PXRs的多态变体，一种cDNA捕获方法将被用于 从肝组织和肝外组织中筛选c DNA文库。瞬变 将进行与细胞色素P3A报告基因共转染实验，以确定 每个PXR的激活配置文件。要确定PXR在中国的重要性 将测试CYP3A诱导、PXR反义构建物的能力 阻断CYP3A诱导；以及具有配体结合域的PXR嵌合体 另一个物种将接受测试，以确定它们是否有能力调节细胞色素P3A的表达 对物种选择性激活剂的反应。要确定协同效应 PXR诱导剂对PXR激活剂介导的细胞色素P3A的诱导作用 肝细胞将用PXR诱导剂、PXR激活剂或 结合；将通过Northern和Western印迹来确定对CYP3A的诱导 分析。将进行定点突变实验，以确定 PXRs在RIF诱导CyP3A中的重要功能残基 和PCN。在实现拟议目标方面取得了重大进展。 编码多种形式的啮齿动物和人类PXR的全长cDNA已经被 与世隔绝。几种化合物被发现显著增加rPXR-1的mRNA量 级别。这些结果支持我们的假设，即多样性和多态 与PXR的诱导性有关的是物种差异，诱导剂 细胞色素P3A诱导具有多样性和个体变异的特点。