SIGNALING OF PREGNANE X RECEPTOR

性状（改编自申请人摘要）：细胞色素P4503A（CYP3A） 酶参与三分之二的药物和其他异生物质的代谢。 已知许多化合物对CYP3A酶的诱导是重要的 许多治疗失败或严重毒性的促成因素。CYP3A 诱导具有明显的种间差异性、结构多样性、 诱导物和个体间变异。CYP3A启动子定位分析 三个顺式反应元件可能参与CYP3A诱导。报告基因 包含其中一个元素的构造可以被孤立体反式激活 受体命名为PXR，和差异 几种化合物对小鼠和人PXR的激活在很大程度上反映了 在体内观察到种属差异。提出的中心假设 研究表明PXR在CYP3A诱导中起决定性作用， 多重性/多态性，沿着PXR的诱导，负责 种间差异、诱导物多样性和个体变异。的 该项目的具体目标是：（1）确定 人类PXR的多样性/多态性;（2）确定PXR的重要性 PXR在CYP3A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CYP3A诱导的影响;以及（4）确定 PXR在利福平（RIF）诱导CYP3A中的重要残基 和双烯醇酮16 α-腈（PCN）。作为研究的一部分， 确定多种形式存在的分子基础， 为了研究人类PXR的多态性变体，将使用cDNA捕获方法来 从肝组织和肝外组织中筛选cDNA文库。瞬态 将进行与CYP3A报告基因的共转染实验，以确定 每个PXR的激活曲线。为了确定PXR的必要性， 将检测CYP3A诱导、PXR反义构建体的能力， 阻断CYP3A诱导;和具有来自 将检测另一种属调节CYP3A表达的能力 对物种选择性激活剂的反应。为了确定协同作用， PXR诱导剂对PXR激活剂介导的CYP3A诱导的影响，大鼠和 肝细胞将用PXR诱导剂、PXR激活剂或 联合;将通过北方和西方印迹法测定CYP3A的诱导 分析。将进行定点诱变实验以确定 PXR在RIF诱导CYP3A中的重要功能残基 的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已经被克隆。 与世隔绝发现几种化合物可显著增加rPXR-1 mRNA 程度.这些结果支持了我们的假设，即多样性和多态性 沿着PXR的诱导作用是造成种间差异的主要原因， CYP3A诱导的多样性和个体差异。

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.