Signaling of the Pregnane X Receptor

描述(申请人提供)：严，冰芳摘要这是一项修订申请，以回应公告NOT-OD-09-058：NIH宣布恢复法案资金可用于竞争性修订申请该修订申请提出了一系列研究，以延长父母拨款的特定目标。父母赠款的重点是阐明与孕烷X受体(PXR)相关的信号事件，PXR是许多化学消除基因表达的主要调节者，例如人类的细胞色素P450 3A4(CYP3A4)和大鼠的CYP3A23。有趣的是，尽管在CYP3A4和CYP3A23近端启动子中都存在一个功能性的PXR元件，但CYP3A23而不是CYP3A4近端启动子对PXR配体有很强的反应。相反，CYP3A4近端的启动子与其他上游PXR元件协调并赋予反式激活活性。序列比较发现，与CYP3A23相比，CYP3A4启动子的PXR元件有30个碱基远离转录起始点。此外，CYP3A4而不是CYP3A23启动子在PXR元件前面有一个HNF3/CEBP位点。另一方面，PXR介导的反式激活可被白介素6(IL-6)等细胞因子显著减弱。IL-6高度诱导的microRNA-511(miR-511)可抑制PXR的反式激活。众所周知，MIR通过与其靶mRNAs的序列配对来沉默基因的表达。本研究旨在验证以下假设：PXR转录本是miR-511的序列特异性靶标，PXR元件的物理位置和HNF3/CEBP位点的存在显著导致CYP3A4近端启动子无法有效地响应PXR反式激活。为了确定物理位置或HNF3/CEBP位点的干扰的潜在作用，将准备突变体来扰乱HNF3/CEBP位点，或将PXR元件重新定位到更接近转录起始点的位置，或两者兼而有之。这些突变体将接受测试，以提高对PXR反式激活的反应能力。为了确定miR-511降低PXR表达的分子作用，将miR-511导入细胞，检测PXR的mRNA水平及其翻译效率。此外，还将定位支持miR-511作用的序列，并确定miR-511在IL-6抑制PXR中的作用。总体而言，修订申请中的研究表明，家长补助金中的原始目标得到了显著扩展。重要的是，这些研究与父母拨款中的研究紧密结合，共同提供了对药物消除基因表达的更全面的理解，这些基因涉及物种依赖的反式激活和炎症过程中表达的变化。 与公共卫生相关：药物消除的能力因其他药物和炎症等疾病状态的共同作用而改变。修订申请提出了一系列研究，以揭示联合用药或炎症介质如何以及在多大程度上改变个人的药物清除能力的潜在机制。

DESCRIPTION (provided by applicant): Yan, Bingfang ABSTRACT This is a revision application in response to the announcement NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications This revision application proposes a set of studies to extend the specific aims of the parent grant. The focus of the parent grant, presented as two specific aims, is to elucidate signaling events associated with the pregnane X receptor (PXR), a master regulator on the expression of many chemical elimination genes such as cytochrome P450 3A4 (CYP3A4) in humans and CYP3A23 in rats. Interestingly, the CYP3A23 but not the CYP3A4 proximal promoter responds robustly to a PXR ligand, although a functional PXR element is present in both CYP3A4 and CYP3A23 proximal promoters. Instead, the CYP3A4 proximal promoter coordinates with other upstream PXR elements and confers transactivation activity. Sequence comparison reveals that the CYP3A4 promoter, compared with CYP3A23, has the PXR element 30 bases farther from the transcription starting site. In addition, the CYP3A4 but not CYP3A23 promoter has an HNF3/CEBP site in front of the PXR element. The PXR-mediated trans- activation, on the other hand, is markedly attenuated by cytokines such as interleukin-6 (IL-6). microRNA-511 (miR-511), highly induced by IL-6, diminishes PXR transactivation. miRs are known to silence gene expression through sequence pairing with their target mRNAs. The proposed studies are designed to test the hypotheses that the PXR transcript is a sequence-specific target of miR-511, and the physical location of the PXR element and the presence of the HNF3/CEBP site contribute significantly to the inability of the CYP3A4 proximal promoter to effectively respond to PXR-transactivation. To define the potential role of the physical location or the interference of the HNF3/CEBP site, mutants will be prepared to disrupt the HNF3/CEBP site, relocate the PXR element closer to the transcription starting site, or both. These mutants will be tested for increased ability to respond to PXR transactivation. To determine the molecular action of miR-511 to decrease PXR expression, cells will be transfected with miR-511 and the levels of PXR mRNA and its translation efficiency will be monitored. In addition, the sequence supporting the action of miR-511 will be located and the role of miR-511 in the suppression of PXR by IL-6 will be established. Overall, the studies in the revision application represent significant expansion of the original aims in the parent grant. Importantly, these studies are closely integrated into the studies in the parent grant, and together, they will provide more comprehensive understanding on the expression of drug elimination genes regarding species-dependent transactivation and altered expression during inflammation. PUBLIC HEALTH RELEVANCE: The capacity of drug elimination is altered by co-administration of other drugs and disease status such as inflammation. The revision application proposes a set of studies to reveal the underlying mechanisms on how and to which extent the drug-eliminating capacity of an individual is altered by co-administered drugs or inflammatory mediators.