Signaling of the Pregnane X Receptor

摘要这是一份针对公告no - od -09-058的修订申请：NIH宣布为竞争性修订申请提供恢复法案资金。这份修订申请提出了一系列研究，以扩展父母资助的具体目标。亲本资助的重点是阐明与妊娠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因子，如人类的细胞色素P450 3A4 （CYP3A4）和大鼠的CYP3A23。有趣的是，CYP3A23而非CYP3A4近端启动子对PXR配体有强烈反应，尽管CYP3A4和CYP3A23近端启动子中都存在功能性PXR元件。相反，CYP3A4近端启动子与其他上游PXR元件协调并赋予转激活活性。序列比较发现，CYP3A4启动子与CYP3A23相比，PXR元件距离转录起始位点远30个碱基。此外，CYP3A4启动子在PXR元件前面有一个HNF3/CEBP位点，而CYP3A23启动子则没有。另一方面，pxr介导的反式激活被白细胞介素-6 （IL-6）等细胞因子显著减弱。microRNA-511 (miR-511)，被IL-6高度诱导，减少PXR的转激活。已知miRs通过与其靶mrna的序列配对来沉默基因表达。拟议的研究旨在验证PXR转录物是miR-511序列特异性靶点的假设，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响应PXR-转激活。为了确定HNF3/CEBP位点的物理位置或干扰的潜在作用，突变体将准备破坏HNF3/CEBP位点，将PXR元件重新定位到更靠近转录起始位点，或两者兼有。将对这些突变体进行测试，以增强对PXR交互激活的反应能力。为了确定miR-511在降低PXR表达中的分子作用，我们将miR-511转染细胞，并监测PXR mRNA的水平及其翻译效率。此外，我们将定位支持miR-511作用的序列，建立miR-511在IL-6抑制PXR中的作用。总体而言，修订申请中的研究代表了父母资助的原始目标的显著扩展。重要的是，这些研究与亲本研究紧密结合，将更全面地了解药物消除基因在炎症过程中与物种依赖性转激活和表达改变有关的表达。

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.