Signaling of the Pregnane X Receptor

说明（申请人提供）：严炳芳摘要这是一份修订申请，以回应公告NOT-OD-09-058：NIH宣布恢复法案基金可用于竞争性修订申请这份修订申请提出了一系列研究，以扩展母基金的特定目标。母基金的重点，提出了两个具体的目标，是阐明与藜芦烷X受体（PXR），对许多化学消除基因的表达，如细胞色素P450 3A 4（CYP 3A 4）在人类和大鼠CYP 3A 23的主调节相关的信号事件。有趣的是，CYP 3A 23而不是CYP 3A 4近端启动子对PXR配体有强烈的反应，尽管在CYP 3A 4和CYP 3A 23近端启动子中都存在功能性PXR元件。相反，CYP 3A 4近端启动子与其他上游PXR元件协调并赋予反式激活活性。序列比较显示，与CYP 3A 23相比，CYP 3A 4启动子具有距离转录起始位点30个碱基的PXR元件。此外，CYP 3A 4启动子而非CYP 3A 23启动子在PXR元件前具有HNF 3/CEBP位点。另一方面，PXR介导的反式激活被细胞因子如白细胞介素-6（IL-6）显著减弱。由IL-6高度诱导的microRNA-511（miR-511）减少PXR反式激活。已知miR通过与其靶mRNA的序列配对来沉默基因表达。这些研究旨在验证以下假设：PXR转录物是miR-511的序列特异性靶点，PXR元件的物理位置和HNF 3/CEBP位点的存在显著导致CYP 3A 4近端启动子无法有效响应PXR反式激活。为了确定HNF 3/CEBP位点的物理位置或干扰的潜在作用，将制备突变体以破坏HNF 3/CEBP位点，将PXR元件重新定位到更靠近转录起始位点，或两者兼而有之。将测试这些突变体对PXR反式激活的响应能力是否增加。为了确定miR-511降低PXR表达的分子作用，将用miR-511转染细胞，并监测PXR mRNA的水平及其翻译效率。此外，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。总的来说，修订申请中的研究代表了母补助金最初目标的重大扩展。重要的是，这些研究与母基金中的研究紧密结合，它们将共同提供对药物消除基因表达的更全面的理解，这些基因在炎症过程中的物种依赖性反式激活和表达改变。 公共卫生相关性：药物消除的能力会因其他药物的联合给药和疾病状态（如炎症）而改变。修订申请提出了一系列研究，以揭示共同给药药物或炎症介质如何以及在何种程度上改变个体的药物消除能力的潜在机制。

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.