Regulation of PXR by cell cycle and phosphorylation

描述（由申请人提供）：药物代谢酶表达的变化是否会改变药物的治疗或毒理学反应，并导致严重的药物不良相互作用？这是美国的一个重大公共卫生问题。细胞色素P450（CYP）是最常见的药物代谢酶，并且人的胆甾烷X受体（hPXR）在激活CYP的表达中起核心作用。CYP 3A 4催化超过50%的临床使用药物的代谢。hPXR活化和CYP 3A 4介导的药物代谢主要发生在肝细胞中。重要的是，CYP（包括CYP 3A 4）的表达在再生肝脏的增殖肝细胞中显著低于正常肝脏中的静止肝细胞。然而，负责增殖肝细胞中药物代谢酶水平降低的分子机制尚不清楚。药物代谢酶的抑制水平可能对药物疗效和毒性产生深远影响。由于肝再生是由大范围的损伤引起的肝损伤触发的，这些损伤影响所有年龄和背景的人，因此研究增殖肝细胞中PXR的调节以预测和预防再生肝患者中的不良药物相互作用非常重要。拟定研究的目的是确定hPXR活性和CYP 3A 4表达在增殖肝细胞中受到抑制的机制。我们的中心假设是Cdks磷酸化并抑制hPXR活性，从而导致增殖肝细胞中hPXR表达减少。进行拟议研究的基本原理是，阐明hPXR活性和CYP 3A 4表达在增殖肝细胞中受到抑制的机制，将为药物代谢的调节提供全新的见解，并提高我们对外源性反应的理解，最终有助于预测药物的准确剂量，并降低药物不良相互作用的风险。我们的长期目标是了解hPXR如何通过正常和患病肝脏中的磷酸化受到细胞信号通路的调节，以便设计更有效的治疗方法。我们计划通过以下4个具体目的来验证我们的中心假设：（1）确定Cdk调节的hPXR磷酸化位点;（2）确定哪些磷酸化位点是Cdk抑制作用的原因，以及磷酸化如何影响hPXR的活性;（3）确定在增殖肝细胞的细胞周期中Cdk抑制hPXR介导的CYP 3A 4表达的程度;（4）测定磷酸化对hPXR活性的体内影响。这些结果有望为增殖肝细胞中药物代谢和处置的调控提供基础性的新信息，也将为药物安全性评价和治疗策略的有效设计奠定基础。 公共卫生相关性：拟议的研究旨在填补药物代谢和处置这一重要领域的知识空白，这将大大提高我们对外源性反应的理解，以及影响所有年龄和背景的人的再生肝脏中不良药物相互作用的风险预测和预防。拟定的研究与公共卫生相关，因为药物不良相互作用对治疗相关的发病率和死亡率有很大影响，这是美国的一个主要公共卫生问题。拟议研究的结果最终将有助于改善人类健康。

DESCRIPTION (provided by applicant): Changes in the expression of drug-metabolizing enzymes can alter the therapeutic or toxicologic response to a drug and lead to serious adverse drug interactions ? a major public health problem in the US. Cytochromes P450 (CYPs) are the most prevalent drug-metabolizing enzymes and the human pregnane X receptor (hPXR) plays a central role in activating the expression of CYP. CYP3A4 catalyzes the metabolism of more than 50% of clinically used drugs. hPXR activation and CYP3A4-mediated drug metabolism occur primarily in hepatocytes. Importantly, expression of CYPs, including CYP3A4, is significantly lower in proliferating hepatocytes of regenerating livers than in quiescent hepatocytes in the normal livers. However, the molecular mechanism responsible for the reduction in levels of drug-metabolizing enzymes in proliferating hepatocytes is unknown. Repressed levels of drug-metabolizing enzymes can have a profound effect on drug efficacy and toxicity. Because liver regeneration is triggered by liver injuries caused by a large range of insults, which affect people of all ages and backgrounds, it is important to study the regulation of PXR in proliferating hepatocytes in order to predict and prevent adverse drug interactions in patients with regenerating livers. The objective of the proposed study is to determine the mechanism by which hPXR activity and CYP3A4 expression are repressed in proliferating hepatocytes. Our central hypothesis is that Cdks phosphorylate and repress hPXR activity, thereby causing the reduction of CYP expression in proliferating hepatocytes. The rationale for conducting the proposed research is that elucidating the mechanisms by which hPXR activity and CYP3A4 expression are repressed in proliferating hepatocytes will provide fundamentally novel insights into the regulation of drug metabolism and improve our understanding of the xenobiotic response, ultimately aid the prediction of the accurate dosage of drugs, and reduce the risk of adverse drug interactions. Our long-term goal is to understand how hPXR is regulated by cellular signaling pathways through phosphorylation in both normal and diseased livers in order to design more effective therapies. We plan to test our central hypothesis by the following 4 Specific Aims: (1) Identify Cdk-regulated phosphorylation sites on hPXR; (2) Determine which phosphorylation sites are causally responsible for the inhibitory effect of Cdks and how the phosphorylation affects the activity of hPXR; (3) Determine the extent to which hPXR-mediated CYP3A4 expression is repressed by Cdk during the cell cycle in proliferating hepatocytes; and (4) Determine the in vivo effect of phosphorylation on the activity of hPXR. The outcomes are expected to provide fundamental novel information on the regulation of drug metabolism and disposition in proliferating hepatocytes, and will also lay the foundation for the effective design of drug safety evaluations and therapeutic strategies. PUBLIC HEALTH RELEVANCE: The proposed studies aim to fill the knowledge gap in an important area of drug metabolism and disposition, which will considerably improve our understanding of xenobiotic responses as well as risk prediction and prevention of adverse drug interactions in regenerating livers that affect people of all ages and backgrounds. The proposed studies are relevant to public health because adverse drug interactions contribute considerably to therapeutic-related morbidity and mortality, a major public health problem in the US. The results from the proposed studies will ultimately contribute to improving the health of human beings.