MRP4 in Drug Sensitivity and Physiology

MRP4 在药物敏感性和生理学中的应用

基本信息

批准号：
7029703
负责人：
JOHN D SCHUETZ
金额：
$ 39.3万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-04-01 至 2008-03-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): ABC transporters are increasingly recognized as genes that play important roles in normal biology and in the therapeutic response to medications. Among the many mammalian ABC transporters that have been identified in the human genome, the functions of some have been deduced because "accidents of nature" produced mutations that render loss of function. However, for many ABC transporters the biological substrate is unknown and no adventitious accident of nature has been identified. One successful approach to identifying function is to develop drug resistant cell lines with resistance due to drug efflux and then identify an ABC transporter that dominantly imparts this resistance by drug efflux. This was the case for the ABC transporter Mrp4 that was an orphan ABC transporter until our studies first determined that it was unique in its ability to transport nucleotide analogs used in HIV therapy. During the last funding period of this application we used cell line model systems to expand Mrp4's biological substrates (e.g., cAMP and conjugated steroids) and the drugs (mercaptopurine nucleotides) it confers resistance. We also discovered that Mrp4 was expressed in the liver and it's expression dramatically increased as an adaptive response to the accumulation of cholestatic bile acids. Our current research will focus on understanding the function of Mrp4 in vivo. I have developed an Mrp4 nullizygous mouse with T cells that have impaired cAMP efflux. In contrast, T cells from Mrp4 +/+ mice readily efflux cAMP. Moreover, these Mrp4 -/- T cells have a defect in proliferation. The relationship between Mrp4 regulation by the second messenger cAMP and the relation with proliferation will be further explored in Aim 1 of the current application. Because our results show that Mrp4 has a role in the transport of cholestatic conjugated bile salts and is upregulated under cholestatic conditions, I will determine if Mrp4 absence renders the liver more susceptible to cholestatic injury. I will also use the Mrp4 -/- animals to identify mechanisms (Aim II) in the liver that compensate under cholestasis, but are independent of Mrp4. I will also determine if nuclear receptors (e.g., the pregnane X receptor, PXR) contribute to Mrp4 upregulation in Aim II by using nuclear receptor knockout models. Finally, to more fully elucidate the signaling processes and cross talk among these Mrp4 activation pathways we will develop an Mrp4-GFP knock-in mouse model in Aim III. Cumulatively, these studies will increase our knowledge of this increasingly important ABC transporter and provide insight into how Mrp4 plays a role in hematopoietic cells and the liver

描述（由申请人提供）：ABC 转运蛋白越来越被认为是在正常生物学和药物治疗反应中发挥重要作用的基因。在人类基因组中已发现的许多哺乳动物 ABC 转运蛋白中，其中一些转运蛋白的功能已被推断出来，因为“自然事故”产生了导致功能丧失的突变。然而，对于许多 ABC 转运蛋白来说，其生物底物是未知的，并且尚未发现自然偶然事件。识别功能的一种成功方法是开发因药物流出而产生耐药性的耐药细胞系，然后鉴定主要通过药物流出赋予这种耐药性的 ABC 转运蛋白。 ABC 转运蛋白 Mrp4 就是这种情况，它是一种孤儿 ABC 转运蛋白，直到我们的研究首次确定它具有独特的转运用于 HIV 治疗的核苷酸类似物的能力。在本申请的最后资助期间，我们使用细胞系模型系统来扩展 Mrp4 的生物底物（例如 cAMP 和缀合类固醇）及其赋予耐药性的药物（巯基嘌呤核苷酸）。我们还发现Mrp4在肝脏中表达，并且作为对胆汁淤积性胆汁酸积累的适应性反应，其表达急剧增加。我们目前的研究重点是了解 Mrp4 在体内的功能。我开发了一种 Mrp4 无效小鼠，其 T 细胞 cAMP 外流受损。相比之下，Mrp4 +/+ 小鼠的 T 细胞很容易流出 cAMP。此外，这些Mrp4 -/- T细胞存在增殖缺陷。第二信使cAMP对Mrp4的调节与增殖的关系将在本申请的目标1中进一步探讨。因为我们的结果表明，Mrp4 在胆汁淤积性结合胆盐的转运中发挥作用，并且在胆汁淤积条件下上调，所以我将确定 Mrp4 的缺失是否会使肝脏更容易受到胆汁淤积性损伤。我还将使用 Mrp4 -/- 动物来鉴定肝脏中在胆汁淤积下进行补偿但独立于 Mrp4 的机制 (Aim II)。我还将通过使用核受体敲除模型来确定核受体（例如孕烷 X 受体，PXR）是否有助于 Aim II 中的 Mrp4 上调。最后，为了更全面地阐明这些 Mrp4 激活途径之间的信号传导过程和串扰，我们将在 Aim III 中开发 Mrp4-GFP 敲入小鼠模型。总而言之，这些研究将增加我们对这种日益重要的 ABC 转运蛋白的了解，并深入了解 Mrp4 如何在造血细胞和肝脏中发挥作用