REGULATION OF APOAI GENE EXPRESSION BY NUCLEAR RECEPTORS

高密度脂蛋白（HDL）及其主要蛋白质成分， 载脂蛋白AI（apoAI）在胆固醇稳态中起主要作用。 了解调节apoAI基因表达的分子机制 在临床上是重要的，因为血浆apoAI和HDL水平降低， 与过早的动脉粥样硬化和冠心病有关。 我们 长期目标是研究apoAI的转录调控 基因，并鉴定调节肝脏中apoAI合成的新信号 和肠。 为了实现这一目标，我们发现， 在apoAI启动子与四个核受体相互作用， 配体阿普-1、ARP-2、ARP-3和HNF-4，并形成异二聚体 9-顺式视黄酸受体α（RXR α）与阿普-1， 全反式视黄酸受体α（RAR α），维生素A D受体（VDR）或过氧化物酶体增殖物激活受体（PPAR）。 ApoAI基因表达被阿普-1抑制，被HNF-4激活， 然而，其他核受体对apoAI的影响 表达是未知的。 具体目标是：1）确定 与apoAI启动子结合的核受体的功能效应 对该基因在肝和肠细胞中的表达的影响。 2)映射 HNF-4和阿普-1的功能结构域，并确定基本的 这些转录因子是这些核受体的靶点。 第三章 为了鉴定调节阿普-1的DNA结合活性的因子， -2和 实现这些目标的实验设计和方法是：1） 表达上述核受体的质粒与 含有在apoAI控制下的CAT报告基因的构建体 启动子区在人肝癌（HepG 2）和人结肠癌（Caco-2）中的表达 2)细胞，使用磷酸钙共沉淀法。 用RXR α、RAR α、VDR和PPAR转染将包括 转染后用9-顺式和全反式视黄酸、维生素A D和氯贝酸或亚油酸。 2)缺失诱变 将HNF-4和阿普-1与共转染实验组合， 用于绘制这些核的激活和抑制结构域， 受体。 蛋白质-蛋白质相互作用测定将用于研究 HNF-4和阿普-1与基础转录因子的相互作用， 包括TATA结合蛋白（TBP）、TFIIB和其他TBP相关的 因子（TAF），以确定这些相互作用中涉及的域。 此外，相互作用克隆，将用于克隆组织特异性 TAF可能与HNF-4或阿普-1相互作用。 3)生化分析和 高效液相色谱法将用于纯化和 表征调节阿普-1的DNA结合活性的因子。 这些研究将加深我们对细胞凋亡分子机制的理解。 apoAI通过核受体进行转录调控，并将揭示新的 调节apoAI合成的信号转导途径 胆固醇平衡的关键 此外，该信息可以提供 开发新的药理学策略的基础， 监测常见的脂质紊乱。

High-density lipoprotein (HDL) and its major protein component, apolipoprotein AI (apoAI), play a major role in cholesterol homeostasis. Knowledge of the molecular mechanisms that regulate apoAI gene expression is clinically important because reduced plasma levels of apoAI and HDL are associated with premature atherosclerosis and coronary heart disease. Our long term objective is to study the transcriptional regulation of the apoAI gene and to identify novel signals that modulate apoAI synthesis in liver and intestine. Towards this goal, we have found that regulatory elements in the apoAI promoter interact with four nuclear receptors with unknown ligands, ARP-1, EAR-2, EAR-3, and HNF-4, and with heterodimers formed between the 9-cis retinoic acid receptor alpha (RXRalpha) and either ARP-1, EAR-2, EAR-3, all -trans retinoic acid receptor alpha (RARalpha), vitamin D receptor (VDR), or peroxisome proliferator-activated receptor (PPAR). ApoAI gene expression is repressed by ARP-1 and activated by HNF-4 and RXRalpha, however, the effects of the other nuclear receptors on apoAI expression are not known. The specific aims are: 1) To determine the functional effects of the nuclear receptors that bind to the apoAI promoter on the expression of this gene in hepatic and intestinal cells. 2) To map the functional domains of HNF-4 and ARP-1, and to identify the basal transcription factors which are targets for these nuclear receptors. 3) To identify the factors that regulate the DNA-binding activity of ARP-1, EAR-2, and EAR-3. The experimental design and methods for achieving these aims are; 1) Cotransfections of plasmids expressing the above nuclear receptors with constructs containing the CAT reporter gene under the control of the apoAI promoter region in human hepatoma (HepG2) and human colon carcinoma (Caco- 2) cells, using the calcium phosphate coprecipitation method. Transfections with RXRalpha, RARalpha, VDR, and PPAR will include posttransfection treatments with 9-cis and all-trans retinoic acid, vitamin D, and clofibric or linoleic acid, respectively. 2) Deletion mutagenesis of HNF-4 and ARP-1 in combination with cotransfection experiments will be employed to map the activation and repression domains of these nuclear receptors. Protein-protein interaction assays will be used to study the interactions of HNF-4 and ARP-1 with basal transcription factors., including the TATA-binding protein (TBP), TFIIB, and other TBP-associated factors (TAFs), an to identify the domains involved in these interactions. Furthermore, interaction cloning, will be used to clone tissue-specific TAFs that may interact with HNF-4 or ARP-1. 3) A biochemical assay and High performance liquid Chromatography will be employed to purify and characterize the factor that regulates the DNA-binding activity of ARP-1. These studies will enhance our understanding of the molecular mechanisms of apoAI transcriptional regulation by nuclear receptors and will reveal novel signal transduction pathways that modulate apoAI synthesis and contribute to cholesterol homeostasis. Furthermore, this information may provide a basis for the development of novel pharmacological strategies for monitoring common lipid disorders.