Identification of Genes Activated by Bile Acids

描述(申请人提供)：鹅去氧胆酸(CDCA)，一种主要 胆汁酸，最近被证明激活法尼醇X受体(FXR)， 核荷尔蒙受体超家族的成员。最近的数据表明， 激活的FXR既控制胆汁酸的生物合成，又控制血脂水平。AS 这样，FXR可能会影响胆结石和/或动脉粥样硬化的发展。 然而，到目前为止，人们对靶基因和 受激活的FXR影响的代谢途径。在第一个具体的 目的：我们建议使用抑制性消减杂交和DNA 用于识别受CDCA激活的FXR调控的基因的微阵列。这些 研究将利用稳定过表达高水平的HepG2和Caco 2细胞 以便更容易地识别FXR靶基因。我们将使用Normal， FXR-/-或VP16-FXR转基因小鼠，用FXR配体治疗，以证明 (I)这些相同的基因在体内被诱导，以及(Ii)FXR的激活 结果降低了血脂水平。在第二个具体目标中，我们将 确定FXRE和其他关键顺式元件在几个启动子中 已在目标1中识别的选定基因，以确认这些 基因是FXR/CDCA的直接靶点。在第三个目标中，我们将产生小鼠 在他们的肝脏中过表达大鼠VP16-FXR(见目标1)。最后，具体地说 目的4，我们将分离来自HepG2和Caco 2细胞的细胞系 表示FXR1、FXR2或FXR3。目的基因的诱导，鉴定 在目标1中，将确定每个FXR亚型，以便测试 假设特定的基因/代谢途径被每个FXR激活 异构体。

DESCRIPTION (provided by applicant): Chenodeoxycholic acid (CDCA), a primary bile acid, has recently been shown to activate the farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily. Recent data suggest that activated FXR controls both bile acid biosynthesis and plasma lipid levels. As such, FXR may affect the development of gallstones and/or atherosclerosis. However at the current time, very little is known about the target genes and metabolic pathways that are affected by activated FXR. In the first specific aim, we propose to use Suppression Subtractive Hybridization and DNA microarrays to identify genes that are regulated by CDCA-activated FXR. These studies will utilize HepG2 and Caco2 cells that stably overexpress high levels of FXR in order to more easily identify FXR-target genes. We will use normal, FXR-/- or VP16-FXR transgenic mice, treated with FXR ligands, to demonstrate (i) that these same genes are induced in vivo and (ii) that activation of FXR results in a decrease in plasma lipids. In the second specific aim, we will identify FXREs and other critical cis elements in the promoters of a few selected genes that have been identified in aim 1, so as to confirm that these genes are direct targets of FXR/CDCA. In the third aim, we will generate mice that overexpress rat VP16-FXR in their livers (see aim 1). Finally, in specific aim 4, we will isolate cell lines derived from HepG2 and Caco2 cells that express either FXR1, FXR2, or FXR3. The induction of target genes, identified in aim 1, by each FXR isoform will be determined in order to test the hypothesis that specific genes/metabolic pathways are activated by each FXR isoforms.