Estrogenic LXR Alpha Response /Cholesterol Homeostasis

雌激素 LXR Alpha 反应/胆固醇稳态

基本信息

批准号：
6614747
负责人：
PHILLIP R KRAMER
金额：
$ 7.28万
依托单位：
TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-05-01 至 2005-04-30
项目状态：
已结题

项目摘要

The objective of this application is to determine the mechanism by which estrogen withdrawal, seen in aging women, increases LXR alpha (a gene necessary for regulating cholesterol homeostasis) transcript levels. My central hypothesis is that the estrogen-bound estrogen receptor (ER) represses AP-1 dependent activation of LXR alpha transcription. This hypothesis is based on our recent findings that a decrease in estrogen significantly increases the level of LXR alpha mRNA/protein in primary macrophages and in the monocytic-macrophage cell line THP-1 in the absence of nascent protein synthesis. In addition, this hypothesis incorporates data from other labs that estrogen, through the ER (predominantly ER beta) can repress transcription at AP-1 consensus sites in macrophage cell lines and that the LXR alpha promoter contains multiple AP-1 binding sites but not an estrogen response element. To test our central hypothesis aim one will determine ER's role in regulating the LXR alpha estrogenic response by quantitating transcription after treating macrophages with antiestrogen (ICI 182,780) and HPTE (ER alpha agonist; ER beta antagonist) in the presence and absence of lX10-8 M 17-beta-estradiol. Aim two will determine transcription factors impacted by estrogen that act directly in regulating LXR alpha gene transcription in macrophages. Luciferase activity driven by an intact or mutated (e.g., AP-1 binding sites) LXR alpha promoter will be measured in transiently transfected macrophages before and after estrogen treatment. Once specific promoter sequences are identified that transduce an estrogen signal the activity and binding of specific proteins (e.g., Jun and Fos family members) to small labeled promoter sequences (about 20 bp) will be determined by western analysis and electrophoretic mobility shift assays (EMSA), requiring nuclear extracts of THP-1, treated with and without estrogen. The expected result will identify AP-1 binding site(s) and identify the individual proteins that make up the AP-1 complex that binds to the AP-1 promoter element required for increased LXR alpha transcription after estrogen depletion in macrophages.

本申请的目的是确定老年女性中雌激素戒断增加 LXR α（调节胆固醇稳态所必需的基因）转录水平的机制。我的中心假设是雌激素结合的雌激素受体 (ER) 抑制 LXR α 转录的 AP-1 依赖性激活。这一假设基于我们最近的发现，即在缺乏新生蛋白质合成的情况下，雌激素的减少会显着增加原代巨噬细胞和单核巨噬细胞系 THP-1 中 LXR α mRNA/蛋白质的水平。此外，该假设还结合了其他实验室的数据，即雌激素通过 ER（主要是 ER beta）可以抑制巨噬细胞系中 AP-1 共有位点的转录，并且 LXR α 启动子包含多个 AP-1 结合位点，但不包含一个 AP-1 结合位点。雌激素反应元件。为了检验我们的中心假设，我们将在存在和不存在 LX10-8 的情况下，通过抗雌激素 (ICI 182,780) 和 HPTE（ER α 激动剂；ER β 拮抗剂）处理巨噬细胞后定量转录，确定 ER 在调节 LXR α 雌激素反应中的作用M 17-β-雌二醇。目标二将确定受雌激素影响的转录因子，这些因子直接调节巨噬细胞中的 LXR α 基因转录。在雌激素治疗之前和之后，在瞬时转染的巨噬细胞中测量由完整或突变（例如，AP-1结合位点）LXR α启动子驱动的荧光素酶活性。一旦确定了特定的启动子序列转导雌激素信号的特定蛋白质的活性和结合（例如，Jun 和 Fos）家族成员）到小标记启动子序列（约 20 bp）将通过蛋白质印迹分析和电泳迁移率变动测定（EMSA）来确定，需要 THP-1 的核提取物，用或不用雌激素处理。预期结果将鉴定 AP-1 结合位点并鉴定构成 AP-1 复合物的各个蛋白质，该复合物与巨噬细胞中雌激素耗尽后增加 LXR α 转录所需的 AP-1 启动子元件结合。