Estrogenic LXR Alpha Response /Cholesterol Homeostasis

本申请的目的是确定在老年妇女中观察到的雌激素戒断增加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AP-1依赖的LXR α转录激活。这一假设是基于我们最近的发现，即在缺乏新生蛋白质合成的情况下，雌激素的减少显著增加了原代巨噬细胞和单核细胞-巨噬细胞系THP-1中LXR α mRNA/蛋白质的水平。此外，这一假说还结合了来自其他实验室的数据，即雌激素通过ER（主要是ER β）可以抑制巨噬细胞系中AP-1共有位点的转录，LXR α启动子含有多个AP-1结合位点，但不含雌激素反应元件。为了检验我们的中心假设，在存在和不存在1 × 10 - 8M 17-β-雌二醇的情况下，通过用抗雌激素（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雌激素应答中的作用。目的二是确定雌激素对巨噬细胞LXR α基因转录的直接调控因子。由完整或突变的（例如，在雌激素处理之前和之后，在瞬时转染的巨噬细胞中测量AP-1结合位点）LXR α启动子。一旦确定了特定的启动子序列 这些信号传递给雌激素信号的活性和特定蛋白质的结合（例如，jun和fos 家族成员）与小的标记启动子序列（约20 bp）的差异将通过蛋白质印迹分析和电泳迁移率变动分析（EMSA）来确定，需要THP-1的核提取物，用和不用雌激素处理。预期结果将鉴定AP-1结合位点，并鉴定构成AP-1复合物的单个蛋白质，该复合物结合巨噬细胞中雌激素耗竭后增加LXR α转录所需的AP-1启动子元件。

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.