Regulation of Aromatase Expression in the Corpus Luteum

描述（由申请人提供）：芳香酶（CYP19A1）是生物合成雌二醇所必需的，雌二醇是正常卵泡和黄体细胞功能所需的类固醇，也是生殖所必需的。芳香酶在卵巢卵泡细胞中的表达主要是由cyp19基因近端启动子中发现的cAMP反应元件样序列（CLS）的激活刺激的。我们已经证明这个启动子也驱动黄体细胞中的芳香化酶表达；然而，在这些细胞中，cAMP和CLS都不参与这种酶的表达。这表明黄体中有其他机制控制雌二醇的产生。另一方面，我们已经证明怀孕大鼠黄体中芳香化酶的表达与黄体核蛋白与近端启动子中发现的激活蛋白3反应元件（AP3-RE）结合的变化相关。AP3-RE的突变，而CLS结合位点的突变，会消除黄体细胞中芳香化酶启动子的活性。基于这些证据，我们假设AP3-RE在黄体细胞雌二醇产生的调节中起关键作用。我们一直无法验证这一假设，因为与AP3-RE结合的蛋白质的身份尚不清楚。本探索性R21应用的目的是鉴定特异性结合AP3-RE的黄体蛋白。将使用两种方法：i)亲和柱纯化蛋白的质谱分析和ii)筛选黄体cDNA表达文库。分子生物学、蛋白质化学和遗传技术目前正在我的实验室使用，或者在UIC的蛋白质组学和信息学服务设施向我提供。一旦确定了AP3-RE结合蛋白，将产生重组GST融合蛋白。纯化的蛋白质将用于开发特异性抗体。我们将利用激活蛋白3表达载体和反义探针研究该蛋白在芳香化酶调控和黄体基因表达中的作用。这个项目将加强我们对控制雌二醇产生的分子机制的理解。

DESCRIPTION (provided by applicant): The aromatase enzyme (CYP19A1) is required for the biosynthesis of estradiol, a steroid needed for normal follicular and luteal cell function and essential for reproduction. Aromatase expression in ovarian follicular cells is stimulated mainly by the activation of a cAMP response element-like sequence (CLS) found in the proximal promoter of the cyp19 gene. We have shown that this promoter also drives aromatase expression in luteal cells; however, in these cells neither cAMP nor CLS are involved in the expression of this enzyme. This suggests that alternative mechanisms control estradiol production in the corpus luteum. On the other hand, we have demonstrated that aromatase expression in corpora lutea of pregnant rats correlates with changes in the binding of luteal nuclear proteins to an activation protein 3 response element (AP3-RE) found in the proximal promoter. Mutation of the AP3-RE, but not of the CLS binding site, abolishes aromatase promoter activity in luteal cells. Based on this evidence, we hypothesize that the AP3-RE plays a key role in the regulation of estradiol production in luteal cells. We have been unable to test this hypothesis because the identity of the protein that binds to AP3-RE is unknown. The aim of this exploratory R21 application is to identify luteal proteins that specifically bind to the AP3-RE. Two approaches will be used: i) mass spectrometry analysis of affinity-column purified proteins and ii) screening of a luteal cDNA expression library. Molecular biology, protein chemistry, and genetic techniques are currently being used in my laboratory or are available to me at the Proteomics and Informatics Services Facility at UIC. Once the AP3-RE binding protein has been identified, recombinant GST fusion proteins will be produced. Purified proteins will be used to develop specific antibodies. Activation protein 3 expression vectors and antisense probes will be used to investigate the role of this protein in aromatase regulation and luteal gene expression. This project will enhance our understanding of the molecular mechanisms that govern estradiol production. PUBLIC HEALTH RELEVANCE: The aim of this proposal is to identify proteins that bind to the promoter of the aromatase gene. This gene encodes an enzyme that catalyzes the synthesis of estradiol. Estradiol is an essential hormone in reproduction and crucial for the normal function of the brain, liver, and bone. Estradiol also stimulates the proliferation of cancer cells. Therefore, this project will have a broad impact on our understanding of the molecular mechanisms that control estradiol production under normal and pathological conditions.