Regulation of Aromatase Expression in the Corpus Luteum

描述（由申请人提供）：芳香酶（CYP 19 A1）是雌二醇生物合成所必需的，雌二醇是正常卵泡和黄体细胞功能所需的类固醇，对生殖至关重要。卵巢滤泡细胞中芳香化酶的表达主要是由cyp 19基因近端启动子中的cAMP反应元件样序列（CLS）激活所刺激的。我们已经表明，这个启动子也驱动芳香化酶在黄体细胞中的表达，然而，在这些细胞中既不cAMP也不CLS参与这种酶的表达。这表明，替代机制控制雌二醇的生产在黄体。另一方面，我们已经证明，芳香化酶在黄体中的表达与黄体核蛋白结合的变化相关的激活蛋白3反应元件（AP 3-RE）中发现的近端启动子。突变的AP 3-RE，但不是CLS结合位点，废除芳香化酶启动子活性在黄体细胞。基于这些证据，我们推测AP 3-RE在调节黄体细胞雌二醇产生中起关键作用。我们一直无法验证这一假设，因为与AP 3-RE结合的蛋白质的身份是未知的。该探索性R21应用的目的是鉴定特异性结合AP 3-RE的黄体蛋白。将使用两种方法：i）亲和柱纯化的蛋白质的质谱分析和ii）黄体cDNA表达文库的筛选。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提供给我。一旦鉴定出AP 3-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。激活蛋白3表达载体和反义探针将用于研究该蛋白在芳香化酶调节和黄体基因表达中的作用。这个项目将提高我们对雌二醇产生的分子机制的理解。 公共卫生相关性：该提案的目的是鉴定与芳香酶基因启动子结合的蛋白质。该基因编码一种催化雌二醇合成的酶。雌激素是生殖过程中的一种必需激素，对大脑、肝脏和骨骼的正常功能至关重要。雌二醇还刺激癌细胞的增殖。因此，该项目将对我们理解在正常和病理条件下控制雌二醇产生的分子机制产生广泛的影响。

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.