Regulation of Aromatase Expression in the Corpus Luteum

说明(申请人提供)：芳香酶(CYP19A1)是雌二醇生物合成所必需的，雌二醇是一种类固醇，对正常的卵泡和黄体细胞功能是必需的，对生殖也是必不可少的。芳香酶在卵泡细胞中的表达主要受cyp19基因启动子近端的cAMP反应元件样序列(CLS)的激活所刺激。我们已经证明，这个启动子也驱动黄体细胞中芳香化酶的表达；然而，在这些细胞中，cAMP和CLS都不参与该酶的表达。这表明，替代机制控制黄体中雌二醇的产生。另一方面，我们已经证明了芳香酶在妊娠大鼠黄体中的表达与黄体核蛋白与近端启动子中的激活蛋白3反应元件(AP3-RE)结合的变化有关。AP3-RE的突变，而不是CLS结合位点的突变，取消了黄体细胞中芳香酶启动子的活性。基于这一证据，我们推测AP3-RE在黄体细胞雌二醇产生的调节中起关键作用。我们一直无法检验这一假设，因为与AP3-RE结合的蛋白质的身份尚不清楚。R21的这一探索性应用的目的是鉴定与AP3-RE特异结合的黄体蛋白。将使用两种方法：i)亲和柱纯化蛋白的质谱分析和ii)黄体表达文库的筛选。分子生物学、蛋白质化学和基因技术目前正在我的实验室中使用，或者我可以在UIC的蛋白质组学和信息学服务设施中获得。一旦AP3-RE结合蛋白被鉴定出来，重组GST融合蛋白就会产生。纯化的蛋白质将被用来开发特定的抗体。激活蛋白3表达载体和反义探针将被用来研究该蛋白在芳香酶调节和黄体基因表达中的作用。这个项目将加深我们对雌二醇产生的分子机制的理解。 公共卫生相关性：这项建议的目的是确定与芳香酶基因启动子结合的蛋白质。该基因编码一种酶，催化合成雌二醇。雌二醇是生殖过程中的一种必不可少的激素，对大脑、肝脏和骨骼的正常功能至关重要。雌二醇还能刺激癌细胞的增殖。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.