Regulation of Aromatase Expression in the Corpus Luteum

黄体中芳香酶表达的调节

基本信息

批准号：
8045278
负责人：
CARLOS OSCAR STOCCO
金额：
$ 19.63万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2012-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8045278
关键词：
Affinity Anabolism Anti-Sense Probes Antibodies Aromatase Binding Binding Proteins Binding Sites Brain CYP19A1 gene Cell physiology Cells Chimeric Proteins Complementary DNA Cyclic AMP Cyclic AMP Response Element DNA Elements Enzymes Estradiol Expression Library Follicular Ovarian Cell Gene Expression Genes Genetic Techniques Granulosa-Lutein Cells Hormones Human Informatics Laboratories Libraries Liver Luteal Cells Mammary Neoplasms Mass Spectrum Analysis Molecular Molecular Biology Mutation Nuclear Proteins Oligonucleotides Ovarian Ovary Play Production Protein Binding Protein Chemistry Proteins Proteomics Rattus Recombinants Regulation Reproduction Response Elements Rodent Role Screening procedure Services Site Steroids Testing Tissues Translations Uterine Neoplasms base bone cancer cell corpus luteum expression vector granulosa cell pregnant promoter protein activation theories

项目摘要

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.

描述（由申请人提供）：雌二醇的生物合成需要芳香酶（CYP19A1），雌二醇是正常卵泡和黄体细胞功能所需的类固醇，并且对于生殖至关重要。卵巢滤泡细胞中芳香酶的表达主要是通过激活 cyp19 基因近端启动子中的 cAMP 反应元件样序列 (CLS) 来刺激的。我们已经证明该启动子还驱动黄体细胞中芳香酶的表达。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，怀孕大鼠黄体中芳香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（而非 CLS 结合位点的突变）会消除黄体细胞中芳香酶启动子的活性。基于这一证据，我们假设 AP3-RE 在黄体细胞雌二醇生成的调节中发挥关键作用。我们无法检验这一假设，因为与 AP3-RE 结合的蛋白质的身份未知。这一探索性 R21 应用的目的是鉴定与 AP3-RE 特异性结合的黄体蛋白。将使用两种方法：i) 亲和柱纯化蛋白的质谱分析和 ii) 黄体 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中使用，或者可以在伊利诺伊大学芝加哥分校的蛋白质组学和信息学服务设施中使用。一旦鉴定出 AP3-RE 结合蛋白，就会产生重组 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酶调节和黄体基因表达中的作用。该项目将增强我们对控制雌二醇产生的分子机制的理解。公共健康相关性：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。雌二醇是生殖过程中必需的激素，对于大脑、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，该项目将对我们对正常和病理条件下控制雌二醇产生的分子机制的理解产生广泛的影响。