DNA sequences impact estrogen and antiestrogen activity

DNA 序列影响雌激素和抗雌激素活性

• 批准号: 8009209
• 负责人: Carolyn M. Klinge
• 金额: $ 10万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8009209
• 关键词: 4-Hydroxy-Tamoxifen; Acetylation; Acute; Address; Aging; Aorta; Apoptosis; Apoptotic; Area; Binding Sites; Biogenesis; Biological; Biological Assay; Breast; Breast Cancer Cell; Calorimetry; Cell Cycle; Cell Cycle Proteins; Cell Nucleus; Cell Proliferation; Cells; Communication; Consumption; Cyclin D1; Cytochrome c Reductase; DNA Maintenance; DNA Polymerase II; DNA Sequence; Estradiol; Estrogen Antagonists; Estrogen Receptor Modulators; Estrogen Receptors; Estrogens; Event; Fluorescence; Gene Expression; Gene Proteins; Gene Targeting; Generations; Genes; Genetic Transcription; Genome; Goals; Heart; Heart Arrest; Heating; Histones; Human; Hypothalamic structure; Investigation; Kidney; Knockout Mice; Lead; Ligands; Liver; Lung; MCF7 cell; Malignant neoplasm of lung; Mammary Gland Parenchyma; Mammary Neoplasms; Mammary gland; Measures; Mediating; Messenger RNA; Mitochondria; Mitochondrial DNA; Molecular; Mus; Nerve Degeneration; Nuclear; Output; Pathway interactions; Play; Prevention; Production; Promoter Regions; Proteins; RNA; Reactive Oxygen Species; Recruitment Activity; Regulation; Reporting; Research; Respiratory Chain; Response Elements; Role; Selective Estrogen Receptor Modulators; Small Interfering RNA; Specimen; Steroids; Stroke; Testing; Time; Tissues; Titrations; Trauma; Tumor Tissue; Uterus; cancer cell; cancer initiation; chromatin immunoprecipitation; cytochrome c oxidase; functional outcomes; in vivo; insight; mitochondrial dysfunction; neoplastic; neoplastic cell; novel; nuclear respiratory factor; ovex (pesticide); promoter; protein expression; public health relevance; research study; respiratory; response; survivin; transcription factor; tumor progression; tumor xenograft

DESCRIPTION (provided by applicant): Although mitochondrial gene expression depends on nuclear genome function and reciprocally "retrograde communication" from mitochondria regulates nuclear gene expression, the mechanisms coordinating these events remain to be clarified. Mitochondria are targets of estrogen action. However, the mechanisms for estrogenic effects on mitochondrial function are not yet defined. The overall goal of this project is to determine the mechanism(s) by which estradiol (E2) and selective estrogen receptor (ER) modulators (SERMs) regulate the transcription of Nuclear Respiratory Factor-1 (NRF-1) and the downstream impact of NRF-1 on estrogen- and SERM- regulated mitochondrial activity in normal and neoplastic cells. NRF-1 is a transcription factor that is critical for integrating nucleo-mitochondrial function by initiating transcription of nuclear-encoded mitochondrial DNA (mtDNA) -specific transcription factors including Tfam (mtDNA maintenance factor). We recently reported that E2 induces NRF-1 transcription in MCF-7 breast cancer cells via ER1-estrogen response element (ERE) activation. In Aim 1 we will test the hypothesis that the direct interaction of ER1 and ER2 with the NRF-1 promoter recruits different coregulators in a ligand- and cell- specific manner. Chromatin immunoprecipitation (ChIP) assays will address this hypothesis. The effect of siNRF-1 on E2-regulated cell cycle and apoptotic proteins and cell cycle entry will be examined. Results will reveal if E2-induced NRF-1 plays a role in E2's anti-apoptotic activity and if there are cell-specific differences in these responses. Additionally, we will examine NRF-1 protein expression by IHC in normal and neoplastic human breast specimens. Experiments in Aim 2 will determine the effect of E2 and SERMs on NRF-1 and downstream target gene expression and ER-NRF-1 promoter interaction in vivo. ChIP assays will determine if E2 increases the direct interaction of ER1, coactivators, and RNA pol II with the NRF-1 promoter in vivo in MCF-7 tumor xenografts and mouse tissues, focusing first on uterus. Aim 3 is the `functional outcome' aim to measure the genes/proteins/mitochondrial activity, including O2 consumption, mt biogenesis, heat generation, i.e., downstream effects of E2-induced NRF-1 expression. Experiments will test the hypothesis that the E2- stimulated increase in NRF-1 will, in turn, increase the expression of NRF-1 regulated genes, e.g., TFAM, that subsequently increase expression of its mtDNA-encoded target genes, e.g., Cytochrome c oxidase subunit I (MTCO1) and anti-apoptotic genes, e.g., survivn. Together the results will define the molecular mechanisms of estrogen action coordinating mitochondrial function. Understanding how E2 and SERMs regulate mitochondrial function could lead to new insights in the prevention and progression of breast and lung cancer. PUBLIC HEALTH RELEVANCE: Although mitochondrial gene expression depends on nuclear genome function and reciprocally by "retrograde communication" mitochondrial activity regulates nuclear gene expression, the mechanisms coordinating these events remain to be clarified. Mitochondrial dysfunction and increased reactive oxygen species mediate the pathophysiologic mechanisms of aging, acute neurodegeneration caused by trauma, stroke, or cardiac arrest; and cancer initiation and tumor progression. The impact of steroids on mitochondrial function "is a new and novel area of investigation" and the proposed study seeks to delineate a new biological pathway of estrogen and antiestrogens/SERMs in regulating nuclear respiratory factor-1 (NRF-1) and its downstream targets in mitochondrial activity.

描述（由申请人提供）：尽管线粒体基因的表达取决于核基因组功能和线粒体的相互“逆行交流”来调节核基因的表达，但协同这些事件的机制仍然待阐明。线粒体是雌激素作用的靶标。然而，尚未定义雌激素作用对线粒体功能的机制。该项目的总体目的是确定雌二醇（E2）和选择性雌激素受体（ER）调节剂（SERMS）调节核呼吸因子1（NRF-1）的转录以及NRF-1对雌激素和雌激素调节的正常细胞中雌激素 - 调节线粒体活性的下游影响的机制。 NRF-1是一种转录因子，对于通过核编码的线粒体DNA（MTDNA）特定转录因子（包括TFAM（包括MTDNA维持因子））的转录来整合核中核功能至关重要。我们最近报道，E2通过ER1-雌激素反应元件（ERE）激活在MCF-7乳腺癌细胞中诱导NRF-1转录。在AIM 1中，我们将检验以下假设：ER1和ER2与NRF-1启动子的直接相互作用以配体和细胞特异性方式募集了不同的核心基因。染色质免疫沉淀（CHIP）测定将解决此假设。将检查SINRF-1对E2调节的细胞周期和凋亡蛋白的影响以及细胞周期进入。结果将揭示E2诱导的NRF-1是否在E2的抗凋亡活性中起作用，以及这些反应中是否存在细胞特异性差异。此外，我们将在正常和肿瘤的人乳房标本中检查IHC的NRF-1蛋白表达。 AIM 2中的实验将确定E2和SERM对NRF-1和下游靶基因表达和ER-NRF-1启动子相互作用的影响。 CHIP分析将确定E2是否增加了MCF-7肿瘤异种移植物和小鼠组织中ER1，共激活因子和RNA POL II与NRF-1启动子的直接相互作用，首先关注子宫。 AIM 3是旨在测量基因/蛋白/线粒体活性的“功能结果”，包括O2消耗，MT生物发生，热产生，即E2诱导的NRF-1表达的下游效应。实验将检验以下假设：NRF-1的E2刺激增加将依次增加NRF-1调节基因的表达，例如TFAM，即随后增加其MTDNA所编码的靶基因的表达，例如胞染色体C氧化酶亚基I（MTCO1）和Anti-apapoptototic，cytotrome c氧化酶亚基I（MTCO1）和抗抗原。结果共同定义了雌激素作用协调线粒体功能的分子机制。了解E2和SERM调节线粒体功能如何导致乳腺癌和肺癌的预防和发展的新见解。公共卫生相关性：尽管线粒体基因的表达取决于核基因组功能，并且通过“逆行交流”线粒体活性相互调节核基因的表达，但协同这些事件的机制仍然待阐明。线粒体功能障碍和增加的活性氧介导了衰老，急性神经变性的病理生理机制，由创伤，中风或心脏骤停引起；以及癌症的启动和肿瘤进展。类固醇对线粒体功能的影响“是一个新的新型研究领域”，拟议的研究试图描绘雌激素和抗雌激素/抗雌激素/Serm在调节核呼吸因子1（NRF-1）中的新生物学途径及其在线粒体活动中的下游靶标。