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: 7895911
• 负责人: Carolyn M. Klinge
• 金额: $ 32.78万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7895911
• 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）：尽管线粒体基因表达取决于核基因组功能并且线粒体的相互“逆行通讯”调节核基因表达，但协调这些事件的机制仍有待阐明。线粒体是雌激素作用的目标。然而，雌激素对线粒体功能影响的机制尚未确定。该项目的总体目标是确定雌二醇 (E2) 和选择性雌激素受体 (ER) 调节剂 (SERM) 调节核呼吸因子 1 (NRF-1) 转录的机制以及下游影响NRF-1 对雌激素和 SERM 调节正常细胞和肿瘤细胞中的线粒体活性。 NRF-1 是一种转录因子，通过启动核编码线粒体 DNA (mtDNA) 特异性转录因子（包括 Tfam（mtDNA 维持因子））的转录，对于整合核线粒体功能至关重要。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。在目标 1 中，我们将测试以下假设：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招募不同的共调节子。染色质免疫沉淀 (ChIP) 检测将解决这一假设。将检查 siNRF-1 对 E2 调节的细胞周期和凋亡蛋白以及细胞周期进入的影响。结果将揭示 E2 诱导的 NRF-1 是否在 E2 的抗凋亡活性中发挥作用，以及这些反应是否存在细胞特异性差异。此外，我们将通过 IHC 检查正常和肿瘤性人类乳腺标本中 NRF-1 蛋白的表达。目标 2 中的实验将确定 E2 和 SERM 对 NRF-1 和下游靶基因表达以及体内 ER-NRF-1 启动子相互作用的影响。 ChIP 测定将确定 E2 是否会增加 MCF-7 肿瘤异种移植物和小鼠组织中 ER1、共激活剂和 RNA pol II 与 NRF-1 启动子的直接相互作用，首先关注子宫。目标 3 是“功能结果”，旨在测量基因/蛋白质/线粒体活性，包括 O2 消耗、mt 生物发生、热量产生，即 E2 诱导的 NRF-1 表达的下游效应。实验将检验以下假设：E2 刺激的 NRF-1 增加反过来会增加 NRF-1 调节基因（例如 TFAM）的表达，随后增加其 mtDNA 编码的靶基因（例如细胞色素 c）的表达氧化酶亚基 I (MTCO1) 和抗凋亡基因，例如 survivn。这些结果将共同确定雌激素作用协调线粒体功能的分子机制。了解 E2 和 SERM 如何调节线粒体功能可能会给乳腺癌和肺癌的预防和进展带来新的见解。公共卫生相关性：尽管线粒体基因表达取决于核基因组功能，并且线粒体活动通过“逆行通讯”相互调节核基因表达，但协调这些事件的机制仍有待阐明。线粒体功能障碍和活性氧增加介导衰老、创伤、中风或心脏骤停引起的急性神经变性的病理生理机制；以及癌症发生和肿瘤进展。类固醇对线粒体功能的影响“是一个新的研究领域”，拟议的研究旨在描绘雌激素和抗雌激素/SERM 在调节核呼吸因子-1 (NRF-1) 及其下游靶标方面的新生物学途径在线粒体活性中。