DNA sequences impact estrogen and antiestrogen activity

描述(申请人提供)：尽管线粒体基因的表达依赖于核基因组功能，线粒体的相互“逆行通讯”调节核基因的表达，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靶点。然而，雌激素对线粒体功能的影响机制尚未明确。本项目的总体目标是确定雌激素(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(S)，以及NRF-1对雌激素和SERM调节的正常细胞和肿瘤细胞线粒体活性的下游影响。NRF-1是一种转录因子，通过启动核编码的线粒体DNA(MtDNA)特异性转录因子包括Tfam(mtDNA维护因子)的转录，对整合核-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)的激活诱导MCF-7乳腺癌细胞NRF-1的转录。在目标1中，我们将检验这样的假设，即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的方式招募不同的协同调节因子。染色质免疫沉淀(CHIP)分析将解决这一假设。我们将研究siNRF-1对E2调节的细胞周期、凋亡蛋白和细胞周期进入的影响。这些结果将揭示E2诱导的NRF-1是否在E2‘S抗细胞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，我们还将通过免疫组织化学方法检测正常和肿瘤人乳腺标本中NRF-1蛋白的表达。Aim 2的实验将确定E2和SERM对NRF-1及其下游靶基因表达的影响，以及体内ER-NRF-1启动子的相互作用。芯片分析将确定在MCF-7肿瘤移植瘤和小鼠组织中，E2是否增加了ER1、辅活化子和RNA polII与NRF-1启动子在体内的直接相互作用，首先集中在子宫。目的3是功能结果，目的是测量基因/蛋白质/线粒体的活性，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。实验将验证这一假设，即E2刺激的NRF-1的增加反过来将增加NRF-1调节的基因的表达，例如TFAM，从而增加其mtDNA编码的靶基因的表达，例如细胞色素C氧化酶I(MTCO1)和抗凋亡基因，例如Survivin。总之，这些结果将确定雌激素作用协调线粒体功能的分子机制。了解E2和SERM是如何调节线粒体功能的，可能会对乳腺癌和肺癌的预防和进展有新的见解。公共卫生相关性：尽管线粒体基因表达依赖于核基因组功能，并通过“逆行通讯”相互作用调节核基因表达，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的增加介导了衰老、创伤、中风或心脏骤停引起的急性神经变性以及癌症的启动和肿瘤进展的病理生理机制。类固醇对线粒体功能的影响是一个新的研究领域，这项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)及其下游靶点线粒体活性方面的新生物途径。

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.