Estrogen Receptor-Coregulator Functional Interactions

描述(申请人提供)：经典的雌激素受体，ER1，是一种配体调节的转录因子，对正常发育、动态平衡和生殖非常重要，也与乳腺癌等病理过程有关。一般认为，共激活子有助于ER1活性的正向调节，而辅阻遏子对与4-羟基三苯氧胺(4HT)等拮抗剂结合的ERs的负性调节起重要作用；后一种相互作用对于确定选择性雌激素受体调节剂(SERM)如4HT的相对拮抗活性至关重要。然而，关于辅抑制子在缺乏药物的情况下调节ER1功能的机制的信息很少，我们最近的结果挑战了广泛接受的观点，即辅抑制子SMRT和NCoR仅对与药理抗雌激素结合的ER1发挥作用。事实上，最近的证据表明，SMRT对雌激素刺激的、依赖于ER1的基因表达具有基因选择性要求，并表明我们需要改变对ER1的辅抑制子调控的想法。因此，本提案的总体目标是全面评估辅抑制子SMRT和NCoR在激动剂和拮抗剂配体存在下对ER1转录活性的调节作用，并评估它们在这些背景下的作用机制。我们计划的研究基于四个关键观察。首先，辅抑制子与ER1结合不需要拮抗剂，辅抑制子与ER1靶基因的相互作用也不需要拮抗剂。第二，SMRT过表达可增强雌激素(E2)刺激的ER1活性，而SMRT表达缺失可抑制E2-ER1活性。第三，SMRT和NCoR可以与有效激活基因表达所需的分子结合。第四，SMRT的一个剪接变异体缺乏其第一个抑制结构域，在组织和细胞系中表达，它作为共激活因子刺激与E2和4HT结合的ER1的活性。这些发现导致假设，在缺乏药理抗雌激素的情况下，SMRT和NCoR在调节ER1功能方面发挥着重要作用，因此这些核心抑制物的生物活性显著有助于雌激素和抗雌激素以基因特异性的方式调节ER1的转录活性。这一假设将在三个具体目标上得到检验。1)确定SMRT和NCoR在调节ER1靶基因表达中的生物学作用。2)鉴定调节ER1转录活性所需的新的SMRT和NCoR功能相互作用。3)阐明SMRT和NCoR及其相关蛋白对ER1靶基因表达的正负调控的分子机制。这些研究将利用最先进的技术来调节SMRT1、SMRT2剪接变异体和NCoR在ER1作用的细胞模型中的表达，开发用于辅阻遏子功能分析的新试剂和细胞系，并确定辅阻遏子控制ER1作用的分子机制。项目简介了解和开发雌激素受体活性的正常和药理学调节对于预防和治疗雌激素敏感的病理如骨质疏松症和乳腺癌对人类健康具有重要意义。我们关于SMRT对ER1转录活性的最大雌激素刺激所需的新发现与目前对ER1功能的辅阻遏子调控的预期不一致，这强烈表明，我们对SMRT/NCoR和ER1之间的功能关系的理解需要发生范式转变。进一步了解辅阻遏子对ER1功能的复杂调控将为在正常(如组织选择性)和病理生理情况(如乳腺癌)中选择性调节ER1的机制提供新的见解，并最终为基于ER的治疗方法的识别和表征提供新的途径。

DESCRIPTION (provided by applicant): The classical estrogen receptor, ER1, is a ligand regulated transcription factor important for normal development, homeostasis and reproduction that also contributes to pathological processes such breast cancer. It is generally accepted that coactivators contribute to positive regulation of ER1 activity, while corepressors are important for negative regulation of ERs bound to antagonists such as 4-hydroxytamoxifen (4HT); the latter interactions are important for determining the relative antagonist activity of selective estrogen receptor modulators (SERMs) such as 4HT. However, there is little information on the mechanisms by which corepressors regulate ER1 function in the absence of pharmacologic agents, and our recent results challenge the widely accepted view that the corepressors SMRT and NCoR exert their effects only on ER1 bound to pharmacological antiestrogens. Indeed, recent evidence points to a gene-selective requirement of SMRT for estrogen-stimulated, ER1-dependent gene expression, and indicates that a change in our thinking about `corepressor' regulation of ER1 is required. Thus, the overall goal of this proposal is to comprehensively evaluate the role of the corepressors SMRT and NCoR with respect to their regulation of ER1 transcriptional activity in the presence of agonist as well as antagonist ligands, and to assess their mechanisms of action in these contexts. Our planned studies are based on four key observations. First, antagonists are not required for corepressors to bind to ER1, nor are they required for corepressor interaction with ER1 target genes. Second, SMRT overexpression can enhance estradiol (E2)-stimulated ER1 activity, while depletion of SMRT expression can inhibit E2-ER1 activity. Third, SMRT and NCoR can bind to molecules required for efficient activation of gene expression. Fourth, a splice variant of SMRT which lacks its first repression domain and is expressed in tissues and cell lines, functions as a coactivator to stimulate the activity of ER1 bound to E2 as well as 4HT. These findings lead to the hypothesis that SMRT and NCoR play important roles in modulating ER1 function in the absence of pharmacological antiestrogens, and that the biological activities of these corepressors therefore contribute significantly to the ability of estrogen as well as antiestrogens to regulate ER1 transcriptional activity in a gene-specific manner. This hypothesis will be tested in three specific aims. 1) Determine the biological roles of SMRT and NCoR in regulating the expression of ER1 target genes. 2) Characterize novel SMRT and NCoR functional interactions required to regulate ER1 transcriptional activity. 3) Elucidate the molecular mechanisms by which SMRT and NCoR and their associated proteins contribute to positive and negative regulation of ER1 target gene expression. These studies will utilize state-of-the-art technologies to regulate the expression of SMRT1, the SMRT2 splice variant and NCoR in cell models of ER1 action, develop new reagents and cell lines for corepressor functional analyses, and determine the molecular mechanisms through which corepressors control ER1 action. Project Narrative Understanding and exploiting normal and pharmacological regulation of estrogen receptor activity is of major significance to human health with respect to both preventing and treating estrogen-sensitive pathologies such as osteoporosis and breast cancer. Our novel findings on the requirement of SMRT for maximal estrogen stimulation of ER1 transcriptional activity are not in line with current expectations regarding corepressor regulation of ER1 function, and this strongly suggests that a paradigm shift in our understanding of the functional relationships between the SMRT/NCoR and ER1 is required. It is anticipated that a greater understanding of the complex control of ER1 function by corepressors will provide novel insights into mechanisms that selectively regulate ER1 in normal (e.g. tissue selectivity) and pathophysiological scenarios (e.g. breast cancer), and ultimately provide new approaches for identification and characterization of ER-based therapeutics.