Estrogen Receptor-Coregulator Functional Interactions

描述（由申请人提供）：经典雌激素受体ER 1是一种配体调节的转录因子，对正常发育、稳态和生殖很重要，也有助于病理过程，如乳腺癌。人们普遍认为，辅激活因子有助于ER 1活性的正向调节，而辅阻遏因子对于ER与拮抗剂如4-羟基他莫昔芬（4-hydroxytamoxifen，4 HT）结合的负向调节很重要;后者的相互作用对于确定选择性雌激素受体调节剂（SERM）如4 HT的相对拮抗剂活性很重要。然而，有很少的信息辅抑制剂调节ER 1功能的药理学药物的情况下的机制，我们最近的研究结果挑战了广泛接受的观点，辅抑制剂SMRT和NCoR发挥其作用，只有在ER 1结合药理学抗雌激素。事实上，最近的证据表明，SMRT对雌激素刺激的ER 1依赖性基因表达的基因选择性要求，并表明我们对ER 1的“辅阻遏物”调节的想法需要改变。因此，本提案的总体目标是全面评估的辅阻遏物SMRT和NCoR的作用，其调节ER 1的转录活性的激动剂和拮抗剂配体的存在下，并评估其在这些情况下的作用机制。我们计划的研究基于四个关键观察。首先，辅阻遏物与ER 1结合不需要拮抗剂，辅阻遏物与ER 1靶基因相互作用也不需要拮抗剂。其次，SMRT过表达可增强雌二醇（E2）刺激的ER 1活性，而SMRT表达缺失可抑制E2-ER 1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的剪接变体缺乏其第一抑制结构域并在组织和细胞系中表达，其作为共激活因子发挥作用以刺激与E2以及4 HT结合的ER 1的活性。这些研究结果导致的假设，SMRT和NCoR在调节ER 1功能的药理学抗雌激素的情况下发挥重要作用，这些辅阻遏物的生物活性，因此有助于显着的雌激素以及抗雌激素调节ER 1的转录活性的能力，在基因特异性的方式。这一假设将在三个具体目标中得到检验。1)确定SMRT和NCoR在调节ER 1靶基因表达中的生物学作用。2)表征调节ER 1转录活性所需的新型SMRT和NCoR功能相互作用。3)阐明SMRT和NCoR及其相关蛋白对ER 1靶基因表达的正调控和负调控的分子机制。这些研究将利用最先进的技术来调节SMRT 1，SMRT 2剪接变体和NCoR在ER 1作用的细胞模型中的表达，开发用于辅助阻遏物功能分析的新试剂和细胞系，并确定辅助阻遏物控制ER 1作用的分子机制。了解和利用雌激素受体活性的正常和药理学调节对于预防和治疗骨质疏松症和乳腺癌等雌激素敏感性疾病对人类健康具有重要意义。我们的新发现的要求SMRT最大的雌激素刺激ER 1转录活性不符合目前的预期辅阻遏物调节ER 1功能，这强烈表明，我们的理解之间的SMRT/NCoR和ER 1的功能关系的范式转变是必要的。预计更深入地了解辅阻遏物对ER 1功能的复杂控制将为在正常（例如组织选择性）和病理生理学情况（例如乳腺癌）中选择性调节ER 1的机制提供新的见解，并最终为ER为基础的治疗方法的鉴定和表征提供新的方法。

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.