Regulation of NFkappaB activity by the Estrogen Receptor

描述(由申请人提供)：该项目的总体目标是通过靶向雌激素受体(ER)减少关键转录因子NF-B的作用的新途径来改善炎症性疾病的预防和治疗。雌激素抑制NFB在炎症性肠病、脓毒症、关节炎、动脉粥样硬化和脂质代谢中的保护作用至关重要，这突显了这一途径的广泛重要性。我们的假设是，调节核因子-B的ER配体诱导内质网的独特构象，促进与基因特异性蛋白复合体的结合，诱导基因选择性辅助调节因子和转录因子的移位。为了研究CBP和Grig1在ER介导的抑制NF-B所需的分子相互作用中的重要性，我们将通过染色质免疫沉淀来测量它们对MCP-1、IL-6、IL-8、MIP-1和TNF-a基因的募集，并分析针对CBP和Grig1的RNA干扰对这些基因表达的影响。我们还将研究ER靶组织中的这些相互作用，包括乳腺、子宫、骨和巨噬细胞。我们认为，共激活因子/转录因子置换是一种基因特有的现象，而其他起作用的转录因子，如c-jun，决定了ER是否与CBP或GRIP1竞争。我们还假设有配体药效团选择性地将CBP与Grig1置换到特定的炎症基因，我们建议用我们的大组NF-B选择性ER配体来测试这一点。具体目的2.为了确定介导核因子-B途径选择性的内质网配体复合体的结构和化学特征，我们将使用X射线结晶学来表征具有途径选择性配体的受体的结构。我们认为，途径选择性配体诱导受体的独特构象不同于激动剂构象。我们最近开发了一种新的技术，可以将核受体配体结合域的结晶加速几个数量级，使我们能够表征整个类别的受体配体复合体，就像我们在这里建议的那样。使用我们基于机器人细胞的筛选核心，我们已经确定了与ER相互作用的辅因子簇，这些辅因子与对核因子-B或雌激素反应元件信号具有选择性的化合物结合。我们还提出了一个基因组规模的siRNA筛选，以寻找ER与核因子-B相互作用所需的基因，提供了一种公正的方法来定义抑制复合体的成分。这些实验的目的是了解配体化学中的细微变化如何改变受体结构和相关的蛋白质复合体，以通过雌激素受体影响信号特异性。每一个领域的进展都将显著提高我们对核受体如何作为分子机器实现信号特异性的理解。由于这一途径在炎症性疾病中的关键重要性，这项工作也有很大的潜力直接影响改进的治疗方法的发展。本项目的总体目标是通过靶向一种新的途径来改善炎症性疾病的预防和治疗，通过这一途径，雌激素可以减少作为炎症介质的关键细胞蛋白--核因子-kappaB的作用。我们已经产生了一些合成雌激素，这些雌激素抑制NFkappaB炎症途径，而不刺激细胞中的其他雌激素反应。我们建议使用这些合成的化合物来了解这一重要抗炎途径的分子细节。

DESCRIPTION (provided by applicant): The overall goal of this project is to improve the prevention and treatment of inflammatory disease through targeting a novel pathway by which the estrogen receptor (ER) reduces the action of a key transcription factor, NF-B. ER suppression of NFB is critical for protective effects of estrogens in inflammatory bowel disease, sepsis, arthritis, atherosclerosis, and lipid metabolism, highlighting the broad importance of this pathway. Our hypothesis is that ER ligands that modulate NF-B induce a unique conformation of ER, facilitating association with gene-specific protein complexes, inducing displacement of gene-selective coregulators and transcription factors. This hypothesis will be pursued in the following Specific Aims: SPECIFIC AIM 1. To investigate the importance of CBP and Grip1 in the molecular interactions required for ER-mediated suppression of NF-B, we will measure their recruitment to the MCP-1, IL-6, IL-8, MIP-1 and TNF-a genes by chromatin immunoprecipitation, and assay the effects of RNA interference targeting CBP and Grip1 on expression of the same genes. We will also examine these interactions in ER target tissues, including breast, uterine, bone, and macrophage cells. We propose that coactivator/transcription factor displacement is a gene specific phenomenon, and that other contributing transcription factors, such as c-jun, determine whether ER competes with CBP or Grip1. We also hypothesize that there are ligand pharmacophores that are selective for displacement of CBP versus Grip1 to specific inflammatory genes, which we propose to test with our large sets of NF-B selective ER ligands. SPECIFIC AIM 2. To define the structural and chemical features of ER-ligand complexes that mediate selectivity for the NF-B pathway, we will characterize the structure of the receptor with pathway-selective ligands, using x-ray crystallography. We propose that pathway-selective ligands induce a unique conformation of the receptor that is distinct from the agonist conformation. We recently developed a novel technique that accelerates the crystallization of the nuclear receptor ligand-binding domain by several orders of magnitude, allowing us to characterize entire classes of receptor ligand complexes, as we propose here. Using our robotic cell based screening core, we have identified clusters of cofactors that interact with ER bound to compounds that are selective for NF-B or estrogen response element signaling. We also propose a genome scale siRNA screen for genes required for ER cross-talk with NF-B, providing an unbiased approach to define components of the repressive complex. The goal of these experiments is to understand how subtle changes in ligand chemistry alter receptor structure, and associated protein complexes, to effect signaling specificity through the estrogen receptor. Advances in each of these areas will significantly improve our understanding of how nuclear receptors act as molecular machines to achieve signaling specificity. Due to the critical importance of this pathway in inflammatory disease, this work also has great potential to directly impact the development of improved therapeutics.The overall goal of this project is to improve the prevention and treatment of inflammatory disease through targeting a novel pathway by which the estrogen hormones reduces the action of a key cellular protein that is a mediator of inflammation, NF-kappaB. We have generated a number of synthetic estrogens that inhibit the NFkappaB inflammatory pathway without stimulating other estrogenic responses in the cell. We propose to use these synthetic compounds to understand the molecular details of this important anti-inflammatory pathway.