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）降低关键转录因子NF-B作用的新途径来改善炎性疾病的预防和治疗。内质网抑制NFB对于雌激素在炎症性肠病、败血症、关节炎、动脉粥样硬化和脂质代谢中的保护作用至关重要，突出了这一途径的广泛重要性。我们的假设是，调节NF-B的内质网配体诱导内质网的独特构象，促进与基因特异性蛋白复合物的结合，诱导基因选择性共调节因子和转录因子的位移。这一假设将在以下具体目标中进行：为了研究CBP和Grip1在er介导的NF-B抑制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法测量它们对MCP-1、IL-6、IL-8、MIP-1和TNF-a基因的募集，并检测靶向CBP和Grip1的RNA干扰对这些基因表达的影响。我们还将研究内质网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/转录因子位移是一种基因特异性现象，其他转录因子，如c-jun，决定了ER是否与CBP或Grip1竞争。我们还假设存在选择性地将CBP与Grip1置换到特定炎症基因的配体药物载体，我们建议用我们的大量NF-B选择性ER配体进行测试。具体目标2。为了确定介导NF-B途径选择性的er配体复合物的结构和化学特征，我们将使用x射线晶体学表征途径选择性配体的受体结构。我们提出，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技术，将核受体配体结合域的结晶速度提高了几个数量级，使我们能够表征整个受体配体复合物的类别，正如我们在这里提出的那样。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相互作用的辅助因子簇，这些辅因子结合的化合物对NF-B或雌激素反应元件信号传导具有选择性。我们还提出了一种基因组尺度的siRNA筛选方法，用于筛选内质网与NF-B串扰所需的基因，为确定抑制复合体的成分提供了一种公正的方法。这些实验的目的是了解配体化学的细微变化如何改变受体结构和相关蛋白复合物，从而通过雌激素受体影响信号特异性。这些领域的进展将大大提高我们对核受体如何作为分子机器来实现信号特异性的理解。由于这一途径在炎症性疾病中至关重要，这项工作也有很大的潜力直接影响改进治疗方法的发展。该项目的总体目标是通过靶向雌激素激素降低炎症介质nf - κ b的关键细胞蛋白的作用的新途径来改善炎症疾病的预防和治疗。我们已经合成了一些抑制NFkappaB炎症通路的合成雌激素，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.