Regulation of NFkappaB activity by the Estrogen Receptor

雌激素受体对 NFkappaB 活性的调节

• 批准号: 7574370
• 负责人: Kendall W Nettles
• 金额: $ 38.18万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7574370
• 关键词: Agonist; Anti-Inflammatory Agents; Anti-inflammatory; Area; Arthritis; Atherosclerosis; Binding; Biological Assay; Breast; CCL2 gene; Cell Line; Cells; Chemicals; Chemistry; Complex; Crystallization; Crystallography; DNA Binding; Data; Development; Disease; Estrogen Receptors; Estrogen receptor positive; Estrogens; Genes; Genome; Goals; Hormones; IL8 gene; Inflammation Mediators; Inflammatory; Inflammatory Bowel Diseases; Interleukin-6; JUN gene; Ligand Binding Domain; Ligands; MCF7 cell; Macrophage Inflammatory Protein-1; Measures; Mediating; Molecular; Molecular Conformation; Molecular Machines; NF-kappa B; Nuclear Receptors; Pathway interactions; Prevention; Proteins; RNA Interference; Receptor Cross-Talk; Recruitment Activity; Regulation; Reporting; Response Elements; Robotics; Screening procedure; Sepsis; Signal Pathway; Signal Transduction; Small Interfering RNA; Specificity; Structure; TNF gene; Techniques; Testing; Therapeutic; Tissues; Transcription Coactivator; Work; base; bone; chromatin immunoprecipitation; cofactor; improved; lipid metabolism; macrophage; novel; p65; pharmacophore; protective effect; protein complex; receptor; receptor binding; research study; response; transcription factor; xenoestrogen

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.

描述（由申请人提供）：该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，雌激素受体（ER）通过该途径降低关键转录因子NF-B的作用。ER抑制NFB对于雌激素在炎症性肠病、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，突出了该途径的广泛重要性。我们的假设是，调节NF-B的ER配体诱导ER的独特构象，促进与基因特异性蛋白质复合物的结合，诱导基因选择性辅助调节因子和转录因子的置换。这一假设将在以下具体目标中得到实现：具体目标1。为了研究CBP和Grip 1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-α基因的募集，并测定靶向CBP和Grip 1的RNA干扰对相同基因表达的影响。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/转录因子的位移是一种基因特异性现象，和其他贡献的转录因子，如c-jun，决定ER是否与CBP或GRIP 1竞争。我们还假设，有配体药效团是选择性的CBP与GRIP 1特定的炎症基因，我们建议测试与我们的大组NF-B选择性ER配体的位移。具体目标2.为了确定ER-配体复合物的结构和化学特征，介导NF-B途径的选择性，我们将使用X射线晶体学表征具有途径选择性配体的受体的结构。我们建议，路径选择性配体诱导的受体，这是从激动剂构象不同的独特构象。我们最近开发了一种新的技术，加速了几个数量级的核受体配体结合域的结晶，使我们能够表征整个类的受体配体复合物，因为我们在这里提出。使用我们的基于机器人细胞的筛选核心，我们已经鉴定了与ER相互作用的辅因子簇，所述辅因子簇与对NF-B或雌激素反应元件信号传导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筛选所需的ER与NF-B的串扰的基因，提供了一个公正的方法来定义组件的阻遏复合物。这些实验的目的是了解配体化学的微妙变化如何改变受体结构和相关的蛋白质复合物，以影响通过雌激素受体的信号特异性。这些领域的进展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。由于该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疗法的发展。该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，通过该途径雌激素降低炎症介质NF-κ B的关键细胞蛋白的作用。我们已经产生了许多合成雌激素，抑制NF κ B炎症通路，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。