NUCLEAR INTEGRATION OF ENDOTHELIAL SIGNALS

内皮信号的核整合

• 批准号: 6327719
• 负责人: Tucker O Collins
• 金额: $ 32.32万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6327719
• 关键词: cAMP response element binding protein; calcium binding protein; gel mobility shift assay; gene induction /repression; genetic promoter element; genetic regulation; genetic transcription; genetically modified animals; human tissue; immunocytochemistry; intermolecular interaction; laboratory mouse; leukocyte adhesion molecules; nuclear factor kappa beta; phosphoprotein phosphatase; selectins; tissue /cell culture; transcription factor; transfection; vascular endothelium

Expression of endothelial-leukocyte adhesion molecule is dramatically induced by inflammatory cytokines. This increased expression occurs at the transcriptional level and requires activation of the nuclear factor-kappaB (NF-kB) system. Recent studies indicate that the p65 component of NF-kB, like many signal dependent transcriptional activators, interacts with the transcriptional co-activator CREB-binding protein (CBP). In this continuing project, we will investigate whether CBP is part of a novel level of nuclear regulation linking diverse signaling systems in endothelial cells and test the hypothesis that competition for limiting amounts of CBP facilitates the activation of some signal dependent genes at the expense of others. In the renewal period we propose to determine if this regulatory system is relevant to the process of endothelial activation during inflammatory responses. In Specific Aim #1, the antagonistic interactions between p65 and other signal-dependent transcription factors occur because for limiting amounts of the common transcriptional co-activator, CBP. These studies will examine the levels of CBP expression in quiescent and cytokine-activated cultured endothelial cells, determine if levels of the co-activator limit NF-kappaB dependent gene expression, and will explore the molecular basis of the antagonistic interaction. In Specific Aim #2, the pathophysiologic consequences of CBP over- expression in endothelial cells will be examined in a transgenic model of endothelial activation. In this defined genetic context, we will determine if CBP over expression increases induction of NF-kappaB dependent genes and diminishes the negative regulatory effects of the activated nuclear receptors on the induction of kappaB-dependent genes. Like the induction process, decreased expression of the endothelial adhesion molecules following cytokine exposure is an active process that occurs at the transcriptional level. This level of negative regulation my be key in preventing inappropriate or prolonged expression of some adhesion molecule genes. In Specific Aim #3, the mechanisms responsible for the post-induction transcriptional repression of E-selectin will continue to be investigated. These studies will determine the role of the dual specificity phosphatases in limiting the expression of this adhesion molecule gene using tools developed in this project, as well as unique cellular and animal reagents developed by collaborators. Collectively, the findings from these studies should provide insights into two new endothelial regulatory systems: first, they should indicate if CBP serves an integration function for interactions between p65 and other classes of CBP dependent transcription factors during cytokine-induced gene expression; and second, they should define the mechanisms which tightly control the post-induction transcriptional repression of the E- selectin gene.

内皮细胞-白细胞粘附分子的表达显着 由炎症细胞因子诱导。这种增加的表达发生在 转录水平，需要激活核因子-kappaB （NF-kB）系统。最近的研究表明 NF-kB 的 p65 成分， 像许多信号依赖性转录激活剂一样，与 转录共激活因子 CREB ​​结合蛋白 (CBP)。在这个 继续项目，我们将调查 CBP 是否是小说的一部分 连接不同信号系统的核调控水平 内皮细胞并检验限制竞争的假设 一定量的 CBP 有助于激活一些信号依赖性基因 以牺牲他人为代价。在更新期间，我们建议确定是否 该调节系统与内皮细胞的过程有关 炎症反应过程中的激活。 在具体目标 #1 中，p65 与其他蛋白之间的拮抗相互作用 信号依赖性转录因子的出现是因为数量有限 常见的转录辅激活因子 CBP。这些研究将 检查静态和细胞因子激活状态下 CBP 表达水平 培养的内皮细胞，确定共激活剂的水平是否限制 NF-kappaB依赖的基因表达，并将探讨其分子基础 的拮抗相互作用。 在具体目标#2 中，CBP 过度的病理生理后果 在内皮细胞中的表达将在转基因模型中进行检查 内皮激活。在这个定义的遗传背景下，我们将确定 如果 CBP 过度表达会增加 NF-κB 依赖性基因的诱导 并减少激活核的负面调节作用 受体对 kappaB 依赖性基因的诱导。 与诱导过程一样，内皮细胞表达减少 细胞因子暴露后的粘附分子是一个活跃的过程 发生在转录水平。这种程度的负面监管我的 是防止某些不当或长期表达的关键 粘附分子基因。 在具体目标#3中，负责诱导后的机制 E-选择素的转录抑制将继续进行研究。 这些研究将确定双特异性磷酸酶的作用 使用工具限制该粘附分子基因的表达 该项目中开发的以及独特的细胞和动物试剂 由合作者开发。 总的来说，这些研究的结果应该提供以下见解： 两个新的内皮调节系统：首先，它们应该表明 CBP 是否 为 p65 和其他物质之间的相互作用提供整合功能 细胞因子诱导过程中 CBP 依赖性转录因子的类别 基因表达；其次，他们应该定义机制 严格控制E-的诱导后转录抑制 选择素基因。