Mechanism of RANTES-mediated Astrocyte Activation

RANTES介导的星形胶质细胞激活机制

• 批准号: 7025798
• 负责人: MARTIN E DORF
• 金额: $ 35.38万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7025798
• 关键词: antibody; astrocytes; biological signal transduction; cAMP response element binding protein; chemokine; cyclic AMP; cytokine receptors; gene targeting; genetically modified animals; granulocyte; immunoregulation; inflammation; interleukin 1; laboratory mouse; leukocyte activation /transformation; monocyte; nuclear factor kappa beta; phosphorylation; protein kinase A; regulatory gene; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): It is now established that stimulation of astrocytes with RANTES induces a storm of proinflammatory chemokines and cytokines. This self-limiting inflammatory cascade may be responsible for prolonging production of inflammatory mediators within the central nervous system. We propose to examine the molecular basis for glial cell responses to the prototype chemokine, RANTES. Specifically, we will define the major signaling requirements for RANTES-mediated astrocyte activation. The proposal lists three specific Aims. The first is to identify the immediate early genes transcribed in this system. Preliminary data suggest that RANTES stimulated astrocytes produce TNF-a and that this cytokine in turn stimulates production of MCP-1 and perhaps other inflammatory mediators. Thus treatment of astrocytes with neutralizing anti-TNF-a Ab blocked induction of MCP-1 transcripts while expression of other chemokines and cytokines remained unaffected. Additional Ab blocking experiments are designed to identify other intermediary factors. Astrocytes derived from TNF-a and IL-1 R knockout mice alone or in combination with neutralizing antibodies will also be used to examine this issue. The role of membrane TNF and its two receptors will also be examined. Aim #2 focuses on the role of p90RSK in signaling. Astrocytes transfected with a dominant negative RSK mutant failed to transcribe a chemokine promoter-luciferase construct indicating a major role for the RSK kinase in regulating the RANTES signal transduction pathway. Our working hypothesis is that activation of a MAP kinase results in RSK phosphorylation, activation, and nuclear translocation. Preliminary data support this hypothesis but leave several questions open including identifying additional upstream signaling components. The tissue specificity of the RANTES-induced signaling pathway will be compared between astrocytes and microglia. The third Aim focuses on defining the factors controlling transcription of chemokines in astrocytes. Using mutagenesis of the chemokine promoter used to drive a luciferase reporter we demonstrated one NF-kB site was critical for inducing transcriptional activity. Additional mutagenesis experiments are designed to identify other promoter elements especially those activated by RSK. In summary, the proposed experiments should provide insights into the role of chemokines in glial biology and expand our vision of the broadening field of chemokine biology.

描述（由申请人提供）：现在已经确定，用RANTES刺激星形胶质细胞会诱导促炎性趋化因子和细胞因子的风暴。这种自限制的炎症性级联反应可能导致中枢神经系统内延长炎症介体的产生。我们建议检查对原型趋化因子rantes的神经胶质细胞反应的分子基础。具体而言，我们将定义RANTES介导的星形胶质细胞激活的主要信号传导要求。 该提案列出了三个具体目标。首先是确定该系统中转录的直接早期基因。初步数据表明，RANTES刺激星形胶质细胞产生TNF-A，而这种细胞因子反过来刺激了MCP-1以及其他炎症介质的产生。因此，用中和抗TNF-A AB对星形胶质细胞进行处理，封闭了MCP-1转录物的诱导，而其他趋化因子和细胞因子的表达仍然不受影响。其他AB阻塞实验旨在识别其他中间因素。单独或与中和抗体结合使用TNF-A和IL-1 R基因敲除小鼠的星形胶质细胞也将用于检查此问题。还将检查膜TNF及其两个受体的作用。 AIM＃2着重于P90RSK在信号传导中的作用。用显性负RSK突变体转染的星形胶质细胞未能转录趋化因子启动子 - 卢西酶构建体，这表明RSK激酶在调节RANTES信号转导途径中起主要作用。我们的工作假设是，MAP激酶的激活导致RSK磷酸化，激活和核易位。初步数据支持这一假设，但留下了几个问题，包括识别其他上游信号传导组件。在星形胶质细胞和小胶质细胞之间将比较兰特斯诱导的信号通路的组织特异性。 第三个目的是定义控制星形胶质细胞中趋化因子转录的因素。使用用于驱动荧光素酶报告基因的趋化因子启动子的诱变，我们证明了一个NF-KB位点对于诱导转录活性至关重要。其他诱变实验旨在识别其他启动子元素，尤其是RSK激活的启动子元素。总而言之，提出的实验应提供有关趋化因子在神经胶质生物学中的作用的见解，并扩大我们对趋化因子生物学拓宽领域的看法。