Mechanism of RANTES-mediated Astrocyte Activation

描述(申请人提供)：现已证实RANTES刺激星形胶质细胞可诱导大量促炎趋化因子和细胞因子的产生。这种自我限制的炎性级联反应可能是导致中枢神经系统内炎性介质产生时间延长的原因。我们建议研究神经胶质细胞对原型趋化因子RANTES的反应的分子基础。具体地说，我们将定义RANTES介导的星形胶质细胞激活的主要信号要求。该提案列出了三个具体目标。第一个是识别在这个系统中转录的即刻早期基因。初步数据表明，RANTES刺激的星形胶质细胞产生肿瘤坏死因子-α，这种细胞因子反过来刺激MCP-1的产生，可能还有其他炎症介质的产生。因此，用中和的抗肿瘤坏死因子-α抗体处理星形胶质细胞可以阻止MCP-1转录本的诱导，而其他趋化因子和细胞因子的表达没有受到影响。此外，还设计了抗体阻断实验，以确定其他中介因素。来自肿瘤坏死因子-α和白介素1受体基因敲除小鼠的星形胶质细胞也将单独或与中和抗体联合使用来研究这一问题。膜上的肿瘤坏死因子及其两种受体的作用也将被检测。 目的#2重点研究p90RSK在信号转导中的作用。转染显性阴性RSK突变体的星形胶质细胞不能转录趋化因子启动子-荧光素酶结构，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激酶的激活会导致RSK的磷酸化、激活和核转位。初步数据支持这一假设，但还有几个问题有待解决，包括识别额外的上游信号组件。RANTES诱导的信号通路的组织特异性将在星形胶质细胞和小胶质细胞之间进行比较。 第三个目标是确定控制星形胶质细胞中趋化因子转录的因素。通过突变用于驱动荧光素酶报告的趋化因子启动子，我们证明了一个核因子-kB位点是诱导转录活性的关键。另外还设计了突变实验来鉴定其他启动子元件，特别是那些被RSK激活的启动子元件。综上所述，拟议中的实验应该能够深入了解趋化因子在胶质生物学中的作用，并扩大我们对趋化因子生物学不断拓宽的领域的视野。

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.