Mechanism of RANTES-mediated Astrocyte Activation

描述（由申请人提供）：现已确定，用RANTES刺激星形胶质细胞可诱导促炎趋化因子和细胞因子风暴。这种自限性炎症级联反应可能是延长中枢神经系统内炎症介质产生的原因。我们建议检查胶质细胞反应的原型趋化因子，RANTES的分子基础。具体来说，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。第一个是鉴定在该系统中转录的立即早期基因。初步数据表明，RANTES刺激星形胶质细胞产生TNF-α，这种细胞因子反过来刺激MCP-1和可能其他炎症介质的产生。因此，用中和性抗TNF-α Ab处理星形胶质细胞阻断了MCP-1转录物的诱导，而其它趋化因子和细胞因子的表达保持不受影响。设计额外的Ab阻断实验以鉴定其他中间因素。单独或与中和抗体组合的源自TNF-α和IL-1 R敲除小鼠的星形胶质细胞也将用于检查该问题。膜TNF和它的两个受体的作用也将被检查。 目的#2关注p90 RSK在信号传导中的作用。显性负性RSK突变体转染的星形胶质细胞未能转录趋化因子启动子-荧光素酶构建体，表明RSK激酶在调节RANTES信号转导途径中的主要作用。我们的工作假设是MAP激酶的激活导致RSK磷酸化、激活和核转位。初步数据支持这一假设，但留下几个问题，包括确定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。 第三个目标是确定星形胶质细胞中控制趋化因子转录的因子。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-κ B位点对于诱导转录活性是至关重要的。设计另外的诱变实验以鉴定其它启动子元件，特别是被RSK激活的那些。总之，拟议的实验应该提供深入了解趋化因子在神经胶质生物学中的作用，并扩大我们的视野，扩大趋化因子生物学领域。

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.