Mechanism of RANTES-mediated Astrocyte Activation

描述（由申请人提供）：现在已经确定，RANTES刺激星形胶质细胞可诱导促炎趋化因子和细胞因子的风暴。这种自我限制的炎症级联反应可能是中枢神经系统内炎症介质产生延长的原因。我们建议研究神经胶质细胞对原型趋化因子RANTES反应的分子基础。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。首先是确定在这个系统中转录的直接早期基因。初步数据表明，RANTES刺激的星形胶质细胞产生TNF-a，而这种细胞因子反过来刺激MCP-1和其他炎症介质的产生。因此，用中和性抗tnf -a抗体处理星形胶质细胞阻断了MCP-1转录物的诱导，而其他趋化因子和细胞因子的表达不受影响。设计了其他Ab阻断实验来确定其他中间因素。来自TNF-a和IL-1 R敲除小鼠的星形胶质细胞单独或与中和抗体结合也将用于研究这一问题。膜TNF及其两种受体的作用也将被检查。

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.