NOVEL PATHWAYS OF ENDOTOXIN SIGNALING

描述：（改编自申请人摘要）：革兰氏阴性败血症 和感染性休克是存活患者的常见死因 创伤和烧伤。 革兰氏阴性杆菌对巨噬细胞的刺激作用 细菌脂多糖（LPS）负责启动一些 导致败血性休克的细胞反应 LPS刺激激活 几种细胞内信号转导途径， 促炎细胞因子产生。 这些细胞因子，尤其是肿瘤细胞因子， 坏死因子（TNF）是败血性休克的主要介质。 在 感染HIV-1的巨噬细胞，LPS刺激也激活病毒基因 表达和复制。 调查人员发现LPS 刺激导致酪蛋白激酶II（CKII）的激活， 转录因子PU.1，以前不知道的分子， 参与LPS诱导的反应。 需要CKII和PU.1 用于响应LPS从HIV-1和TNF启动子的最大表达。 研究人员的研究表明，LPS刺激迅速 上调CKII的酶活性并诱导CKII易位 从细胞质到细胞核。 CKII的核转位代表了 CKII可以选择性磷酸化核的独特机制 LPS刺激后的底物。 CKII的一种底物是PU.1， 原癌基因反式激活因子Ets家族成员。 的 研究人员发现，由CKII引起的PU.1磷酸化上调了其 反式激活功能，但不是其DNA结合活性。 而 已知转录因子NF-κ B对于LPS-应答性是必需的 HIV-1长末端重复序列（LTR）和TNF启动子， 发现NF-kB和PU.1都是最大表达所必需的。 遗传 研究表明，PU.1通过与HIV-LTR结合激活HIV-LTR转录， 病毒LTR内的NF-κ B基序。 这种效应是启动子特异性的 因为PU.1不能激活所有NF-κ B依赖性启动子。 因此 似乎是NF-kB基序的一个子集，可以与PU.1相互作用。 最 研究已经确定了LPS诱导的NF-kB基序中的必需NF-kB基序， 基因还没有考虑到其他因子可以结合到 这些图案在体内。 研究者假设LPS诱导的 HIV-LTR以及TNF和其他基因的表达受到以下因素的调节： PU. 1与NF-κ B基序的结合。 这一长期目标 该项目的目的是确定CKII和PU.1如何调节HIV-1的生长和 介导炎症和败血性休克的细胞因子的产生。 的 具体目标是：（1）确定控制CKII激活的机制 LPS刺激后的核转位，（2）确定 LPS诱导的巨噬细胞反应由PU.1和CKII介导，（3） 确定PU.1上的哪些CKII磷酸化位点赋予启动子特异性 识别该转录因子，和（4）确定机制， 该PU.1利用NF-κ B基序来激活转录。

DESCRIPTION: (adapted from applicant's abstract): Gram-negative septicemia and septic shock are common causes of death in patients who survive traumatic and burn injuries. Stimulation of macrophages by gram-negative bacterial lipopolysaccharide (LPS) is responsible for initiating some of the cellular responses that lead to septic shock. LPS stimulation activates several intracellular signal transduction pathways that induce pro-inflammatory cytokine production. These cytokines, most notably tumor necrosis factor (TNF), are the primary mediators of septic shock. In macrophages infected with HIV-1, LPS stimulation also activates viral gene expression and replication. The investigator has discovered that LPS stimulation leads to the activation of casein kinase II (CKII) and the transcription factor PU.1, molecules which were not previously known to participate in LPS-inducible responses. In turn CKII and PU.1 are required for maximal expression from the HIV-1 and TNF promoters in response to LPS. The investigator's studies have shown that LPS stimulation rapidly upregulates the enzymatic activity of CKII and induces CKII translocation from the cytosol to the nucleus. Nuclear translocation of CKII represents a unique mechanism by which CKII can selectively phosphorylate nuclear substrates following LPS stimulation. One substrate for CKII is PU.1, a member of the Ets family of proto-oncogene transactivating factors. The investigator discovered that PU.1 phosphorylation by CKII upregulates its trans-activation function, but not its DNA-binding activity. While the transcription factor NF-kB is known to be essential for LPS-responsiveness of the HIV-1 long terminal repeat (LTR) and TNF promoter, the investigator found that both NF-kB and PU.1 are required for maximal expression. Genetic studies demonstrate that PU.1 activates HIV-LTR transcription by binding to NF-kB motifs within the viral LTR. This effect was promoter-specific because PU.1 did not activate all NF-kB-dependent promoters. Thus, there appears to be a subset of NF-kB motifs that can interact with PU.1. Most studies which have identified essential NF-kB motifs within LPS-inducible genes have not considered the possibility that other factors can bind to these motifs in vivo. The investigator hypothesizes that LPS-inducible expression of the HIV-LTR, as well as TNF and other genes, is regulated by the binding of PU.1 to NF-kB motifs. The long term objective of this project is to determine how CKII and PU.1 regulate both HIV-1 growth and the production of cytokines that mediate inflammation and septic shock. The specific aims are to (1) define the mechanisms that control CKII activation and nuclear translocation upon LPS stimulation, (2) determine which LPS-inducible macrophage responses are mediated by PU.1 and CKII, (3) determine which CKII phosphorylation sites on PU.1 confer promotor-specific recognition on this transcription factor, and (4) determine the mechanism by which PU.1 utilizes NF-kB motifs to activate transcription.