NOVEL PATHWAYS OF ENDOTOXIN SIGNALING

描述：(改编自申请者摘要)：革兰氏阴性败血症 和感染性休克是存活患者的常见死亡原因 创伤和烧伤。革兰氏阴性菌对巨噬细胞的刺激作用 细菌脂多糖(LPS)负责启动一些 导致感染性休克的细胞反应。内毒素刺激激活 几条细胞内信号转导通路可诱导 促炎细胞因子的产生。这些细胞因子，尤其是肿瘤 肿瘤坏死因子是感染性休克的主要介质。在……里面 巨噬细胞感染HIV-1，内毒素刺激也激活病毒基因 表达和复制。调查人员发现，内毒素 刺激导致酪蛋白激酶II(CKII)的激活 转录因子PU.1，以前未知的分子 参与内毒素诱导的反应。依次需要CKII和PU.1 以最大限度地表达HIV-1和肿瘤坏死因子启动子对内毒素的反应。 研究人员的研究表明，内毒素刺激迅速 上调CKII的酶活性并诱导CKII易位 从胞浆到细胞核。CKII的核移位代表着一种 CKII选择性磷酸化核的独特机制 脂多糖刺激后的底物。CKII的一种底物是PU.1，a 原癌基因反式激活因子ETS家族的成员。这个 研究人员发现，CKII对PU1的磷酸化上调了其 反式激活功能，但不具有DNA结合活性。而当 已知转录因子核因子-kB在内毒素应答中起重要作用 HIV-1长末端重复序列和肿瘤坏死因子启动子的研究人员 发现核因子-kB和PU1都是最大限度表达所必需的。遗传 研究表明PU.1通过与HIV-LTR结合来激活HIV-LTR转录 病毒LTR中的核因子-kB基序。这种效应是启动子特有的。 因为PU.1不能激活所有依赖于NF-kB的启动子。因此，在那里 似乎是NF-kB基序的一个子集，可以与PU.1相互作用。多数 已确定内毒素诱导的必需核因子-kB基序的研究 基因还没有考虑到其他因素可以结合的可能性 活体中的这些模体。研究人员假设内毒素是可诱导的 HIV-LTR以及肿瘤坏死因子和其他基因的表达受 PU1与核因子-kB基序的结合。这样做的长期目标是 该项目旨在确定CKII和PU.1如何调节HIV-1的生长和 产生调节炎症和感染性休克的细胞因子。这个 具体目标是(1)定义控制CKII激活的机制 和内毒素刺激下的核移位，(2)确定哪一个 内毒素诱导的巨噬细胞反应由PU1和CKII介导，(3) 确定PU1上哪些CKII磷酸化位点是启动子特异性的 对该转录因子的识别，以及(4)通过以下方式确定其机制 其中PU.1利用核因子-kB基序激活转录。

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.