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Activation of phospholipiase D in endotoxin signaling : its molecular mechanism and function

内毒素信号传导中磷脂酶D的激活：其分子机制和功能

基本信息

批准号：
11672204
负责人：
NISHIJIMA Masahiro
金额：
$ 2.24万
依托单位：
National Institute of Infectious Diseases
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672204/
关键词：
LPS diacylglycerol hospholipase D RhoA NF-κB Taxol TLR4 MD-2 マクロファージ Toll-like receptor (TLR)NF-κB CD14 リピドA リポ多糖 NF-kB ジアシルグリセロール

项目摘要

We have previously reported that exposure of mouse macrophage-like J774.1 cells to lipopolysaccharide (LPS) induces rapid production of the cellular diacylglycerol (DAG) from phosphatidycholine (PC) mediated by phospholipase D(PLD) / phosphatidate phosphohydrolase (PAP) activity and this DAG production was a crucial step in the NF-κB. activation in J774.1 cells. PLD activity is regulated by small G proteins, protein kinase C and other factors. In this paper, we have examined whether LPS-induced NF-κB activation was regulated by these PLD activators, especially RhoA, because it was recently reported that members of the Rho family of GTPases activated the NF-κB. When digitoninpermeabillized J774.1 cells were stimulated in the presence of GTPγS, the NF-κB activation was enhanced ; on the other hand, the LPS-induced NF-κB activation was blocked in the presence of Clostridium botulinum C3 exoenzyme, which is a RhoA specific ADP-ribosylation factor and inhibits the function was increased. Wh … More en the membrane was pretreated with C3 exoenzyme, the LPS-induced PLD activation was blocked, because ADP-ribosylated RhoA could not associated with PLD. Our results indicated that LPS stimulus activated the membrane translocation of RhoA and the results indicated that LPS stimulus activated the membrane translocation of RhoA and the assembly of active RhoA/PLD signaling complexes on membranes in J774.1 cells, and that the DAG production from PC by the PLD triggered the NF-κB activation.Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice, but not in humans. The LPS-mimetic activity of Taxol is not observed in LPS-hyporesponsive C3H/HeJ mice which possess a point mutation in Toll-like receptor 4 (TLR4) ; therefore, TLR4 appears to be involved in both Taxol and LPS signaling. In addition, TLR4 was recently shown to physically associate with MD-2, a molecule that confers LPS-responsiveness on TLR4. Here we examined whether or not TLR4/MD-2 complex mediates a Taxol-induced signal by using transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, both mouse TLR4 and mouse MD-2 and both mouse MD-2 and mouse TLR4 lacking the cytoplasmic portion. Our results demonstrated that coexpression of mouse TLR4 and mouse MD-2 was required for Taxol-responsiveness, and that the TLR4/MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice. We also found that mouse MD-2, but not human MD-2, is involved in Taxol-signaling, suggesting that MD-2 is responsible for the species-specific responsiveness to Taxol. Less

我们之前报道过，小鼠巨噬细胞样J774.1细胞暴露于脂多糖（LPS）诱导磷脂酰胆碱（PC）通过磷脂酶D(PLD) /磷脂酰磷酸水解酶（PAP）活性快速产生细胞二酰基甘油（DAG），这种DAG的产生是NF-κB的关键步骤。激活J774.1细胞。PLD活性受小G蛋白、蛋白激酶C等因素调控。在本文中，我们研究了lps诱导的NF-κB激活是否受这些PLD激活剂，特别是RhoA的调节，因为最近有报道称GTPases的Rho家族成员激活了NF-κB。gtp - γ s刺激洋地黄不透J774.1细胞时，NF-κB活化增强；另一方面，lps诱导的NF-κB活化在肉毒杆菌C3外切酶（Clostridium botulinum C3 exoenzyme， RhoA特异性adp -核糖基化因子）存在下被阻断，抑制功能增强。进一步用C3外酶预处理后，由于adp核基化的RhoA与PLD不相关，lps诱导的PLD活化被阻断。结果表明，LPS刺激激活了J774.1细胞中RhoA的膜易位，激活了活性RhoA/PLD信号复合物在膜上的组装，PLD由PC产生DAG触发了NF-κB的激活。紫杉醇是一种从植物中提取的抗肿瘤药物，它在小鼠体内可以模拟脂多糖（LPS）的作用，但在人体中却没有。在toll样受体4 （TLR4）点突变的脂多糖低反应C3H/HeJ小鼠中，未观察到紫杉醇的模拟脂多糖活性；因此，TLR4似乎参与了紫杉醇和LPS的信号传导。此外，TLR4最近被证明与MD-2有物理关联，MD-2是一种赋予TLR4 lps响应性的分子。本研究利用小鼠pro-B细胞系Ba/F3的转化子，分别表达小鼠TLR4、小鼠TLR4和小鼠MD-2以及缺乏细胞质部分的小鼠MD-2和小鼠TLR4，研究了TLR4/MD-2复合物是否介导紫杉醇诱导的信号。我们的研究结果表明，小鼠TLR4和小鼠MD-2的共表达是紫杉醇响应性的必要条件，并且TLR4/MD-2复合物是小鼠紫杉醇和LPS信号转导的共享分子。我们还发现小鼠MD-2参与了紫杉醇信号传导，而不是人类MD-2，这表明MD-2负责对紫杉醇的物种特异性反应。少