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

我们曾报道，小鼠巨噬细胞样J774.1细胞暴露于脂多糖可诱导磷脂酰胆碱(PC)在磷脂酶D/磷脂酸盐磷酸水解酶(PAP)活性的介导下迅速产生细胞二酰甘油，这种二酰甘油的产生是J774.1细胞中核因子-κB活化的关键步骤。PLD活性受小G蛋白、蛋白激酶C等因素的调节。在这篇文章中，我们研究了脂多糖诱导的NF-κB的激活是否受这些PLD激活剂，尤其是RhoA的调节，因为最近有报道说Rho家族的成员激活了NF-κB。当洋地黄渗透的J774.1细胞在GTPγS的存在下被刺激时，NF-κB的激活被增强；另一方面，内毒素诱导的NF-κB的激活被肉毒杆菌C3外酶阻断，这是一种RhoA特异性的腺苷二磷酸核糖化因子，抑制作用增强。WH…此外，C3胞外酶可阻断脂多糖诱导的PLD激活，因为ADP核糖化的RhoA不能与PLD结合。我们的结果表明，脂多糖刺激激活了RhoA的膜转位，并激活了J774.1细胞膜上活性的RhoA的膜转位和活性的RhoA/PLD信号复合体的组装，并且PLD从PC产生的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，它在小鼠体内模拟脂多糖的作用，而在人类中不能。在具有Toll样受体4(TLR4)点突变的低反应性C3H/HeJ小鼠中，未观察到紫杉醇的类脂多糖活性，因此TLR4似乎同时参与了紫杉醇和脂多糖的信号转导。此外，TLR4最近被证明与MD-2物理上相关，MD-2是一种赋予TLR4上的内毒素反应性的分子。在这里，我们研究了TLR4/MD-2复合体是否通过只表达小鼠TLR4的小鼠Pro-B细胞系BA/F3的转化子、小鼠TLR4和小鼠MD-2以及小鼠MD-2和小鼠TLR4缺乏胞浆部分的转化子来介导紫杉醇诱导的信号。结果表明，小鼠TLR4和小鼠MD-2的共表达是紫杉醇应答所必需的，TLR4/MD-2复合体是小鼠紫杉醇和内毒素信号转导的共同分子。我们还发现，参与紫杉醇信号转导的是小鼠的MD-2，而不是人的MD-2，这表明MD-2负责紫杉醇的物种特异性反应。较少

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