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

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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