Toll-like Receptor 4 Signaling Regulates Cytosolic Phospholipase A2 Activation and Lipid Generation in Lipopolysaccharide-stimulated Macrophages

Abstract

Inflammatory lipid mediators such as prostaglandins and leukotrienes play crucial roles in the pathogenesis of bacterial lipopolysaccharide (LPS)-induced inflammation. Cytosolic phospholipase A 2 (cPLA 2 ) is a key enzyme in the generation of pro-inflammatory lipid mediators. Here, we found that Toll-like receptor 4 (TLR4) is essential for LPS-induced cPLA 2 activation and lipid release. Inhibition of TLR4 protein expression by TLR4 small interfering RNA or neutralization of TLR4 by the specific antibody against TLR4/MD2 blocked cPLA 2 phosphorylation and cPLA 2 -hydrolyzed arachidonic acid release. Furthermore, activation of the TLR4 signaling pathway by LPS regulated cPLA 2 activation and lipid release. cPLA 2 phosphorylation and cPLA 2 -hydrolyzed lipid release were significantly impaired when TLR4 adaptor protein, either MyD88 or TRIF, was knocked down in LPS-stimulated macrophages. Similarly, LPS-induced arachidonate release was inhibited in cells transfected with a dominant-negative MyD88 or TRIF construct. Subsequently, cPLA 2 activation could be suppressed by inhibition of the TLR4 adaptor protein-directed p38 and ERK MAPK pathways. These findings suggest that, in LPS-induced inflammation, the TLR4-mediated MyD88-and TRIF-dependent MAPK pathways result in cPLA 2 activation and production of pro-inflammatory lipid mediators.

Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages

“…We and others have shown in monocytes and macrophages that cPLA 2 ␣ phosphorylation might be caused by various MAPKs (39,95). We found here that HA-induced cPLA 2 ␣ activation is associated with ERK1/2, p38, and JNK activation (Fig.…”

“…3, B and C). Interestingly, the time course of ERK1/2 phosphorylation was different from other studies showing TLR4 involvement (39,96). Even though the peak phosphorylation of all three MAPKs was at 30 min, ERK1/2 phosphorylation tended to be sustained up to 6 h, both in murine macrophages and in human monocytes (Fig.…”

“…cPLA 2 ␣ is activated by intracellular calcium elevation and phosphorylation at serine residues (22). Several pro-inflammatory mediators, such as IL-1␤, TNF␣, IFN␥, phosphatidylinositol phosphate, ceramide 1-phosphate, sphingosine 1-phosphate, and LPS, have been reported to activate cPLA 2 ␣ in various cells through different pathways (35)(36)(37)(38)(39)(40). However, to date there is no evidence of HA-dependent lipid mediator activation.…”

Simvastatin and a Plant Galactolipid Protect Animals from Septic Shock by Regulating Oxylipin Mediator Dynamics through the MAPK-cPLA2 Signaling Pathway

“…19-HETE and prostanoids PGE 2 and PGF 2α are proinflammatory metabolites of AA; both prostanoids are produced from COX-2 and the subsequent action of specific inducible prostanoid synthases (40). Pretreatment with simvastatin or dLGG were characterized most significantly by the elevated levels of LXA 4 in the liver, while dLGG posttreatment can also increase EPA metabolites, resolvin D1 and 15-HEPE, and DHA and its metabolites, 17-HDoHE, 10,17-DiHDoHe.…”

MyD88–BLT2-dependent cascade contributes to LPS-induced interleukin-6 production in mouse macrophage

“…34 The MyD88-dependent pathway of LPS-TLR4 signaling was found to activate MAPKs, including extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase, with p38 being thought to be required for LPS-induced activation of cPLA 2 and lipid release. 25 MyD88-deficient mice show resistance to LPS-induced responses, including cytokine production by macrophages, and MyD88 − / − macrophages do not produce IL-6 in response to LPS. 35 Consistent with these previous observations, we found that transfection of Raw 264.7 cells with MyD88 siRNA inhibited LPS-induced p38 MAPK phosphorylation (Figure 6a) and that the p38-specific inhibitor SB203580 attenuated the LPS-induced phosphorylation of cPLA 2 and upregulation of IL-6 mRNA (Figures 6b and c).…”