ABSTRACT
Lipopolysaccharide (LPS) is the component of Gram-negative bacteria that activates Toll-like receptor 4 (TLR4) to trigger proinflammatory responses. We examined the involvement of Lyn tyrosine kinase in TLR4 signaling of macrophages, distinguishing its catalytic activity and intermolecular interactions. For this, a series of Lyn-GFP constructs bearing point mutations in particular domains of Lyn were overexpressed in RAW264 macrophage-like cells or murine peritoneal macrophages, and their influence on LPS-induced responses was analyzed. Overproduction of wild-type or constitutively active Lyn inhibited production of TNF-α and CCL5/RANTES cytokines and down-regulated the activity of NFκB and IRF3 transcription factors in RAW264 cells. The negative influence of Lyn was nullified by point mutations of Lyn catalytic domain or Src homology 2 (SH2) or SH3 domains or of the cysteine residue that undergoes LPS-induced palmitoylation. Depending on the cell type, overproduction of those mutant forms of Lyn could even up-regulate LPS-induced responses, and this effect was reproduced by silencing of endogenous Lyn expression. Simultaneously, the Lyn mutations blocked its LPS-induced accumulation in the raft fraction of RAW264 cells. These data indicate that palmitoylation, SH2- and SH3-mediated intermolecular interactions, and the catalytic activity of Lyn are required for its accumulation in rafts, thereby determining the negative regulation of TLR4 signaling.
Subject(s)
Membrane Microdomains/enzymology , src-Family Kinases/genetics , src-Family Kinases/metabolism , Animals , Cell Line , Chemokine CCL5/metabolism , Green Fluorescent Proteins , Interferon Regulatory Factor-3/metabolism , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Macrophages, Peritoneal/metabolism , Male , Membrane Microdomains/metabolism , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
LPS binds sequentially to CD14 and TLR4/MD2 receptor triggering production of proinflammatory mediators. The LPS-induced signaling is controlled by a plasma membrane lipid PI(4,5)P2 and its derivatives. Here, we show that stimulation of murine peritoneal macrophages with LPS induces biphasic accumulation of PI(4,5)P2 with peaks at 10 and 60-90 min that were still seen after silencing of TLR4 expression. In contrast, the PI(4,5)P2 elevation was abrogated when CD14 was removed from the cell surface. To assess the contribution of CD14 and TLR4 to the LPS-induced PI(4,5)P2 changes, we used HEK293 transfectants expressing various amounts of CD14 and TLR4. In cells with a low content of CD14 and high of TLR4, no accumulation of PI(4,5)P2 occurred. With an increasing amount of CD14 and concomitant decrease of TLR4, 2 peaks of PI(4,5)P2 accumulation appeared, eventually approaching those found in LPS-stimulated cells expressing CD14 alone. Mutation of the signaling domain of TLR4 let us conclude that the receptor activity can modulate PI(4,5)P2 accumulation in cells when expressed in high amounts compared with CD14. Among the factors limiting PI(4,5)P2 accumulation are its hydrolysis, phosphorylation, and availability of its precursor, PI(4)P. Inhibition of PLC and PI3K or overexpression of PI4K IIα that produces PI(4)P promoted PI(4,5)P2 elevation in LPS-stimulated cells. The elevation of PI(4,5)P2 was dispensable for TLR4 signaling yet enhanced its magnitude. Taken together, these data suggest that LPS-induced accumulation of PI(4,5)P2 that maximizes TLR4 signaling is controlled by CD14, whereas TLR4 can fine tune the process by affecting the PI(4,5)P2 turnover.