title: Contribution of CD14 and TLR4 to changes of the PI(4,5)P2 level in LPS-stimulated cells.
abstract: 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)P2and its derivatives. Here, we show that stimulation of murine peritoneal macrophages with LPS induces biphasic accumulation of PI(4,5)P2with peaks at 10 and 60-90 min that were still seen after silencing of TLR4 expression. In contrast, the PI(4,5)P2elevation 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)P2changes, 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)P2occurred. With an increasing amount of CD14 and concomitant decrease of TLR4, 2 peaks of PI(4,5)P2accumulation 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)P2accumulation in cells when expressed in high amounts compared with CD14. Among the factors limiting PI(4,5)P2accumulation 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)P2elevation in LPS-stimulated cells. The elevation of PI(4,5)P2was dispensable for TLR4 signaling yet enhanced its magnitude. Taken together, these data suggest that LPS-induced accumulation of PI(4,5)P2that maximizes TLR4 signaling is controlled by CD14, whereas TLR4 can fine tune the process by affecting the PI(4,5)P2turnover.
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Płóciennikowska
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Aneta
authors.initials: A
authors.lastname: Hromada-Judycka
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Justyna
authors.initials: J
authors.lastname: Dembińska
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Paula
authors.initials: P
authors.lastname: Roszczenko
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Ciesielska
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw, Poland k.kwiatkowska@nencki.gov.pl.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kwiatkowska
mesh_terms: D000818:Animals; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D054878:Lipoylation; D008213:Lymphocyte Activation; D008262:Macrophage Activation; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015778:Minor Histocompatibility Antigens / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000096:biosynthesis*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D034622:RNA Interference; D013047:Specific Pathogen-Free Organisms; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D015778:Minor Histocompatibility Antigens; D016328:NF-kappa B; D019269:Phosphatidylinositol 4,5-Diphosphate; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; C440499:lipopolysaccharide, Escherichia coli O111 B4; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase
keywords: inflammation; macrophages; nuclear factor κB; phosphatidylinositol 4-monophosphate; phospholipase C