title: Kdo2-lipid A, a TLR4-specific agonist, induces de novo sphingolipid biosynthesis in RAW264.7 macrophages, which is essential for induction of autophagy.
abstract: Activation of RAW264.7 cells with a lipopolysaccharide specific for the TLR4 receptor, Kdo(2)-lipid A (KLA), causes a large increase in cellular sphingolipids, from 1.5 to 2.6 × 10(9) molecules per cell in 24 h, based on the sum of subspecies analyzed by "lipidomic" mass spectrometry. Thus, this study asked the following question. What is the cause of this increase and is there a cell function connected with it? The sphingolipids arise primarily from de novo biosynthesis based on [U-(13)C]palmitate labeling, inhibition by ISP1 (myriocin), and an apparent induction of many steps of the pathway (according to the distribution of metabolites and microarray analysis), with the exception of ceramide, which is also produced from pre-existing sources. Nonetheless, the activated RAW264.7 cells have a higher number of sphingolipids per cell because KLA inhibits cell division; thus, the cells are larger and contain increased numbers of membrane vacuoles termed autophagosomes, which were detected by the protein marker GFP-LC3. Indeed, de novo biosynthesis of sphingolipids performs an essential structural and/or signaling function in autophagy because autophagosome formation was eliminated by ISP1 in KLA-stimulated RAW264.7 cells (and mutation of serine palmitoyltransferase in CHO-LYB cells); furthermore, an anti-ceramide antibody co-localizes with autophagosomes in activated RAW264.7 cells versus the Golgi in unstimulated or ISP1-inhibited cells. These findings establish that KLA induces profound changes in sphingolipid metabolism and content in this macrophage-like cell line, apparently to produce sphingolipids that are necessary for formation of autophagosomes, which are thought to play important roles in the mechanisms of innate immunity.
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
authors.forename: Kacee
authors.initials: K
authors.lastname: Sims
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Haynes
authors.forename: Samuel
authors.initials: S
authors.lastname: Kelly
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Allegood
authors.forename: Elaine
authors.initials: E
authors.lastname: Wang
authors.forename: Amin
authors.initials: A
authors.lastname: Momin
authors.forename: Martina
authors.initials: M
authors.lastname: Leipelt
authors.forename: Donna
authors.initials: D
authors.lastname: Reichart
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Glass
authors.forename: M Cameron
authors.initials: MC
authors.lastname: Sullards
authors.forename: Alfred H
authors.initials: AH
authors.lastname: Merrill
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D006056:Golgi Apparatus / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007113:Immunity, Innate / Q000187:drug effects* / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D010588:Phagosomes / Q000276:immunology / Q000378:metabolism; D051102:Serine C-Palmitoyltransferase / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D013107:Sphingolipids / Q000096:biosynthesis* / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000819:agonists* / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: C506188:Kdo2-lipid A; D008070:Lipopolysaccharides; D013107:Sphingolipids; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D051102:Serine C-Palmitoyltransferase; C436412:Prss28 protein, mouse; D012697:Serine Endopeptidases