Kdo2-Lipid A, a TLR4-specific Agonist, Induces de Novo Sphingolipid Biosynthesis in RAW264.7 Macrophages, Which Is Essential for Induction of Autophagy

Donna Reichart,Elaine Wang,Amin Momin,Martina Leipelt,Samuel Kelly,M Cameron Sullards,Kacee Sims,Alfred H Merrill,Christopher K Glass,Jeremy C Allegood,Christopher A Haynes

doi:10.1074/jbc.m110.170621

Abstract

Activation of RAW264.7 cells with a lipopolysaccharide specific for the TLR4 receptor, Kdo2-lipid A (KLA), causes a large increase in cellular sphingolipids, from 1.5 to 2.6 × 109 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-13C]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.

Highlights

As part of the characterization of the mouse macrophage lipidome by the LIPID MAPS Consortium, the major sphingolipid subspecies of the mouse macrophage-like cell line, RAW264.7, have been quantified from the first detectable biosynthetic intermediate through the branch point where Cer is partitioned into SM, ceramide 1-phosphate (Cer-1-P), GlcCer, and GalCer using liquid chromatography electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS) [18]
The Consortium has investigated the effects of activation of these cells with Kdo2-lipid A (KLA), a chemically defined substructure of bacterial lipopolysaccharide (LPS) that is recognized by the TLR4 receptor [19]
KLA Inhibits Growth and Increases the Size of RAW264.7 Cells—In contemplating why the RAW264.7 cells acquire such substantial amounts of additional SL upon activation, as well as why the 13C-labeled SL of the control cells decrease on a per cell basis between 12 and 24 h (Fig. 7A), it occurred to us that this might be related to cell growth, i.e. that SL biosynthesized by growing cells will be distributed into multiple daughter cells upon division, whereas growth-inhibited cells that continue to synthesize SL will accumulate them unless turnover is more rapid

Summary

Introduction

As part of the characterization of the mouse macrophage lipidome by the LIPID MAPS Consortium, the major sphingolipid subspecies of the mouse macrophage-like cell line, RAW264.7, have been quantified from the first detectable biosynthetic intermediate (sphinganine) through the branch point where Cer is partitioned into SM, Cer-1-P, GlcCer, and GalCer using liquid chromatography electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS) [18]. ISP1 reduced the KLA-induced being higher for KLA-stimulated RAW264.7 cells because sph- elevation of Cer somewhat, completely eliminated the increase inganine is the first detectable pathway intermediate, followed in HexCer, and decreased SM (reflecting SM turnover by DHCer and Cer (Fig. 1).

Results

Conclusion