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. 2010 Dec 3;285(49):38568-79.
doi: 10.1074/jbc.M110.170621. Epub 2010 Sep 27.

Kdo2-lipid A, a TLR4-specific agonist, induces de novo sphingolipid biosynthesis in RAW264.7 macrophages, which is essential for induction of autophagy

Kacee Sims  1 , Christopher A Haynes, Samuel Kelly, Jeremy C Allegood, Elaine Wang, Amin Momin, Martina Leipelt, Donna Reichart, Christopher K Glass, M Cameron Sullards, Alfred H Merrill Jr
Affiliations

Kdo2-lipid A, a TLR4-specific agonist, induces de novo sphingolipid biosynthesis in RAW264.7 macrophages, which is essential for induction of autophagy

Kacee Sims et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
KLA induces substantial increases in the amounts of multiple cellular sphingolipids. RAW264.7 cells were incubated with vehicle control (PBS) or KLA (100 ng/ml). Following 24 h of treatment, cells were harvested for lipid extraction and analysis by LC ESI-MS/MS. The number of million molecules per cell for each sphingolipid species is shown for control (upper number) and KLA (lower number)-treated cells with heat map coloration to illustrate the fold change of each subspecies. Data represent the means (n = 9).
FIGURE 2.
FIGURE 2.
KLA induces time-dependent increases in ceramide and dihydroceramide. RAW264.7 cells were incubated with vehicle control (PBS) or KLA (100 ng/ml). Following treatment, cells were harvested at the indicated time points for lipid extraction and analysis by LC ESI-MS/MS. A, amounts of the major chain length subspecies of DHCer. Data represent the means (n = 9). B, amounts of total DHCer (a summation of all chain lengths) in KLA versus control conditions. Data represent the means ± S.E. (n = 9); *, p ≤ 0.05; **, p ≤ 0.001. C, amounts of the major chain length subspecies of Cer. Because of the difficulties in visualization, the quantities of ceramide subspecies in control treated cells at the 24-h time point are as follows: C16 (9 pmol/μg DNA); C18 (0.4 pmol/μg DNA); C20 (0.2 pmol/μg DNA); C22 (1.9 pmol/μg DNA); C24:1 (5.4 pmol/μg DNA); C24 (5.5 pmol/μg DNA); C26:1 (0.2 pmol/μg DNA); and C26 (0.1 pmol/μg DNA). Data represent the means ± S.E. (n = 9). D, amounts of total Cer (a summation of all chain lengths) in KLA versus control conditions. Data represent the means ± S.E. (n = 9); *, p ≤ 0.05; **, p ≤ 0.001.
FIGURE 3.
FIGURE 3.
KLA induces time-dependent increases in ceramide metabolites. RAW264.7 cells were incubated with vehicle control (PBS) or KLA (100 ng/ml). Following treatment, cells were harvested at the indicated time points for lipid extraction and analysis by LC ESI-MS/MS. Amounts of the major chain length subspecies of Cer metabolites as follows: sphingomyelins (A), monohexosylceramides (B), and ceramide 1-phosphates (C) are shown. Data represent the means (n = 9).
FIGURE 4.
FIGURE 4.
KLA alters the amount of sphingoid bases and sphingoid base phosphates. RAW264.7 cells were incubated with vehicle control (PBS) or KLA (100 ng/ml). Following treatment, cells were harvested at the indicated time points for lipid extraction and analysis by LC ESI-MS/MS. A, amounts of sphingoid bases, sphinganine and sphingosine. Data represent the means (n = 9). B, amounts of sphingoid base phosphates, sphinganine 1-phosphate and sphingosine 1-phosphate. Data represent the means ± S.E. (n = 9); *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005; ****, p ≤ 0.001.
FIGURE 5.
FIGURE 5.
KLA increases mRNA expression of genes involved in sphingolipid biosynthesis. Gene expression data for the sphingolipid metabolic enzymes following 24 h of treatment with KLA (100 ng/ml) using the KEGG-based microarray analysis tool (GenMAPP version 2.1) that has been modified to display this pathway more completely. The coloration is log2-fold change in expression following treatment with KLA (100 ng/ml) for 24 h from microarray data generated by the LIPID MAPS Consortium. The sphingolipid metabolites and genes (with the gene abbreviations shown in boxes or enzyme names where gene names are ambiguous) are given for the condensation of serine and palmitoyl-CoA to form 3-ketosphinganine (3-ketoSa) by serine palmitoyltransferase (SPT), which is reduced to sphinganine (Sa), acylated to dihydroceramides (DHCer) by (DH)Cer synthases (CerS), and incorporated into more complex DH-sphingolipids (the 1-phosphate (DHCer-1-P) sphingomyelins (DHSM), glucosylceramides (DHGlcCer), galactosylceramides (DHGalCer)) or desaturated to Cer followed by headgroup addition. Also included are a number of the catabolic genes, e.g. sphingomyelinases (SMPD), ceramidases (ASAH), sphingosine kinases (SphK) for the formation of sphinganine 1-phosphate (Sa1P) and sphingosine 1-phosphate (So1P), and phosphatases for the reverse reaction and the lyase that cleaves sphingoid base 1-phosphates to ethanolamine phosphate (EP), hexadecanal (C16:0al), and hexadecenal (C16:1al).
FIGURE 6.
FIGURE 6.
Effect of ISP1 on sphingolipid biosynthesis in KLA-treated RAW264.7 cells. RAW264.7 cells were incubated for 24 h with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μm), or KLA + ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA. Following treatment, cells were harvested for lipid extraction and analysis by LC ESI-MS/MS. A, change in the amounts of Cer, HexCer, and SM for cells treated with KLA or KLA + ISP1. Data represent the means ± S.E. (n = 3). B, amounts of the major chain length subspecies of ceramide in cells treated with KLA + ISP1. Data represent the means (n = 3).
FIGURE 7.
FIGURE 7.
Analysis of de novo sphingolipid biosynthesis in RAW264.7 cells by [13C]palmitate labeling. RAW264.7 cells were incubated with 0.1 mm [U-13C]palmitic acid (as the BSA complex) with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μm), or KLA+ ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA for the times shown. The appearance of 13C in newly synthesized sphingolipids was quantified by mass spectrometry as described under supplemental “Materials and Methods”. A, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer subspecies. Data represent the means ± S.E. (n = 3). B, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species in cells treated with control (left) or KLA (right), Data represent the means (n = 3). Because of the difficulties in visualization, the quantities of ceramide subspecies in control treated cells at the 24-h time point are as follows: C16 (7.6 pmol/μg DNA); C18 (0.7 pmol/μg DNA); C20 (0.3 pmol/μg DNA); C22 (3.2 pmol/μg DNA); C24:1 (6 pmol/μg DNA); C24 (6.4 pmol/μg DNA); C26:1 (0.2 pmol/μg DNA); C26 (0.1 pmol/μg DNA). C, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species (left) and 12C (unlabeled) Cer species (right) in cells treated with KLA + ISP1. Data represent the means (n = 3).
FIGURE 8.
FIGURE 8.
KLA reduces cell number and increases cell size in RAW264.7 cells. A, μg of DNA per dish following treatment with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μm), or KLA+ ISP1 for various times. Data represent the means ± S.E. (n = 3). B, representative bright field images with measured cellular diameter for control and KLA-treated cells. For each experimental condition, 225 cells were analyzed. Data represent the means. The relative cellular diameter (C) and relative cellular surface area (D) distribution patterns are shown. For each experimental condition, 225 cells were analyzed.
FIGURE 9.
FIGURE 9.
KLA induces autophagy in RAW264.7 cells. RAW264.7 cells stably expressing GFP-LC3 were incubated for various times with vehicle control (PBS) or KLA (100 ng/ml). A, representative images. B, number of cells displaying GFP-LC3 puncta (upper panel) and the number of GFP-LC3 puncta/cell (lower panel) were quantified using ImageJ. For each experimental condition, a minimum of 260 cells/experiment was counted. Data represent mean ± S.E. (n = 3); # (n = 2); *, p ≤ 0.05; **, p ≤ 0.01.
FIGURE 10.
FIGURE 10.
KLA-induced autophagy is dependent on de novo sphingolipid biosynthesis. RAW264.7 cells stably expressing GFP-LC3 were incubated for 24 h with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μm), or KLA + ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA. A, representative images. B, number of cells displaying GFP-LC3 puncta (upper panel) and the number of GFP-LC3 puncta/cell were quantified using ImageJ. For each experimental condition, a minimum of 195 cells/experiment were counted. Data represent mean ± S.E. (n = 4). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
FIGURE 11.
FIGURE 11.
KLA promotes the co-localization of ceramide with the autophagosome. RAW264.7 cells stably expressing GFP-LC3 were incubated for 24 h with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μm), or KLA + ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA. A, following treatment, the extra- and intracellular Cer was stained for all conditions. Representative images. B, number of cells displaying GFP-LC3 puncta co-localized with ceramide (upper panel) was quantified using ImageJ. For each experimental condition, a minimum of 150 cells/experiment were analyzed for ceramide co-localization. Data represent mean ± S.E. (n = 4). Lower panel, number of autophagic cells displaying GFP-LC3 puncta co-localized with Cer was quantified using ImageJ. For each experimental condition, a minimum total of 40 autophagic cells were analyzed for Cer co-localization. Data represent mean ± S.E. (n = 4). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. C, RAW264.7 cells stably expressing GFP-LC3 were incubated for 24 h with vehicle control (PBS) or KLA (100 ng/ml). Following treatment, the Golgi complex and endoplasmic reticulum were stained using antibodies against GM130 and BiP, respectively.
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