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

Sims K, Haynes CA, Kelly S, Allegood JC, Wang E, Momin A, Leipelt M, Reichart D, Glass CK, Sullards MC, Merrill AH - J. Biol. Chem. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.

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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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.
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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.

Mentions: To determine whether de novo SL biosynthesis is required for the induction of autophagy by KLA, RAW264.7 cells stably expressing GFP-LC3 were treated with ISP1 for 1 h, and then KLA was added, and the numbers of autophagosomal puncta were analyzed (Fig. 10). By both visual inspection (Fig. 10A) and quantitative analysis of the confocal images (Fig. 10B), it is evident that inhibition of de novo SL biosynthesis by ISP1 blocked the appearance of GFP-LC3-associated autophagosomes. This is doubly interesting because it not only links de novo SL biosynthesis with KLA-induced autophagy but also establishes that the elevation of Cer due to SL turnover (which was minimally affected by ISP1) (Figs. 6B and 7C) is not sufficient to induce autophagy.


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

Sims K, Haynes CA, Kelly S, Allegood JC, Wang E, Momin A, Leipelt M, Reichart D, Glass CK, Sullards MC, Merrill AH - J. Biol. Chem. (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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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.
Mentions: To determine whether de novo SL biosynthesis is required for the induction of autophagy by KLA, RAW264.7 cells stably expressing GFP-LC3 were treated with ISP1 for 1 h, and then KLA was added, and the numbers of autophagosomal puncta were analyzed (Fig. 10). By both visual inspection (Fig. 10A) and quantitative analysis of the confocal images (Fig. 10B), it is evident that inhibition of de novo SL biosynthesis by ISP1 blocked the appearance of GFP-LC3-associated autophagosomes. This is doubly interesting because it not only links de novo SL biosynthesis with KLA-induced autophagy but also establishes that the elevation of Cer due to SL turnover (which was minimally affected by ISP1) (Figs. 6B and 7C) is not sufficient to induce autophagy.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.

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.

Show MeSH