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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 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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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.

Mentions: SL biosynthesis de novo originates in the endoplasmic reticulum (ER) (56) followed by trafficking of Cer (and other metabolites) to the Golgi complex (GC) by vesicular and ceramide transporter-mediated processes (57). Because the ER and Golgi are thought to be membrane components for autophagosome formation (58–61), it is possible that de novo biosynthesized Cer and/or other SL are incorporated into autophagosomes for structural and/or signaling purposes. To determine whether Cer is associated with autophagosomes, we examined RAW264.7 cells using an anti-Cer antibody that has been previously characterized for subcellular localization studies (26–31). In unstimulated RAW264.7 cells, this antibody co-localized mainly in the perinuclear Golgi region, as has been previously observed for other cells using anti-Cer antibodies (26) and fluorescent Cer analogs (Fig. 11A) (62). However, following addition of KLA, the immunofluorescence clearly shifts to punctate vesicles that co-localize with the autophagosomal marker, GFP-LC3 (Fig. 11A). Furthermore, inhibition of de novo SL biosynthesis with ISP1 had no noticeable effect on the anti-Cer antibody immunofluorescence associated with the GC (in control or KLA-treated cells) but blocked the KLA-induced co-localization of Cer with autophagosomes (Fig. 11A). The quantitative analysis of multiple confocal images shows that this suppression was highly significant (Fig. 11B) whether expressed as the total number of cells with GFP-LC3 puncta co-localized with Cer (Fig. 11B, upper panel) or as the number of autophagic cells (i.e. those cells with five or more GFP-LC3 puncta) with GFP-LC3 puncta co-localized with Cer (Fig. 11B, lower panel). The lack of effect of ISP1 on the GC localization is not surprising because Cer from SL turnover is known to reach the GC by retrograde trafficking (63, 64). Thus, these findings are strongly suggestive that the anti-Cer antibody immunofluorescence reflects Cer (and possibly related compounds such as DHCer) incorporation into autophagosomes in KLA-activated RAW264.7 cells.


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 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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Related In: Results  -  Collection

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Show All Figures
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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.
Mentions: SL biosynthesis de novo originates in the endoplasmic reticulum (ER) (56) followed by trafficking of Cer (and other metabolites) to the Golgi complex (GC) by vesicular and ceramide transporter-mediated processes (57). Because the ER and Golgi are thought to be membrane components for autophagosome formation (58–61), it is possible that de novo biosynthesized Cer and/or other SL are incorporated into autophagosomes for structural and/or signaling purposes. To determine whether Cer is associated with autophagosomes, we examined RAW264.7 cells using an anti-Cer antibody that has been previously characterized for subcellular localization studies (26–31). In unstimulated RAW264.7 cells, this antibody co-localized mainly in the perinuclear Golgi region, as has been previously observed for other cells using anti-Cer antibodies (26) and fluorescent Cer analogs (Fig. 11A) (62). However, following addition of KLA, the immunofluorescence clearly shifts to punctate vesicles that co-localize with the autophagosomal marker, GFP-LC3 (Fig. 11A). Furthermore, inhibition of de novo SL biosynthesis with ISP1 had no noticeable effect on the anti-Cer antibody immunofluorescence associated with the GC (in control or KLA-treated cells) but blocked the KLA-induced co-localization of Cer with autophagosomes (Fig. 11A). The quantitative analysis of multiple confocal images shows that this suppression was highly significant (Fig. 11B) whether expressed as the total number of cells with GFP-LC3 puncta co-localized with Cer (Fig. 11B, upper panel) or as the number of autophagic cells (i.e. those cells with five or more GFP-LC3 puncta) with GFP-LC3 puncta co-localized with Cer (Fig. 11B, lower panel). The lack of effect of ISP1 on the GC localization is not surprising because Cer from SL turnover is known to reach the GC by retrograde trafficking (63, 64). Thus, these findings are strongly suggestive that the anti-Cer antibody immunofluorescence reflects Cer (and possibly related compounds such as DHCer) incorporation into autophagosomes in KLA-activated RAW264.7 cells.

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