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. 2010 Jul 2;285(27):21103-13.
doi: 10.1074/jbc.M109.080671. Epub 2010 Mar 17.

Studies on the role of acid sphingomyelinase and ceramide in the regulation of tumor necrosis factor alpha (TNFalpha)-converting enzyme activity and TNFalpha secretion in macrophages

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Studies on the role of acid sphingomyelinase and ceramide in the regulation of tumor necrosis factor alpha (TNFalpha)-converting enzyme activity and TNFalpha secretion in macrophages

Krasimira A Rozenova et al. J Biol Chem. .

Abstract

Acid sphingomyelinase (ASMase) has been proposed to mediate lipopolysaccharide (LPS) signaling in various cell types. This study shows that ASMase is a negative regulator of LPS-induced tumor necrosis factor alpha (TNFalpha) secretion in macrophages. ASMase-deficient (asm(-/-)) mice and isolated peritoneal macrophages produce severalfold more TNFalpha than their wild-type (asm(+/+)) counterparts when stimulated with LPS, whereas the addition of exogenous ceramides or sphingomyelinase reduces the differences. The underlying mechanism for these effects is not transcriptional but post-translational. The TNFalpha-converting enzyme (TACE) catalyzes the maturation of the 26-kDa precursor (pro-TNFalpha) to an active 17-kDa form (soluble (s)TNFalpha). In mouse peritoneal macrophages, the activity of TACE was the rate-limiting factor regulating TNFalpha production. A substantial portion of the translated pro-TNFalpha was not processed to sTNFalpha; instead, it was rapidly internalized and degraded in the lysosomes. TACE activity was 2-3-fold higher in asm(-/-) macrophages as compared with asm(+/+) macrophages and was suppressed when cells were treated with exogenous ceramide and sphingomyelinase. Indirect immunofluorescence analyses revealed distinct TNFalpha-positive structures in the close vicinity of the plasma membrane in asm(-/-) but not in asm(+/+) macrophages. asm(-/-) cells also had a higher number of early endosomal antigen 1-positive early endosomes. Experiments that involved inhibitors of TACE, endocytosis, and lysosomal proteolysis suggest that in the asm(-/-) cells a significant portion of pro-TNFalpha was sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis, it was recycled to the plasma membrane and processed to sTNFalpha.

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Figures

FIGURE 1.
FIGURE 1.
Cytokine levels in LPS-injected asm+/+ and asm−/− mice. Mice were injected intraperitoneally with LPS (5.8 mg/kg body weight) or saline. Serum was collected at the indicated times, and the levels of TNFα (A) and IL-1β (B) were measured by ELISA. Data for LPS-injected asm+/+ (filled circles) and asm−/− (open circles) are shown. The levels of the cytokines in saline-injected animals were below the detection levels. Data for individual animals are shown and are the average of identical measurements done in triplicate. The group average ± S.E. is shown on the side. The significance of the main effects of genotype and LPS (***, p < 0.001; **, p < 0.01, *, p < 0.05) and the interaction effect (###, p < 0.001) are based on two-way ANOVA with Bonferroni post-test analysis. The insets are representation of the data from asm+/+ mice on a smaller scale.
FIGURE 2.
FIGURE 2.
Effect of ASMase, bacterial SMase, and ceramide on TNFα production in primary macrophages. TNFα levels were measured by ELISA in the medium of peritoneal macrophages from asm+/+ (filled symbols) or asm−/− (open symbols). A, cells treated with LPS (100 ng/ml, circles) or phosphate-buffered saline (triangles) for the indicated times. B, cells treated with LPS for 4 h in the presence of bacterial SMase, C2-, C16-ceramide at the indicated concentrations. Control cells were treated with 0.1% ethanol. Data are means ± S.E., n = 3 (A) or n =2 (B). Statistical significance of the main effect (***, p < 0.001) and the interaction effect (###, p < 0.001; #, p < 0.05) was calculated by two-way ANOVA. The statistical significance of bacterial SMase and ceramide effects was calculated by one-way ANOVA (*, p < 0.05; ***, p < 0.001).
FIGURE 3.
FIGURE 3.
Stimulation of IRAK-1, ERK, and TNFα mRNA in asm+/+ and asm−/− macrophages. Peritoneal macrophages from asm+/+ and asm−/− mice were treated with LPS (10 ng/ml) for the indicated times. A–D, activation of IRAK-1 (A and C) and ERK1/2 (B and D). Analyses were done using Western blotting and antibodies against IRAK-1 and phosphorylated ERK1/2. Data are representative of three independent experiments. β-Actin levels were used to control for uniform loading. E, stimulation of TNFα mRNA. mRNA levels were determined by real time PCR. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level was used for normalization. Data are presented as mean ± S.E. of three independent experiments. Statistical significance of the treatment effect was calculated (***, p < 0.001) based on two-way ANOVA.
FIGURE 4.
FIGURE 4.
Post-translational processing of TNFα. Peritoneal macrophages were isolated from asm+/+ and asm−/− mice (A–C) or from asm+/+ mice (D and E) and treated with LPS (10 ng/ml) for the indicated times. Ammonium chloride (10 mm) was added to the culture medium 1 h after stimulation with LPS as indicated. TNFα levels were determined in cell extracts (A and D) or medium (B, C, and E) by Western blotting. Cyclophilin A (Cycloph. A) levels were used to control for uniform loading. The data shown in A and B were compiled from three different experiments, each covering specific time intervals of stimulation with LPS and each repeated at least twice using different macrophage preparation from two to three mice in each genotype. For quantification purposes, the data were normalized for the respective values in asm+/+ macrophages and are means ± S.D. ***, p < 0.001; *, p < 0.05.
FIGURE 5.
FIGURE 5.
TACE activity, expression, and subcellular localization in asm+/+ and asm−/− cells. A and B, test for linearity of TACE activity assay with protein and time. TACE activity was measured in vitro in detergent-free lysates from RAW264.7 cells. C and D, effect of ASMase deficiency on activity of TACE. asm+/+ and asm−/− macrophages were treated with saline (C) or with a combination of C2-ceramide (Cer) (60 μm) or vehicle (0.1% ethanol) (D). Statistical significance (p < 0.001) of the genotype-based difference in TACE activity is designated ###. TACE activity was measured in detergent-free lysates using fluorigenic substrate, and the fluorescence was monitored over time on a microplate reader. The data are means ± S.D. (n = 3). E, subcellular localization of TACE. TACE was visualized by indirect immunofluorescence in permeabilized asm+/+ and asm−/− macrophages using antibodies against mouse TACE and fluorescent microscopy. Hoechst 33258 was used for staining the nuclei. Scale bar represents 20 μm. F, effect of ASMase deficiency on TACE mRNA and protein expression. TACE mRNA levels were determined by real time PCR in asm+/+ and asm−/−macrophages that were treated with phosphate-buffered saline or LPS for the indicated times. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level was used for normalization. Data are presented as means ± S.E. of three independent experiments. Inset, Western blot analysis in human fibroblasts from healthy individuals or patients with Niemann-Pick disease, type A. β-Actin levels were used to control for uniform loading. G, effect of nonionic detergents on TACE activity. asm+/+ macrophages were treated with LPS (10 ng/ml) (filled bars) or vehicle (open bars) for 4 h. The activity of TACE was measured using fluorigenic substrate and cell lysates (5 μg of protein per assay) in the absence or presence of Nonidet P-40 (NP-40) or Triton X-100 at the indicated concentrations. Data shown are average ± S.D., n = 3. Statistical significance of detergent and LPS effects was determined by two-way ANOVA. Asterisk designates the main effect of detergent (***, p < 0.001; **, p < 0.01), and ns (nonsignificant) refers to the significance of the interaction effect.
FIGURE 6.
FIGURE 6.
Subcellular localization of TNFα in asm+/+ and asm−/− macrophages. Macrophages from asm+/+ and asm−/− mice were treated with LPS (10 ng/ml) in the presence or absence of TAPI-1 (20 μm) and NH4Cl (10 mm) for 4 h. A–C, staining for TNFα (A and B) and early endosomes (C). TNFα was visualized in permeabilized (A) and nonpermeabilized (B) cells using antibodies against TNFα and confocal microscopy. The arrows indicate TNFα-positive vesicles proximal to the plasma membrane that are visible in asm−/− cells. Antibodies against the endosomal marker EEA1 were used to visualize early endosomes (C). Transmitted light images (B and C) show cell morphology. The scale bar represents 10 μm. D, suppression of TNFα secretion by TACE inhibitor. Western blot analyses of TNFα in cell culture medium from macrophages stimulated with LPS and indicated inhibitors.
FIGURE 7.
FIGURE 7.
Co-localization of TNFα and EEA1 in asm+/+ and asm−/− macrophages. Peritoneal asm+/+ and asm−/− macrophages were treated with LPS (10 ng/ml) for 4 h. Permeabilized cells were probed sequentially for TNFα (red) and EEA1 (green). Images were collected using confocal microscopy and merged (yellow). Only merged images are shown (A). The TNFα/EEA1 co-localization in a single cell for each panel is depicted by arrows. For quantification purposes, the bulk fluorescence visible in the center of each cell was excluded, and the number of TNFα- positive (B), EEA1-positive (C), and double-positive (D) vesicles (with size bigger than 4 pixels) was counted in 10 cells of each slide. Data shown are average ± S.E. Statistical significance of the main effect (***, p < 0.001; **, p < 0.01; *, p < 0.05) and the interaction effect (###, p <0.001; ##, p < 0.01; and #, p < 0.05) was determined by two-way ANOVA. The scale bar represents 20 μm.
FIGURE 8.
FIGURE 8.
Effect of inhibition of endocytosis on sTNFα secretion in asm+/+ and asm−/− macrophages. Macrophages from asm+/+ and asm−/− mice were treated for 4 h with LPS (10 ng/ml) in the presence and absence of Dynasore (80 μm), chlorpromazine (2.5 μg/ml), or monodansylcadaverine (MDC) (50 μm) added 1 h after LPS stimulation. sTNFα in the medium was measured by ELISA. Statistical significance of the main effect (***, p < 0.001; **, p < 0.01) and interaction effect (###, p < 0.001) was determined by two-way (for Dynasore treatment) and one-way (for chlorpromazine and monodansylcadaverine treatments) ANOVA.
FIGURE 9.
FIGURE 9.
Effect of exogenous sphingomyelinase on sTNFα and EEA1 in asm−/− macrophages. Peritoneal macrophages from asm+/+ and asm−/− mice were treated for 4 h with 10 ng/ml LPS alone or in combination with 0.1 unit/ml bacterial sphingomyelinase (bSMase). A and B, levels of TNFα in medium. TNFα secretion was monitored by Western blotting analyses of cell culture medium. The graph represents the mean values ± S.D. of intensity of TNFα bands from three independent experiments. Statistical significance of the main effect (**, p < 0.01) and the interaction effect of the treatment and the genotype (#, p < 0.05) is shown based on two-way ANOVA. C, visualization of the early endosomes. Immunofluorescence images depicting EEA1-positive early endosomes (green) in permeabilized macrophages. PBS, phosphate-buffered saline.

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