Acid sphingomyelinase recruits palmitoylated CD36 to membrane rafts and enhances lipid uptake

Abstract

CD36 palmitoylation increases its membrane localization and is required for CD36-mediated uptake of oxidized low-density lipoprotein (oxLDL). Acid sphingomyelinase (ASMase) is transported to the plasma membrane, where it promotes lipid raft clustering, facilitating membrane protein anchoring for biological functions. We investigated the effects of oxLDL on CD36 palmitoylation and explored the role of ASMase in CD36 membrane translocation. We found that oxLDL increased CD36 palmitoylation and drives its intracellular trafficking from the endoplasmic reticulum to the plasma membrane lipid rafts in macrophages. Affinity purification followed by mass spectrometry analysis identified CD36 bound to ASMase in the plasma membrane. The CD36/ASMase binding was enhanced by oxLDL treatment. Genetic ablation and pharmacological inhibition of ASMase reduced CD36 recruitment to lipid rafts and inhibited CD36 intracellular signaling and lipid uptake. Moreover, inhibiting Sortilin to block ASMase intracellular trafficking and reduce membrane ASMase also caused a sharp decrease in the amount of membrane CD36. In addition, ASMase overexpression dramatically promoted palmitoylated CD36 membrane localization but not CD36 without palmitoylation, in which the modification was inhibited by 2-bromopalmitate (2-BP) treatment or point mutation at the palmitoylation site. Moreover, ASMase knockout inhibited CD36 membrane recruitment both in peritoneal macrophages and in the aorta, and attenuated lipid accumulation in atherosclerotic plaques in mice. Finally, we found oxLDL activated extracellular signal-regulated kinase1/2 (ERK1/2)/specificity protein (SP1) signaling, upregulating ASMase transcription and promoting sphingomyelin catabolism. Therefore, these data demonstrate that ASMase expression induced by oxLDL is required for palmitoylated CD36 membrane translocation during foam cell formation in macrophages.

Keywords: CD36; acid sphingomyelinase; foam cell; lipid raft; palmitoylation.

Figure 1

OxLDL increases CD36 palmitoylation and membrane translocation.A, RAW264.7 macrophages are incubated with the indicated concentrations of oxLDL for 24 h. CD36 palmitoylation is determined using the acyl biotin exchange (ABE) method. HAM, hydroxylamine. B, CD36 palmitoylation levels in cells treated with LDL (50 μg/ml), oxLDL (50 μg/ml) for 24 h. C, subcellular fractions are separated by discontinuous sucrose gradient ultracentrifugation in cells incubated with or without 50 μg/ml oxLDL. CD36 distribution is detected by Western blot. Fractions 4 and 5, enriched in flotillin-1, contain lipid rafts (LR). Transferrin receptor (TfR) was used as non-raft marker. D, immunofluorescence staining shows CD36 (red) and CTxB (green) in left; CD36 (red) and Caln (Calnexin, green) in right. E, colocalization of CD36 with CTxB, Caln and TfR. Data were from six separated fields with three repeats. F, immunofluorescence staining shows CD36 (red) and TfR (green). G and H, CD36 palmitoylation in oxLDL-treated BMDMs and human THP-1 cells. I and J, subcellular location of CD36 in oxLDL-treated BMDMs and human THP-1 cells. Data are analyzed by one-way ANOVA followed by Tukey’s post hoc test in (A and B) (n = 3), by Student's t test in (C, G–J) (n = 3), and by two-way ANOVA in (E) (n = 6). Mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant.

Figure 2

CD36 interacts with ASMase in membrane of oxLDL-induced cells.A, schematic representation of the steps for identification of CD36-associated proteins in the membrane. RAW264.7 cells transfected with Flag-CD36 are incubated with oxLDL (50 μg/ml). Plasma LR are isolated, and CD36-associated proteins are purified using an anti-Flag affinity column. The protein complex is analyzed by mass spectrometry. B, immunoblotting analysis of presence of ASMase in eluates immunoprecipitated with anti-Flag-CD36 antibodies. Blank vector used as control. C, cells transfected with Flag-CD36 and HA-ASM undergo immunoprecipitation with anti-Flag agarose gel, followed by immunoblotting with CD36 and ASMase antibodies. D, co-immunoprecipitation assay shows the association of endogenous CD36 and ASMase. E and F, purified GST-CD36 is incubated with HA-ASM and cell lysates, and the complexes are subjected to a pull-down assay. G and H, schematic diagram of full-length and deletion mutants of ASMase, and the domain responsible for interaction with CD36 was identified by using co-immunoprecipitation. I, immunofluorescence staining shows Flag-CD36 (red) and HA-ASM (green). Percentages of Flag-CD36/HA-ASM colocalization cells in total cells were from eight separated fields. J and K, Flag-CD36 and HA-ASM intensities in randomly selected 30 cells. L, Spearman analysis for the relationship between CD36 and ASMase intensities. M, cell lysate were centrifugated and fractionized. The ASMase in the raft and nonraft fractions were detected by Western blot. Representative blots and images were from three independent experiments. Data are analyzed by Student's t test in (C, I–K, M), by one-way ANOVA followed by Tukey’s post hoc test in (D) and by Spearman Correlation Analysis in (L), mean ± SD, n = 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 3

ASMase promotes lipid recruitment of palmitoylated CD36 during foam cell formation.A, the ABE assay measures CD36 palmitoylation in oxLDL-incubated peritoneal macrophages isolated from wild-type and ASMase−/− mice (Student's t test, n = 6 mice per group). B, LR localization of CD36 in wild-type and ASMase deficient macrophages (Student's t test, n = 6 mice per group). C, LR localization of CD36 in RAW264.7 macrophages with ASMase knockdown via shRNA (two-way ANOVA with Tukey’s test, n = 3). D, RAW264.7 macrophages are stained with anti-CD36 antibody (red) and FITC-CTxB (green) (two-way ANOVA with Tukey’s test, n = 3). E, Western blot analysis of CD36 localization in LR in oxLDL-treated cells treated with ASM-IN-1 (3 μM), an ASMase inhibitor, for 24 h. (one-way ANOVA with Tukey’s test, n = 3). F, Western blot analysis of CD36 localization in LR in oxLDL-treated cells transfected with adenovirus-mediated ASMase expressing vector. (Student's t test, n = 3). G and H, CD36 palmitoylation and LR localization in oxLDL-treated cells treated with recombinant ASMase (20 μg/ml) for 24 h and with or without MβCD (5 mmol/L) for 2 h. (one-way ANOVA with Tukey’s test, n = 3). I, immunofluorescence staining shows CD36 (red) and FITC-CTxB (green) in oxLDL-treated cells treated with ASMase and ASMase + MβCD (one-way ANOVA with Tukey’s test, n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant.

Figure 4

Reduced membrane AMSase inhibits CD36-mediated downstream signaling in RAW264.7 macrophages.A, co-immunoprecipitation assay measures CD36/Fyn/Lyn association in cells with ASMase knockdown. Cells are transfected with sh-ASMase and incubated with oxLDL for 24 h (Student's t test, n = 3). B–D, phosphorylation levels of Fyn, Lyn, and JNK1/2 are analyzed in oxLDL-treated cells with ASMase knockdown (Student's t test, n = 3). E, cells transfected with sh-ASMase are incubated with DiI-oxLDL (30 μg/ml) for 6 h. DiI-oxLDL uptake is measured by flow cytometry (Student's t test, n = 3). F, cells transfected with sh-ASMase are incubated with oxLDL for 24 h and stained with Oil Red O (Student's t test, n = 3). G, total cholesterol is measured in cells transfected with sh-ASMase and incubated with oxLDL (Student's t test, n = 3). H, LR localization of CD36 and ASMase is analyzed in cells incubated with oxLDL with or without Sortilin inhibitor AF38469 (one-way ANOVA followed by Tukey’s post hoc test, n = 3). I, cells incubated with oxLDL with or without AF38469 are stained with anti-CD36 antibody (red) and FITC-CTxB (green). J, DiI-oxLDL uptake is measured in cells incubated with or without AF38469 (Student's t test, n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5

Reduced CD36 palmitoylation inhibits ASMase-mediated CD36 LR localization.A, CD36 palmitoylation is analyzed in cells treated with oxLDL and with or without 2-BP. B, CD36 LR localization is measured in oxLDL-incubated cells treated with ASMase or ASMase + 2-BP. C, CD36 palmitoylation is analyzed in oxLDL-induced cells transfected with Flag-wCD36 (wild-type) or Flag-mCD36 (mutant). D, CD36 LR localization is measured in cells transfected with Flag-wCD36 or Flag-mCD36, following oxLDL incubation. E and F, RAW264.7 macrophages were stimulated with oxLDL and treated as indicated. The cells were then stained with CD36 and CTxB, and percentage of cells with CD36/CTxB co-localization in total cells were counted. G, inhibited association of ASMase and CD36 in LR after CD36 palmitoylation were blocked by 2-BP (left) or transfection with mCD36 (right) in oxLDL-treated cells. H, immunofluorescence staining assay shows reduced co-localization of ASMase with CD36 of palmitoylation inhibited by 2-BP or mutation in oxLDL-treated cells. CD36 appears red and ASMase appears green. All data are analyzed by Student's t test, mean ± SD, n = 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6

ASMase deficiency reduces CD36 LR recruitment and aortic lipid accumulation in atherosclerotic plaques ASM+/+ApoE−/− and ASM−/−ApoE−/− mice are fed a high-fat diet beginning at 8 weeks of age for 16. A, peritoneal macrophages isolated from ASM+/+ApoE−/− and ASM−/−ApoE−/− mice are incubated with DiI-oxLDL for 6 h, and DiI-oxLDL uptake is measured by flow cytometry. B, intracellular cholesterol levels in peritoneal macrophages are quantified. C, peritoneal macrophages are incubated with oxLDL for 24 h and stained with Oil Red O. D, whole aorta from ASM+/+ApoE−/− and ASM−/−ApoE−/− mice are stained with Oil Red O to assess plaque formation. E, CD36 LR localization is analyzed in macrophages isolated from high-fat diet ASM+/+ApoE−/− and ASM−/−ApoE−/− mice. F and G, Oil Red O staining of aortic root sections is performed in ASM+/+ApoE−/− and ASM−/−ApoE−/− mice. Positive staining in total plaque area was calculated. H and I, sections of the aortic roots are stained with HE and anti-CD68 antibodies. All data are analyzed by Student's t test, mean ± SD, n = 6. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 7

OxLDL increases ASMase expression and promotes sphingomyelin (SM) catabolism.A, schematic illustration of SM catabolism. SM is hydrolyzed by ASMase to generate ceramide and phosphatidylcholine. Ceramidases convert ceramides into sphingosine and fatty acids. B, Western blot detection of ASMase expression is performed in cells treated with varying concentrations of oxLDL (50–120 μg/ml; one-way ANOVA followed by Tukey’s post hoc test, n = 3). C, RT-qPCR analysis measures ASMase mRNA levels in oxLDL (50 μg/ml) treated macrophages. D, ASMase enzymatic activity is assessed in cells treated with oxLDL at concentration of 50 μg/ml. E, OxLDL-treated cells are seeded on coverslip and incubated with SM-binding protein, lysenin-His (1 μg/ml). Membrane SM is detected using immunofluorescence staining and quantified by flow cytometry. F–H, total cellular levels of SM and ceramides, and SM/ceramide ratio are measured by LC-MS/MS in oxLDL-treated cells (50 μg/ml). I and J, lipidomics analysis of specific SM and ceramide species in cells treated with oxLDL (50 μg/ml). All comparisons were conducted with Student's t test except in (B) (mean ± SD, n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant.

Figure 8

ASMase is upregulated by activating ERKs/Sp-1 signaling in oxLDL-induced macrophages.A, schematic illustration shows the ASMase promoter containing an SP-1 binding site. Primers P1 and P2 are used for the ChIP assay. The primer sequences used are listed in the Experimental procedures section. B, the ChIP assay detects SP-1 binding to the ASMase promoter (two-way ANOVA followed by Tukey’s post hoc test). C, EMSA detects SP-1/ASMase promoter binding. D, luciferase reporter assay of truncated ASMase promoter activity. Student's t test. E, Western blot analysis measures SP-1 phosphorylation. Macrophages are treated with oxLDL and incubated with SCH772984, VX-702, or SP600125, which are specific inhibitors of ERK1/2, p38, and JNK1/2, respectively (one-way ANOVA followed by Tukey’s post hoc test). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant, mean ± SD, n = 3.

Figure 9

Model for ASMase-mediated palmitoylated CD36 membrane rafts recruitment.