LAMTOR/Ragulator regulates lipid metabolism in macrophages and foam cell differentiation

Abstract

Late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) is a scaffold protein complex that anchors and regulates multiprotein signaling units on late endosomes/lysosomes. To identify LAMTOR-modulated endolysosomal proteins, primary macrophages were derived from bone marrow of conditional knockout mice carrying a specific deletion of LAMTOR2 in the monocyte/macrophage cell lineage. Affymetrix-based transcriptomic analysis and quantitative iTRAQ-based organelle proteomic analysis of endosomes derived from macrophages were performed. Further analyses showed that LAMTOR could be a novel regulator of foam cell differentiation. The lipid droplet formation phenotype observed in macrophages was additionally confirmed in MEFs, where lipidomic analysis identified cholesterol esters as specifically downregulated in LAMTOR2 knockout cells. The data obtained indicate a function of LAMTOR2 in lipid metabolism.

Keywords: LAMTOR; foam cells; macrophages; organelle proteomics; ragulator; transcriptomics.

Figure 1

Experimental design. (A) Schematic illustration of transcriptomics and endosome proteomics experimental setup. Hematopoietic stem cells from LAMTOR2‐deficient (LMCLAMTOR2−/−) and control (LMCLAMTOR2+/+) mice were extracted and differentiated in vitro with recombinant M‐CSF for 7 days. For transcriptomic analysis, macrophages whole cells lysates were prepared, RNA was extracted and hybridized onto an Affymetrix GeneChip, and microarrays gene expression profiling was performed. For LE proteomics, BMDM were fed with LB for 15 min followed by 60 min chase without LB and LE/LYS were separated on density gradients. Differentially expressed proteins were quantified by iTRAQ labeling and LC‐MS/MS. (B) Surface expression of F4/80, CD11b, and Gr1. BMDM from LMCLAMTOR2+/+ and LMCLAMTOR2−/− mice were stained for F4/80, CD11b, and Gr1 and the surface expression was determined as shown in the histogram (blue: LMCLAMTOR2+/+, red: LMCLAMTOR2−/−, gray: isotype control). Right panels: dot plot showing macrophages double positive for CD11b and F4/80. (C) Immunoelectron microscopic analysis of macrophages with internalized LB. LB within LAMP1‐positive LE/LYS compartments (arrows) as seen in LMCLAMTOR2+/+ and LMCLAMTOR2−/− BMDM, incubated for 15 min with LB and chased for 60 min; n, nucleus; pm, plasma membrane; visualization of primary antibodies with Nanogold‐conjugates plus silver enhancement (Nano+SE); scale bars, 500 nm. Right panel, LB within a LE compartment (asterisk) with distinct Cathepsin D immunolabeling (double arrows; visualization with 5 nm gold conjugates); LMCLAMTOR2−/− BMDM incubated with LB for 30 min without chase; scale bar, 200 nm.

Figure 2

LAMTOR2 regulates downstream mTORC1 signaling and key enzymes of lipid metabolism. (A) Knockout efficiency of LAMTOR2 in macrophages derived from LMCLAMTOR2+/+ and LMCLAMTOR2−/− mice. (B) Western blot analyses of mTORC1 targets—phospho‐p70S6K and downstream phospho‐S6. (C) Western blotting of SOAT1 and MGLL. Whole BMDM cell lysates were analyzed and actin was used as loading control. Relative values (shown as fold change, n = 3) were normalized to actin as loading control and to total p70S6K and total S6 for corresponding phosphorylated proteins.

Figure 3

LAMTOR2 regulates foam cell differentiation. (A) Confocal microscopy analysis of LMCLAMTOR2+/+ and LMCLAMTOR2−/− foam cells. Cells were fixed and stained with BODIPY 493/503 and Hoechst. Scale bar, 20 μm. (B) Relative fluorescence intensity quantification expressed as ratio of integrated density of BODIPY 493/503 to Hoechst fluorescent signal (n = 10 images containing at least 100 cells for each cell type, P = 0.0009).

Figure 4

Lipid droplets are reduced in LAMTOR2−/− MEFs. (A) LAMTOR2f/−, LAMTOR2−/−, and LAMTOR2 reconstituted by expression of LAMTOR2‐GFP MEFs were fixed, lipid droplets were stained with Oil Red O and pictures were taken with a confocal fluorescence microscope. (B) Lipid droplet count per cell of the indicated cell types. Student's t‐test P < 0.001, ***.

Figure 5

LAMTOR2 regulates cellular lipid composition. (A) Thin‐layer chromatography of whole cell lysates from LMCLAMTOR2+/+ and LMCLAMTOR2−/− BMDM. Foam cells were cultured in the presence of 50 μg·mL⁻¹ acetylated LDL. (B) Molar abundance (%) of membrane lipids and storage lipids in LAMTOR2−/− MEFs versus control LAMTOR2f/− MEFs and LAMTOR2−/− MEFs reconstituted by expression of LAMTOR2‐GFP, analyzed by shotgun lipidomics. Student's t‐test P < 0.001, ***. (C) More detailed view zooming in on lipid classes.