Subcellular Localization and Role of Lipin-1 in Human Macrophages

Martín Valdearcos, Esperanza Esquinas, Clara Meana, Luis Gil-de-Gómez, Carlos Guijas, Jesús Balsinde and María A. Balboa
J Immunol May 15, 2011, 186 (10) 6004-6013; DOI: https://doi.org/10.4049/jimmunol.1003279

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Human lipin-1α localizes to intracellular membranes. Human macrophages transfected with lipin-1α–EGFP (A), HA–lipin-1α (B), lipin-1α–D678E–EGFP (C), or with lipin-1α–EGFP and afterwards treated with 10 ng/ml leptomycin B for 24 h (D) were stained with DAPI (blue) and analyzed by confocal microscopy as described in Materials and Methods. Inserts in C and D are magnifications of selected regions of the cells. The cells shown in the pictures are representative of many analyzed. Scale bars, 10 μm.

  • FIGURE 2.
    FIGURE 2.

    Human lipin-1α associates with LDs. A, Human macrophages transfected with lipin-1α–EGFP (green) were stained with 100 ng/ml Nile Red (red) and analyzed by confocal microscopy as described in Materials and Methods. B, Z-stack fluorescence series from the bottom to the upper part of a cell (1–7). Nuclei are stained with DAPI (blue). The image in the lower right panel is a tridimensional image generated from 15 Z-stacks using the confocal software (8). Cells shown are representative of many cells analyzed. Original magnification ×600.

  • FIGURE 3.
    FIGURE 3.

    Analysis of the association of endogenous lipin-1α expression with LDs. A, Cell homogenates from U937 cells (lane 1) or HEK-293 untransfected (lane 2) or transfected (lanes 3 and 4) with lipin-1α–EGFP were analyzed by immunoblot using an Ab against lipin-1 (lanes 1–3) or an Ab against EGFP (lane 4). The arrowhead points to the endogenous protein and the arrow points to the construct lipin-1α–EGFP. B, Equivalent amounts of total cellular proteins (H) or from the cytosol (C), membrane (M), or LD-enriched fraction were analyzed by immunoblot using the antiserum against lipin-1 or against ADRP, as indicated. C, PCR analysis of mRNA expression of lipin-1α and lipin-1β in human macrophages.

  • FIGURE 4.
    FIGURE 4.

    Human lipin-1β is not associated with LDs. Human macrophages transfected with lipin-1β–EGFP (green) were immunostained with Abs against ADRP (red) (A) or stained with Nile Red (red) (B) and analyzed by confocal microscopy as described in Materials and Methods. Lower images are magnifications of selected areas. Cells shown are representative of many cells analyzed. Original magnification ×600.

  • FIGURE 5.
    FIGURE 5.

    Human lipin-1α associates with the cytoplasmic side of LDs membranes. Live human macrophages transfected with lipin-1α–EGFP were analyzed under the confocal microscope. Fluorescence was recorder before (0 s) and after permeabilization with 20 μM digitonin (80 s) and after 4 μM trypsin treatment for 2 s, as indicated. Images in the left column show green fluorescence while images in the right column show light transmission. Original magnification ×600.

  • FIGURE 6.
    FIGURE 6.

    Human lipin-1α colocalizes with ADRP, and TIP47 and does not change its localization after cellular activation in LDs. Human macrophages transfected with lipin-1α–EGFP (green) were immunostained with Abs against ADRP (red) (A), or treated for 18 h with 200 μM oleic acid and immunostained with Abs against TIP47 (Alexa 555, red) (B), or treated with 1 μM ionomycin, 80 nM PMA, 100 ng/ml LPS, 200 μM oleic acid, or vehicle for 1 h as indicated and stained with with 100 ng/ml Nile Red (red) (C). Cells were then analyzed by confocal microscopy. A colocalization mask was generated by the confocal software (Bio-Rad) to show colocalization pixels (A, B). The cells shown are representative of many analyzed. Original magnification ×600.

  • FIGURE 7.
    FIGURE 7.

    Depletion of lipin-1 does not decrease TAG synthesis in human macrophages. A, Human macrophages were treated with a siRNA negative control (20 nM, filled symbols) or with a siRNA against lipin-1 (20 nM, open symbols). After 48 h, the cells were treated with [14C]oleate (1 μM) for the indicated times, and radioactivity incorporated in TAG was measured as described in Materials and Methods. B, Disappearance of lipin-1 from cell homogenates after cell treatment with siRNA against lipin. C, Peritoneal macrophages obtained from heterozygous (+/−, black bar) or fld animals (gray bar) were treated with [14C]oleate (1 μM) for 1 h, and radioactivity incorporated in TAG was determined. The results are shown as means ± SEM of three independent determinations. Experiments are representative of at least three different ones.

  • FIGURE 8.
    FIGURE 8.

    Lipin-1 expression levels influence LD size and number in macrophages. Human macrophages (A, B) treated with a negative control siRNA or with a siRNA against lipin-1 for 48 h, or peritoneal macrophages from heterozygous (+/−) or fld animals (C–E) were treated with 100 μM oleic acid for 18 h. Cells were then stained with BODIPY493/503 and analyzed by confocal microscopy (A, C). Fluorescence from human macrophages was also analyzed by flow cytometry. Original magnification ×600. Gray plots are from cells without oleic acid treatment and the open plots represent the oleic acid-treated cells (B). The size (D) and number (E) of LDs from peritoneal macrophages from heterozygous (filled bars) or fld animals (open bars) in untreated cells or treated with 100 μM oleic acid (as indicated) were evaluated under the microscope from >600 cells are shown as means ± SEM. *p < 0.05.

  • FIGURE 9.
    FIGURE 9.

    Fatty acid distribution of TAG in LDs (A) and whole macrophages (B). Human macrophages were transfected with 20 nM siRNA negative control (open bars) or 20 nM siRNA against lipin-1 (filled bars) for 48 h. Afterwards, the analysis of TAG fatty acids from LDs (A) or whole cells (B) was carried out by gas chromatography/mass spectrometry after converting the fatty acid glyceryl esters into fatty acid methyl esters. Data are expressed as means ± range of a representative experiment with duplicate determinations.

  • FIGURE 10.
    FIGURE 10.

    Lipin-1 regulates AA mobilization in human macrophages. A, Human macrophages transfected with 20 nM siRNA negative control (open bars) or 20 nM siRNA against lipin-1 (gray bars) for 48 h were labeled with [3H]AA and treated with 100 μM oleic acid, 1 mg/ml opsonized zymosan, or vehicle (control) for 6 h, as indicated. Release of [3H]AA was assayed as described in Materials and Methods. B, Transfected macrophages were stimulated with 1 mg/ml opsonized zymosan for the indicated periods of time, and cellular protein was analyzed by immunoblot using specific Abs for total cPLA2α or the Ser505 phosphorylated form, as indicated. Relative quantification of the phosphorylated cPLA2α bands is shown in the lower panel, normalized with respect to the total cPLA2α content. C, Transfected macrophages were stimulated with 1 mg/ml opsonized zymosan for 18 h and cellular supernatants were assayed for PGE2 content by ELISA. Data are expressed relative to the response observed in control untreated cells and are shown as means ± SEM of three independent determinations.

  • FIGURE 11.
    FIGURE 11.

    Mass spectrometry analysis of AA-containing glycerophospholipids in human macrophages. Human macrophages were transfected with 20 nM siRNA negative control (open bars) or 20 nM siRNA against lipin-1 (gray bars) for 48 h. Afterwards, the analysis of AA-containing phospholipids was carried out by ion-trap mass spectrometry. Data are expressed as means ± range of a representative experiment with duplicate determinations. Shorthand notation of glycerophospholipids (abscissa) follows the guidelines proposed by Fahy et al. (62).

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