Research ArticleBiochemistry

Endogenous retinoid X receptor ligands in mouse hematopoietic cells

See allHide authors and affiliations

Science Signaling  31 Oct 2017:
Vol. 10, Issue 503, eaan1011
DOI: 10.1126/scisignal.aan1011
  • Fig. 1 Natural RXRA ligands in mouse hematopoietic cells in vivo.

    (A) Schema for bone marrow transplant procedure. Kit+ cells isolated from the bone marrow of UAS-GFP mice using magnetic-activated cell sorting (MACS) were transduced with one of the indicated retroviruses and then injected into lethally irradiated recipient mice. After 6 weeks of engraftment, recipient mice were sacrificed, and their hematopoietic cells harvested and analyzed. (B) Representative fluorescence-activated cell sorting (FACS) showing mCherry and GFP intensity in bone marrow cells (BM), peripheral blood (PB), spleen cells, and peritoneal macrophages from mice transplanted with UAS-GFP bone marrow Kit+ cells that were transduced with Gal4-RXRA or Gal4-RXRA-ΔAF2. Data shown are from individual representative recipient mice. (C) Ratio of GFP+mCherry+ cells relative to total mCherry+ cells in bone marrow cells, peripheral blood, spleen, and peritoneal macrophages from mice transplanted with UAS-GFP bone marrow Kit+ cells transduced with Gal4-RXRA (circles; n = 6 recipient mice) or RXRA-ΔAF2 (squares; n = 3 recipient mice). (D) The percentage of CD11b+ (circles), CD71+ (squares), and B220+ (triangles) cells in the bone marrow GFP+mCherry+ cell population from the recipient Gal4-RXRA mice (n = 6). Error bars represent SDs between individual mice. *P < 0.05; ***P < 0.001, t test with Welch’s correction.

  • Fig. 2 G-CSF or PHZ treatment increased UAS-GFP reporter output in hematopoietic cells.

    (A) Representative FACS showing mCherry and GFP intensity in whole bone marrow cells from mice that were treated with bexarotene (Bex), G-CSF, or PHZ after receiving UAS-GFP bone marrow Kit+ cells transduced with Gal4-RXRA or RXRA-ΔAF2. Data shown are from individual representative recipient mice. (B) Ratio of GFP+mCherry+ cells to total mCherry+ cells and (C) MFI in whole bone marrow cells from recipient mice transplanted with UAS-GFP bone marrow Kit+ cells transduced with Gal4-RXRA; n = 21 (no treatment control), 9 (bexarotene), 11 (G-CSF), and 10 (PHZ). Error bars represent SDs between individual recipient mice. *P < 0.05; ***P < 0.001, analysis of variance (ANOVA) with Tukey’s multiple comparisons test. ns, not significant.

  • Fig. 3 RXRA in G-CSF–induced granulopoiesis and HSC mobilization.

    (A) WBC and (B) NE counts of C57BL6 mice treated with or without G-CSF and bexarotene as indicated. n = 16 (no treatment control), 20 (G-CSF), and 19 (G-CSF + bexarotene). (C) Absolute number of KLS cells and (D) Kit+LinSca-1 (progenitor) cells and (E) CFU in 10 μl of peripheral blood from C57BL6 mice treated with or without G-CSF and bexarotene as indicated. n = 11 (no treatment control), 15 (G-CSF), and 15 (G-CSF + bexarotene). (F) WBC and (G) NE count of RXR-KO and RXR-WT mice treated with G-CSF. (H) CFU and (I) absolute number of KLS cells and Kit+LinSca-1 (progenitors) cells in 10 μl of peripheral blood of RXR-KO and RXR-WT mice. n = 3 RXR-KO and 4 RXR-WT mice. Error bars represent SDs between individual mice. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA with Tukey’s multiple comparisons test.

  • Fig. 4 RXRA reporter activated by mouse plasma.

    (A) Ratio of GFP+mCherry+ cells to total mCherry+ cells in UAS-GFP bone marrow Kit+ cells transduced with Gal4-RXRA retrovirus and treated in culture with the RXRA agonists bexarotene or 9-cis-RA, the RXRA antagonist HX531, or mouse plasma as indicated. Two-way t test compared against untreated control. (B) Ratio of GFP+mCherry+ cells to total mCherry+ cells in UAS-GFP bone marrow Kit+ cells transduced with Gal4-RXRA retrovirus and treated in culture with plasma from mice that had been treated with PHZ, G-CSF, or vehicle control. Error bars represent SDs between measurements of plasma obtained from individual mice (n = 3 mice per treatment group). ANOVA with Tukey’s multiple comparisons compared results obtained at each plasma concentration. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 5 Effect of vitamin A deficiency and fatty acid deficiency on plasma RXRA ligands.

    (A) Plasma concentration of retinol in VAD and VADC mice as determined by MS. n = 5 VADC and 5 VAD mice. (B) Ratio of GFP+mCherry+ cells to total mCherry+ cells in UAS-GFP bone marrow Kit+ cells transduced with Gal4-RXRA retrovirus and treated with plasma from VAD or VADC mice with and without PHZ treatment. n = 4 VAD, 4 VAD + PHZ, 5 VADC, and 5 VADC + PHZ. (C) Plasma concentration of the fatty acid C16:1 in mice fed an NF or NFC diet as determined by MS. n = 5 NF and 4 NFC. (D) Ratio of GFP+mCherry+ cells to total mCherry+ cells in UAS-GFP bone marrow Kit+ cells transduced with Gal4-RXRA retrovirus and treated with plasma from NF or NFC mice with and without PHZ treatment. n = 3 NFC, 2 NFC + PHZ, 3 NF, and 4 NF + PHZ. Error bars represent SDs between measurements of plasma obtained from separate mice. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA with Tukey’s multiple comparisons compared results obtained at each plasma concentration.

  • Fig. 6 Serum by extraction with immiscible solvents.

    (A) Percentage of GFP+ cells in 293T-FXP reporter cells treated with serum from mouse, hamster, rabbit, rat, guinea pig, or goat as indicated. Similar results were observed with two additional lots of mouse and goat serum (not shown). (B) Percentage of GFP+ cells in 293T-FXP reporter cells treated with hamster serum or with mouse serum extracted with the indicated ratios of hexane/isopropanol. (C) Percentage of GFP+ cells in 293T-FXP cells reporter treated with 9-cis-RA or with mouse serum extracted with the indicated ratios of methanol/H2O. For each panel, error bars indicate SDs between three biological replicates.

  • Fig. 7 MS comparison of plasma and serum concentrations of long-chain fatty acids C24:4 and C24:5.

    (A to F) Concentrations of the fatty acids C24:4 and C24:5 in (A) plasma from VADC mice treated with or without PHZ (n = 4 VADC and 5 VADC + PHZ mice); (B) plasma from VAD mice treated with or without PHZ (n = 4 VAD and 4 VAD + PHZ mice); (C) plasma from NF and NFC mice (n = 4 NFC and 5 NF mice); (D) plasma from NF and NFC mice treated with PHZ (n = 3 NF and 2 NFC + PHZ mice); (E) commercially available mouse and goat serum, n = 3 independent extractions of serum and MS analysis, with one lot from each species analyzed by MS; (F) mouse serum extracted with 9:1 and 1:1 methanol/H2O (n = 3 separate extractions of serum and MS analysis). (G) Concentrations of C24:4 and C24:5 extracted from Flag-Gal4-RXRA immunoprecipitated from 293T-FXP cells treated with mouse serum for the indicated durations. (n = 1 immunoprecipitation, extraction, and MS analysis per time point). (H) Concentrations of C24:4 and C24:5 extracted from Flag-Gal4-RXRA immunoprecipitated from 293T-FXP cells treated with or without mouse serum or goat serum for 12 hours. n = 3 independent experiments. (I and J) Quantification of (I) C24:4 and (J) C24:5 in peripheral blood and bone marrow from mice treated with PHZ or vehicle [phosphate-buffered saline (PBS)] by MS. n = 2 PBS-treated mice and 2 PHZ-treated mice. Error bars represent SDs of individual mice or biological triplicate experiments as indicated. *P < 0.05; **P < 0.01; ***P < 0.001, t test.

  • Fig. 8 Transactivation of RXRA by C24:5.

    (A and B) In silico models of C24:5 docked in RXRA in an active configuration [(A) PDB: 1DKF] or an inactive configuration [(B) PDB: 1XDK]. Blue, nitrogen; red, oxygen; green, carbon in the lipid; gray, hydrogen and carbon in the protein. (C) Transactivation of UAS-GFP bone marrow Kit+ cells transduced with Gal4-RXRA and treated in culture with commercially synthesized C24:5 or C24:4. Error bars indicate SDs of independent triplicates. (D) Transactivation of multimerized DR1 promoter by full-length RXRA and indicated ligands. Error bars indicate SDs of independent triplicates compared by t test. ATRA, all-trans retinoic acid; Luc, luciferase. (E and F) TR-FRET showing RXRA binding to PGC1α (E) and D22 (F) coactivator peptides in the presence of the indicated concentrations of 9-cis-RA, DHA, C24:5, or C24:4. Error bars represent SDs of independent triplicate experiments. EC50 determined by fitting the data to a log(agonist) variable slope with four parameters. *P < 0.05; **P < 0.01; ***P < 0.001.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/503/eaan1011/DC1

    Fig. S1. Ex vivo validation of the UAS-GFP reporter transgene.

    Fig. S2. Reporter analysis.

    Fig. S3. Analysis of RXRA-ΔAF2 and RARA to natural ligands in mouse sera.

    Fig. S4. Sixteen different docking configurations of C24:5 in RXRA (1XDK).

    Fig. S5. Sixteen different docking configurations of C24:4 in RXRA (1XDK).

    Fig. S6. Comparison of liquid chromatography–MS/MS peaks of C24:5.

    Table S1. Analysis of long-chain fatty acids in serum samples by MS.

  • Supplementary Materials for:

    Endogenous retinoid X receptor ligands in mouse hematopoietic cells

    Haixia Niu, Hideji Fujiwara, Orsola di Martino, Gayla Hadwiger, Thomas E. Frederick, María P. Menéndez-Gutiérrez, Mercedes Ricote, Gregory R. Bowman, John S. Welch*

    *Corresponding author. Email: jwelch{at}wustl.edu

    This PDF file includes:

    • Fig. S1. Ex vivo validation of the UAS-GFP reporter transgene.
    • Fig. S2. Reporter analysis.
    • Fig. S3. Analysis of RXRA-ΔAF2 and RARA to natural ligands in mouse sera.
    • Fig. S4. Sixteen different docking configurations of C24:5 in RXRA (1XDK).
    • Fig. S5. Sixteen different docking configurations of C24:4 in RXRA (1XDK).
    • Fig. S6. Comparison of liquid chromatography–MS/MS peaks of C24:5.
    • Table S1. Analysis of long-chain fatty acids in serum samples by MS.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.16 MB


    Citation: H. Niu, H. Fujiwara, O. di Martino, G. Hadwiger, T. E. Frederick, M. P. Menéndez-Gutiérrez, M. Ricote, G. R. Bowman, J. S. Welch, Endogenous retinoid X receptor ligands in mouse hematopoietic cells. Sci. Signal. 10, eaan1011 (2017).

    © 2017 American Association for the Advancement of Science

Stay Connected to Science Signaling

Navigate This Article