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. 2017 Oct 31;10(503):eaan1011.
doi: 10.1126/scisignal.aan1011.

Endogenous Retinoid X Receptor Ligands in Mouse Hematopoietic Cells

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Free PMC article

Endogenous Retinoid X Receptor Ligands in Mouse Hematopoietic Cells

Haixia Niu et al. Sci Signal. .
Free PMC article

Abstract

The retinoid X receptor α (RXRA) has been implicated in diverse hematological processes. To identify natural ligands of RXRA that are present in hematopoietic cells, we adapted an upstream activation sequence-green fluorescent protein (UAS-GFP) reporter mouse to detect natural RXRA ligands in vivo. We observed reporter activity in diverse types of hematopoietic cells in vivo. Reporter activity increased during granulocyte colony-stimulating factor (G-CSF)-induced granulopoiesis and after phenylhydrazine (PHZ)-induced anemia, suggesting the presence of dynamically regulated natural RXRA ligands in hematopoietic cells. Mouse plasma activated Gal4-UAS reporter cells in vitro, and plasma from mice treated with G-CSF or PHZ recapitulated the patterns of reporter activation that we observed in vivo. Plasma from mice with dietary vitamin A deficiency only mildly reduced RXRA reporter activity, whereas plasma from mice on a fatty acid restriction diet reduced reporter activity, implicating fatty acids as plasma RXRA ligands. Through differential extraction coupled with mass spectrometry, we identified the long-chain fatty acid C24:5 as a natural RXRA ligand that was greatly increased in abundance in response to hematopoietic stress. Together, these data suggest that natural RXRA ligands are present and dynamically increased in abundance in mouse hematopoietic cells in vivo.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 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 MACS were transduced with one of the indicated retroviruses, then injected into lethally irradiated recipient mice. After 6 weeks of engraftment, recipient mice were sacrificed and their hematopoietic cells analyzed. (B) Representative 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 Kit+ bone marrow cells that were transduced with Gal4-RXRA or Gal4-RXRA-ΔAF2. (C) Ratio of GFP+mCherry+ cells relative to total mCherry+ cells in BM, PB, spleen, and peritoneal macrophages from mice transplanted with Kit+ bone marrow cells transduced with Gal4-RXRA (circles, N = 6 recipient mice) or RXRAΔAF2 (squares, N = 3 recipient mice). (D) The percentage of CD11b+ (circles), CD71+ (squares), and B220+ (triangles) cells in bone marrow GFP+mCherry+ from the recipient Gal4-RXRA mice (N = 6). Error bars represent standard deviation between individual mice. * P < 0.05, *** P < 0.001, T-test with Welch’s correction.
Figure 2
Figure 2. UAS-GFP reporter output increased in hematopoietic cells following GCSF and PHZ treatment
(A) mCherry and GFP intensity in whole bone marrow cells from recipient mice that were transplanted as in Fig. 1A and then treated with or without bexarotene, GCSF, or phenylhydrazine (PHZ). (B and C) Summary results of the ratio of GFP+mCherry+ cells to total mCherry+ cells (B) or mean fluorescence intensity (MFI) (C) in whole bone marrow cells from recipient mice transplanted with UAS-GFP Kit+ bone marrow cells transduced with Gal4-RXRA; N = 21 (Gal4-RXRA recipient mice without treatment), 9 (Gal4-RXRA recipient mice treated with bexarotene), 11 (Gal4-RXRA recipient mice treated with GCSF), 10 (Gal4-RXRA recipient mice treated with PHZ). Error bars represent standard deviation between individual recipient mice. * P < 0.05, *** P < 0.001, ANOVA with Tukey’s multiple comparison test.
Figure 3
Figure 3. RXRA in GCSF-induced granulopoiesis and hematopoietic stem cell mobilization
(A) White blood cell (WBC) count of C57BL6 mice treated with or without GCSF and bexarotene as indicated. (B) Neutrophil (NE) count of C57BL6 mice treated with or without GCSF and bexarotene as indicated. (C) Absolute number of Kit+Lin-Sca-1+ (KLS) cells in 10 μl peripheral blood of C57BL6 mice treated with or without GCSF and bexarotene as indicated. (D) Absolute number of Kit+Lin-Sca-1- (progenitors) cells in 10 μl peripheral blood of C57BL6 mice treated with or without GCSF and bexarotene as indicated. (E) Colony forming units in 10 μl peripheral blood of C57BL6 mice treated with or without GCSF and bexarotene as indicated. (F) WBC count of Rxraflox/flox × Rxrbflox/flox × Mx1-Cre (RXR-KO) mice and Rxraflox/flox × Rxrbflox/flox (RXR-WT) mice treated with GCSF. (G) NE count of RXR-KO mice and RXR-WT mice treated with GCSF. (H) Colony number generated from 10 μl peripheral blood of RXR-KO mice and RXR-WT mice. (I) Absolute number of Kit+Lin-Sca-1+ (KLS) cells and Kit+Lin-Sca-1- (progenitors) cells in 10 μl peripheral blood of RXR-KO mice and RXR-WT mice. A – B, control mice, N = 16; GCSF treated mice, N = 20; GCSF + bexarotene, N = 19 mice. C – E, control mice, N = 11; GCSF treated mice, N = 15; GCSF + bexarotene treated mice, N = 15. F – I, RXR-KO mice, n = 3; WT mice, n = 4. Error bars represent standard deviation between individual mice. * P < 0.05, ** P < 0.01, *** P < 0.001, ANOVA with Tukey’s multiple comparison test.
Figure 4
Figure 4. RXRA reporter activated by mouse plasma
(A) UAS-GFP bone marrow Kit+ cells were transduced with Gal4-RXRA retrovirus and treated in culture with RXRA agonist (bexarotene and 9-cis-RA), antagonist (HX531), or mouse plasma as indicated, and the ratio of GFP+mCherry+ cells to total mCherry+ cells was determined. Cells were treated in a total volume of 200 μl media. Two way T-test compared against untreated control. (B) UAS-GFP bone marrow Kit+ cells were transduced with Gal4-RXRA retrovirus and treated in culture with plasma from indicated mice and the ratio of GFP+mCherry+ cells to total mCherry+ cells was determined. Error bars represent standard deviation between measurement of plasma obtained from individual mice (N = 3 mice per group). ANOVA with Tukey’s multiple comparisons compared results obtained at each plasma concentration. * P < 0.05, ** P < 0.01, *** P < 0.001.
Figure 5
Figure 5. Effect of vitamin A deficiency (VAD) and fatty acid deficiency (NF) on plasma RXRA ligands
(A) Plasma concentration of retinol in VAD mice and control mice by mass spectrometry. Control mice, N = 5; VAD mice, N = 5. (B) UAS-GFP bone marrow Kit+ cells were transduced with Gal4-RXRA retrovirus and treated with plasma from VAD mice and control mice which were treated with or without PHZ. VAD: plasma from vitamin A deficient mice. VADC: plasma from mice treated with a control diet. VAD mice, N = 4; VAD + PHZ mice, N = 4; VADC mice, N = 5; VADC + PHZ mice, N = 5. (C) Plasma concentration of the fatty acid C16:1 in NF mice and control mice determined by mass spectrometry. NF mice, N = 5; NFC mice, N = 4. (D) The concentration of RXR ligands in fatty acid deficient mice was determined by treating reporter cells as in (B). NF: plasma obtained from mice treated with a fatty acid deficient diet. NFC: plasma from mice treated with a fatty acid control diet. NFC mice, N = 3; NFC + PHZ mice, N = 2; NF mice, N = 3; NF + PHZ mice, n = 4. Error bars represent standard deviation 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.
Figure 6
Figure 6. Serum by extraction with immiscible solvents
(A) Percentage of GFP+ cells in 293T-FXP reporter cells treated with or without serum from mouse, hamster, rabbit, rat, guinea pig or goat as indicated. (B) Percentage of GFP+ cells in 293T-FXP reporter cells treated with or without mouse serum extracted with hexane/isopropanol in different ratios. (C) Percentage of GFP+ cells in 293T-FXP cells reporter treated with or without mouse serum extracted with methanol/H2O in different ratios. Error bars indicate standard deviation between triplicate analysis.
Figure 7
Figure 7. Mass spectrometry comparison of plasma and serum concentrations of long chain fatty acids C24:4 and C24:5
The concentration of C24:4 and C24:5 were determined in: (A) plasma from VADC mice treated with or without PHZ; VADC mice, N = 4; VADC + PHZ mice, N = 5; (B) plasma from VAD mice treated with or without PHZ; VAD mice, N = 4; VAD + PHZ mice, N = 4; (C) plasma from NF mice and NFC mice; NFC mice, N = 4; NF mice, N = 5; (D) plasma from NF mice and NFC mice treated with PHZ; NF mice, N = 3; NFC + PHZ, N = 2. (E) serum from mouse and goat analyzed in triplicate; (F) mouse serum extracted by methanol:H2O in 9:1 and 1:1 ratio and analyzed in triplicate. (G) Concentrations of C24:4 and C24:5 extracted from Flag-Gal4-RXRA following immunoprecipitation from 293T-FXP cell extracts treated with mouse serum for 0, 3, 6, 9, 12, or 24 hours, single determination performed at each time point. (H) Concentrations of C24:4 and C24:5 extracted from Flag-Gal4-RXRA following immunoprecipitation of 293T-FXP cells treated with or without mouse serum or goat serum for 12 hrs, analyzed in triplicate (I). Quantification of the fatty acid C24:4 in peripheral blood (PB) and bone marrow (BM) from mice treated with or without PHZ. PBS treated mice, N = 2; PHZ treated mice, N = 2. (J) Quantification of the fatty acid C24:5 in peripheral blood (PB) and bone marrow (BM) from mice treated with or without PHZ by mass spectrometry. PBS treated mice, N = 2; PHZ treated mice, N = 2. Error bars represent standard deviation of individual mice or biological triplicate experiments as indicated. * P < 0.05, ** P < 0.01, *** P < 0.001, T-test.
Figure 8
Figure 8. Transactivation of RXRA by C24:5
(A-B) In silico models of C24:5 docked in RXRA in an active configuration (A: PDB 1DKF) or and inactive configuration (B: PDB 1XDK) (C) Transactivation of UAS-GFP Kit+ bone marrow cells transduced with Gal4-RXRA and treated in culture with synthesized fatty acid C24:5 or C24:4. Error bars indicate standard deviation of independent triplicates. (D) 293T cells were transfected with DR1x3-tk-Luc and CMX-RXRA plasmids and treated as indicated. Error bars indicate standard deviation of independent triplicates compared by T-test. (E and F) TR-FRET of RXRA binding to indicated co-activator peptides and indicated concentrations of ligands. Error bars represent standard deviation 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.

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