Research ArticleBiochemistry

Endogenous retinoid X receptor ligands in mouse hematopoietic cells

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Sci. Signal.  31 Oct 2017:
Vol. 10, Issue 503, eaan1011
DOI: 10.1126/scisignal.aan1011

Endogenous RXRA ligands in hematopoietic cells

Like other nuclear receptors, retinoid X receptor α (RXRA) stimulates the transcription of target genes in a ligand-dependent manner. Both vitamin A–derived retinoic acids and fatty acids have been implicated as endogenous ligands for RXRA. Using a reporter system for detecting RXRA activation in vivo, Niu et al. found that RXRA activity increased in hematopoietic cells when mice were subjected to treatments that stimulated myeloid cells. Plasma from these mice also stimulated RXRA activation, even if the mice were deficient in vitamin A but not if the mice were deficient in fatty acids. Mass spectrometry and other biochemical methods identified the long-chain fatty acid C24:5 as the most likely endogenous ligand for RXRA in this context. These findings establish fatty acids as dynamically controlled natural ligands for RXRA in hematopoietic cells.

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.

INTRODUCTION

Retinoid X receptors (RXRs) are members of the nuclear receptor superfamily (1). Like other nuclear receptors, the transcriptional activity of RXRs is ligand-dependent. Ligand binding results in a conformational shift of the terminal α helix [activation function 2 (AF2) domain], which displaces bound corepressors and facilitates the binding of coactivators.

Diverse molecules have been implicated as natural RXR ligands, including both retinoic acids and fatty acids (2). It is unknown whether any of these are present in hematopoietic cells in physiologically relevant quantities or whether they are dynamically regulated during hematopoietic stress. The vitamin A derivative 9-cis retinoic acid (9-cis-RA) has been described as a natural RXR ligand (3, 4), yet several groups have reported 9-cis-RA to be absent or below detectable limits in testis, liver, heart, lung, and serum (5, 6). Another vitamin A metabolite, 9-cis-13,14-dihydroretinoic acid (9-cis-13,14-DHRA), was reported to be an endogenous RXR ligand in mouse serum, brain, and liver, although it is unclear whether it is present in hematopoietic cells (7). Using a luciferase reporter assay, Lengqvist et al. (8) showed that unsaturated fatty acids such as docosahexaenoic acid (DHA; C22:6), docosapentaenoic acid (DPA; C22:5), and arachidonic acid (AA; C20:4) activate RXRα (RXRA) in vitro, whereas saturated fatty acids such as arachidic acid (C20:0) and stearic acid (C18:0) do not.

Three separate genes encode three RXR isoforms—RXRA, RXRβ (RXRB), and RXRγ (RXRG)—at least one of which is present in every mammalian cell (9). These subtypes are highly conserved, with nearly identical ligand-binding pocket conformations (1). RXRs bind to DNA as either a homodimer (10) or a heterodimer (1); the heterodimeric partners of RXR include retinoic acid receptors (RARs), thyroid hormone receptors (TRs), the vitamin D receptor (VDR), peroxisome proliferator–activated receptors (PPARs), liver X receptors, the pregnane X receptor, and the constitutive androstane receptor (1115). By heterodimerizing with these partners, RXRs participates in diverse essential biological processes, such as development, metabolism, cell differentiation, and cell death.

RXRs play important roles in hematopoiesis. RXRA abundance increases upon monocytic differentiation (16). Ectopic overexpression of RXRA in hematopoietic stem and progenitor cells inhibits granulopoiesis by impairing proliferation and differentiation, whereas expression of a dominant negative form of RXRA promotes the generation of late-stage granulocytes in vitro (17). Rxra binds to the promoter of Epo, the gene encoding the cytokine erythropoietin (EPO), and promotes its transcription during the embryonic day 9.5 (E9.5) to E11.5 phase of fetal liver erythropoiesis before subsequently being supplanted by hepatocyte nuclear factor 4–dependent transcription of Epo from E11.5 onward (18). Rxrs also participate in osteoclast differentiation and postnatal bone remodeling (19). Conditional deletion of both Rxra and Rxrb in mouse hematopoietic cells (Rxrg is not expressed in hematopoietic cells) generates giant, nonresorbing osteoclasts, increases bone mass in male mice, and protects female mice from osteoporotic bone loss after ovariectomy. No information is available about the presence or distribution of natural ligands of RXRA in hematopoietic cells.

Here, we demonstrate that natural RXRA ligands are present in mouse hematopoietic cells and plasma in vivo and are predominantly active in myeloid cells. We find that concentrations of RXRA ligands are dynamically increased in response to myeloid stress, such as exposure to granulocyte colony-stimulating factor (G-CSF) and phenylhydrazine (PHZ). Moreover, we identify the long-chain fatty acid C24:5 as an endogenous RXRA ligand that undergoes dynamic increases in concentration during mouse hematopoietic stress.

RESULTS

Adaptation of Gal4–upstream activation sequence reporter to detect RXRA ligands in vivo

To determine whether natural RXRA ligands are present in hematopoietic cells in vivo, we used transgenic upstream activation sequence–green fluorescent protein (UAS-GFP) reporter mice (20). UAS promoter sequences are recognized by the yeast transcription factor Gal4 and are not activated by mammalian proteins. When the modular Gal4 DNA binding domain (Gal4 DBD) is fused to the RXRA ligand-binding domain (RXRA LBD) and retrovirally expressed in UAS-GFP bone marrow Kit+ cells (Gal4-RXRA), the reporter should respond to intracellular ligands that bind to and transactivate RXRA. We included an internal ribosomal entry site (IRES)–mCherry cassette in the Gal4-RXRA retroviral vectors to identify cells that had been transduced (fig. S1A). In mouse bone marrow Kit+ cells transduced with Gal4-RXRA retrovirus and cultured ex vivo, this system was sensitive to the natural RXRA agonist 9-cis-RA and to the synthetic RXRA agonist bexarotene (fig. S1, B and C). The median effective concentration (EC50) of 9-cis-RA was 1.3 nM, and the EC50 of bexarotene was 5.1 nM, similar to previously reported results (3), thus validating the reporter assay.

Detection of RXRA ligands in mouse hematopoietic cells in vivo

To determine whether natural RXRA ligands are present in hematopoietic cells in vivo, bone marrow Kit+ cells from UAS-GFP transgenic mice were transduced with Gal4-RXRA retrovirus and then transplanted into lethally irradiated recipient mice (Fig. 1A). Six weeks after the transplantation, the recipient mice were sacrificed, and bone marrow, peripheral blood, spleen, and peritoneal macrophages were collected and analyzed by flow cytometry. We found GFP+mCherry+ cells in all four tissue types, suggesting the presence of natural RXRA ligands in hematopoietic cells (Fig. 1, B and C). The RXRA terminal helix (AF2 domain) is required for ligand-dependent transcription, and deletion of this domain acts as a negative control (21). We verified that the Gal4-RXRA and Gal4-RXRA-ΔAF2 fusion proteins were present in bone marrow Kit+ cells and NIH-3T3 cells after transduction with the corresponding retrovirus (fig. S2A). Because long-chain fatty acids have been implicated as natural ligands for both RXRs and PPARs (8, 22), we also verified the specificity of the reporter and found that it was not activated by PPAR ligands when cells were transduced with Gal4-RXRA (fig. S2, B and C). GFP+mCherry+ cells were not observed in UAS-GFP mice transplanted with Gal4-RXRA-ΔAF2, suggesting that the GFP+mCherry+ cells observed after Gal4-RXRA transplantation resulted from RXRA ligand binding and not from heterodimeric cross-talk or nonspecific reporter activity. Reporter activity was not uniform across all hematopoietic populations. Subgate analysis using lineage cell markers revealed that GFP+mCherry+ cells are biased toward CD11b+ myeloid lineage cells (Fig. 1D).

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.

RXRA reporter output after G-CSF and PHZ treatment

Given the roles of RXRA in myeloid maturation and the myeloid bias in GFP+mCherry+ cells observed in UAS-GFP transplants, we evaluated the effect of myeloid stress on natural RXR ligands in hematopoietic cells. We again transduced UAS-GFP bone marrow Kit+ cells with Gal4-RXRA retrovirus and transplanted these cells into recipient mice. After engraftment, we treated transplanted mice with G-CSF to induce granulopoiesis or with PHZ to induce hemolytic anemia and erythropoiesis. After PHZ treatment, we also observed granulopoiesis, with the white blood cell (WBC) peak at 18 to 22,000/μl, as has been noted elsewhere (23). As a positive control, we also treated transplanted mice with the synthetic RXRA agonist bexarotene. Compared to the mice without treatment, we found an increase in the proportion of GFP+mCherry+ cells in total bone marrow cells after G-CSF or PHZ treatment (Fig. 2, A and B). In addition, we found that the single-cell abundance of GFP, indicated by mean fluorescence intensity (MFI), also increased (Fig. 2C). In comparison, GFP+mCherry+ cells were not detected in Gal4-RXRA-ΔAF2 transplanted mice after G-CSF or PHZ treatment (Fig. 2A). These data suggest that intracellular concentrations of natural RXRA ligands increase in hematopoietic cells during G-CSF–induced granulopoiesis and PHZ-induced erythropoiesis, although concentrations of these natural ligands do not result in the maximum RXRA activation achieved by a potent pharmacologic agent (bexarotene).

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.

Participation of RXRA in G-CSF–induced granulopoiesis and hematopoietic stem cell mobilization

Because bexarotene treatment induced greater activation of the UAS-GFP reporter than did granulopoiesis stem cell factor (GSCF) treatment (both in terms of the absolute proportion of GFP+mCherry+ cells and the MFI of the GFP+mCherry+ cells), we determined whether bexarotene could augment the effects of G-CSF on granulopoiesis and hematopoietic stem cell (HSC) mobilization. We found that bexarotene increased both the WBC count and neutrophil (NE) count after G-CSF treatment, as compared to G-CSF treatment alone (Fig. 3, A and B). After G-CSF treatment, bexarotene did not affect the absolute number of immunologically defined stem and progenitor populations in the peripheral blood [Kit+LinSca-1+ (KLS) stem cells (Fig. 3C) or Kit+LinSca-1 progenitor cells (Fig. 3D)], but it did increase the number of functional stem and progenitor cells, as measured by colony-forming units (CFUs; Fig. 3E).

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.

To determine whether RXR activity is essential for G-CSF–induced granulopoiesis and HSC mobilization and function, we treated Mx1-Cre Rxraflox/floxRxrbflox/flox [RXR–knockout (KO)] mice and Rxraflox/floxRxrbflox/flox [RXR–wild type (WT)] mice with G-CSF (19). We observed a nonsignificant decrease in the WBC and NE counts and peripheral blood CFUs between RXR-KO and RXR-WT mice after G-CSF treatment (Fig. 3, F to H), but RXR-KO mice had fewer peripheral blood Kit+LinSca-1 progenitor cells after G-CSF treatment compared to RXR-WT mice (Fig. 3I). These data demonstrate that Rxra and Rxrb signaling contributes to G-CSF–induced granulopoiesis and HSC mobilization but is not essential.

Natural RXRA ligands in mouse plasma

To determine whether natural RXRA ligands are exclusively intracellular or are also present in plasma, we transduced UAS-GFP bone marrow Kit+ cells with Gal4-RXRA retrovirus and treated the cultured cells ex vivo with mouse plasma. We found that plasma activated the reporter in a dose-dependent manner (Fig. 4A), and the ligands in 6 μl of plasma (3% of the total culture volume) activated the reporter as much as did 100 nM 9-cis-RA.

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.

Furthermore, we found that UAS-GFP reporter output increased when Kit+ cells were treated with plasma from mice treated with PHZ or GSCF (Fig. 4B), consistent with our previous in vivo results (Fig. 2B), and we did not observe an increase after 5-fluorouracil (5-FU) treatment, which causes global hematopoietic aplasia followed by stem cell expansion and hematopoietic recovery (fig. S3A). As expected, reporter activity was absent in cells transduced with Gal4-RXRA-ΔAF2 (fig. S3B) and was not observed in cells transduced with Gal4-RARA (fig. S3C), again excluding the possibility of reporter activation through heterodimeric cross-talk or nonspecific activation of the reporter. Although both G-CSF and PHZ induced granulopoiesis (23) and increased UAS-GFP reporter activity in our assays, the response after PHZ treatment was modestly greater than that after G-CSF treatment (Fig. 4B). Therefore, we focused our subsequent ligand identification efforts primarily on plasma from mice treated with PHZ.

Dietary vitamin A deficiency and fatty acid restriction

To determine whether the reporter activity induced by plasma might be due to vitamin A derivatives (for example, 9-cis-RA or 9-cis-13,14-DHRA) (3, 4, 7), we analyzed plasma from vitamin A–deficient (VAD) mice (24). Before PHZ treatment, we confirmed vitamin A deficiency in these mice using mass spectrometry (MS) detection of plasma retinol (Fig. 5A). Unexpectedly, activation of the GFP reporter by plasma from VAD mice was not statistically different from activation by plasma from VAD control mice (VADC), suggesting that prolonged vitamin A deficiency did not cause a reduction in plasma ligands that activate RXRs in hematopoietic cells. Furthermore, plasma from VAD mice treated with PHZ and plasma from VADC mice treated with PHZ activated the GFP reporter equally, suggesting that natural RXR ligands can be generated in mouse plasma in the absence of vitamin A (Fig. 5B).

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.

To determine whether the RXRA ligands that increase after PHZ treatment are fatty acids, such as DHA or AA (8), we fed mice a no-fat (NF) diet or NF control (NFC) diet for 4 weeks, a period not associated with sequelae because of essential fatty acid deficiency (25). Unlike vitamin A, nonessential fatty acids can be synthesized in vivo. After 4 weeks on the NF diet, the plasma concentration of palmitoleic acid C16:1 (a common glyceride constituent) was reduced about twofold (Fig. 5C). Unlike plasma from mice fed the VAD diet, plasma from mice fed the NF diet stimulated less GFP activation than did plasma from mice on the control diet (Fig. 5D). This presumably reflects reduced concentrations of RXR ligands in the plasma both at baseline and after PHZ treatment. These data suggest that natural RXR ligands in mouse plasma either are essential fatty acids present in the diet or require intake of essential fatty acids for their synthesis.

Identification of plasma RXRA ligands

To identify RXRA ligands by MS, we generated a stable 293T cell line that contains the UAS-GFP reporter and the fusion protein Flag–Gal4 DBD–RXRA LBD (293T-FXP). These cells produce GFP when treated with either plasma or serum and are amenable to greater experimental throughput of serum detection and subsequent pull-down studies. We tested commercially available serum from a wide range of mammals on these reporter cells. We found that mouse and hamster serum had abundant RXRA ligands that activated the GFP reporter, rabbit and rat serum had moderate concentrations of RXRA ligands, and guinea pig and goat serum had undetectable concentrations of RXRA ligands (Fig. 6A), indicating that baseline RXRA activation may differ between species. To characterize the serum-available RXRA ligands, we extracted mouse serum with different mixtures of immiscible solvents. We found that the reporter-activating ligands were maximally extracted in the organic phase at ratios of hexane/isopropanol (3:2) and methanol/H2O (9:1), consistent with them being mildly nonpolar lipids (Fig. 6, B and C).

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.

By MS, we first quantified the concentrations of diverse fatty acids under conditions we previously observed to elicit different UAS-Gal4 reporter activity. On the basis of our reporter results (Figs. 4 to 6), we anticipated that the RXRA ligands would meet the following requirements: They should show at least a threefold difference in concentration between mouse and goat serum; a fivefold difference between mouse serum extracted with 9:1 methanol/H2O and extracted with 1:1 methanol/H2O; a 1.5-fold difference between VAD plasma and VAD-PHZ plasma; a 1.5-fold difference between NF plasma and NF-PHZ plasma; and a twofold difference between NFC plasma and NFC-PHZ plasma. Several fatty acids fulfilled one or more of these criteria (Fig. 7, A to F and table S1).

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.

Second, we assessed fatty acids that bound to Flag-Gal4-RXRA during immunoprecipitation. We immunoprecipitated Flag-tagged RXRA from 293T-FXP cells that had been treated with mouse or goat serum, extracted the immunoprecipitated RXRA with hexane/isopropanol (3:2), and analyzed the extraction by MS. Only six fatty acids bound to RXRA, in a manner that depended on the duration of cell exposure to serum: C22:3, C22:4, C22:6, C24:4, C24:5, and C24:6 (Fig. 7G and table S1). Third, we compared the concentrations of lipids from mouse serum versus goat serum that could be immunoprecipitated with Flag-Gal4-RXRA and found that C24:4 and C24:5 were three times higher in mouse serum pull-down samples versus goat serum pull-down samples (Fig. 7H). Fourth, we analyzed peripheral blood cells and bone marrow cells from mice treated with PHZ or vehicle control by MS. We found that the concentrations of C24:4 and C24:5 increased threefold in peripheral blood cells and increased twofold in bone marrow cells after PHZ treatment (Fig. 7, I and J). These data suggest that in mouse hematopoietic cells and in mouse serum, the long-chain fatty acids C24:4 and C24:5 could be candidate RXRA ligands, with concentrations that increase in response to PHZ-induced hematopoietic stress.

Binding of long-chain fatty acids C24:4 and C24:5 to RXRA

We investigated the possibility that C24:4 and C24:5 could potentially dock within the RXRA ligand-binding pocket. We modeled docking of these lipids using Surflex-Dock (26) and crystal structures of RXRA containing oleic acid [Protein Data Bank (PDB): 1DFK, which has the AF2 domain in an inactive configuration] or 9-cis-RA (PDB: 1XDK, which has the AF2 domain in an active configuration) (2729). Our results identified several plausible docked poses within the RXRA LBD that could result from the torsional mobility of these lipids, and both lipids could be docked within either configuration of the AF2 domain (Fig. 8, A and B, and figs. S4 and S5). In all predicted configurations, the carboxyl group interacted with the Arg321 side chain. Furthermore, previously reported ligand-contacting side chains could be modeled with close (<3 Å) distances of C24:4 and C24:5 (for example, L331, A332, V347, I350, C437, H440, L441, and F444) (2729).

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.

We also measured the effect of commercially synthesized C24:4 and C24:5 on GFP fluorescence in primary UAS-GFP Kit+ cells transduced with the Gal4-RXRA retrovirus. By MS, the peak of the commercially synthesized C24:5 compound was identical to the inferred C24:5 plasma peak from mouse plasma (fig. S6). Application of C24:4 did not activate the GFP reporter, whereas application of C24:5 activated the GFP reporter in a dose-dependent manner (Fig. 8C). We also observed that C24:5 could activate a direct repeat 1 (DR1)–luciferase reporter (containing three multimerized repeats of the RXRA DR1 sequence) when cells were transduced with full-length RXRA and that low-dose all-trans retinoic acid augmented the response to the lipid, as has been observed with DHA (Fig. 8D) (8).

Finally, to determine whether the long-chain fatty acid C24:5 can bind to and activate RXRA directly, we performed fluorescence resonance energy transfer (FRET)–based coactivator binding assays. C24:5 induced binding of RXRA to both PGC1 and D22 peptides in a dose-dependent manner, with an EC50 of 94 and 712 nM, comparable to the results observed with DHA (86 and 366 nM) but lower than the results observed with 9-cis-RA (17 and 79 nM) (Fig. 8, G and H). C24:4 did not induce RXRA binding to either PGC1 or D22 (Fig. 8, E and F). These data implicate the long-chain fatty acid C24:5 as a natural ligand of RXRA in mouse, which undergoes dynamic increases in concentrations in response to hematopoietic stress.

DISCUSSION

RXRA is a ligand-dependent transcription factor that participates in several essential biological processes, including development, metabolism, and hematopoiesis. Although multiple RXRA-deficient mouse models have been used to study its function, these studies have not addressed whether endogenous RXRA ligands are present or whether their concentrations in hematopoietic cells are altered in response to stimuli in vivo. Other groups have used a UAS-Gal4 reporter system to explore natural RXRA ligands in the developing mouse spinal cord, although none have examined the presence of natural RXRA ligands in hematopoiesis (30). By using a similar in vivo reporter system, we found that natural RXRA ligands are present and dynamically increase in concentration in mouse hematopoietic cells and plasma after hematopoietic stress induced by G-CSF or PHZ. Finally, we identified the long-chain fatty acid C24:5 as a natural RXRA ligand in mice. 9-cis-RA was initially identified as a high-affinity, highly active ligand for RXRA in vitro (3, 4). However, it has been the low-affinity long-chain fatty acids, such as DHA (8), that have been implicated as natural ligands in vivo. Our data further support a role for low-affinity long-chain fatty acids as natural ligands for RXRA and demonstrate that these lipids may be dynamically regulated in vivo.

RARA-RXRA heterodimers play important roles in hematopoiesis and leukemogenesis (31, 32). Most of the patients with acute promyelocytic leukemia (APL) express the fusion protein promyelocytic leukemia–RARA, and at least 10 additional proteins have been identified as RARA fusion partners in APL patients (33). Using our in vivo reporter strategy, we did not detect natural RARA ligands in hematopoietic cells in vivo (20) or in mouse plasma (fig. S3C). Instead, we observed evidence of natural ligands of RXRA in both hematopoietic cells and plasma and found that reporter activity increased during myeloid stress, such as that caused by G-CSF or PHZ treatment (Figs. 2 and 4), but not after stem cell stress, such as that induced by 5-FU (fig. S3A). We observed no reporter activity when bone marrow cells were transduced with a form of RXRA lacking the AF2 domain, which is required for ligand-dependent activation of the reporter, suggesting that the increased reporter activity after induction of myeloid stress was due to increased concentrations of natural RXRA ligands. Because this deletion only prevents ligand-dependent activation of the reporter, it does not control for alternative, ligand-independent effects on RXRA that could result from G-CSF or PHZ treatment, and this limitation must be acknowledged, although increased concentrations of RXRA ligands DHA and C24:5 were observed in parallel by MS.

RXRs are central members of the nuclear receptor superfamily (1) and can function as homodimers (10) or heterodimers (1115). PPAR-RXRs are permissive heterodimers that can be activated by either PPAR ligands or RXR ligands (34). In contrast, TR-RXR and VDR-RXR are nonpermissive heterodimers, which only can be activated in the presence of TR ligands or VDR ligands, respectively (35). PPARs participate in self-renewal of hematopoietic stem and progenitor cells (36, 37), TRs influence proliferation and differentiation of erythroid progenitors (38), and VDR deficiency promotes survival of hematopoietic stem and progenitor cells in the spleen (39). Both RXRs and PPARs can be activated by long-chain fatty acids. However, we did not observe activation of the reporter when cells were treated with PPAR ligands (8, 22), suggesting that the UAS-Gal4 system was responding specifically to RXRA ligands (fig. S2, B and C).

We found that RXRA reporter activity increased during GSCF-induced granulopoiesis. Through studies with RXR-KO mice, we found that RXR activation contributed to G-CSF–induced mobilization of HSCs (although it was not absolutely required) and that G-CSF–induced mobilization of HSCs was augmented by the addition of pharmacologic concentrations of RXR ligands (Fig. 3, A to E).

The vitamin A derivatives 9-cis-RA and 9-cis-13,14-DHRA have been proposed as natural RXRA ligands in mouse serum, brain, and liver (3, 4, 7). However, when we examined plasma in a VAD mouse model, in which plasma retinol concentrations were undetectable (Fig. 5A), we nevertheless detected activation of the RXRA reporter, consistent with the presence of RXRA ligands (Fig. 5B). These data suggest that the predominant plasma RXRA ligands both under baseline conditions and after PHZ treatment are unlikely to be vitamin A derivatives.

In contrast, multiple groups have suggested that fatty acids might serve as activating RXR ligands. The crystallographic analysis of a constitutively active mutant form of mouse RXRA (RXRAF318A) revealed a continuous U-shaped electron density in the ligand-binding cavity, suggesting the presence of a bound ligand and consistent with a fatty acid of 16 or 18 carbon atoms (27). C18:1 can activate the RXRA reporter ex vivo but with low efficiency (8). We detected fatty acid C18:1 in mouse serum and found that it was more abundant in mouse than in goat serum, but its concentration was not affected by PHZ treatment (table S1). Moreover, we found that C18:1 could be extracted by methanol/H2O (1:1), yet the products extracted by methanol/H2O (1:1) did not activate the RXRA reporter (Fig. 6C). The unsaturated fatty acids C20:4, C22:5, and C22:6 have been identified as Rxr ligands in mouse brain (8). We detected C22:5 in both mouse and goat serum, although goat serum did not activate the RXRA reporter (Fig. 6A and table S1). We also found that C22:5 did not coimmunoprecipitate with RXRA when the reporter cells were incubated with serum, although C22:6 did (table S1). However, PHZ treatment did not consistently affect C22:6 concentrations in VAD mice and NF mice (table S1), suggesting that C22:6 may contribute to basal RXR activation but does not appear to increase in concentration in response to PHZ. In contrast, C24:5 was consistently detected by MS under conditions that lead to activation of the RXRA reporter and was dynamically increased in plasma concentration after PHZ treatment. Commercially synthesized C24:5 activated the RXRA reporter, activated a DR1-luciferase reporter when coexpressed with full-length RXRA, and induced dose-dependent coactivator binding to purified RXRA (Figs. 7 and 8), suggesting that this lipid is a natural RXRA ligand that increases in plasma concentration in response to hematopoietic signals.

In summary, we observed evidence of activating natural ligands for RXRA in mouse hematopoietic cells, as well as in plasma and serum. Concentrations of these ligands were increased after G-CSF– or PHZ-induced myeloid stress, and we identified C24:5 as a likely natural RXRA ligand that undergoes dynamic increases in concentration during myeloid stress. These data do not address further important questions: Which pathways lead to alterations in serum concentrations of natural RXRA ligands? Do differences in diet augment or diminish RXRA signaling and the subsequent consequences on inflammation and bone metabolism? Does intervention with RXRA agonists improve stem cell mobilization or augment G-CSF–induced NE count recovery after chemotherapy? Additional studies will be required to further define the function of RXRs and the outcome of their in vivo regulation.

MATERIALS AND METHODS

Reagents

Bexarotene was from LC Laboratories. PHZ, 5-FU, corn oil, hexane, and isopropanol were from Sigma-Aldrich. G-CSF was from Amgen. GW6471, GW7647, pioglitazone, and tesaglitazar were from Tocris. Anti-mouse CD11b (M1/70)–BV421, anti-mouse c-Kit (2B8)–BV421, anti-mouse B220 (RA3-6B2)–phycoerythrin (PE)–Cy7 were from BD Biosciences. Anti-mouse CD8 (53 to 6.7)–eFluor 450, anti-mouse CD71 (R17217)–eFluor 450, anti-mouse Sca-1 (D7)–APC (allophycocyanin), anti-mouse Gr-1 (RB6-8C5)–APC, anti-mouse CD4 (GK1.5)–APC, anti-mouse Ter119 (TER119)–APC, anti-mouse c-Kit (2B8)–PE-Cy7, anti-mouse CD19 (eBio1D3)–PE-Cy7, anti-mouse Ter119 (TER119)–PE-Cy7, anti-mouse CD127 (ATR34)–PE-Cy7, anti-mouse CD8 (53-6.7)–PE-Cy7, anti-mouse CD4 (RM4-5)–PE-Cy7, and anti-mouse CD3e (145-2C11)–PE-Cy7 were from eBioscience. Sera from mouse, hamster, rabbit, rat, guinea pig, and goat were obtained from Equitech-Bio Inc., and sera were obtained while animals were maintained on a standard diet. C24:4 [(9Z,12Z,15Z,18Z,21Z)-tetracosa-9,12,15,18,21-tetraenoic acid] and C24:5 [(9Z,12Z,15Z,18Z,21Z)-tetracosa-9,12,15,18,21-pentaenoic acid] were synthesized from Avanti Polar Lipids Inc. The DR1-luciferase reporter and pBABE-RXRA plasmids were gifts from V. Arora, Washington University.

Hematopoietic cell culture

Mouse bone marrow Kit+ cells were isolated using an autoMACS Pro (Miltenyi Biotec) per the manufacturer’s protocol. Kit+ cells were plated in progenitor expansion medium [RPMI 1640, 15% fetal bovine serum (FBS), SCF (50 ng/ml), interleukin-3 (IL-3; 10 ng/ml), Fms-related tyrosine kinase 3 (Flt3; 25 ng/ml), thrombopoietin (Tpo; 10 ng/ml), l-glutamine (2 mM), sodium pyruvate (1 mM), Hepes buffer (10 mM), penicillin/streptomycin (100 U/ml), and β-mercaptoethanol (50 μM)] overnight and transduced by spinfection with polybrene (10 μg/ml) and 10 mM Hepes at 2400 rpm, 30°C for 90 min in an Eppendorf 5810R centrifuge. Fluorescence was detected on a FACS scan or Gallios instrument (Beckman Coulter).

Mice

UAS-GFP and RXR-KO mice were bred as described (19, 20). RXR deletion was induced by injecting RXR-KO mice intraperitoneally with polyinosinic:polycytidylic acid (pI:pC) (300 μg per mouse); four doses were given every other day. RXR deletion was confirmed by polymerase chain reaction 4 weeks after mice were treated with pI:pC. Bexarotene was administrated by oral gavage, suspended in sterile corn oil, 1 mg per mouse per day for 3 days, and mice were sacrificed at day 4. G-CSF was administrated by subcutaneous injection, 125 μg/kg every 12 hours for nine injections, and mice were sacrificed 2 hours after the last injection. All mice were cared for in the experimental animal center of Washington University School of Medicine. The Washington University Animal Studies Committee approved all animal experiments.

VAD mouse model

VAD mice were generated as described (24). Briefly, friend virus B (FVB) female mice were fed with VAD diet (TD.86143; Teklad) or vitamin A control diet (TD.91280; Teklad) during pregnancy. The offspring received the same diet for at least 12 weeks. Plasma concentrations of retinol were undetectable by MS before experimental intervention.

Diet-restricted fatty acid mouse model

A diet-restricted fatty acid mouse model was generated as described (25). Briefly, FVB mice were fed with NF diet (TD.03314; Teklad) and NFC diet (TD.130321; Teklad) for 4 weeks before experimental intervention. Plasma concentrations of palmitoleic acid (C16:1) were evaluated by MS before experimental intervention.

Retrovirus production

Retrovirus production was performed as described (40). Calcium chloride transfection and low-passage 293T cells were used for virus packaging. 293T cells (5 × 106) were seeded in 10-cm dishes in Dulbecco’s modified Eagle’s medium (high glucose) + 10% FBS 18 to 24 hours before transfection and were grown to 80% confluence. Twelve-microgram MSCV–Gal4 DBD–RXRA LBD–IRES–mCherry (Gal4-RXRA), MSCV–Gal4 DBD–RXRA LBD ΔAF2–IRES–mCherry (Gal4-RXRA ΔAF2), or MSCV–Gal4 DBD–RARA LBD–IRES–mCherry (Gal4-RARA), 8 μg of Ecopak, and 155 μl of 2 M CaCl2 were mixed, and the volume was adjusted to 1.25 ml by adding H2O. Hepes buffer (2×; 1.25 ml) was drop-wise added to the mixture. The mixture was incubated for 20 min at room temperature and then drop-wise added onto 293T cells. Fresh medium was changed after 12 hours transfection. Virus was collected at 48 and 72 hours and stored at −80°C.

Bone marrow transplantation

Femurs, tibias, and pelvises were isolated from 6- to 8-week-old UAS-GFP mice. Bone marrow cells were collected by centrifuging bones at 6000 rpm for 2 min. Red blood cells were lysed in ACK Buffer (NH4Cl, 150 mM; KHCO3, 10 mM; Na2EDTA, 0.1 mM) on ice for 5 min. Kit+ cells were isolated by MACS using autoMACS Pro (Miltenyi Biotec) per the manufacturer’s protocol. Kit+ cells were cultured in transplant medium [RPMI 1640 + 15% FBS + mSCF (20 ng/ml) + mFlt3L (25 ng/ml) + mIL3 (10 ng/ml) + Tpo (10 ng/ml) + 50 μM β-mercaptoethanol] overnight and transduced with Gal4-RXRA or Gal4-RXRA-ΔAF2 retrovirus by spinfection with polybrene (10 μg/ml) and 10 mM Hepes at 2400 rpm, 30°C for 90 min in an Eppendorf 5810R centrifuge. Kit+ cells were spinfected with the same virus for a second time after 24 hours. Transduced cells were injected to lethally irradiated recipient mice 2 hours after the second spinfection. After 6 weeks of engraftment, mice were sacrificed and analyzed.

Flow cytometry

Bone marrow, peripheral blood, spleen, and peritoneal macrophages were collected from the bone marrow of bone marrow transplant recipient mice after engraftment (typically ~6 weeks). For hematopoietic stem and progenitor cell analysis, cells were stained with Lineage-PE-Cy7, c-Kit–BV421, and Sca-1–APC. For myeloid lineage analysis, cells were stained with c-Kit–PE-Cy7, CD11b-BV421, and Gr-1–APC. For erythroid lineage analysis, cells were stained with c-Kit–PE-Cy7, CD71–eFluor 450, and Ter119-APC. For lymphoid lineage analysis, cells were stained with B220-PE-Cy7, CD4-APC, and CD8–eFluor 450. Fluorescence was detected using a Gallios flow cytometer (Beckman Coulter).

Plasma or serum extraction

Fifty microliters of mouse plasma or serum was mixed with 500 μl of hexane/isopropanol or methanol/H2O and vortexed vigorously for 30 s. After centrifugation at 12,000 rpm for 5 min, the upper phase (organic phase) was collected into a new tube and stored at −80°C for later analysis.

Pull-down assays

We generated a line of 293T cells stably expressing UAS-GFP and 3× Flag–Gal4 DBD–RXRA LBD–IRES–Puro (293T-FXP). 293T-FXP (1 × 106 cells per well) were seeded in six-well plates and treated with mouse serum or goat serum. Cells were collected and lysed with radioimmunoprecipitation assay buffer (50 mM tris-HCl, 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% SDS, 1 mM EDTA, and proteinase inhibitor) at 0, 3, 6, 9, 12, and 24 hours. 293T-FXP cell lysate was incubated with prewashed anti-Flag magnetic beads and rotated overnight at 4°C. Beads were washed with tris-buffered saline and Tween 20 solution (TBST) three times. Washed beads were resuspended in 50 μl of TBST and then extracted with hexane/isopropanol (3:2). The organic phase was collected and stored in −80°C for later analysis.

Mass spectrometry

For retinol analysis, 100 μl of plasma extraction sample was used. Before MS analysis, 50 ng of retinol-d6 as the internal standard was added to each sample. The solvents in the sample were dried under a stream of nitrogen. The dried sample was redissolved in 1 ml of ethanol/H2O (1:1) for MS analyses.

For fatty acids analysis, 100 μl of plasma extraction sample or 400 μl of pull-down sample was used for DMAPA (dimethylaminopropylamine) derivatization for improving MS sensitivity of fatty acids. Before the derivatization, 50 ng (10 ng for the pull-down sample) of AA-d8 as the internal standard was added to each sample. The solvents in the sample were dried under a stream of nitrogen. To derivatize the sample, 50 μl of 100 mM EDC [N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride] and 50 μl of 50 mM DMAPA and 50 mM DMAP (4-dimethylaminopyridine) were added to the dried fatty acid samples and heated at 50°C for 30 min. The samples were dried under nitrogen and then dissolved in 1 ml of ethanol/H2O (1:1) for MS analysis.

In silico ligand docking analysis

Docking of C24:5 and C24:4 against crystal structures of RXRA (PDB: 1DKF and 1XDK) was performed with Surflex-Dock (26). MOL2 files for each compound were generated from SMILES (simplified molecular-input line-entry system) strings using Open Babel (41). Surflex-Dock receptor protomols were generated with a threshold of 0.25 and a bloat of 2.0 and subsequently docked using the default “-pgeom” docking accuracy parameter set. Residues lining the binding pocket were defined as those within 4 Å of oleic acid (PDB: 1DKF) or 9-cis-RA (PDB: 1XDK). Images were generated in Pymol (version 1.8.6.0; Schrodinger).

Luciferase detection

293T cells were transfected with pBABE-RXRA in combination with DR1x3-TK-luciferase using Lipofectamine 2000 (Invitrogen). Six hours after transfection, the cells were collected and plated into a 48-well plate in 1% bovine serum albumin (BSA) medium in triplicate. DHA, fatty acid 24:5, bexarotene, or 9-cis-RA were added to the cells as a BSA complex (BSA, Fraction V, fatty acid–free, Calbiochem), with equal amounts of BSA added to all samples (42). After a 40-hour incubation, the cells were harvested and assayed for luciferase (luciferase assay system with reporter lysis buffer; Promega) in a Beckman Coulter LD400 plate reader.

Time resolution–FRET assay

The assay was performed using LanthaScreen TR-FRET Retinoic X Receptor alpha Coactivator assay (Invitrogen). Reaction mixture contained 10 nM RXRA LBD–GST (glutathione S-transferase), 5 nM terbium-labeled anti-GST antibody, 500 nM fluorescein-PGC1α or D22 peptide, 5 mM dithiothreitol, 1% dimethyl sulfoxide, and testing compounds. Twenty microliters of the reaction mixture was analyzed after 1-hour incubation at room temperature and analyzed using a Synergy2 plate reader (BioTek Instruments), using a 340-nm filter with a 30-nm bandwidth, and an emission filter was centered at 520 nm with a 25-nm bandwidth and at 495 nm with a 10-nm bandwidth. The TR-FRET ratio was calculated by dividing the emission signal at 520 by the emission signal at 495.

Data analysis

Flow cytometry data were analyzed with FlowJo software version 10. Statistical analysis was performed using Prism (GraphPad). ANOVA and t tests were performed, as appropriate. FRET studies were analyzed using a sigmoidal dose response with variable slope. All studies were performed in triplicate, unless otherwise indicated. Error bars represent SDs. Data points without error bars have SDs below GraphPad’s limit to display.

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.

REFERENCES AND NOTES

Acknowledgments: We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, MO for the use of the Flow Cytometry Core. The Siteman Cancer Center is supported in part by a National Cancer Institute Cancer Center Support Grant P30 CA91842. We thank the High-Throughput Screening Center at Washington University School of Medicine in St. Louis, MO, and the MS facility at Washington University. We thank D. Laflamme for technical assistance and F. Gao for statistical assistance. Funding: This work was supported by NIH R01 HL128447 (J.S.W.), NIH P50 CA171963 (project 1; J.S.W.), and grants from the Spanish Ministry of Economy and Competitiveness (SAF2015-64287R and SAF2015-71878-REDT) (M.R.). The MS facility at Washington University is supported by NIH P30 DK020579 (D. Ory). Author contributions: J.S.W., H.N., and M.R. designed the experiments, performed the experiments, and wrote the manuscript. H.F., O.d.M., G.H., M.P.M.-G., T.E.F., and G.R.B. designed and performed the experiments. Competing interests: The authors declare that they have no competing interests. Data and materials availability: MS data are available at the University of California, San Diego Metabolomics Workbench (www.metabolomicsworkbench.org; accession: hhfuji11_20170922_072445.
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