-) mice - PubMed (original) (raw)

ROS-mediated amplification of AKT/mTOR signalling pathway leads to myeloproliferative syndrome in Foxo3(-/-) mice

Safak Yalcin et al. EMBO J. 2010.

Abstract

Reactive oxygen species (ROS) participate in normal intracellular signalling and in many diseases including cancer and aging, although the associated mechanisms are not fully understood. Forkhead Box O (FoxO) 3 transcription factor regulates levels of ROS concentrations, and is essential for maintenance of hematopoietic stem cells. Here, we show that loss of Foxo3 causes a myeloproliferative syndrome with splenomegaly and increased hematopoietic progenitors (HPs) that are hypersensitive to cytokines. These mutant HPs contain increased ROS, overactive intracellular signalling through the AKT/mammalian target of rapamycin signalling pathway and relative deficiency of Lnk, a negative regulator of cytokine receptor signalling. In vivo treatment with ROS scavenger N-acetyl-cysteine corrects these biochemical abnormalities and relieves the myeloproliferation. Moreover, enforced expression of Lnk by retroviral transfer corrects the abnormal expansion of Foxo3(-/-) HPs in vivo. Our combined results show that loss of Foxo3 causes increased ROS accumulation in HPs. In turn, this inhibits Lnk expression that contributes to exaggerated cytokine responses that lead to myeloproliferation. Our findings could explain the mechanisms by which mutations that alter Foxo3 function induce malignancy. More generally, the work illustrates how deregulated ROS may contribute to malignant progression.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Myeloproliferative-like syndrome in Foxo3−/− mice. (A) Representative whole-mount (upper panels) and histological sections (lower panels) of spleens from 11-week-old wild-type (+/+) and Foxo3−/− (−/−) mice. Increased extramedullary myeloid hematopoiesis in the red pulp and minimal depletion of marginal zone lymphocytes with the retention of the T-cell regions in Foxo3−/− spleen, as compared with the wild-type are shown. (B) Total number of bone marrow (_n_=32) and spleen cells (_n_=12) is shown, Student's _t_-test. (C) Representative FACS plots of FSC versus SSC of bone marrow, spleen and blood of wild-type and Foxo3−/− mice are shown. Percentages of FSChighSSChigh (granulocytic) cells are marked. (D) Total number of bone marrow cells in each lineage is plotted. Total number of bone marrow erythroid (TER 119, _n_=11), B (B220, _n_=14) and T cells (CD3, _n_=11) and neutrophils (Gr-1/Mac-1, _n_=11) is shown. (E) Total number of cells in each lineage of the spleen, TER 119 (_n_=8), B220 (_n_=8), CD3 (_n_=11) and Gr-1/Mac-1 (_n_=11) (Student's _t_-test). The analyses are from at least four independent experiments.

Figure 2

Enhanced hematopoietic progenitor activity in Foxo3−/− mice. (A) Progenitor-derived colonies were measured in the bone marrow, spleen and blood of wild-type and Foxo3−/− mice. The analyses are from four independent experiments in each of which two to three animals were either pooled or analysed independently. (B) Frequency (left panel) of highly enriched myeloid progenitor Lin− IL7Rα− Sca-1− c-Kit+ compartment (right panel) in wild-type and Foxo3−/− bone marrow is shown (_n_=5 in each group, Student's _t_-test). The frequency of Sca-1− c-Kit+ cells within Lin− IL7Rα−-gated cells is shown (the frequency of Lin− IL7Rα− Sca-1− c-Kit+ cells within bone marrow is 1.3±0.11% for wild type and 2.2±0.2% for Foxo3−/−). One representative of three independent experiments is shown. (C) CFU-S-derived colonies formed in the spleen were measured 12 days after in vivo injection of 105 wild-type or Foxo3−/− bone marrow cells into lethally irradiated hosts; representative of two independent experiments, _n_=5 in each group is shown, Student's _t_-test. (D) Colony-forming cell ability of wild-type and Foxo3−/− progenitors was measured after plating 105 cells in semi-solid methylcellulose cultures in three replicates in the presence of limiting doses of the indicated cytokines (colonies of 20 or more cells were counted after 8 days and the numbers of colonies present at each cytokine concentration were calculated as percentages of the number formed in the highest concentration of the indicated cytokine). Mean+s.e.m. of three independent experiments, each pool of two to three mice. Student's _t_-test; *P<0.05, **P<0.01.

Figure 3

Enhanced ROS accumulation in Foxo3−/− primitive myeloid progenitors. Representative FACS profile of bone marrow Lin− IL7Rα− Sca-1− c-Kit+ that contain 98% of all myeloid progenitor cells (Akashi et al, 2000) (left panel). Frequency of c-Kit+ Sca1− cells within Lin− IL7Rα− cells is shown. Endogenous ROS concentrations were measured in freshly isolated Lin− IL7Rα− Sca-1− c-Kit+ cells (right panel) from wild-type or Foxo3−/− mice treated daily in vivo with NAC (100 mg/kg) or PBS for 15 days; fold change in mean fluorescence intensity (MFI) of ROS in gated subpopulations (ROS-hi using - - - - - - - gate), as compared with control wild-type cells treated with PBS is shown as mean±s.e.m., _n_=3; Student's _t_-test. One of two independent experiments is shown.

Figure 4

mTOR mediates the enhancement of Foxo3−/− hematopoietic progenitor cell compartment. (A) Western blot analysis of phosphorylation of signalling proteins in lineage-negative bone marrow cells isolated from wild-type and Foxo3−/− mice (_n_=4). Mice were administered daily with NAC (100 mg/kg) or PBS in vivo for 3 days, after which lineage-negative cells were isolated, serum- and cytokine starved for 2 h and stimulated with IL-3 (20 ng/ml) for the indicated time points (0, 10 and 30 min) in vitro in the absence or presence of NAC (100 μM) before preparing the whole cell extract; representative immunoblot of three independent experiments is shown. (B) Number of CFU-Sd12-derived colonies formed in the spleens of lethally irradiated mice reconstituted with 105 wild-type or Foxo3−/− bone marrow cells detected after 12 days during which mice were administered either rapamycin (Rapa; 4 mg/kg) or vehicle (Veh) intraperitoneally for 5 days a week. Results shown are mean±s.e.m. (_n_=5 in each group, Student's _t_-test). One representative of three independent experiments is shown. Representative spleen from each group is shown in the lower panel. (C) ROS levels (right panel) and phosphorylated AKT protein kinase (left panel) were measured by FACS in Lin− IL7Rα− Sca-1− c-Kit+ bone marrow cells of wild-type and Foxo3−/− mice treated daily with NAC or PBS for 2 weeks in vivo, after which isolated cells were serum- and cytokine starved for 2 h and stimulated with IL-3 (20 ng/ml) in vitro for 10 min. Results are mean±s.e.m. of percentage of Lin− IL7Rα− Sca-1− c-Kit+ bone marrow cells that express phosphoAKT (pAKT), as detected by FACS (_n_=6 in each group, Student's _t_-test). Endogenous ROS-hi levels were measured in Lin− IL7Rα− Sca-1− c-Kit+ bone marrow cells of the same mice as in the left panel, at the end of the 2 week in vivo treatment (right panel), and shown as fold change in MFI of experimental as compared with control wild-type cells (_n_=6 in each group, Student's _t_-test). Animals were analysed individually. One of two independent experiments is shown.

Figure 5

NAC treatment corrects the expansion of Foxo3−/− primitive multipotential hematopoietic progenitor compartment in vivo. CFU-S-derived colonies were measured 12 days after injection of 105 wild-type or Foxo3−/− bone marrow cells into lethally irradiated hosts and treated daily with NAC (100 mg/kg) or PBS. One representative of three independent experiments is shown (_n_=5 in each group, Student's _t_-test). Representative spleen of each group is shown in the bottom panel.

Figure 6

NAC treatment ameliorates myeloproliferative syndrome in Foxo3−/− mice in vivo. (A) Mice were treated daily with NAC (100 mg/kg) or PBS and their total number in the bone marrow and spleen was measured after 15 days (_n_=3 in each group). (B) Frequency of myeloid (Mac-1 and Gr-1 positive) and B (B220 positive) cells in the bone marrow of mice from A (_n_=3 in each group). (C) Percentage of BrdU- (upper) and annexin-V-binding positive (lower) cells in wild-type and Foxo3−/− Lin− IL7Rα− Sca-1− c-Kit+ cells was analysed by flow cytometry after 15 days in vivo of NAC (100 mg/kg) or PBS treatment of mice from A (_n_=3 in each group). (D) Wild-type or Foxo3−/− mice were treated daily with NAC (100 mg/kg) or PBS for 2 weeks after which bone marrow cells were isolated and injected (5 × 105 cells) into lethally irradiated hosts in the absence of any further treatment. CFU-S-derived colonies were counted in the hosts 12 days after cell injection, _n_=5 in each group; Student's _t_-test. Representative spleen from each group is shown in the lower left panel. ROS-hi concentrations were measured in an aliquot of Lin− IL7Rα− Sca-1− c-Kit+ cells isolated from each mouse 15 days after in vivo NAC or PBS treatment. ROS levels are shown as fold change in MFI as compared to control wild type cells (bottom right panel). One representative of two independent experiments is shown.

Figure 7

Deregulated expression of modulators of cytokine signalling in Foxo3−/− hematopoietic progenitor cells. (A) QRT–PCR expression analysis of cytokine receptor signalling regulatory genes in lineage-negative wild-type and Foxo3−/− cells. Quantification of target genes is relative to β-actin. Results are mean±s.e.m. of duplicate analysis of at least three cDNAs, each generated from a pool of two to three wild-type or Foxo3−/− mice; Student's _t_-test. (B) Western blot analysis of endogenous Lnk protein in total bone marrow cells isolated from two wild-type and two Foxo3−/− mice; representative immunoblot of three independent experiments is shown. (C) QRT–PCR analysis of Foxo3 (upper panel) and Lnk (lower panel) expression in mononuclear cells derived from 5-FU-treated wild-type and Foxo3−/− mice, transduced with MSCV-IRES-GFP (MIG) vector control or MIG-Foxo3; results are mean±s.e.m. of three independent experiments.

Figure 8

Decreased expression of Lnk is critical for increased activity of primitive hematopoietic progenitor cell compartment in Foxo3-deficient mice. (A) Flow cytometry profile of GFP expression in wild-type or Foxo3−/− bone marrow lineage-negative cells derived from 5-FU-treated mice and transduced with MIG encoding for Lnk (MIG-Lnk; FACS profile was almost identical with MIG vector control). (B) QRT–PCR analysis of Lnk expression in FACS-sorted retrovirally transduced GFP+ gated cells from A; results are mean±s.e.m. of three independent experiments; Student's _t_-test (C) Number of CFU-S-derived colonies formed in the spleens of lethally irradiated hosts (_n_=5 in each group) reconstituted with 5-FU-treated bone marrow mononuclear cells transduced with retroviral vector MIG or MIG-Lnk. FACS-sorted GFP-positive (3 × 104)-transduced primitive hematopoietic wild-type or Foxo3−/− cells (from A, B) were injected into lethally irradiated hosts and spleen colonies were counted 12 days later. Results shown as mean±s.e.m. (_n_=5 mice); Student's _t_-test. Representative spleens are shown in the lower panel. One representative of three independent experiments is shown. (D) Quantification of phosphorylated AKT, mTOR and S6 proteins in mononuclear cells derived from 5-FU-treated wild-type or Foxo3−/− mice transduced with MIG vector control or MIG-Lnk in response to IL-3. The percentage of cells with phoshorylated AKT, mTOR or S6 proteins was measured in GFP+ gated cells. Results shown are mean of two independent experiments.

Figure 9

Regulation of Lnk expression is mediated by ROS in Foxo3−/− primitive myeloid progenitor cells. (A) QRT–PCR gene expression analysis of regulators of cytokine signalling in Lin− IL7Rα− Sca-1− c-Kit+ bone marrow cells from mice (_n_=3) treated in vivo for 3 consecutive days with NAC (100 mg/kg) or PBS. One representative of three independent experiments is shown. (B) Western blot analysis of endogenous Lnk protein in total bone marrow cells isolated from wild-type and Foxo3−/− mice daily treated with NAC (100 mg/kg) or PBS in vivo for 3 days. A representative immunoblot of two independent experiments is shown. (C) QRT–PCR analysis of Lnk expression in total bone marrow cells isolated from wild-type and Foxo3−/− mice and treated in vitro with rapamycin (2 μM) or vehicle for 24 h (_n_=3).

Figure 10

Model for Foxo3 regulation of hematopoietic progenitor cell activity. In hematopoietic progenitors, cytokine receptor signalling generates ROS that further activate receptor signalling (AKT/mTOR), leading to cell proliferation, increased production of ROS and ultimate induction of Foxo3 nuclear localization once ROS are accumulated above certain threshold. Signalling is modulated by cytokine receptor regulators including Lnk, a negative regulator. In Foxo3−/− hematopoietic progenitors, decreased expression of Lnk, associated with significant increase in ROS accumulation, enhance cytokine-mediated signalling, leading to myeloproliferation.

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ROS-mediated amplification of AKT/mTOR signalling pathway leads to myeloproliferative syndrome in Foxo3(-/-) mice - PubMed (original) (raw)