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• W2127659799 abstract "Article26 November 2010free access ROS-mediated amplification of AKT/mTOR signalling pathway leads to myeloproliferative syndrome in Foxo3−/− mice Safak Yalcin Safak Yalcin Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Dragan Marinkovic Dragan Marinkovic Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Sathish Kumar Mungamuri Sathish Kumar Mungamuri Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Xin Zhang Xin Zhang Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Wei Tong Wei Tong Division of Hematology, Children's Hospital of Philadelphia and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Rani Sellers Rani Sellers Department of Pathology, Albert Einstein College of Medicine, New York, NY, USA Search for more papers by this author Saghi Ghaffari Corresponding Author Saghi Ghaffari Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA Black Family Stem Cell Institute, New York, NY, USA Department of Regenerative & Developmental Biology, New York, NY, USA Division of Hematology, Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Safak Yalcin Safak Yalcin Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Dragan Marinkovic Dragan Marinkovic Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Sathish Kumar Mungamuri Sathish Kumar Mungamuri Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Xin Zhang Xin Zhang Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Wei Tong Wei Tong Division of Hematology, Children's Hospital of Philadelphia and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Search for more papers by this author Rani Sellers Rani Sellers Department of Pathology, Albert Einstein College of Medicine, New York, NY, USA Search for more papers by this author Saghi Ghaffari Corresponding Author Saghi Ghaffari Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA Black Family Stem Cell Institute, New York, NY, USA Department of Regenerative & Developmental Biology, New York, NY, USA Division of Hematology, Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Author Information Safak Yalcin1, Dragan Marinkovic1, Sathish Kumar Mungamuri1, Xin Zhang1, Wei Tong2, Rani Sellers3 and Saghi Ghaffari 1,4,5,6 1Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA 2Division of Hematology, Children's Hospital of Philadelphia and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA 3Department of Pathology, Albert Einstein College of Medicine, New York, NY, USA 4Black Family Stem Cell Institute, New York, NY, USA 5Department of Regenerative & Developmental Biology, New York, NY, USA 6Division of Hematology, Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA *Corresponding author. Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA. Tel.: +1 212 659 8271; Fax: +1 212 803 6740; E-mail: [email protected] The EMBO Journal (2010)29:4118-4131https://doi.org/10.1038/emboj.2010.292 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Oxidative stress, broadly defined as an imbalance between generation and detoxification of reactive oxygen species (ROS), is deleterious to cells and implicated in a number of degenerative diseases and malignancies (reviewed in Beckman and Ames, 1998). In addition, excess accumulation of ROS impacts cellular aging, whereas the ability to resist oxidative stress is associated with evolutionary conserved enhanced longevity (Beckman and Ames, 1998). Although ROS are considered to be toxic byproducts of cellular metabolism, increasing evidence support the notion that ROS have a critical role in normal cellular signalling. In particular, ROS are generated by cytokine signalling and impact the function of a rapidly expanding list of numerous effectors (reviewed in Thannickal and Fanburg, 2000). How these activities affect normal and pathological physiology is not fully understood. ROS are particularly deleterious to hematopoietic stem cells, specifically as they age (Ito et al, 2004; Miyamoto et al, 2007; Tothova et al, 2007; Yalcin et al, 2008; and reviewed in Ghaffari, 2008). A tightly controlled balance between hematopoietic stem and progenitor cell compartments maintains normal blood cell homeostasis throughout life. Alterations of this balance result in various disorders including leukaemias or bone marrow failure. For instance, myeloproliferative disorders are a group of hematopoietic malignancies whose incidence increase with age, exhibit enhanced proliferation and survival of one or more myeloid lineage cells that arises from an unbalanced expansion of hematopoietic myeloid progenitor cells (Tefferi and Gilliland, 2007). The Forkhead FoxO family of transcription factors are critical regulators of oxidative stress and exert this function at least partly by upregulating the expression of several anti-oxidant enzymes (Kops et al, 2002; Nemoto and Finkel, 2002; Murphy et al, 2003; Marinkovic et al, 2007; Tothova et al, 2007; Yalcin et al, 2008). FoxO1, FoxO3 and FoxO4 are wildly expressed while FoxO6 is predominantly expressed in neuronal tissues. Loss of a single FoxO leads to distinct phenotypes in mice, underscoring their diverse non-redundant functions in vivo (Castrillon et al, 2003; Hosaka et al, 2004). In particular, female Foxo3-deficient mice exhibit a premature infertility associated with ovarian follicle depletion early on in life (Castrillon et al, 2003; Hosaka et al, 2004). In addition, both Foxo3−/− hematopoietic stem and erythroid cell compartments exhibit enhanced susceptibility to oxidative stress (Marinkovic et al, 2007; Miyamoto et al, 2007; Yalcin et al, 2008). FoxOs also regulate cellular responses to genotoxic stress, consistent with a tumour suppressor function (Paik et al, 2007). In response to stress such as DNA damage or oxidative stress, FoxOs induce cell cycle arrest, repair damaged DNA or initiate apoptosis by modulating genes that control these processes (Brunet et al, 1999; Dijkers et al, 2000; Medema et al, 2000; Nakamura et al, 2000; Tran et al, 2002; Alvarez et al, 2003; Ghaffari et al, 2003; Marinkovic et al, 2007; Yalcin et al, 2008). FoxO genes are also found at chromosomal breakpoints in certain cancers, including acute myeloid leukaemias (FoxO3 and FoxO4) (Borkhardt et al, 1997; Hillion et al, 1997). Moreover, FoxO3 regulates the expression and activity of ataxia telangiectasia-mutated protein kinase, suggesting an important role in the maintenance of genomic stability (Tsai et al, 2008; Yalcin et al, 2008). Function of FoxO is restrained primarily by the phosphoinositide-3-kinase (PI3-kinase)/AKT signalling pathway (Biggs et al, 1999; Brunet et al, 1999; Dijkers et al, 2000; Kashii et al, 2000; Nakae et al, 1999; Rena et al, 1999; Tang et al, 1999; and reviewed in Greer and Brunet, 2008). The AKT serine threonine protein kinase regulates a wide range of metabolic processes through phosphorylation of numerous effectors, including FoxO and mammalian target of rapamycin (mTOR) (Gingras et al, 1998; Brunet et al, 1999; Inoki et al, 2002; Manning et al, 2002) a kinase that stimulates cell growth and proliferation through multiple effectors including ribosomal S6 kinase (S6K1) and the eukaryotic initiation factor 4E-binding protein. In response to cytokines, growth factors or oncoproteins, activated AKT kinase directly phosphorylates FoxO on three conserved residues, resulting in their nuclear exclusion and subsequent degradation (Biggs et al, 1999; Brunet et al, 1999; Matsuzaki et al, 2003; Plas and Thompson, 2003; Hu et al, 2004). In contrast, stress stimuli, or inhibition of PI3-kinase/AKT signalling pathway by growth factor/cytokine withdrawal, induce FoxO's nuclear localization, thereby enhancing their transcriptional activity (Essers et al, 2004; Lehtinen et al, 2006; van der Horst et al, 2006). The PI3-kinase/AKT signalling pathway is activated in numerous human and animal malignancies, although how this contributes to the pathogenesis of these diseases is not entirely clear (Ugo et al, 2004; Bellacosa et al, 2005; Dai et al, 2005; Yilmaz et al, 2006; Zhang et al, 2006). In addition to AKT, a number of kinases regulate the activity of FoxO both positively and negatively. In addition to phosphorylation, FoxO proteins are subject of several other post-translational modifications such as acetylation, methylation and ubiquitination whose combined integrated signals determine the activity of FoxOs. Recent findings have established a critical function for FoxO family members in the regulation of normal and malignant hematopoietic stem cell activity (Miyamoto et al, 2007; Tothova et al, 2007; Yalcin et al, 2008; Naka et al, 2010). In particular, Foxo3's suppression of ROS is essential for the maintenance of hematopoietic stem cell quiescence and homeostasis (Miyamoto et al, 2007; Yalcin et al, 2008). In addition, abnormal repression of Foxo3 has been implicated in the pathogenesis of myeloproliferative disorders and other haematological malignancies (Ghaffari et al, 2003; Komatsu et al, 2003; Fernandez de Mattos et al, 2004; Essafi et al, 2005). Despite these findings, of the entire scope of Foxo3 functions, regulation of hematopoietic progenitors (HPs) is not fully defined (Miyamoto et al, 2007; Yalcin et al, 2008). Here, we show that loss of Foxo3 results in a myeloproliferative syndrome in mice. We further demonstrate that increased ROS accumulation in Foxo3−/− primitive myeloid progenitors activates the cytokine-induced AKT/mTOR signalling pathway and expands Foxo3-deficient primitive myeloid progenitors. Accordingly, this myeloproliferative syndrome is ameliorated by systemic administration of ROS scavengers. Moreover, Lnk (SH2B3), a negative regulator of cytokine signalling, is directly implicated in this process. Our combined data indicate that Foxo3 modulates HP homeostasis by controlling cytokine-dependent production of, and response to, ROS. These cumulative findings illustrate new mechanisms through which deregulated ROS could contribute to the development of malignancies. Results Foxo3−/− mice exhibit a myeloproliferative syndrome Foxo3−/− mice display increased white blood cell counts, with an increased circulating neutrophils (P<0.03) and monocytes (P<0.01), and a concomitant reduction of circulating lymphocytes (P<0.005) and red blood cells (Table I; Marinkovic et al, 2007). These anomalies of the peripheral blood are associated with myeloproliferative syndrome (Figure 1). Foxo3-deficient mice exhibit an enlarged spleen (Figure 1A), increased number of splenocytes (Figure 1B) and extramedullary hematopoiesis (Figure 1C, D and E, Supplementary Figure 1A), with increased frequency of erythrocytic and granulocytic lineages (Supplementary Figure 1). Concomitantly, the bone marrow is hypocellular (Figure 1B) with decreased production of mature B and erythroid cells (Figure 1D; Supplementary Figure 1; Marinkovic et al, 2007). Histopathology analysis corroborated these findings, showing increased extramedullary hematopoiesis containing erythroid and myeloid cells in the spleen and liver of Foxo3−/− mice (Figure 1A and Supplementary Figure 2). 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. Download figure Download PowerPoint Table 1. Comparison of blood parameters of Foxo3+/+ and Foxo3−/− mice Parameters Foxo3+/+ Foxo3−/− P-value WBC (× 1000/μl) 9.45±1.13 13.48±1.16 0.01 Neutrophils (%) 8.98±1.66 13.80±1.76 0.03 Lymphocytes (%) 83.69±1.98 75.55±2.09 0.005 Monocytes (%) 2.98±0.58 6.87±1.59 0.01 Eosinophils (%) 2.70±0.25 2.33±0,39 0.21 Basophils (%) 1.34±0.32 0.95±0.09 0.14 Results for wild type (n=10) and Foxo3−/− (n=21) blood are shown as mean±s.e.m. The analyses are from at least three independent experiments. In agreement with a myeloproliferative syndrome, the myeloid progenitor compartment is significantly enhanced in the bone marrow, spleen and peripheral blood of Foxo3-deficient mice (Figure 2A and Supplementary Figure 3). In particular, myeloid colony-forming unit-granulocyte-macrophage-derived colonies are increased in numbers and size (Supplementary Figure 3 and data not shown). 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. Download figure Download PowerPoint Similarly, the size of primitive myeloid progenitor pool was enhanced in Foxo3−/− bone marrow (Figure 2B). The compartment of colony-forming-spleen day 12 (CFU-Sd12)-derived colonies was also increased (P<0.002; Figure 2C), further supporting an expansion of the early myeloid progenitors. In addition, Foxo3−/− HP cells were overly sensitive to cytokines (Figure 2D) and generated significantly larger-size colonies in vitro (Supplementary Figure 3 and data not shown), which are the hallmarks of myeloproliferative disorders (Ghaffari et al, 1999; Levine and Gilliland, 2008). These data suggest that Foxo3 suppresses HP production and proliferation by inhibiting cytokine signalling. These findings were surprising, as Foxo3−/− hematopoietic stem cells are not highly cycling in vivo and do not generate excessive number of HPs in culture (Yalcin et al, 2008), suggesting that these observations were not simply the result of a highly proliferative hematopoietic stem cell compartment in Foxo3−/− mice. ROS-mediated amplification of AKT/mTOR signalling pathway enhances primitive HP compartment in Foxo3−/− mice Foxo3 suppresses ROS in many cell types, including in hematopoietic cells, by regulating a programme of anti-oxidant gene expression (Kops et al, 1999; Nemoto and Finkel, 2002; Marinkovic et al, 2007; Miyamoto et al, 2007; Yalcin et al, 2008). Accordingly, Foxo3-mutant lineage-negative bone marrow cells exhibited reduced expression of several anti-oxidant enzyme genes (Supplementary Figure 4). In addition, ROS were significantly overaccumulated in different Foxo3−/− subpopulations of lineage-negative cells enriched for myeloid progenitors (Figure 3 and Supplementary Figure 5). ROS concentrations were highly enhanced (approximately 1.6-fold, P<0.02; Figure 3) in Foxo3−/− Lin− IL7Rα− Sca-1− c-Kit+ cells, a population that encompasses all myeloid progenitors (Akashi et al, 2000), and increased significantly in freshly isolated Foxo3−/− common myeloid progenitors (CMP), as compared with their wild-type counterparts (approximately 1.2-fold, P<0.03; Supplementary Figure 5). CMP is a highly pure population of hematopoietic cells giving rise to megakaryocyte/erythrocyte and granulocyte/monocyte progenitors (Akashi et al, 2000). Similar results were obtained from analysis of ROS accumulation in total Foxo3-deficient bone marrow depleted from lineage-restricted cells (data not shown). In vivo treatment of mice with ROS scavenger N-acetyl-cysteine (NAC, 100 mg/kg), normalized the levels of ROS in Foxo3−/− Lin− IL7Rα− Sca-1− c-Kit+ cell population (Figure 3), supporting the specificity of ROS measurement. Interestingly, these experiments revealed two distinct populations of ROS-containing cells (ROS-high or ROS-hi and ROS-low) in primitive myeloid progenitors of both wild-type and Foxo3−/− origin. The significant increase of ROS observed in Foxo3−/− Lin− IL7Rα− Sca-1− c-Kit+ cell population was entirely in ROS-hi fraction (Figure 3). This ROS-hi subpopulation may be the one that elicits cellular responses, such as increased cell cycle or apoptosis, to oxidative stress in primitive myeloid progenitors. 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. Download figure Download PowerPoint To investigate the mechanisms of enhanced myeloproliferation caused by loss of Foxo3, we interrogated cytokine-mediated activation of principal signalling pathways in bone-marrow-derived HPs. Freshly isolated bone marrow cells were depleted of mature lineages by immunoselection (Lin− cells), and subjected to cytokine starvation followed by stimulation with interleukin-3 (IL-3). Myeloid progenitors constitute a significant majority of Lin− cells expressing IL-3 receptor at the steady state. To our surprise, IL-3 stimulation of Foxo3−/− Lin− cells led to hyperphosphorylation of AKT, mTOR and mTOR substrate S6K1 (Figure 4A). In contrast, STAT5 proteins, another effector of IL3 signalling, were not affected in these cells (Figure 4A). Similar results showing specific hyperactivation of the AKT/mTOR pathway in Foxo3−/− cells were obtained with other cytokines such as erythropoietin (Epo, data not shown). In agreement with in vivo overactivation of AKT/mTOR signalling pathway mediating enhanced generation of early myeloid progenitors in Foxo3−/− mice, in vivo administration of the mTOR inhibitor rapamycin resulted in significant reduction of Foxo3−/−-derived CFU-Sd12, as compared with controls in lethally irradiated hosts (Figure 4B). 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. Download figure Download PowerPoint Normal cytokine signalling, including signalling by IL-3 (Sattler et al, 1999), is mediated in part by ROS in in vitro cultured cells (Thannickal and Fanburg, 2000; Finkel, 2003). Thus, we investigated whether abnormal increase of ROS contributes to the hyperactivation of AKT/mTOR signalling pathway caused by loss of Foxo3 in primary cells in vivo. We treated mice with the ROS scavenger NAC (100 mg/kg), and tested IL-3 signalling responses by examining phosphorylation of downstream targets AKT, mTOR and S6K1 (Figure 4A). In vivo treatment with NAC specifically reduced the intensity of IL-3-mediated phosphorylation of AKT and mTOR in Foxo3-mutant cells enriched for HPs. Surprisingly, reduction in phospho-mTOR in response to NAC did not impact phosphorylation of the mTOR target S6K1 in Foxo3 mutants, as compared with normal hematopoietic cells (Figure 4A). Although the mechanism of lack of pS6K1 response to NAC in Foxo3-mutant cells is not clear, it is possible that the residual pmTOR kinase activity, despite the presence of NAC (Figure 4A, lanes 11 and 12), is sufficient for phosphorylation of S6K1 to the same extent as controls, especially if the activity of a phospho-S6K1-specific phosphatase is reduced in Foxo3−/− cells. NAC treatment did not alter STAT5 activity, as determined by phosphorylation on tyrosine 694 or serine 726 (Figure 4A). Interestingly, NAC treatment specifically attenuated IL-3-mediated phosphorylation of AKT and S6K1 in wild-type HPs (Figure 4A), suggesting that ROS participate in specific cytokine signalling pathways in primary bone marrow cells in vivo. Next, we asked whether this data remains valid in populations of lineage-negative bone marrow cells that contain all myeloid but not lymphoid progenitors. We found that AKT was hyperphosphorylated in response to IL-3 in Foxo3−/− myeloid (Lin− IL7Rα− Sca-1− c-Kit+) progenitors, as measured by flow cytometric analysis of intracellular pAKT (Figure 4C). NAC treatment reduced pAKT significantly in this highly enriched population of myeloid progenitors, in both wild-type and Foxo3−/− mice (Figure 4C, Supplementary Figure 6). As anticipated, in vivo treatment with NAC reduced ROS concentrations significantly in Foxo3−/− Lin− IL7Rα− Sca-1− c-Kit+ bone marrow cells (Figure 4C, right panel). Importantly, and in agreement with the results above, in vivo administration of NAC normalized the number and the size of multipotential Foxo3−/−-derived CFU-Sd12 in lethally irradiated hosts without any significant impact on wild-type CFU-Sd12 (P<0.0003, n=5; Figure 5). Taken together, these results indicate that ROS specifically amplify cytokine-mediated AKT/mTOR signalling pathway to stimulate the expansion of HPs in Foxo3−/− mice. 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. Download figure Download PowerPoint To investigate whether accumulation of ROS in myeloid progenitors contributes to the pathogenesis of the myeloproliferative syndrome, we subjected wild-type and Foxo3-deficient mice to a short, 15-day treatment with NAC. Interestingly, in vivo NAC administration treated some of the myeloproliferative symptoms in Foxo3-deficient mice (Figure 6, Supplementary Figure 6). The in vivo treatment with NAC normalized the total number of bone marrow and spleen cells in Foxo3−/− mice (Figure 6A). This treatment also had a specific and significant effect on the frequency and total number of immature myeloid (Mac-1/Gr-1 positive) cells in the bone marrow without any impact on B cells (Figure 6B and Supplementary Figure 6). 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 th" @default.
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• W2127659799 date "2010-11-26" @default.
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• W2127659799 title "ROS-mediated amplification of AKT/mTOR signalling pathway leads to myeloproliferative syndrome in Foxo3−/− mice" @default.
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