Manual de Ceremonial y Protocolo UNIVERSIDAD TECNICA FEDERICO SANTA MARIA Los actos y ceremonias que se realizan en la USM, requieren estar dotados de uni- formidad y revestidos de la solemnidad correspondiente a su prestigio e imagen institucional. Por ello, la Dirección General de Comunicaciones (DGC) presenta el Manual de Ceremonial y Protocolo, con el objetivo de ordenar y orientar su elabora-
Regbio.yeditepe.edu.trHEMATOPOIESIS AND STEM CELLS Meis1 regulates the metabolic phenotype and oxidant defense of hematopoieticstem cells *Fatih Kocabas,1 *Junke Zheng,2,3 Suwannee Thet,1 Neal G. Copeland,4 Nancy A. Jenkins,4 Ralph J. DeBerardinis,5Chengcheng Zhang,2 and Hesham A. Sadek1 1Department of Internal Medicine, Division of Cardiology, and 2Departments of Physiology and Developmental Biology, UT Southwestern Medical Center, Dallas, TX; 3Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine, Shanghai, China; 4The Methodist Hospital Research Institute, Houston, TX; and 5Departments of Pediatrics and Genetics, UT Southwestern Medical Center, Dallas, TX The role of Meis1 in leukemia is well
amined the effect of loss of Meis1 on HSC
duction, and apoptosis of HSCs. Finally,
established, but its role in hematopoietic
function and metabolism. Inducible Meis1
we demonstrate that the effect of Meis1
stem cells (HSCs) remains poorly under-
deletion in adult mouse HSCs resulted in
knockout on HSCs is entirely mediated
stood. Previously, we showed that HSCs
loss of HSC quiescence, and failure of
through reactive oxygen species where
use glycolytic metabolism to meet their
bone marrow repopulation after transplan-
treatment of the Meis1 knockout mice
energy demands. However, the mecha-
tation. While we previously showed that
with the scavenger N-acetylcystein re-
nism of regulation of HSC metabolism,
Meis1 regulates Hif-1␣ transcription in
stored HSC quiescence and rescued
and the importance of maintaining this
vitro, we demonstrate here that loss of
HSC function. These results uncover an
distinct metabolic phenotype on HSC
Meis1 results in down-regulation of both
important transcriptional network that
function has not been determined. More
Hif-1␣ and Hif-2␣ in HSCs. This resulted
regulates metabolism, oxidant defense,
importantly, the primary function of Meis1
in a shift to mitochondrial metabolism,
and maintenance of HSCs. (Blood. 2012;
in HSCs remains unknown. Here, we ex-
increased reactive oxygen species pro-
Hematopoietic stem cells (HSCs) are defined by their abilities to ish NAD⫹. Anaerobic glycolysis produces 18 times less ATP than self-renew and to differentiate into all blood cell types.1,2 Much of mitochondrial oxidative phosphorylation,12 which may be well the advancement in HSC therapy is credited to decades of suited for quiescent cells, but certainly cannot sustain cells with pioneering work that led to the development of HSC enrichment techniques based on staining of cell-surface antigens or vital dyes The energy advantage of mitochondrial oxidative phosphoryla- followed by fluorescence-activated cell sorting (FACS).3-5 How- tion over glycolysis is, unfortunately, not without deleterious ever, little is known about metabolic characteristics of HSCs, its consequences, as the mitochondrion is considered a major source regulation, or how the metabolic phenotype may influence HSC of reactive oxygen species (ROS) production.13,14 ROS are believed to be important mediators of aging, and of numerous degenerative In 1978, the concept of the special microenvironment, or niche, diseases, including HSC dysfunction and senescence.15 In fact, of HSCs was introduced.6 Since then, it has become clear that the within the HSC compartment, the repopulation capacity is local- niche plays a crucial role in self-renewal and differentiation of ized to only those HSCs with low levels of free radicals.16 HSCs.7,8 One of the hallmarks of the HSC niche is its low oxygen Therefore, the glycolytic metabolic phenotype of HSCs may not tension, hence the term "hypoxic niche."9 Numerous studies only protect them against hypoxic insults, but may also serve to indicate that this low oxygen environment is not only tolerated by minimize oxidant damage that result from mitochondrial oxidative HSCs, but is also essential for their function.10 We recently demonstrated that HSCs rely on glycolysis and have lower rates of Hypoxia-inducible factor-1␣ (Hif-1␣) is a major transcriptional oxygen consumption,11 which may be crucial for survival of HSCs regulator of hypoxic response. Hif-1␣ mediates the metabolic within hypoxic bone marrow niches.
switch from aerobic mitochondrial metabolism, to anaerobic cytoplas- In the mitochondria, oxygen is used as the terminal electron mic glycolysis17-19 by increasing both the expression,20 and kinetic acceptor for the respiratory chain, and in the absence of oxygen the rate21 of key glycolysis enzymes. Moreover, Hif-1␣ inhibits the use proton gradient generated by the respiratory chain collapses and of pyruvate by the mitochondria,22,23 and inhibits mitochondrial mitochondrial ATP production declines. Under these hypoxic or biogenesis.24 Takubo and colleagues recently demonstrated that anoxic conditions, energy production is derived from cytoplasmic Hif-1␣ is enriched in HSCs, and that loss of Hif-1␣ results in HSC glycolysis through the fermentation of glucose, and in the final step dysfunction,25 while our group recently showed that Meis1 is required of anaerobic glycolysis, pyruvate is converted to lactate to replen- for optimum transcriptional activation of Hif-1␣ in HSCs ex vivo.11 Submitted May 30, 2012; accepted September 11, 2012. Prepublished online The publication costs of this article were defrayed in part by page charge as Blood First Edition paper, September 20, 2012; DOI 10.1182/blood-2012- payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement'' in accordance with 18 USC section 1734.
*F.K. and J.Z. contributed equally to this project.
The online version of this article contains a data supplement.
2012 by The American Society of Hematology BLOOD, 13 DECEMBER 2012 䡠 VOLUME 120, NUMBER 25 BLOOD, 13 DECEMBER 2012 䡠 VOLUME 120, NUMBER 25 Meis1, which is a 3-amino-acid loop extension homeodomain lineages was also performed to confirm multilineage reconstitution as protein, plays an important role in leukemogenesis as well as previously described.11 normal hematopoiesis. Meis1 was first identified as a common viral For analyzing LT-HSCs in the peripheral blood by flow cytometry, integration site in myeloid leukemic cells of BXH-2 mice,26 and it peripheral blood of Meis1⫺/⫺ or Meis1⫹/⫹ mice was collected by retro-orbital bleeding and stained with LT-HSCs markers as described in the is also frequently up-regulated in human primary acute myeloid previous paragraph.
leukemia (AML) and acute lymphoblastic leukemia (ALL) The cell-cycle analysis with Hoechst 33342 and pyronin Y staining was samples.27 Moreover, overexpression of Meis1 accelerates the performed as we described.36 Samples were immediately analyzed by flow initiation of AML in murine models.28,29 In normal hematopoiesis, cytometry (FACSAria; BD Biosciences). To examine the apoptosis, Meis1 is expressed in the most primitive hematopoietic populations Lin⫺Kit⫹Sca-1⫹ cells were stained with PE-conjugated anti–annexin V and and is down-regulated on differentiation.30-32 Targeted Meis1 7AAD according to the manufacturer's manual (BD Pharmingen). To study knockout causes lethality by embryonic day 14.5 with multiple the apoptosis in LT-HSCs, bone marrow cells were stained for HSC markers hematopoietic and vascular defects.33,34 Moreover, Pbx-1, a cofac- Sca-1-PE/Cy5.5, C-Kit-APC, CD34-PE, and Flk2-PE after lineage deplet- tor of Meis1, has been shown to regulate self-renewal of HSCs by ing and stained with FITC-conjugated anti–annexin V according to the maintaining their quiescence.35 However, the role of Meis1 regulat- manufacturer's manual (eBioscience).
ing the function and metabolism if HSCs remain poorly understood.
In the current report, we show that Meis1 regulates both HSC Competitive reconstitution analysis
metabolism and oxidant stress response, through transcriptional The indicated numbers of CD45.2 donor cells from Meis1⫹/⫹ or Meis1⫺/⫺ regulation of Hif-1␣ and Hif-2␣, respectively.
mice were mixed with 1 ⫻ 105 freshly isolated CD45.1 competitor BMcells and the mixture was injected intravenously via the retro-orbital routeinto each of a group of 6- to 8-week-old CD45.1 mice previously irradiatedwith a total dose of 10 Gy. To measure reconstitution of transplanted mice, peripheral blood was collected at the indicated time points posttransplanta-tion, and the presence of CD45.1⫹ and CD45.2⫹ cells in lymphoid and Mouse breeding and genotyping
myeloid compartments was measured as described.37 Meis1 knockout (KO) mice were genotyped with Meis1 For1: 5⬘-CCAAAGTAGCCACCAATATCATGA-3⬘ and Meis1 Rev: 5⬘-AGCGT- CACTTGGAAAAGCAATGAT-3⬘ primers. Wild-type (WT) allele is de-termined by a 332-bp-long PCR product and mutant allele determined by BM Lin⫺ cells were labeled with 5- (and -6) carboxyfluorescein succinimi- a 440-bp long PCR product on 1.2% agarose gel. HSC-specific deletion of dyl ester (CFSE), and 3 ⫻ 106 cells were transplanted into indicated strains Meis1 was achieved by following crosses of Meis1f/f with Scl-Cre-ERT of lethally irradiated mice. After 16 hours, the total number of CFSE⫹ cells mice.4 Scl-Cre mice were genotyped using Scl-Cre-ER primer 1: 5⬘- in the BM, spleen, or liver was determined by flow cytometry. When GAACCTGAAGATGTTCGCGAT-3 and Scl-Cre-ER primer 2: 5⬘- CFSE⫹ LSK (CFSE⫹Lin⫺Sca1⫹Kit⫹) cells (HSCs) were analyzed, the BM ACCGTCAGTACGTGAGATATC-3. To generate Meis1⫺/⫺ mice, Meis1f/ cells were stained with a biotinylated lineage cocktail followed by f;Scl-Cre-ERT⫹ mice were injected intraperitoneally with tamoxifen streptavidin-PE/Cy5.5, anti–Sca-1–PE, and anti-Kit–APC before analysis, (40 mg/kg, T5648-1G; Sigma-Aldrich) daily for 14 days. We used age- matched tamoxifen-injected Meis1⫹/⫹;Scl-Cre-ERT(⫹) or Meis1f/f;Scl-Cre-ERT(⫺) mice as controls (Meis1⫹/⫹ mice). Genotyping of Cre-deleted Meis1 locus (Meis1 exon8 deleted) was performed using Meis1 For2:5⬘-CATTGACTTAGGTGTATGGGTGTC-3⬘ and Meis1 Rev: 5⬘- Normal BM cells were diluted to the indicated concentration in IMDM with AGCGTCACTTGGAAAAGCAATGAT-3⬘ primers. Cre-deleted Meis1 2% FBS, and were then seeded into methylcellulose medium M3434 locus gives rise to a 261-bp product while wt and mutant (nondeleted) (StemCell Technologies), for CFU-GM and BFU-E colony formation shows no amplicon.
according to the manufacturer's instructions.
Dr Joseph A. Garcia (UT Southwestern) kindly provided the Hif-1␣ floxed mice. HSC-specific deletion of Hif-1␣ was achieved by following crosses of Hif-1␣f/f with Scl-Cre-ERT mice and tamoxifen injections(40 mg/kg for 14 days). We used age-matched tamoxifen-injected Hif-1␣⫹/⫹; Total RNA was isolated from Meis1⫺/⫺ and Meis1⫹/⫹ HSCs using the Scl-Cre-ERT(⫹) or Hif-1␣f/f;Scl-Cre-ERT(⫺) mice as controls (Hif-1␣⫹/⫹ mice).
RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions.
cDNA was synthesized using SuperScript II RT (Invitrogen). Predesignedprimers (Table 1) from the National Institutes of Health (NIH) mouse primer depot (http://mouseprimerdepot.nci.nih.gov/) were ordered from Donor bone marrow (BM) cells were isolated from 8- to 12-week-old Integrated DNA Technologies. Real-time PCR was performed with Syber- Meis1⫹/⫹ or Meis1⫺/⫺ mice (or Hif-1␣⫹/⫹ and Hif-1␣⫺/⫺ mice).
Green (Applied Biosystems) on an ABI Prism 7700 Sequence Detector Lin⫺Sca1⫹Kit⫹Flk2⫺CD34⫺ cells (long-term HSCs [LT-HSCs]) were (Applied Biosystems). ␤-actin was used as control to normalize results.
isolated by staining with a biotinylated lineage cocktail (anti-CD3, anti-CD5, anti-B220, anti–Mac-1, anti-Gr-1, anti-Ter119; StemCell Technolo- PCR array
gies) followed by streptavidin-PE/Cy5.5, anti–Sca-1–FITC, anti–Kit-allophycocyanin (APC), anti–Flk-2–PE, and anti-CD34–PE. For analyzing The Mouse Hypoxia Signaling Pathway RT2 Profiler PCR Array was repopulation of mouse HSCs, peripheral blood cells of recipient CD45.1 performed as described previously.11 RNA was extracted from Meis1⫺/⫺ mice were collected by retro-orbital bleeding, followed by lysis of red blood and Meis1⫹/⫹ LT-HSCs (Lin⫺Sca1⫹Kit⫹Flk2⫺CD34⫺cells) using the cells and staining with anti-CD45.2–FITC, anti-CD45.1–PE, anti- RNeasy Mini Kit (QIAGEN) according to manufacturer's instructions.
Thy1.2–PE (for T-lymphoid lineage), anti-B220–PE (for B-lymphoid cDNA was retrotranscribed by using SuperScript II RT (Invitrogen). Mouse lineage), anti–Mac-1–PE, or anti–Gr-1–PE (cells costaining with anti- Real-Time Syber Green PCR Master Mix (SuperArray) and Cell Cycle Mac–1 and anti–Gr-1 were deemed to be of the myeloid lineage) primer sets (SABiosciences) used for real-time PCR on an ABI Prism monoclonal antibodies (BD Pharmingen). The "percent repopulation" 7700 Sequence Detector (Applied Biosystems). The data were analyzed shown in all figures was based on the staining results of anti-CD45.2–FITC using the ⌬⌬Ct method. Fold change was calculated as difference in gene and anti-CD45.1–PE. In all cases, FACS analysis of the above-listed expression between Meis1⫺/⫺ and Meis1⫹/⫹ LT-HSC samples.
BLOOD, 13 DECEMBER 2012 Meis1 REGULATES Hif-1 䡠 VOLUME 120, NUMBER 25 ␣ AND Hif-2␣ IN HSCs Table 1. List of primers used for real-time PCR
Generation of luciferase reporter vectors
Measurement of ROS
Conserved Meis1 motifs in Hif-2␣ gene were determined using a genome Bone marrow cells from Meis1⫹/⫹ and Meis1⫺/⫺ mice were isolated as browser (http://genome.ucsc.edu/). A 779-bp-long DNA fragment con- described in "Flow cytometry." After lineage depletion (lineage depletion taining conserved Meis1 sites (located next to start codon sequence, human kit; BD Biosciences), cells were incubated with 1M 5-(and-6)-carboxy- chr2:46 525 052-46 525 064) from the Hif-2␣ promoter was amplified by 2⬘,7⬘-dichlorofluorescein diacetate (carboxy-DCFDA; Invitrogen) for PCR from mouse genomic DNA with the following primers: conserved 30 minutes in a 37°C water bath in the dark. Then, cells were stained for Meis1 site: pHif-2␣-F, 5⬘-GGGCTAAACGGAACTCCAGG-3⬘ and pHif- HSC markers Sca-1-PE/Cy5.5, C-Kit-APC, CD34-PE, and Flk2-PE and 2␣-R, 5⬘-CATAGGAACGCTCTCGGAAAGAC-3⬘. PCR fragments were assayed with a flow cytometer.
subcloned into the pCR2.1-TOPO vector (Invitrogen) according to themanufacturer's instructions. pHif2a-TOPO and E1b-pGL2 vectors were NAC administration in vivo
digested with XhoI and KpnI. Then, the PCR fragments containing con-served Meis1 sites were cloned into E1b-pGL2 to generate the pHif2a- Six-week-old Meis1f/f;Scl-Cre-ERT⫹ mice were injected intraperitoneally pGL2 luciferase reporter vector. To test Meis1 site specificity, Meis1- with tamoxifen (40 mg/kg) daily for 14 days. After tamoxifen injections, binding sites (TGAC) at the TGACAGCTGACAA (Meis1 binding site is
animals were treated daily for 2 weeks with N-acetyl-L-cysteine (NAC; indicated in bold) sequence were mutated to TGGCGGCCGCCAA (NotI
100 mg/kg; Sigma-Aldrich) by subcutaneous administration or provided in site insertion) using iProof High-Fidelity DNA Polymerase (Bio-Rad) from drinking water (500 mg ⫻ kg⫺1 ⫻ d⫺1 in drinking water as described the Hif-2␣-pGL2 vector with the following primers: Mut-Hif2a-F, 5⬘- previously39,40) and were subsequently analyzed by flow cytometry. We AAgcggccgcCAAGGAGAAAAAAAGGTAAGCGGG-3⬘ and Mut- used age-matched tamoxifen-injected Meis1⫹/⫹;Scl-Cre-ERT(⫹) or Hif2a-R, 5⬘-AAgcggccgcCATTGTCGCCGTGGCCCTC-3⬘. The Hif-2␣- Meis1f/f;Scl-Cre-ERT(⫺) mice as controls (Meis1⫹/⫹ mice).
Mut-pGL2 reporter was generated after NotI digestion and ligation of thePCR product. Transcriptional activation of Hif-2␣ by Meis1 was evaluated Chromatin immunoprecipitation assay
using a luciferase reporter system (Promega) as described previously.11 Chromatin immunoprecipitation assay (ChIP) assays were performed to evaluate the in vivo binding of Meis1 to its consensus sequence in the Hif-2␣ gene. Theassays were done in Kasumi-1 cells, a hematopoietic progenitor cell line, using Metabolic assays are carried out as described previously11 with some the ChIP kit (Upstate) as described previously.11 Meis1 antibody (Santa Cruz Biotechnology) and normal goat IgG (Santa Cruz Biotechnology) were used.
The DNA isolated from input chromatin fragments and from the precipitated Oxygen consumption assays
chromatin fragments by anti-Meis1 antibody or control IgG was subjected toreal-time PCR using primers flanking the consensus Meis1-binding sites on Meis1⫹/⫹ and Meis1⫺/⫺ HSCs were separated flowcytometrically as described in "Flow cytometry." Equal numbers of cells (5-10 ⫻ 104 cells/ well) were incubated for 6 hours in the provided 384-well plate (BDOxygen Biosensor System) and sealed to prevent air exchange beforemeasurement. Culture media lacking cells was used as a negative control Measurement of blood cell counts (CBC/differential)
and sodium sulfite (100mM) was used as a positive control. Oxygen Approximately 50 L of peripheral blood collected by retro-orbital bleed- consumption is presented as relative units.
ing from Meis1⫹/⫹ and Meis1⫺/⫺ mice post 1 month and 3 months oftamoxifen injections in K3-EDTA tubes. Samples are submitted to Univer- ATP assays
sity of Texas Southwestern Medical Center Animal Resources Center Meis1⫹/⫹ and Meis1⫺/⫺ HSCs were sorted as described and centrifuged at (UTSW ARC) Diagnostic Laboratory and analyzed with the HemaVet 1200g for 10 minutes. At least 50 000 cells were used for each single ATP 950FS analyzer. The following parameters are reported for the sample measurement. Fifty microliters of ATP standards (10⫺6-10⫺12M) and 50 L submitted: white blood count (WBC), neutrophil count, lymphocyte count, of cell lysates were quantified using the ATP Bioluminescence Assay Kit monocyte count, eosinophil count, basophil count, red blood cell count, CLS II (Roche) using Fluostar Optima plate reader (BMG Labtech).
hemoglobin, HCT, MCV, MCH, MCHC, platelet count, and MPV.
Finally, data were normalized to cell count and protein content.
Generation of the Meisf/f;Scl-Creⴙ; rtTA; TRE-Hif-1␣ mice and
Glycolytic flux assay
rescue analysis by viral Hif-1␣ and Hif-2␣ overexpression
13C-lactate production, end product of glycolysis, was measured as To determine the role of Hif-1␣ in Meis1 KO HSCs, inducible HSC- described previously11 using glycolytic flux medium supplemented with specific, Hif-1␣ transgenic mice were generated. The transgenic construct 10mM D-[1-6-13C]-glucose (Cambridge Isotope Labs) to allow up to all of was made by cloning Hif-1␣ cDNA (Open Biosystems) into the pTRE- the glucose-derived lactate pool to be labeled on C-3. A minimum of Tight vector (Clontech), which makes it responsive to rtTA regulatory 50 000 cells were cultured in 40 L of flux medium overnight. Then, the proteins in the Tet-On system. We crossed TRE-Hif-1␣ mice with rtTA mice cells were pelleted and supernatant collected and prepared for gas which has a stop-codon flanked by loxP sequence. Cre-mediated removal of chromatography–mass spectrometry. Lactate abundance was determined the stop region by deletion of the loxP flanked sequence allows expression by monitoring m/z at 117 (unenriched), 118 (lactate containing 13C from of rtTA as described.41 Therefore, sequential administration of tamoxifen glucose), and 119 (internal standard) as described previously.11 will result in Meis1 deletion, and concomitant deletion of the stop-codon,
BLOOD, 13 DECEMBER 2012 䡠 VOLUME 120, NUMBER 25 Figure 1. Meis1 Deletion in LT-HSCs results in apoptosis and loss of quiescence. (A) Left panel: Representative flow cytometry profile of LT-HSCs (Lin⫺Sca-
1⫹Kit⫹Flk2⫺CD34⫺) of bone marrow (BM) cells are shown for control Meis1⫹/⫹ and mutant Meis1⫺/⫺ mice. Numbers in the FACS plots indicate percentages among total
BM cells. Right panel: Quantification of LT-HSCs demonstrates significantly higher number of HSCs in Meis1⫺/⫺ BM (n ⫽ 6). (B) The in vitro methylcellulose colony formation
assay was performed at the time of sacrifice after tamoxifen injections with BM cells of control and Meis1⫺/⫺ mice. CFU-GEMM colonies representing most undifferentiated
progenitors type of colonies derived from Meis1⫹/⫹ and Meis1⫺/⫺ BM cells demonstrates decreased percentage of CFU-GEMM. Quantification of BFU-E and CFU-GM colonies
derived from Meis1⫺/⫺ cells shows no differences (n ⫽ 3). (C) Left panel: Representative FACS analysis of Pyronin Y/Hoecst staining on LT-HSCs (Lin⫺Sca-
1⫹Kit⫹CD150⫹CD48⫺) of Meis1⫹/⫹ and Mesi1⫺/⫺ mice. Numbers in the FACS plots indicate percentages among LT-HSCs. Right panel, The quantification of G0, G1, or S/G2/M
phase in Meis1⫹/⫹ and Meis1⫺/⫺ LT-HSCs (n ⫽ 6). (D) Quantification of apoptosis in Meis1⫹/⫹ and Meis1⫺/⫺ LT-HSCs (n ⫽ 3). (E) Quantification of LT-HSCs in peripheral blood
(PB) of Meis1⫹/⫹ and Meis1⫺/⫺ mice (n ⫽ 6); *P ⬍ .05, **P ⬍ .01.
which allows for Hif-1␣ overexpression on administration of doxycycline.
We generated Meisf/f;Scl-Cre⫹; rtTA; TRE-Hif-1␣ mice by a series of crosses to overexpress in Hif-1␣ in HSC-specific manner after 14 days oftamoxifen (1 mg/d/mice) and providing doxycycline in drinking water Deletion of Meis1 in LT-HSCs
(500 mg/L). HSCs from these mice were either used for in vivo repopula-tion studies, or for in vitro colony forming assay.
Because the global loss of Meis1 is embryonic lethal,33,34 we sought We also used a viral strategy to overexpress Hif-1␣ or Hif-2␣ in HSCs to pursue an inducible deletion of Meis1 to study the role of Meis1 (generated by cloning into XZ201 vector) and lentivirus-expressing Hif-2␣ in adult HSCs. Meis1fl/fl mice with loxp flanking exon 8 were (kindly provided by Dr Joseph Garcia, UT Southwestern). Fetal liver cells crossed with transgenic mice expressing the tamoxifen-inducible were harvested from Meis1f/f;Scl-Cre⫹ pups at P1 and infected with eitherlentivirus-expressing Hif-2␣ or retrovirus-expressing Hif-1␣ with IRES- Cre recombinase under the control of stem cell leukemia (Scl) HSC GFP. Then, infected cells were transplanted into CD45.1⫹ recipient host enhancer, which drives deletion in HSCs.4 Upon tamoxifen treat- mice. The repopulation was measured 2 months posttransplantation before ment, exon 8 is deleted which results in the loss of Meis1 induction of deletion of Meis1 with tamoxifen injections. The repopulation expression in HSCs (supplemental Figure 1A, available on the analysis performed at indicated time points post-tamoxifen treatments Blood Web site; see the Supplemental Materials link at the top of either measuring GFP⫹ cells or CD45.2⫹ donor cells (n ⫽ 3-5).
the online article). To verify deletion of Meis1, we performed Finally, in a separate set of experiments, we used cobalt chloride (CoCl2 100M), which is a known stabilizer of both Hif-1␣ and Hif-2␣, in genotyping analysis and quantitative RT-PCR in peripheral blood methocult culture after deletion of Meis1 to examine the effects of cells and phenotypic LT-HSCs (Lin⫺Sca1⫹Kit⫹Flk2⫺CD34⫺) in stabilization of Hif-1␣ and Hif-2␣ on colony formation assay after Meis1 the bone marrow after tamoxifen treatment. Meis1 in peripheral deletion. After 14 days of tamoxifen injection into aged matched Meis1⫹/⫹; blood cells was deleted 14 days after tamoxifen treatment, and Scl-Cre⫹ (Control) and Meis1f/f;Scl-Cre⫹ (Meis1 KO) mice, we performed Meis1 mRNA level in LT-HSCs was markedly decreased to colony-forming assays using Methocult medium supplemented with 100M approximately 10% of control values (supplemental Figure 1B).
CoCl2. Same number of bone marrow cells (20 ⫻ 103 cells/plate) wasplated as recommended by manufacturer and colonies were quantified after Meis1 is required for the maintenance of LT-HSCs
12 days of culture.
To explore the role of Meis1 in LT-HSCs, we first examined the frequencies of phenotypic LT-HSCs in control (Meis1⫹/⫹) and Results are expressed as mean ⫾ SEM, and a 2-tailed Student t test was Meis1 conditional KO (Meis1⫺/⫺) mice. As shown in Figure 1A, used to determine the level of significance. P ⬍ .05 was considered 7 days after tamoxifen treatment, there was a 4.5-fold increase of HSC frequency in Meis1⫺/⫺ mice compared with Meis1⫹/⫹ controls
BLOOD, 13 DECEMBER 2012 Meis1 REGULATES Hif-1 䡠 VOLUME 120, NUMBER 25 ␣ AND Hif-2␣ IN HSCs Figure 2. Impaired repopulation in Meis1ⴚ/ⴚ LT-HSCs. Repopulation assays: (A) LT-HSCs (Lin⫺Sca1⫹Kit⫹Flk2⫺CD34⫺; 150 cells) from either control (Meis1⫹/⫹) or mutant
Meis1⫺/⫺ CD45.2 mice were transplanted into irradiated CD45.1 hosts in competition with BM from CD45.1 mice (1 ⫻ 105 cells). Quantification of flow cytometry profile of
peripheral blood of bone marrow recipient mice up to 16 weeks for percentage of CD45.2⫹ cells demonstrates total loss of bone marrow reconstitution of Meis1⫺/⫺ LT-HSCs
(n ⫽ 5). (B) Repopulation assay with whole bone marrow from either control Meis1⫹/⫹ or mutant Meis1⫺/⫺ CD45.2 mice were transplanted into irradiated CD45.1 mice
demonstrates significantly impaired repopulation in mice transplanted with Meis1⫺/⫺ cells (n ⫽ 5). (C) Analysis of repopulation after second bone marrow transplantation (BMT)
from first BMT mice demonstrates complete loss of repopulation (n ⫽ 5). Homing assays: (D) BM Lin⫺ cells (3 ⫻ 106 cells) were transplanted into irradiated mice and quantified
for CFSE⫹ cells in different tissues. Quantification of percentage of CFSE⫹ cells in BM, spleen, and liver show no difference between Meis1⫹/⫹ and Meis1⫺/⫺ mice (n ⫽ 5).
(E) Quantification of percentage of CFSE⫹ LSK cells in BM of Meis1⫹/⫹ and Meis1⫺/⫺ mice (n ⫽ 5). (F) Quantification of repopulation of lineages for Meis1⫺/⫺ using whole bone
marrow from first BMT mice demonstrates no defects in lineage repopulation (n ⫽ 5); *P ⬍ .05, **P ⬍ .01.
(0.0092% vs 0.002%). FACS analysis revealed robust reduction of To further evaluate the function of Meis1 in HSCs in vivo, we granulocyte-monocyte progenitors (GMPs) and common myeloid performed competitive bone marrow transplantation with Meis1⫹/⫹ progenitors (CMPs) and mild reduction in the total number of and Meis1⫺/⫺ HSCs. Two weeks after tamoxifen treatment, megakaryocyte/erythroid progenitors (MEPs; supplemental Figure 150 Lin⫺Sca1⫹Kit⫹Flk2⫺CD34⫺ Meis1⫹/⫹ or Meis1⫺/⫺ CD45.2 1C). Meanwhile, by using a colony-forming assay, we demon- HSCs, together with 1 ⫻ 105 CD45.1 competitors, were adminis- strated that Meis1⫺/⫺ mice had much lower primitive myeloid tered into CD45.1 recipients through retro-orbital injection. Repopu- progenitor cells (CFU-GEMM), but no change in percentage of lation was examined at 4, 10, 16 weeks after transplantation.
differentiated myeloid progenitor cells (CFU-GM), or erythroid Strikingly, as shown in Figure 2A, we could not detect any progenitor cells (BFU-E; Figure 1B, supplemental Figure 1D). No engraftment with Meis1⫺/⫺ HSC donors, indicating a severe difference was detected in the distribution of T, B, myeloid, and impairment of HSC repopulation ability after loss of Meis1. A erythroid lineages either in BM or peripheral blood of Meis1⫺/⫺ repeat transplantation experiment with total BM cells showed (supplemental Figure 1E-F).
similar results (Figure 2B). The impaired repopulation of Meis1- The increase of HSC frequency may result from cell- deficient LT-HSCs (Figure 2A) and progressive decline in repopu- autonomously accelerated proliferation or the compensatory effect lation of Meis1-deficient cells in primary transplant recipients of increased apoptosis or mobilization in Meis1⫺/⫺ HSCs. To (Figure 2B) suggested a defect in self-renewal. Therefore, we address this concern, we first examined the cell cycle of HSCs by performed secondary transplantation experiments to confirm the using Hoechest 33342 and pyronin Y staining, and we found that reduction of function Meis1-deficient LT-HSCs in the bone marrow only approximately 71% of Meis1⫺/⫺ LT-HSCs were in the G0 of primary transplant recipients. Eight weeks after primary trans- compartment, which is much lower than that of Meis1⫹/⫹LT-HSCs plantation, total bone marrow from primary recipients from whole (⬃ 83%; Figure 1C). This indicated that Meis1⫺/⫺ HSCs were transplanted mice were harvested and transplanted into secondary much less quiescent and prone to proliferation. Next, we examined recipients. Meis1-deficient cells from primary recipients (0.5- apoptosis status in LT-HSCs (Lin⫺Sca1⫹Kit⫹Flk2⫺CD34⫺) by 1 ⫻ 106 cells) were unable to repopulate secondary recipients using annexin V. We detected more apoptotic cells in Meis1⫺/⫺ (Figure 2C). The decreased repopulation may result from defects in LT-HSCs compared with Meis1⫹/⫹ counterpart (Figure 1D, supple- HSC maintenance, or homing, or increased apoptosis. To exclude mental Figure 1G for LSK cells). Finally, to examine HSC the possibility that the decreased engraftment is caused by defect of mobilization, we performed LT-HSC staining in peripheral blood in homing in Meis1⫺/⫺ HSCs, we labeled Meis1⫹/⫹ or Meis1⫺/⫺ Lin⫺ Meis1⫹/⫹ and Meis1⫺/⫺ mice, which did not show any difference in cells with CFSE and injected into lethally irradiated recipients.
HSC frequency (Figure 1E). These data suggest that the increased Sixteen hours later, we examined CFSE⫹ or LSK CFSE⫹ cells in apoptosis in Meis1⫺/⫺ LT-HSCs may result in increase of HSC spleen, liver, and BM. No significant difference in homed total frequency and decrease of quiescence in BM.
CFSE⫹ or LSK CFSE⫹ cells was detected between Meis1⫹/⫹and
BLOOD, 13 DECEMBER 2012 䡠 VOLUME 120, NUMBER 25 Figure 3. Metabolic regulation of LT-HSCs by Meis1. (A) RT-PCR histogram demonstrates the significant down-regulation of Hif-1␣ and Hif-2␣ (EPAS1), but not Hif-3␣ after
Meis1 deletion in LT-HSCs (n ⫽ 3). (B) Measurement of oxygen consumption rate for 6 hours demonstrates significantly higher aerobic phosphorylation in Meis1⫺/⫺ LT-HSCs
compared with Meis1⫹/⫹ LT-HSCs (n ⫽ 3). (C) Quantification of labeled lactated in glycolytic flux assay demonstrates that Meis1⫺/⫺ LT-HSCs are less glycolytic (n ⫽ 3).
(D) Measurement of reactive oxygen species (ROS) in LT-HSCs as determined by quantification of the percentage of DCFDA⫹ LT-HSCs in Meis1⫹/⫹ and Meis1⫺/⫺ mice
(n ⫽ 3). (E) RT-PCR of p16 and p19 demonstrates association of higher level of ROS with up-regulation of p16 and p19 in Meis1⫺/⫺ LT-HSCs (n ⫽ 3). (F) Figure shows
conserved consensus Meis1 motifs found on Hif-2␣ (EPAS1) gene. Note the duplex Meis1-binding motifs found next to each other and conserved till Opossum. (G) Luciferase
reporter assays demonstrate dose-dependent transcriptional activation of Hif-2␣ by Meis1 (n ⫽ 3). (H) Real-time PCR with primers flanking the consensus Meis1-binding
sequence after ChIP assay demonstrating in vivo binding of Meis1 to Hif-2␣ promoter (n ⫽ 3); *P ⬍ .05, **P ⬍ .01.
Meis1⫺/⫺ donors (Figure 2D-E). In addition, quantification of hypoxia-inducible factors (Hif-1␣, Hif-2␣, and Hif-3␣) are regu- lineage repopulation of Meis1⫹/⫹ and Meis1⫺/⫺ donors demon- lated by Meis1 in LT-HSCs. Quantitative real-time PCR analysis strates no lineage-specific defects (Figure 2F). Taken together, demonstrated that mRNA levels of both Hif-1␣ and Hif-2␣ were these data provide strong evidence that Meis1 plays a crucial role in down-regulated but no change in Hif-3␣ levels was observed in the maintenance of HSCs and prevention of apoptosis in HSCs.
Meis1⫺/⫺ LT-HSCs (Figure 3A). Moreover, we found that severaldownstream targets of Hif-1␣ and/or Hif-2␣ are down-regulated in Role of Meis1 in regulating Hif-1␣ for glycolytic metabolism of
Meis1⫺/⫺ LT-HSCs as determined by quantitative PCR (qPCR) array (supplemental Figure 2). To explore the role of Hif-1␣ in the We have shown previously that the Meis1 gene acts upstream of regulation of metabolism of LT-HSCs, we used the same inducible Hif-1␣ by regulating its transcriptional activity by an enhancer Cre system to delete Hif-1␣ specifically in HSCs (supplemental located in the first intron of Hif-1␣.11 We further examined whether Figure 3A). Hif-1␣fl/fl mice with loxp flanking exon 2 were crossed BLOOD, 13 DECEMBER 2012 Meis1 REGULATES Hif-1 䡠 VOLUME 120, NUMBER 25 ␣ AND Hif-2␣ IN HSCs with transgenic mice expressing the tamoxifen-inducible Cre activation of Hif-2␣ in LT-HSCs. We also confirmed the in vivo recombinase under the control of stem cell leukemia (Scl) HSC binding of Meis1 to its conserved sequences in the Hif-2␣ gene by enhancer. Upon tamoxifen treatment, exon 2 is deleted resulting in ChIP assays in Kasumi1 cells as determined by real-time PCR after the loss of Hif-1␣ expression in HSCs (supplemental Figure 3A).
immunoprecipitation with Meis1 antibody (Figure 3H).
To verify deletion of Hif-1␣, we performed quantitative RT-PCR inLT-HSCs (Lin⫺Sca1⫹Kit⫹ Flk2⫺CD34⫺) in bone marrow after Effect of ROS scavenging on the Meis1ⴚ/ⴚ phenotype
tamoxifen treatment. Hif-1␣ mRNA level in LT-HSCs was dramati- To study effects of ROS observed in Meis1⫺/⫺ mice, we used NAC, cally decreased, approximately 400-fold lower than that of control an antioxidant, in an attempt to rescue the Meis1 phenotype by (Hif-1⫹/⫹) after 14 days of tamoxifen treatment (supplemental scavenging of ROS in Meis1 HSCs. After 14 days of tamoxifen Figure 3B). We showed previously that LT-HSCs primarily rely on injection, we administered NAC intraperitoneally for 12 days cytoplasmic glycolysis rather than mitochondrial oxidative phos- (Figure 4A). Mice where then harvested and HSCs were isolated phorylation.11 However, the role of Hif-1␣ in HSC metabolism was for analysis of HSC frequency, HSC cell-cycle status, apoptosis not examined before. Here, we demonstrate that HSC-specific rate, ROS levels, and expression of redox-sensitive cell-cycle deletion of Hif-1␣ results in increased mitochondrial respiration as regulators p16Ink4a and p19Arf. Here, we show that NAC administra- shown by increased oxygen consumption (supplemental Figure tion rescues the Meis1⫺/⫺ phenotype. We found that frequency of 3C), and decreased glycolytic flux measured by the rate of HSCs in Meis1⫺/⫺ mice become similar to Meis1⫹/⫹ mice (Figure glucose-derived 13C lactate production (supplemental Figure 3D).
4B). Flow cytometric analysis of cell cycle of Meis1⫹/⫹ and To further examine the role of Hif-1␣ in HSCs, we examined Mesi1⫺/⫺ LT-HSCs, which were both injected with NAC, shows expression of Hif-2␣ and Hif-3␣ in Hif-1␣⫺/⫺ LT-HSCs. We found similar numbers of G that deletion of Hif-1␣ in LT-HSCs results in a profound increase in 0 cells in Meis1⫺/⫺ LT-HSCs (Figure 4C).
Quantification of apoptosis in Meis1⫹/⫹ and Meis1⫺/⫺ mice shows Hif-2␣ mRNA levels (⬎ 120-fold), with no significant change in an increased apoptosis in Meis1⫺/⫺ mice; however, this increase in the levels of Hif-3␣ mRNA (supplemental Figure 3E). This marked the number of apoptotic cells was not statistically significant compensatory up-regulation of Hif-2␣ in Hif-1␣⫺/⫺ LT-HSCs is in (P ⫽ .051; Figure 4D). In addition, quantification of ROS after stark contrast to the down-regulation of Hif-2␣ in the Meis1⫺/⫺ NAC treatments restored ROS to Meis1⫹/⫹ control levels in Meis1⫺/⫺ LT-HSCs (Figure 4E). Finally, scavenging ROS restored HSC-specific deletion of Hif-1␣ also demonstrated hematopoi- the transcript levels of p16Ink4a and p19Arf in HSC to Meis1⫹/⫹ etic defects similar (supplemental Figure 4) to global deletion control levels (Figure 4F). While these effects are most likely (Mx-1-Cre) in the bone marrow shown by Takubo and colleagues,25 attributable to the antioxidant effect of NAC, other unknown including the increased frequency of HSCs (supplemental Figure mechanisms of NAC may also contribute to this rescue.
4A), decreased myeloid progenitors (supplemental Figure 4B), loss To further evaluate the effect of ROS in engraftment defect of quiescence (supplemental Figure 4C), and similar multilineage observed after Meis1 deletion, we performed another bone marrow defects (supplemental Figure 4F-G).
transplantation from Meis1⫹/⫹ and Meis1⫺/⫺ mice treated with and Meis1 is a transcriptional activator of Hif-2␣
without NAC. Two weeks after tamoxifen treatment and NACadministration, 1 ⫻ 105 BM cells from Meis1⫹/⫹ or Meis1⫺/⫺ Down-regulation of Hif-1␣ and Hif-2␣ in Meis1⫺/⫺ LT-HSCs CD45.2, together with 1 ⫻ 105 CD45.1 competitors, were injected (Figure 3A) led us to examine their metabolic profile and ROS into CD45.1 recipients through the retro-orbital complex (Figure levels. We found that Meis1⫺/⫺ LT-HSCs have increased rates of 4G). After bone marrow transplantation, NAC is provided in oxygen consumption (Figure 3B) and decreased glycolytic flux drinking water for 2 weeks and injected daily for 2 more weeks.
(Figure 3C). In addition, Meis1⫺/⫺ LT-HSCs showed significantly Repopulation was examined at 4 weeks posttransplantation. As higher ROS levels compared with control Meis1⫹/⫹ LT-HSCs shown in Figure 4H, scavenging of ROS by NAC administration (Figure 3D). This increased ROS was associated with increased restored engraftment defects in Meis1⫺/⫺ donors. In conclusion, expression of p16Ink4a and p19Arf in Meis1⫺/⫺ LT-HSCs, which are scavenging ROS, through administration of NAC, rescues the known to induce HSC senescence and apoptosis, respectively Meis1 phenotype in HSC by decreasing levels of ROS, thereby (Figure 3E). Therefore, the increased ROS production in Meis1⫺/⫺ normalizing p16Ink4a and p19Arf expression and restoring HSC LT-HSCs is compounded by the down-regulation of the master quiescence and repopulation defects.
antioxidant gene Hif-2␣. Moreover, measurement of peripheralblood counts of Meis1⫺/⫺ mice post 1 month and 3 months of Effect of HIFs on stem cell function in Meis1ⴚ/ⴚ phenotype
tamoxifen treatments demonstrates decreased red blood cells,white blood cells, as well as platelets, similar to the phenotype Meis1 KO BM cells treated with 100M CoCl2 resulted in observed in Hif-2␣⫺/⫺ mice42 (supplemental Figure 5). To deter- restoration of total number of colonies to the WT levels (supplemen- mine the mechanism of down-regulation of Hif-2␣ in Meis1⫺/⫺ tal Figure 6A). Moreover, we found that Hif-1␣ overexpression LT-HSCs, we identified 2 Meis1 consensus-binding motifs in the using the Meis1f/f;Scl-Cre⫹; rtTA; TRE-Hif-1␣ mice strategy, promoter of the Hif-2␣ gene (Figure 3F). Using a Hif-2␣–pGL2 which resulted in marked up-regulation of Hif-1␣ in HSCs, reporter which includes the conserved Meis1-binding sites, we resulted in restoration of the BFU-E and total number of colonies to demonstrate a dose-dependent activation of Hif-2␣ by Meis1 the WT levels, as well as partial restoration of number of mixed expression vector (CMV-Meis1; Figure 3G). In addition, this colonies (CFU-GEMM) which is a measure of most undifferenti- activation demonstrates dependence on binding of Meis1 to its ated hematopoietic progenitors (supplemental Figure 6C). In consensus-binding sequences in the Hif-2␣ promoter because addition, we attempted to rescue repopulation defect in Meis1⫺/⫺ mutation of the seed sequences (Hif-2␣–Mut-pGL2) completely HSCs using viruses that express Hif-1␣ and Hif-2␣ (supplemental abolished the activation of Hif-2␣ by Meis1. Given down- Figure 6D) or using Meisf/f;Scl-Cre⫹; rtTA; TRE-Hif-1␣ (supple- regulation of Hif-2␣ in Meis1⫺/⫺ LT-HSCs (Figure 3A), these mental Figure 6E); however, these strategies failed to rescue the in results indicate that Meis1 is required for optimal transcriptional vivo reconstitution capacity up to 4 months after Meis1 deletion. In
BLOOD, 13 DECEMBER 2012 䡠 VOLUME 120, NUMBER 25 Figure 4. Effect of ROS scavenging on the Meis1ⴚ/ⴚ phenotype. (A) Schematic of NAC administration. We performed daily IP injections for tamoxifen for 14 days followed
by daily NAC injections up to 12 days. (B) Flow cytometry profile of LT-HSCs (Lin⫺Sca-1⫹Kit⫹Flk2⫺CD34⫺) of Meis1⫹/⫹ and Meis1⫺/⫺ mice after 12 days NAC administration.
Note the number of HSCs in Meis1⫺/⫺ mice is now similar to Meis1⫹/⫹ values (n ⫽ 3). (C) Left panel, FACS plot of Pyronin Y/Hoechst staining of LT-HSCs. Right panel:
Quantification of flow cytometric analysis of cell cycle of Meis1⫹/⫹ and Mesi1⫺/⫺ LT-HSCs demonstrates restored numbers of G0 cells in Meis1⫺/⫺ cells which indicates restored
quiescence of LT-HSCs (n ⫽ 3). (D) Quantification of apoptosis in Meis1⫹/⫹ and Meis1⫺/⫺ LSK cells (Lin⫺Sca-1⫹Kit⫹) showing persistent trend toward an increase in the
number of apoptotic cells, which was not statistically significant (P ⫽ .059; n ⫽ 3). (E) Quantification of ROS in Meis1⫹/⫹ and Meis1⫺/⫺ LT-HSCs (Lin⫺Sca-1⫹Kit⫹Flk2⫺CD34⫺)
after NAC treatment showing only a modest increase in ROS in Meis1⫺/⫺ HSCs (n ⫽ 3). (F) Real-time PCR of HSCs isolated from Meis1⫹/⫹ and Meis1⫺/⫺ HSCs after NAC
treatment demonstrating no change in p16 and p19 transcripts (n ⫽ 3). (G) Schematic of NAC administration and bone marrow transplantations. We performed daily IP
injections for tamoxifen and NAC for 14 days followed by bone marrow transplantation. Then, NAC is provided in drinking water for 2 weeks and administrated another
2 weeks. Repopulation was examined at 4 weeks after transplantation. (H) Analysis of repopulation after NAC treatments of BMTs from Meis1⫹/⫹ and Meis1⫺/⫺ mice
demonstrates restoration of repopulation defect after Meis1 deletion (n ⫽ 5). (I) Schematic of proposed model is demonstrating how Meis1 regulates metabolism and
maintenance of HSCs through its role on Hif-1␣ and Hif-2␣.
retrospect, these results are not entirely surprising in light of recent Hif-2␣, and as a result Meis1 deletion results in HSC dysfunction results demonstrating a narrow beneficial dose range of Hif-1␣ on and apoptosis.
HSC function, where overstabilization of Hif-1␣ results in worsen-ing HSC function43 which might explain worsening of the HSCrepopulation defect in Meis1 KO HSCs in vivo (supplemental Figure 6D-E).
In summary, we demonstrate that Meis1 deletion results in a In the current report, we demonstrate that Meis1 functions up- shift in HSC metabolism toward oxygen consumption, with the stream of a transcriptional network that regulates HSC metabolism resultant increase in ROS production. This phenotype is com- and oxidant defense. The Meis1 deletion-induced metabolic shift pounded by down-regulation of the oxidant stress response gene and oxidant injury is compounded by the down-regulation of BLOOD, 13 DECEMBER 2012 Meis1 REGULATES Hif-1 䡠 VOLUME 120, NUMBER 25 ␣ AND Hif-2␣ IN HSCs Hif-2␣, which is in stark contrast to the marked up-regulation of phenotype after Meis1 deletion, there certainly may be additional Hif-2␣ in HSC after Hif-1␣ deletion. This phenotype is associated ROS modulators that are targeted by Meis1.
with up-regulation of p16Ink4a and p19Arf, loss of quiescence, In the current report, we highlight the role of Meis1 in a increased apoptosis, and marked HSC dysfunction.
transcriptional network that regulates HSC metabolism and antioxi- Endothelial PAS domain protein 1 (EPAS1), also known as dant defense. These results implicate Meis1 as an important Hif-2␣,44 is closely related to Hif-1␣ in structure and is likewise regulator of the redox state of HSCs, which may be echoed by its activated during hypoxia.20 While Hif-1␣ is a master regulator of role in leukemogenesis. Therefore, it would be important for future metabolism, Hif-2␣ is a master regulator of oxidant stress re- studies to determine the role of Meis1 in regulation of leukemia sponse45 and is induced by ROS.46 It is involved in regulation of stem cell metabolism, survival, and self-renewal. Our findings numerous antioxidant genes that minimize the oxidant damage that suggest that HSCs are endowed with redundant mechanisms for results from mitochondrial respiration.45 Hif-2␣⫺/⫺ mice are pancy- regulation of their metabolic phenotype, rather than being solely topenic,42,47 and have high levels of oxidative stress,45 which dependent on environmental signals, such as the hypoxic microen-vironment. Deciphering the role of these transcriptional networks suggests that Hif-2␣ is required for normal hematopoiesis. Our in regulating HSC fate and function may provide valuable clues for results indicate that Hif-2␣ is markedly up-regulated after Hif-1␣ understanding HSC disease and malignancies.
deletion, which is an indication of a robust antioxidant response toHif-1␣ deletion, likely secondary to a shift toward oxidativemetabolism, with the subsequent increase in ROS.46 In stark contrast, we show that Hif-2␣ is down-regulated after Meis1 KO inHSCs, which may partially explain the severity of the Meis1⫺/⫺ The authors thank Dr Keith Humphries, Michelle Miller, and Patty phenotype compared with the Hif-1␣⫺/⫺ phenotype.
Rosen for sharing some initial data on characterization of the KO Delicate control of ROS levels in HSCs is crucial for HSC line genotyping, Dr Joachim R. Goethert at Universitaetsklinikum maintenance, where elevated ROS levels in HSCs is associated Essen for providing the Scl-Cre-ERT mice, and Dr Joseph A. Garcia with defects in HSC self-renewal and increased apoptosis. Regula- for Hif-1␣ floxed mice.
tion of ROS in HSCs is a highly complex process that involves This work is supported by grants from the American Heart regulation of the metabolic phenotype of HSCs, as well as Association (AHA; GIA12060240 [H.A.S.]), the Gilead Research regulation of antioxidant defense mechanisms. The contribution of Scholars Program in Cardiovascular Disease (H.A.S.), National oxidative metabolism to ROS production has been extensively Institutes of Health (NIH) grant R01HL115275 (H.A.S.), NIH studied.48 It is estimated that 2% of all electrons flowing through grant K01 CA 120099 (C.Z.), and Cancer Prevention and Research the mitochondrial respiratory chain result in the formation of Institute of Texas (CPRIT) RP100402 (C.Z.).
oxygen free radicals. Electrons leaking from the respiratory chaininteract with oxygen, partially reducing it to superoxide anion (O2⫺䡠).48 Even though O2⫺䡠 itself is not a strong oxidant, it is theprecursor of most other ROS.14 ROS overwhelm the natural Contribution: F.K. designed and performed the research, analyzed antioxidant defense mechanisms over time, and result in wide- data, and wrote the manuscript; J.Z., S.T., N.G.C., N.A.J., and spread cellular damage.48 ROS can also induce the cell-cycle R.J.D. performed research and analyzed data; and C.Z. and H.A.S.
regulators p16Ink4a and p19Arf, which cause loss of quiescence and designed and supervised research and wrote the manuscript.
apoptosis of HSCs.49 Our results indicate that Meis1 regulates ROS Conflict-of-interest disclosure: The authors declare no compet- production in HSC through regulation of both the metabolic ing financial interests.
phenotype (through Hif-1␣) and oxidant defense mechanisms The current affiliation for F.K. is Texas Institute of Biotechnol- (through Hif-2␣). Finally, although our ROS scavenging studies ogy, Edu&Res, North American College, Houston, TX.
indicate that the effect of loss of Meis1 on HSC cell cycle and Correspondence: Hesham A. Sadek, MD, PhD, Department of engraftment defect are entirely mediated through an increase in Internal Medicine, Division of Cardiology, UT Southwestern ROS production, rescue studies using Hif-1␣ and/or Hif-2␣ Medical Center, NB10.222, 6000 Harry Hines Blvd, Dallas, TX overexpression or stabilization failed to fully rescue the phenotype.
75390; e-mail: [email protected]; or Chengcheng These results support previous reports which indicate that the Zhang, PhD, Departments of Physiology and Developmental Biology, precise dose of Hif-1␣ is necessary for optimal HSC function.25 UT Southwestern Medical Center, NB10.222, 6000 Harry Hines Blvd, Although our results implicate Hif-1␣ and Hif-2␣ in the HSC Dallas, TX 75390; e-mail: [email protected]
1. Abramson S, Miller RG, Phillips RA. The identifi- contribute to adult hematopoiesis. Blood. 2005; the neighbors: stem cells and their niche. Cell. cation in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid 5. Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, 9. Eliasson P, Jonsson JI. The hematopoietic stem systems. J Exp Med. 1997;145(6):1567-1579.
Terhorst C, Morrison SJ. SLAM family receptors cell niche: low in oxygen but a nice place to be.
2. Jordan CT, McKearn JP, Lemischka IR. Cellular distinguish hematopoietic stem and progenitor J Cell Physiol. 2010;222(1):17-22.
and developmental properties of fetal hematopoi- cells and reveal endothelial niches for stem cells.
10. Cipolleschi MG, Dello Sbarba P, Olivotto M. The etic stem cells. Cell. 1990;61(6):953-963.
role of hypoxia in the maintenance of hematopoi- 3. Osawa M, Hanada K, Hamada H, Nakauchi H.
6. Schofield R. The relationship between the spleen etic stem cells. Blood. 1993;82(7):2031-2037.
Long-term lymphohematopoietic reconstitution by colony-forming cell and the haemopoietic stem 11. Simsek T, et al. The distinct metabolic profile of a single CD34-low/negative hematopoietic stem cell. Blood Cells. 1978;4(1-2):7-25.
hematopoietic stem cells reflects their location in 7. Spradling A, Drummond-Barbosa D, Kai T. Stem a hypoxic niche. Cell Stem Cell. 2010;7(3):380- 4. Go¨thert JR, Gustin SE, Hall MA, et al. In vivo fate- cells find their niche. Nature. 2001;414(6859):98- tracing studies using the Scl stem cell enhancer: 12. Semenza GL. Life with oxygen. Science. 2007; embryonic hematopoietic stem cells significantly 8. Fuchs E, Tumbar T, Guasch G. Socializing with BLOOD, 13 DECEMBER 2012 䡠 VOLUME 120, NUMBER 25 13. Miquel J, et al. Mitochondrial role in cell aging.
level is essential for hematopoietic stem cells.
neic transplantation. Cell Stem Cell. 2011;9(2): Exp Gerontol. 1980;15(6):575-591.
Cell Stem Cell. 2010;7(3):391-402.
14. Turrens JF. Mitochondrial formation of reactive 26. Moskow JJ, et al. Meis1, a PBX1-related homeo- 38. Xu Y, et al. SUMO-specific protease 1 regulates oxygen species. J Physiol. 2003;552(Pt 2):335- box gene involved in myeloid leukemia in BXH-2 the in vitro and in vivo growth of colon cancer mice. Mol Cell Biol. 1995;15(10):5434-5443.
cells with the upregulated expression of CDK in- 15. Ergen AV, Goodell MA. Mechanisms of hemato- hibitors. Cancer Lett. 2011;309(1):78-84.
27. Rozovskaia T, et al. Upregulation of Meis1 and poietic stem cell aging. Exp Gerontol. 2010;45(4): HoxA9 in acute lymphocytic leukemias with the 39. Barresi MJ, et al. The development of the Can- t(4:11) abnormality. Oncogene. 2001;20(7):874- berra symptom scorecard: a tool to monitor thephysical symptoms of patients with advanced tu- 16. Jang YY, Sharkis SJ. A low level of reactive oxy- mours. BMC Cancer. 2003;3:32.
gen species selects for primitive hematopoietic 28. Pineault N, et al. Differential and common leuke- stem cells that may reside in the low-oxygenic 40. Li HY, et al. Reprogramming induced pluripotent mogenic potentials of multiple NUP98-Hox fusion niche. Blood. 2007;110(8):3056-3063.
stem cells in the absence of c-Myc for differentia- proteins alone or with Meis1. Mol Cell Biol. 2004; tion into hepatocyte-like cells. Biomaterials. 2011; 17. Elvidge GP, et al. Concordant regulation of gene expression by hypoxia and 2-oxoglutarate- 29. Fischbach NA, et al. HOXB6 overexpression in dependent dioxygenase inhibition: the role of 41. Belteki G, et al. Conditional and inducible trans- murine bone marrow immortalizes a myelomono- HIF-1alpha, HIF-2alpha, and other pathways.
gene expression in mice through the combinato- cytic precursor in vitro and causes hematopoietic J Biol Chem. 2006;281(22):15215-15226.
rial use of Cre-mediated recombination and tetra- stem cell expansion and acute myeloid leukemia cycline induction. Nucleic Acids Res. 2005;33(5): 18. Hagg M, Wennstrom S. Activation of hypoxia- in vivo. Blood. 2005;105(4):1456-1466.
induced transcription in normoxia. Exp Cell Res. 30. Argiropoulos B, Yung E, Humphries RK. Unravel- 42. Scortegagna M, et al. The HIF family member ing the crucial roles of Meis1 in leukemogenesis EPAS1/HIF-2alpha is required for normal hema- 19. Maxwell PJ, et al. HIF-1 and NF-kappaB-mediated and normal hematopoiesis. Genes Dev. 2007; topoiesis in mice. Blood. 2003;102(5):1634-1640.
upregulation of CXCR1 and CXCR2 expression pro- 43. Takubo K, et al. Regulation of the HIF-1alpha motes cell survival in hypoxic prostate cancer cells.
31. Argiropoulos B, Humphries RK. Hox genes in he- level is essential for hematopoietic stem cells.
matopoiesis and leukemogenesis. Oncogene.
Cell Stem Cell. 2010;7(3):391-402.
20. Wang GL, et al. Hypoxia-inducible factor 1 is a 44. Tian H, McKnight SL, Russell DW. Endothelial basic-helix-loop-helix-PAS heterodimer regulated 32. Imamura T, et al. Frequent co-expression of PAS domain protein 1 (EPAS1), a transcription by cellular O2 tension. Proc Natl Acad Sci U S A.
HoxA9 and Meis1 genes in infant acute lympho- factor selectively expressed in endothelial cells.
blastic leukaemia with MLL rearrangement. Br J Genes Dev. 1997;11(1):72-82.
21. Marin-Hernandez, Gallardo-Pe´rez JC, Ralph SJ, 45. Scortegagna M, et al. Multiple organ pathology, Rodríguez-Enríquez S, Moreno-Sa´nchez R. HIF- metabolic abnormalities and impaired homeosta- 1alpha modulates energy metabolism in cancer 33. Azcoitia V, et al. The homeodomain protein Meis1 sis of reactive oxygen species in Epas1⫺/⫺ cells by inducing over-expression of specific gly- is essential for definitive hematopoiesis and vas- mice. Nat Genet. 2003;35(4):331-340.
colytic isoforms. Mini Rev Med Chem. 2009;9(9): cular patterning in the mouse embryo. Dev Biol.
46. Wiesener MS, et al. Induction of endothelial PAS domain protein-1 by hypoxia: characterization 22. Kim JW, et al. HIF-1-mediated expression of py- 34. Hisa T, et al. Hematopoietic, angiogenic and eye and comparison with hypoxia-inducible factor- ruvate dehydrogenase kinase: a metabolic switch defects in Meis1 mutant animals. EMBO J. 2004; 1alpha. Blood. 1998;92(7):2260-2268.
required for cellular adaptation to hypoxia. Cell 47. Scortegagna M, et al. HIF-2alpha regulates mu- 35. Ficara F, et al. Pbx1 regulates self-renewal of rine hematopoietic development in an 23. Papandreou I, et al. HIF-1 mediates adaptation to long-term hematopoietic stem cells by maintain- erythropoietin-dependent manner. Blood. 2005; hypoxia by actively downregulating mitochondrial ing their quiescence. Cell Stem Cell. 2008;2(5): oxygen consumption. Cell Metab. 2006;3(3):187- 48. Orrenius S, Gogvadze V, Zhivotovsky B. Mito- 36. Zheng J, et al. Angiopoietin-like protein 3 sup- chondrial oxidative stress: implications for cell 24. Zhang H, et al. HIF-1 inhibits mitochondrial bio- ports the activity of hematopoietic stem cells in death. Ann Rev Pharmacol Toxicol. 2007;47:143- genesis and cellular respiration in VHL-deficient the bone marrow niche. Blood. 2011;117(2):470- renal cell carcinoma by repression of C-MYC ac- 49. Ito K, et al. Reactive oxygen species act through tivity. Cancer Cell. 2007;11(5):407-420.
37. Zheng J, et al. Ex vivo expanded hematopoietic p38 MAPK to limit the lifespan of hematopoietic 25. Takubo K, et al. Regulation of the HIF-1alpha stem cells overcome the MHC barrier in alloge- stem cells. Nat Med. 2006;12(4):446-451.
2012 120: 4963-4972 originally published doi:10.1182/blood-2012-05-432260online September 20, 2012
Meis1 regulates the metabolic phenotype and oxidant defense of
hematopoietic stem cells
Fatih Kocabas, Junke Zheng, Suwannee Thet, Neal G. Copeland, Nancy A. Jenkins, Ralph J.
DeBerardinis, Chengcheng Zhang and Hesham A. Sadek
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Dopage au quotidien Un document de base d'Addiction Suisse 1. Introduction A en croire les médias, on assiste à une augmentation de la tendance à recourir aux artifices les plus divers pour augmenter sa performance au travail et dans la vie privée. Même des personnes en parfaite santé n'hésitent pas à intervenir dans le fonctionnement de leur corps. Une pratique qui n'a rien de nouveau. Dans le domaine du physique ce souci d'optimisation, nourri par l'obsession contemporaine de la beauté et de la performance, se traduit déjà, du moins dans certains milieux, par la chirurgie esthétique, la médecine sportive et les médicaments « lifestyle » (par exemple les produits anti-âge). En matière de sport, le dopage a déjà provoqué un vaste débat éthique et philosophique, non seulement en raison des risques qu'il comporte pour la santé, mais surtout du manque de fair-play dont les sportifs de compétition font preuve, de l'avis général, en se procurant ainsi des avantages injustifiés sur leurs adversaires. Cette controverse a débouché sur l'établissement de règles concernant l'utilisation des produits dopants. En revanche, la discussion sur l'emploi de ces derniers dans le cadre de la vie privée et professionnel e ne fait que commencer.