Health Articles
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].
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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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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-
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.
