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Brief Report
HEMATOPOIESIS AND STEM CELLS

Erk1 and Erk2 are required for maintenance of hematopoietic stem cells
and adult hematopoiesis
Gordon Chan,1 Shengqing Gu,1,2 and Benjamin G. Neel1,2
1

Campbell Family Cancer Research Institute, Ontario Cancer Institute and Princess Margaret Hospital, and 2Department of Medical Biophysics, University Health
Network, University of Toronto, Toronto, ON, Canada

Extracellular signal-regulated kinase 1 (Erk1) and Erk2 play crucial roles in cell survival,
proliferation, cell adhesion, migration, and differentiation in many tissues. Here, we
report that the absence of Erk1 and Erk2 in murine hematopoietic cells leads to bone
Erk1/2 are required for the
maintenance of hematopoietic marrow aplasia, leukopenia, anemia, and early lethality. Mice doubly-deficient in Erk1
and Erk2 show rapid attrition of hematopoietic stem cells and immature progenitors in
stem cells and immature
a cell-autonomous manner. Reconstitution studies show that Erk1 and Erk2 play
progenitors in vivo.
redundant and kinase-dependent functions in hematopoietic progenitor cells. Moreover, in cells transformed by the oncogenic KRasG12D allele, the presence of either Erk1 or Erk2 with intact kinase activity is
sufficient to promote cytokine-independent proliferation. (Blood. 2013;121(18):3594-3598)

Key Points

Introduction
Extracellular signal-regulated kinase 1 (Erk1) and Erk2 (Erk/12)
encode serine/threonine kinases belonging to the mitogen-activating
protein kinase family, and play critical roles in transmitting signals
from a plethora of cytokines and growth factors.1 ERK1 and ERK2
are .80% identical, and both phosphorylate a myriad of substrates,
many of which are cell line- and agonist-specic.1-3 Erk12/2 mice
are viable and fertile,4,5 whereas Erk22/2 mice are embryonic lethal
due to placental defects.6-9
ERK1/2 activation appears to be required for the survival and
differentiation of many types of hematopoietic cells,2,3,10 but most
studies of ERK1/2 function in hematopoiesis have used pharmacologic blockade of mitogen-activated protein kinase kinase 1
(MEK1) and MEK2, the kinases that activate ERK1/2. Genetic
experiments argued that Erk1 is required for T-cell development,4
although this observation is controversial.5,9,11 T-cellspecic
deletion of Erk2 also leads to defective T-lymphocyte maturation,
suggesting that in some cellular contexts, ERK1 cannot compensate for ERK2.9 By contrast, either Erk1 or Erk2 is sufcient for
normal B-cell development,12 although this process is markedly
impaired in Erk1/2 doubly-decient B cells.12 Together, these
observations suggest that ability of Erk1 and Erk2 to compensate
for one another might be cell-type specic. To date, the role of
Erk1/2 in hematopoietic stem cell (HSC) function remains to be
characterized. Here, we show that Erk1/2 are required in a cellautonomous manner for the maintenance of HSCs.

accord with guidelines approved by the animal welfare committee of the

University Health Network.

Results and discussion

Erk12/25 and Erk2ox/ox9 mice were crossed to Mx1-Cre mice (The Jackson
Laboratory, Bar Harbor, ME), as indicated. All mice were maintained in

To assess ERK1 and ERK2 function in hematopoiesis, we generated

Mx1-Cre;Erk2ox/ox and Mx1-Cre Erk12/2;Erk2ox/ox mice in a
C57BL/6J background and induced Cre recombinase expression by
administration of polyinosinic-polycytidylic acid (pIpC). Hematopoiesis in untreated or pIpC-treated Erk2ox/ox (hereafter, control)
mice was indistinguishable from that in wild-type C57BL/6J animals
(data not shown). Most induced Mx1-Cre Erk12/2;Erk2ox/ox
(hereafter, Erk12/2Erk2D/D) mice became moribund or died 30 to
40 days after pIpC injection (Figure 1A), whereas Erk12/2 and
induced Mx1-Cre;Erk2ox/ox (hereafter, Erk2D/D) mice were indistinguishable from controls. Erk12/2Erk2D/D mice were leukopenic, severely anemic, and had markedly reduced numbers of bone
marrow (BM) cells (Figure 1B-D and supplemental Figure 1A,
available on the Blood website). There also was a trend toward
decreased numbers of mature myeloid, B cells, and erythroid cells
in the BM of Erk12/2Erk2D/D mice, although this difference did not
reach statistical signicance (supplemental Figure 1B).
Next, we examined the HSC-enriched Lin2Sca11c-Kit1 (LSK)
compartment and early progenitors in control and Erk-decient BM.
Erk12/2 and Erk2D/D mice showed normal numbers of LSK cells,
common myeloid progenitors (CMPs), granulocyte-macrophage
progenitors (GMPs), and megakaryocte-erythrocyte progenitors
(MEPs), but all of these progenitors were reduced markedly
in Erk12/2Erk2D/D BM (Figure 1E). Analysis of a more highly

Submitted December 27, 2012; accepted February 21, 2013. Prepublished

online as Blood First Edition paper, February 26, 2013; DOI 10.1182/blood2012-12-476200.

The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked advertisement in accordance with 18 USC section 1734.

The online version of this article contains a data supplement.

2013 by The American Society of Hematology

Methods

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BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18

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BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18

Erk1 AND Erk2 IN MURINE HEMATOPOIESIS

3595

Figure 1. Deletion of Erk1 and Erk2 leads to fatal BM failure and loss of HSC. (A) Kaplan-Meier survival analysis of a cohort of Erk12/2 (n 5 23), Mx1-Cre;Erk flox/flox (n 5 21),
Mx1-Cre Erk12/2;Erk2 flox/flox (n 5 22), and littermate control (n 5 18) mice following pIpC treatment. Erk12/2Erk2D/D mice have a median lifespan of 30 days; control mice remain
healthy for .120 days. *P , .05, log-rank test. (B) Hematoxylin and eosinstained sections from humeri of control, Erk12/2, Erk2D/D, and Erk12/2Erk2D/D animals 30 days after
receiving their last dose of pIpC. (C-D) White blood cell count (WBC) and hematocrit (HCT) of control, Erk12/2, Erk2D/D, and Erk12/2Erk2D/D mice (*P , .05, ANOVA). (E) Absolute
number (mean 6 SEM, *P , .05, ANOVA) of LSK, CMP (Lin2Sca12cKit1CD341FcgRlo), GMP (Lin2Sca12cKit1CD341FcgRhi), MEP (Lin2Sca12cKit1CD342FcgR2/lo) of control
(n 5 6), Erk12/2 (n 5 5), Erk2D/D (n 5 5), and Erk12/2Erk2D/D mice (n 5 6). *P , .05, ANOVA. (F) Absolute number (mean 6 SEM, * P , .05, Student t test) of signaling
lymphocyte activation molecule (SLAM)-HSC from control (n 5 6), Erk12/2 (n 5 5), Erk2D/D (n 5 5), and Erk12/2Erk2D/D mice (n 5 6). *P , .05, ANOVA.

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3596

CHAN et al

BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18

Figure 2. Requirement of Erk1 and Erk2 in HSCs and immature progenitors. (A) Single SLAM-HSC from of control, Erk12/2, Erk2D/D, and Erk12/2Erk2D/D mice were
sorted into 60-well plates (1 cell per well) and cultured in media containing SCF, Tpo, Flt3l, and IL-11. Proliferation of each clone was evaluated microscopically over 4 days.
Representative data from 1 of 4 experiments with similar results are shown. The average percentages (6SEM) of surviving clones from the 4 experiments are shown at the
top of each panel. *P , .05, ANOVA. (B) Lethally irradiated CD45.11 recipients (6 mice per group) were transplanted with 106 BM cells from control, Erk12/2, Erk2D/D, and
Erk12/2Erk2D/D mice, along with 105 wild-type CD45.11 cells. After 5 weeks of engraftment, chimeric mice were treated with 5 doses of pIpC, and the percentage of donorderived peripheral blood cells (Total), CD31 (T cells), B2201 (B cells), and Gr11 (myeloid cells) were determined flow cytometry. *P , .05, ANOVA. (C) Control, Erk12/2,
Erk22/2, and Mx1-Cre Erk12/2Erk2flox/flox mice were treated with pIpC for 14 days before their BM cells (5 3 105) were harvested and mixed with equal numbers of wild-type

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BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18

enriched HSC compartment (LSKCD1501CD482, SLAM-HSC) also

revealed a marked reduction in the frequency and the absolute cell
number of these cells in Erk12/2Erk2D/D mice (Figure 1F and data not
shown), indicating a defect at the earliest stage of adult hematopoiesis.
Thus, either Erk1 or Erk2 is sufcient to maintain hematopoiesis, but
deletion of both Erk1 and Erk2 results in rapid hematopoietic failure
and depletion of HSCs and progenitors.
Next, we investigated the ability of Erk-decient HSCs to
respond to cytokines that promote HSC maintenance. SLAM-HSCs
were isolated from control and Erk-mutant mice 10 days after pIpC
treatment, and placed at 1 cell per well in a medium that preserves
multipotency in vitro.13,14 Mutant cells harvested at this time point
all showed complete ablation of the appropriate Erk messenger
RNA (supplemental Figure 2A-B). After 4 days of culture, .85%
of cells from control, Erk12/2, Erk2D/D mice were viable and had
shown substantial proliferation. By contrast, Erk12/2Erk2D/D cells
underwent at most 1 or 2 cell divisions, and only ;40% remained at
the end of the experiment (Figure 2A). These data indicate that
compound loss of Erk1 and Erk2 is incompatible with the proliferation and survival of HSCs in vitro.
To test whether the requirement for Erk1 and Erk2 in
hematopoiesis is cell-autonomous, we transferred uninduced
Erk2ox/ox, Erk12/2, Mx1-Cre;Erk2ox/ox, and Mx1-Cre Erk12/2;
Erk2ox/ox BM cells, together with wild-type congenic CD45.1expressing carrier BM, into lethally irradiated CD45.1 animals.
Recipients were maintained for 5 weeks to allow establishment of
steady-state hematopoiesis. These mice were then injected with
pIpC, and the ratio of CD45.11 vs CD45.21 cells in the peripheral
blood was monitored. All mice showed comparable levels of
CD45.2 engraftment before pIpC treatment (Figure 2B). Moreover,
the percentage of donor chimerism of the B-cell and myeloid
lineages in recipients of control, Erk12/2, and Erk2D/D BM cells did
not differ signicantly. By contrast, there was a signicant reduction
in the percentage of donor-derived T cells in recipients of Erk12/2
or Erk2D/D BM cells, with Erk2D/D BM cells recipients showing
a more pronounced defect. Finally, mice that had received Mx1-Cre
Erk12/2;Erk2ox/ox BM cells showed profound attrition of donorderived T, B, and myeloid cells following pIpC treatment, indicating
that the defect in HSC maintenance in the absence of ERK1 and
ERK2 is cell-autonomous.
To further assess the effects of Erk deciency on the functional
competence of HSCs, we performed competitive reconstitution
studies. In these experiments, control or Erk-decient BM cells were
mixed with CD45.11 BM cells at 1:1 ratio before they were injected
to lethally irradiated CD45.11 recipients. Beginning 8 weeks after
transplantation, CD45.2-derived cells in the peripheral blood of
recipient mice were quantied. Recipients of Erk12/2 and Erk2D/D
BM cells showed a reduced proportion of donor-derived T and
B cells (Figure 2C). However, the frequencies of donor-derived
myeloid cells in recipients of control, Erk12/2, and Erk2D/D BM cells
were similar. By contrast, animals that received Erk12/2Erk2D/D BM
cells had almost no contribution of donor-derived cells from any
lineage in their peripheral blood. Thus, single Erk knockout HSCs
and myeloid progenitors are not disadvantaged under competitive

Erk1 AND Erk2 IN MURINE HEMATOPOIESIS

3597

conditions, but deletion of both Erk isoforms leads to gross attrition

of all hematopoietic lineages.
Although Erk2 is known to play a crucial role in T-cell
development and activation,9,11 the role of Erk1 in T-lineage cells
remains controversial.4,5,9,11 Importantly, the development and maintenance of Erk1-decient T cells in an Erk1-sufcient background
has never been examined. Our data suggest that both Erk1 and Erk2
are necessary for normal T-cell development/maintenance in vivo.
Furthermore, our data indicate that the development and maintenance
of Erk1- and Erk2-decient B cells are diminished in the presence
of wild-type B cells, indicating that B lymphocytes lacking a single
ERK isoform have a subtle functional impairment not revealed in the
noncompetitive setting.
Isoform-specic functions of Erk1 and Erk2 remain controversial.2,3 Moreover, ERK1 and ERK2 are known to have kinaseindependent functions.15 To ask whether Erk1 and Erk2 have unique
functions in hematopoietic cells and whether catalytic activity is
required for hematopoietic progenitor function, we constructed
retroviral vectors coexpressing a green uorescent protein (GFP)
Cre fusion protein and either the hygromycin resistance gene (hygro),
Erk1, Erk2, Erk1K72R, or Erk2K52R. Erk1K72R and Erk2K52R encode
kinase-defective mutants of ERK1 and ERK2.16 Lineage-negative
(lin2) BM cells from Erk12/2Erk2ox/ox mice were infected with
these viruses, and GFP1 cells were puried by uorescence-activated
cell sorter (FACS) and plated in liquid medium containing IL-3, IL-6,
and stem cell factor (SCF) (Figure 2D). In infected cells, GFP-Cre
excises the endogenous Erk2 alleles, generating Erk12/2 Erk2null
cells, while concomitantly, different Erk isoforms are expressed under the control of the same viral promoter. We observed nearly
complete loss of cell proliferation in Erk1 2/2 Erk2ox/ox cells
coexpressing GFP-Cre and hygro. Coexpression of wild-type Erk1
or Erk2 with GFP-Cre restored cell proliferation to a similar extent.
In sharp contrast, cells coexpressing kinase-defective Erk1 or Erk2
showed minimal proliferation. Thus, Erk1 and Erk2 have redundant,
kinase-dependent functions in hematopoietic progenitors.
Pharmacologic blockade of MEK attenuates KRasG12D-evoked
myeloproliferative disease in mice.17 However, the specic role(s)
of Erk1 and Erk2 in KRasG12D-transformed hematopoietic cells
remained be to be addressed. Notably, Erk2 has allele-specic effects
on Ras-induced epithelial-to-mesenchymal transformation,18 raising
the possibility that a single ERK isoform might be important for
KRAS-evoked transformation of hematopoietic cells. To test this
possibility, we infected lin2 BM cells from Erk12/2Erk2ox/oxLSLKRasG12D mice with the retroviral vectors described in the previous
paragraph. In infected cells, Cre expression promotes simultaneous
deletion of Erk2 and expression of KRasG12D under the control of its
endogenous promoter. GFP1 cells were puried by FACS and plated
in media containing no cytokines to assess KRas G12D -evoked
cytokine-independent proliferation. Comparable cell proliferation
was observed in cells coexpressing either wild-type Erk1 or Erk2
(Figure 2E). By contrast, cells expressing catalytically defective ERK1
or ERK2 showed a complete loss of KRasG12D-evoked cytokineindependent proliferation. Thus, myeloid cell transformation by
oncogenic KRAS requires catalytically active ERK1 or ERK2.

Figure 2 (continued) CD45.11 cells and intrafemorally injected into lethally irradiated CD45.11 recipients (5 mice per group). After 8 weeks of engraftment, the percentage of
donor-derived peripheral blood cells (Total), CD31 (T cells), B2201 (B cells), and Gr11 (myeloid cells) were determined flow cytometry. *P , .05, ANOVA. (D) Primary lin2 BM cells
from Erk12/2;Erk2 flox/flox mice were transduced with the indicated retroviruses, and FACS-purified GFP1 cells were cultured in Iscove modified Dulbecco medium containing IL-3,
IL-6, and SCF. MIG-Cre vectors coexpress the hygromycin resistance gene (hygro), Erk1, Erk2, Erk1KR, and Erk2KR. Relative cell proliferation in each experiment is normalized to
that of Erk2. Results are shown as mean 6 SEM of 4 independent experiments. *P , .05, ANOVA. (E) Lin2 BM cells from Erk12/2;Erk2 flox/flox;LSL-KrasG12D mice were transduced
with retroviruses as described in panel D, and GFP1 cells were cultured in IMDM medium containing no cytokines. Cells were harvested and proliferation was determined. Cell
proliferation in each experiment is normalized to that of Erk2 and results are shown as mean 6 SEM of 4 independent experiments. *P , .05, ANOVA.

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3598

BLOOD, 2 MAY 2013 x VOLUME 121, NUMBER 18

CHAN et al

In summary, we found that deletion of both Erk genes results in

the rapid loss of HSCs and progenitors, leading to leukopenia,
anemia, and early lethality in mice. Similar data were reported
recently by Staser et al.19 Together, these results are consistent with
previous ndings reported by us and others that hematopoieticspecic loss of Ptpn11 (encoding Src homology 2 domain containing
phosphatase 2 [SHP2]), a positive regulator of RAS/ERK signaling,
also leads to attrition of HSCs and progenitors.14,20

Acknowledgments
The authors thank Tharsan Velauthapillai for expert assistance with
ow cytometry.
This work was supported by National Institutes of Health grants
RO1 CA114945 and R37CA49132 (B.G.N.) and was also funded

in part by the Ontario Ministry of Health and Long Term Care

(OMOHLTC) and the Princess Margaret Hospital Foundation.
B.G.N. is a Canada Research Chair, Tier 1.

Authorship
Contribution: G.C. designed research, performed experiments,
analyzed data, and wrote the paper; S.G. performed experiments
and analyzed data; and B.G.N. designed research, analyzed data,
and wrote the paper.
Conict-of-interest disclosure: The authors declare no competing nancial interests.
Correspondence: Gordon Chan, Ontario Cancer Institute, 101
College St, TMDT Room 8-301, Toronto, ON M5G 1L7, Canada;
e-mail: gordon.chan@uhnresearch.ca

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2013 121: 3594-3598

doi:10.1182/blood-2012-12-476200 originally published
online February 26, 2013

Erk1 and Erk2 are required for maintenance of hematopoietic stem cells
and adult hematopoiesis
Gordon Chan, Shengqing Gu and Benjamin G. Neel

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