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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1567-1578
By
From the Institute of Molecular and Cellular Biosciences, The
University of Tokyo; the First Department of Internal Medicine, Tokyo
Medical and Dental University; and Institute of Life Science, Kurume
University, Kurume, Japan.
Erythropoietin (EPO) and its cell surface receptor (EPOR) play a
central role in proliferation, differentiation, and survival of
erythroid progenitors. Signals induced by EPO have been studied extensively by using erythroid as well as nonerythroid cell lines, and
various controversial results have been reported as to the role of
signaling molecules in erythroid differentiation. Here we describe a
novel approach to analyze the EPO signaling by using primary mouse
fetal liver hematopoietic cells to avoid possible artifacts due to
established cell lines. Our strategy is based on high-titer retrovirus
vectors with a bicistronic expression system consisting of an internal
ribosome entry site (IRES) and green fluorescent protein (GFP). By
placing the cDNA for a signaling molecule in front of IRES-GFP,
virus-infected cells can be viably sorted by fluorescence-activated
cell sorter, and the effect of expression of the signaling molecule can
be assessed. By using this system, expression of cell-survival genes
such as Bcl-2 and Bcl-XL was found to enhance erythroid colony
formation from colony-forming unit-erythroid (CFU-E) in
response to EPO. However, their expression was not sufficient for
erythroid colony formation from CFU-E alone, indicating that EPO
induces signals for erythroid differentiation. To examine the role of
EPOR tyrosine residues in erythroid differentiation, we introduced a
chimeric EGFR-EPOR receptor, which has the extracellular domain of the
EGF receptor and the intracellular domain of the EPOR, as well as a
mutant EGFR-EPOR in which all the cytoplasmic tyrosine residues are
replaced with phenylalanine, and found that tyrosine residues of EPOR
are essential for erythroid colony formation from CFU-E. We further
analyzed the function of the downstream signaling molecules by
expressing modified signaling molecules and found that both JAK2/STAT5
and Ras, two major signaling pathways activated by EPOR, are involved
in full erythroid differentiation.
TWO TYPES OF erythropoiesis are known.
Primitive erythropoiesis occurs first in the yolk sac blood islands,
and definitive erythropoiesis begins at the aorta-gonad-mesonephros
(AGM) region and shifts to the fetal liver, spleen, and bone marrow.
Primitive erythropoiesis does not require erythropoietin (EPO), but
definitive erythropoiesis depends on EPO.1 Mature
definitive erythrocytes are generated from stem cells through several
steps of differentiation, and progenitors of a distinct stage have been
defined by their ability to make colonies in methylcellulose, ie,
burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid
(CFU-E), and mature erythrocyte. The progenitors proliferate and
differentiate in vitro into erythrocytes in the presence of growth
factors. BFU-E cells are the earliest progenitor of erythropoiesis and give rise to a large colony (burst) of greater than 500 erythrocytes by
7 to 10 days in methylcellulose culture. BFU-E is stimulated by
interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor
(GM-CSF), and stem cell factor (SCF), which are not specific to
erythropoiesis. As they differentiate, the BFU-E cells become sensitive
to EPO. CFU-E cells represent a late stage of erythroid progenitor,
which corresponds to proerythroblasts, and are sensitive to EPO in
vitro and in vivo. CFU-E gives rise to an erythrocyte colony of 8 to 64 cells in 2 days.2,3
EPO is a lineage-restricted cytokine required for survival,
proliferation, and differentiation of committed erythroid progenitor cells.4 EPO exerts its function through the EPO receptor
(EPOR), a member of the class I cytokine receptor family. Both EPO and EPOR are essential for the production of red blood cells, and knock out
of either EPO or EPOR genes results in embryonic lethality at around
13.5 days postcoitum (dpc) due to the lack of definitive erythropoiesis. Interestingly, even in the absence of EPO or EPOR, almost normal numbers of BFU-E are generated, indicating that EPO and
EPOR are not required for the commitment of hematopoietic stem cells to
the erythroid lineage but are required for the terminal differentiation
of these committed progenitors.5-7 Therefore, downstream
signaling pathways from EPOR are important for the terminal
differentiation. Janus kinases (JAKs) play an important role in signal
transduction via cytokine and growth factor receptors. JAK2-deficient
embryos are anemic and die at around 12.5 dpc. Although primitive
erythrocytes are found normally, definitive erythropoiesis is absent in
the mutant mice. Compared with EPOR-deficient mice, phenotype of JAK2
deficiency is more severe. BFU-E and CFU-E colonies from fetal liver
cells of JAK2-deficient embryos are completely absent.8,9
It has been a central question in hematopoiesis whether the commitment
for differentiation is instructive or stochastic, ie, whether
extracellular signals instruct the cells to commit to a particular cell
lineage or the commitment is a stochastic process and cytokines simply
serve as growth/survival factors for the committed cells that express
the cognate receptor. Although it is still a question whether or not
novel molecules yet to be identified initiate the commitment of
hematopoietic stem cells to differentiation, it is clear that EPO plays
a major role in the late stages of erythroid differentiation as
described above. A number of different experiments were performed to
address the signaling mechanism for cell survival and differentiation.
However, the results were rather controversial. To test the role of
cytokine on the commitment, Fairbairn et al10
constitutively expressed Bcl-2 in an IL-3-dependent multipotential
hematopoietic cell line, FDCP-Mix. Bcl-2 not only suppressed apoptosis,
but it also induced multilineage hematopoietic differentiation in the
absence of IL-3,10 indicating that the cells undergo
multilineage differentiation in the absence of any cytokines if a
cell-survival signal is provided. In contrast, targeted expression of
Bcl-2 in the erythroid lineage in transgenic mice did not induce
erythroid differentiation in the absence of EPO.11
Previously, we and others have analyzed the signaling pathways from the
EPOR that lead to erythroid differentiation of EPO-responsive cell
lines and found that STAT5 is involved in the EPO-dependent erythroid
differentiation12-14; however, Chretien et al15
and Pless et al16 described that STAT5 is not required for
the erythroid differentiation. One reason for such a major difference
could be the cell lines that were used by different investigators. As the nature of cell lines can be variable, it would be ideal to use a
more physiological experimental system to address the role of signaling
molecules in erythroid differentiation. By using high-titer retrovirus
vectors, it is possible to introduce genes of interest to the
progenitors in the primary culture. In fact, retrovirus-mediated gene
transfer of EPOR into EPOR-deficient fetal liver cells restored EPO
responsiveness.6
Here we describe a role of signaling molecules activated by the EPOR in
the erythroid differentiation of fetal liver cells. We have used a
bicistronic expression system in which the gene to be expressed is
placed upstream of the internal ribosome entry site (IRES) and the
green fluorescent protein (GFP) gene. Virus-infected cells are
collected by fluorescence-activated cell sorter (FACS) as a
GFP-positive cell population and analyzed their potential for erythroid
differentiation. We show that both JAK2-STAT5 and Ras pathways
contribute to the erythroid differentiation from CFU-E.
Cells.
The retrovirus packaging cell line BOSC23 for the production of
ecotropic retrovirus was maintained in Dulbecco's
modified Eagle's medium (DMEM) containing the GPT selection reagent as described previously.17 Ba/F3 cells stably expressing the
Plasmids.
pBabe plasmids containing the EGFR-EPOR wild-type or Null mutant were
constructed by inserting the EGFR-EPOR fragments into the pBabeXc
vector. pMX EGFR-JAK2 was constructed by inserting the EGFR-JAK2
fragment19 into the pMX vector. The pMX/IRES-GFP vector
(pMIG) was constructed by inserting polioma virus IRES and EGFP
(Clontech, Palo Alto, CA) into pMX vector (M. Takeuchi and
A.Miyajima, unpublished observation, April 1997). pMIG containing Bcl-2, Bcl-XL, Production of retrovirus stock.
BOSC23 cells were seeded onto 100-mm dishes 1 day before transfection.
Transfection was performed using the LipofectAmine Plus reagent
(GIBCO-BRL, Rockville, MD) according to the manufacturer's protocol.
Cells were cultured for 48 hours, and the supernatant was used for
infection of target cells.
Infection of recombinant retrovirus.
For the infection of Ba/F3 cells, 2 × 105 cells were
incubated with 1 mL of the retrovirus stock for 6 hours in the presence of 10 µg/mL Polybrene (Sigma Chemical Co, St Louis, MO) and IL-3. Then, 2 mL of fresh DMEM/10% fetal calf serum (FCS) containing 10 µg/mL Polybrene and IL-3 was added to the culture and incubation was
continued overnight. Medium was changed to RPMI/10% FCS containing 10 ng/mL IL-3 on the next day. For the infection of fetal liver cells, 2 × 106 cells were incubated with 10 mL of retrovirus
stock solution for 24 hours in the presence of 4 µg/mL Polybrene, 10 ng/mL IL-3, 100 ng/mL IL-6 (provided by Ajinomoto, Tokyo,
Japan), and 100 ng/mL SCF (provided by Kirin Brewery,
Tokyo, Japan). Then the medium was changed to RPMI/15% FCS containing
IL-3, IL-6, and SCF. After a 24-hour incubation, the cells were
collected and analyzed for receptor expression or GFP fluorescence. The
positive cells (30% to 50%) were sorted by FACS Vantage
(Becton Dickinson).
CFU-E assay.
One milliliter of the culture mixture containing 2 × 104 cells GFP-positive cells, Gene transfer into primary fetal liver cells by retrovirus.
Previously, we used SKT6 cells established from the spleen cells of the
spleen focus-forming virus-infected mouse and showed that the tyrosine
residues in the cytoplasmic domain of the EPOR are essential for the
EPO-dependent erythroid differentiation and that activation of STAT5 is
involved in the differentiation.14 To analyze EPOR
signaling in erythroid terminal differentiation in a more physiological
system, we expressed various signaling molecules in a primary culture
of the fetal liver cells by retrovirus. Retrovirus with cDNA encoding a
signaling molecule is prepared by packaging the retrovirus construct in
BOSC23 cells and is infected to the fetal liver cells isolated from
embryos at 13.5 dpc. IL-3, IL-6, and SCF are included in the culture
during infection to support immature hematopoietic progenitors.
Selection of the retrovirus-infected cell population can be achieved by
FACS using the antireceptor antibody in the case of receptor gene
transduction. To select the cells infected with the retrovirus encoding
intracellular signaling molecules, in which no such antibodies are
available for selection, we have used a bicistronic expression system
(Fig 1). By placing an IRES and a selection
marker gene (GFP) downstream of the gene to be expressed, both genes
are cotranscribed as a single mRNA. GFP, which is downstream of the
gene to be expressed, is translated by reentry of ribosomes at the
IRES. Thus, GF-positive cells are expected to express virus-encoded
genes as well. Our vector encodes the GFP gene derived from the jelly
Aequorea vibtoria.20 GFP emits bright green light
when it is exposed to ultraviolet or blue light without additional
proteins, substrates, or cofactors, and GFP expression can be monitored
in living cells. After sorting the virus-infected cells, the cells are
plated in the presence or absence of a cytokine, and erythroid colony
formation from CFU-E is evaluated.
Suppression of apoptosis is not the only function of EPOR leading to
erythroid colony formation.
EPO maintains cell survival of erythroid progenitors by inducing
expression of cell-survival genes including Bcl-2 and
Bcl-XL.21 If EPO simply serves as a growth/survival factor
for the committed cells that express EPOR, a survival signal may be
sufficient for the induction of terminal erythroid differentiation. To
test this possibility, we expressed anti-apoptotic genes, Bcl-2 and
Bcl-XL, in fetal liver cells by the bicistronic expression system
described above and examined the effect on erythroid colony formation
from CFU-E. First, the activity of Bcl-2 or Bcl-XL on cell survival was
confirmed in Ba/F3 cells. Ba/F3 cells were infected with retrovirus containing either Bcl-2 or Bcl-XL cDNA, and the virus-infected GFP-positive cells were sorted by FACS. The GFP-positive cells were
cultured in the absence of any cytokine, and the cell viability was
monitored by the Trypan blue exclusion assay. Ba/F3 cells expressing
Bcl-2 or Bcl-XL survived longer than the control cells in the absence
of IL-3 (Fig 2A).
Tyrosine residues of EPOR are required for erythroid colony formation
from CFU-E.
We then analyzed EPOR signaling leading to erythroid differentiation
using mutant EPOR in fetal liver cells. To distinguish the exogenous
EPOR from the endogenous EPOR, we used the chimeric EGFR-EPOR in which
the extracellular domain of the EGF receptor (EGFR) was fused to the
intracellular domain of the EPOR.14 The fetal liver cells
infected with the retrovirus encoding the chimeric receptor were
examined for EGFR expression using anti-EGFR antibody, and
virus-infected cells (P fraction in Fig 3A)
were sorted by FACS. There was no difference in fluorescein
isothiocyanate (FITC) fluorescence between the wild-type chimeric
receptor-expressing cells and the Null chimeric receptor-expressing
cells (Fig 3B), suggesting that there is no significant difference in
receptor numbers between these cell populations. The collected cells
were cultured in the presence of EPO or EGF, and the number of CFU-E colonies was counted. As shown in Fig 3C, EGF induced CFU-E as efficiently as EPO, indicating that the chimeric receptor is capable of
inducing erythroid colony formation from CFU-E in fetal liver cells. In
contrast, the chimeric receptor EGFR-EPOR Null, in which all tyrosine
residues within the cytoplasmic domain of the EPOR are substituted with
phenylalanine, failed to form an erythroid colony in response to EGF
(Fig 3C). The results clearly indicate that the tyrosine residues of
the EPOR are essential for EPO-induced erythroid differentiation.
Essential role of JAK2 in the formation of erythroid colonies from
CFU-E.
It was previously shown that JAK2 is essential for EPOR-mediated cell
proliferation.22 We asked whether the activation of JAK2 is
also essential for the erythroid colony formation from CFU-E. By using
the bicistronic expression system, we expressed a dominant negative
JAK2,
Role of Ras in erythroid colony formation from CFU-E.
The Ras-Raf-MAPK pathway is activated by binding of EPO to the EPOR and
is known to be involved in cytokine-mediated cell proliferation,
differentiation, and cell survival depending on the cell
type.27 We examined the role of Ras in the erythroid colony
formation from CFU-E by expressing an active form of Ras (RasV12) or a
dominant negative form of Ras (RasN17) using our bicistronic expression
system. Fetal liver cells were infected with the retrovirus encoding
either RasV12 or RasN17, or with the empty virus vector, and the
virus-infected GFP-positive cells (P fraction in
Fig 5A) were isolated by FACS. The sorted
cells were cultured in the presence or absence of EPO, and the number of CFU-E colonies was counted. As shown in Fig 5B, both the active form
and the dominant negative form of Ras inhibited erythroid colony
formation from CFU-E. These results will be discussed below.
Role of STAT5 in erythroid colony formation from CFU-E.
Our previous results obtained with EPO-responsive SKT6 cells suggest
that STAT5 plays an important role in erythroid
differentiation.14 To evaluate the role of STAT5 in
erythroid colony formation from CFU-E, we expressed a dominant negative
form of STAT5 in the fetal liver cells and examined its effect on the
erythroid colony formation from CFU-E.
Analysis of signaling pathways is generally conducted by using cell
lines, and numerous signaling molecules have been identified. However,
studies using cell lines often provide controversial results as to the
physiological role of each signaling molecule. Such controversial
results are most likely reflecting the difference of cell lines used
for each experiment. In principle, because cell lines are generated as
a result of alteration of the cellular program of proliferation
and/or differentiation, each cell line may have acquired an
abnormal characteristic during a process of establishing the cell line.
Moreover, phenotype of a cell line can be variable during the culture,
and descendants of the same original cell sometimes exhibit quite
different phenotype. Erythroid progenitor cell lines are not the
exception, and each cell line might have stopped the differentiation at
a specific step. This may be a major reason for the discrepancy among
various investigators as to the role of cytokines and their signaling
molecules in erythroid differentiation. To overcome these potential
problems intrinsic to cell lines, it is ideal to use an in vivo
experimental system to address the physiological role of signaling
molecules. In this study, we have used primary fetal liver cells and a
high-efficiency gene transfer system using retrovirus. As described
above, a significant fraction of the fetal liver cells can be infected
with the recombinant retrovirus, and the effect of expression of the
virus-encoded gene can be assessed by colony-forming assays. This type
of approach was already applied for dissecting EPOR signaling to
erythroid colony formation from CFU-E,31,32 though the
analysis was limited to EPOR mutants. By using the IRES-GFP bicistronic
expression system, we have extended this type of approach to the
functional analysis of signaling molecules, which lacks any selection
reagents such as specific antibody. 33,34 Although in this
study we have analyzed only the erythroid colony-formation from CFU-E, it should be also applicable for evaluation of the role of such signaling molecules in CFU-C, CFU-S, or even long-term reconstitution of the entire hematopoietic cells. We have successfully detected a
GFP-positive virus-infected cell population in spleens of lethally irradiated mice after reconstituting with fetal liver cells infected with retrovirus encoding GFP (D.C. and A.M., unpublished
results, April 1997). Because a CFU-E colony is formed as a result of
the proliferation and differentiation of a CFU-E, and because we could not detect difference in CFU-E colony size, our current results are
unable to discriminate the possibilities of whether the signaling molecules expressed by retrovirus affect proliferation or
differentiation, or both.
We thank T. Sekiguchi for cell sorting; Dr H. Wakao for Ba/F3 cells
expressing Submitted August 10, 1998; accepted October 23, 1998.
Supported in part by the grants from the Ministry of Education,
Culture, Sports, and Science (Monbushou); Core Research for Evolutional
Science and Technology (CREST) of Japan Science and Technology
Corporation, Riken; the Toray Research Foundation; and Uehara Research
Foundation. D.C. is supported by Fellowships in Cancer Research of
Japan Society for the Promotion of Science for Young Scientists.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Atsushi Miyajima, PhD, Institute of
Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; e-mail:
miyajima{at}ims.u-tokyo.ac.jp.
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Top
Abstract
Introduction
-casein 344 promoter Luciferase construct were established by
transfecting the 
JAK2, JAB, RasV12, RasN17, STAT5b, 
-minimum essential medium
(MEM) (GIBCO BRL), 1.2% (1,500 centipoieses)
methylcellulose (Nacalai Tesque, Kyoto, Japan), 1%
bovine serum albumin (BSA) fraction V (Sigma), 30% FCS (Hyclone,
Logan, UT), 10
4 mol/L mercaptoethanol (Sigma), and 2 U/mL human EPO (provided by Kirin Brewery) and 10 ng/mL IL-3 were
incubated at 37°C in a humidified atmosphere with 5%
CO2 in air. The optimal concentration of EPO for CFU-E
formation was determined to be 2 U/mL, and we used EPO at 2 U/mL
throughout our studies. CFU-E colonies were counted after 2 days of
incubation using an inverted microscope. About 40 CFU-E
colonies were observed from 2 × 104
virus-vector-infected GFP-positive cells.
JAK2 or JAB or the virus vector and GFP-positive cells were
collected by FACS. P shows GFP-positive cell populations used for the
CFU-E assays. (B) Sorted GFP-positive cells were subjected to in vitro
colony assays. The relative CFU-E colony numbers were calculated as in
Fig 
