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Plenary paper
Granulocyte colony-stimulating factor regulates myeloid differentiation through
CCAAT/enhancer-binding protein ⑀
Hideaki Nakajima and James N. Ihle
Granulocyte colony-stimulating factor
(G-CSF) is a major cytokine that regulates
proliferation and differentiation of myeloid cells, although the underlying
mechanisms by which G-CSF controls
myeloid differentiation are largely unknown. Differentiation of hematopoietic
cells is regulated by lineage-specific transcription factors, and gene-targeting
studies previously revealed the critical
roles of CCAAT/enhancer-binding protein
(C/EBP) ␣ and C/EBP⑀, respectively, in
the early and mid-late stages of granulocyte differentiation. The expression of
C/EBP⑀ in 32Dcl3 cells and FDCP1 cells
expressing mutant G-CSF receptors was
examined and it was found that G-CSF
up-regulates C/EBP⑀. The signal for this
expression required the region containing the first tyrosine residue of G-CSF
receptor. Dominant-negative signal transducers and activators of transcription 3
blocked G-CSF–induced granulocytic differentiation in 32D cells but did not block
induction of C/EBP⑀, indicating that these
proteins work in different pathways. It
was also found that overexpression of
C/EBP⑀ greatly facilitated granulocytic differentiation by G-CSF and, surprisingly,
that expression of C/EBP⑀ alone was sufficient to make cells differentiate into
morphologically and functionally mature
granulocytes. Overexpression of c-myc
inhibits differentiation of hematopoietic
cells, but the molecular mechanisms of
this inhibition are not fully understood. In
32Dcl3 cells overexpressing c-myc that
do not differentiate by means of G-CSF,
induction of C/EBP⑀ is completely abrogated. Ectopic expression of C/EBP⑀ in
these cells induced features of differentiation, including changes in nuclear morphologic characteristics and the appearance of granules. These data show that
C/EBP⑀ constitutes a rate-limiting step in
G-CSF–regulated granulocyte differentiation and that c-myc antagonizes G-CSF–
induced myeloid differentiation, at least
partly by suppressing induction of
C/EBP⑀. (Blood. 2001;98:897-905)
© 2001 by The American Society of Hematology
Introduction
Development of hematopoietic cells is controlled by lineagespecific transcription factors.1 In the myeloid lineage, a family of
CCAAT/enhancer-binding proteins (C/EBPs) plays a key role in
differentiation. C/EBP␣ is expressed in early myeloid progenitors,
and its expression decreases as these progenitors differentiate into
mature granulocytes.2 In turn, C/EBP⑀ is up-regulated at the
promyelocyte or myelocyte stage and expressed thereafter.3,4
C/EBP␤ and C/EBP␦ are expressed basically in all stages of
myeloid differentiation, although their expression is somewhat
higher in mature myeloid cells.2
Corresponding to a broad pattern of expression, mice deficient
in C/EBP␣ have multiple abnormalities, including neonatal death
due to impaired glycogenesis and lung abnormalities.5,6 During
hematopoiesis, these mice have complete differentiation arrest at
the myeloid progenitor stage, resulting in an absence of mature
neutrophils and eosinophils in peripheral blood.7 The critical role
of C/EBP␤ in the immune response was revealed in studies in
C/EBP␤-deficient mice, which have macrophage dysfunction,
impaired bacterial killing, and lymphoproliferative disorders.8
C/EBP⑀ knockout mice have impaired granulopoiesis and myelodysplasia.9 Although these mice produce mature granulocytes, they
are morphologically and functionally abnormal, with hyposegmented nuclei and an impaired oxidative burst. In addition, bone
marrow hyperplasia resembling myelodysplasia becomes apparent
after 3 months. Importantly, these knockout mice have no developmental defects in other hematopoietic lineages, including the
erythrocyte, megakaryocyte, and lymphocyte. Thus, C/EBP␣ and
C/EBP⑀ are critical, nonredundant regulators in the early and
late stages of myelopoiesis, respectively, and their sequential,
coordinated expression orchestrates the complex gene regulation involved in producing functional mature neutrophils and
eosinophils.
Granulocyte colony-stimulating factor (G-CSF) plays a pivotal
role in myeloid development. Mice deficient in either G-CSF or its
receptor genes have a reduced neutrophil count (⬃ 20% of
normal).10,11 Despite the fundamental role of G-CSF in granulopoiesis, there is controversy regarding whether G-CSF acts as an
active differentiation inducer or just a survival factor for committed
granulocyte precursors. In the latter model, the differentiation
pathways of each precursor are internally predetermined in a
stochastic fashion and cytokines simply prevent them from undergoing apoptosis by means of their cognate pre-expressed receptor
(stochastic model). This model hypothesis is supported by the
observation that a hematopoietic progenitor cell line overexpressing bcl-2 has spontaneous differentiation to multiple lineages
without adding any cytokines.12 On the other hand, many studies
From the Howard Hughes Medical Institute, Department of Biochemistry, St
Jude Children’s Research Hospital, Memphis, TN; and the University of
Tennessee Medical School, Memphis, TN.
Reprints: Hideaki Nakajima, Blood Center, Keio University School of
Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; e-mail:
[email protected].
Submitted January 2, 2001; accepted April 16, 2001.
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 U.S.C. section 1734.
Supported by Cancer Center CORE grant CA21765, grant RO1 DK42932 to
J.N.I., grant PO1 HL53749, and the American Lebanese Syrian Associated
Charities.
BLOOD, 15 AUGUST 2001 䡠 VOLUME 98, NUMBER 4
© 2001 by The American Society of Hematology
897
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898
NAKAJIMA and IHLE
showing that cytokines such as erythropoietin (Epo) and thrombopoietin induce differentiation by inducing specific factors, such as
signal transducers and activators of transcription (STAT) or cyclindependent kinase (CDK) inhibitors,13,14 support the former model,
which grants cytokines an active role in cellular differentiation. Yet
none of those studies linked the signals from cytokine receptor to
activation of transcriptional regulators that is crucial for differentiation to a specific lineage, and they therefore failed to identify the
precise role of cytokines in hematopoietic differentiation.
The differentiation process is often accompanied by cell-cycle
arrest and down-regulation of c-myc, a critical component of
cellular proliferation15,16 that drives transition from G1 to S by
activating its target genes, such as ornithine decarboxylase and
Cdc25B.17,18 In general, differentiation and proliferation are mutually exclusive processes and enforced expression of c-myc inhibits
differentiation by both preventing irreversible withdrawal from the
cell cycle and inhibiting commitment that leads to a terminally
differentiated state.19-21 However, the precise mechanism by which
c-myc prevents cells from committing to a specific differentiation
pathway is unclear. One possibility is that c-myc suppresses
expression of specific genes, such as transcription factors that
critically regulate differentiation to specific lineage. For example,
in preadipocytes, c-myc suppresses differentiation to mature adipocytes by inhibiting transcription of C/EBP␣, a key regulator of
adipogenesis, and overexpression of C/EBP␣ overrides this suppression.22 Likewise, in myeloid cells, ectopic expression of c-myc in
32Dcl3 cells suppresses granulocytic differentiation by G-CSF,
although the mechanism by which this occurs is unknown.
In this study, we found that G-CSF induces C/EBP⑀ in 32Dcl3
cells and that this induction strongly correlates with granulocytic
differentiation. The signal for this induction depends on the first
tyrosine (Tyr) residue of G-CSF receptor but does not depend on
STAT3. We also found that expression of C/EBP⑀ alone is sufficient
for morphologic and functional differentiation in 32Dcl3 cells
and that ectopic expression of C/EBP⑀ overcomes the differentiation block in 32D/myc cells. Our data show the essential,
rate-limiting role of C/EBP⑀ in G-CSF–induced myeloid differentiation, and support the concept that the balance of c-myc and
C/EBP⑀ determines whether cells proliferate or differentiate into
granulocytes.
Materials and methods
Cell lines
The study used 32Dcl3 cells and FDCP1 cells expressing mutant G-CSF
receptor. Cells were cultured in RPMI1640 supplemented with 10%
fetal-bovine serum (FBS; Gibco BRL, Rockville, MD), 100 U/mL penicillin G, 100 ␮g/mL streptomycin, 2 mM L-glutamine, and 25 U/mL
recombinant murine interleukin (IL)–3 at 37°C in a humidified atmosphere
with 5% carbon dioxide. FDCP1 cells expressing various G-CSF receptor
truncation mutants were described previously.23
Plasmids and establishment of 32D transfectants
The expression vector pcEpsilon32 containing human C/EBP⑀ under the
control of cytomegalovirus promoter was a generous gift from Dr K. G.
Xanthopoulos (National Institutes of Health, Bethesda, MD). Dominantnegative STAT3 was created by polymerase chain reaction using Pfu
polymerase (Stratagene, La Jolla, CA) and subcloned into EcoRI and
BamHI sites of pcDNA3 (Invitrogen, Carlsbad, CA). The integrity of
amplified sequences was confirmed by DNA sequencing. To obtain 32D
cells stably expressing C/EBP⑀ or dominant-negative STAT3, 1 ⫻ 107
32Dcl3 cells were electroporated with 20 ␮g expression vector and selected
BLOOD, 15 AUGUST 2001 䡠 VOLUME 98, NUMBER 4
in the presence of G418 (750 ␮g/mL) for 14 days. The clones were obtained
by limiting dilution, and expression of C/EBP⑀ was examined using
Western blot analysis (see below). Three independent clones that showed
high levels of expression were analyzed further. All clones had essentially
the same phenotype, and representative data from each experiment are
shown here. For transfection into 32D/myc cells, a pcZeo-CEBP⑀ expression vector was constructed by subcloning full-length complementary DNA
(cDNA) of C/EBP⑀ excised from pcEpsilon32 by HindIII and BamHI into
the same sites of pcDNA3-Zeo vector (Invitrogen). Transfection was done
as described above, and selection with 750 ␮g/mL zeomycin was started
after 48 hours of transfection. The morphologic characteristics of transfected cells were examined after 6 days of transfection.
Northern blot analysis
Total RNA was extracted from 1 ⫻ 107 cells by using RNAzol-B according
to the manufacturer’s protocol (Tel-test, Friendswood, TX). The RNA
samples (20 ␮g/lane) were separated on 1.0% formaldehyde-denaturing
agarose gel and transferred to Hybond N⫹ membrane (Amersham,
Piscataway, NJ). Full-length cDNA of C/EBP⑀, myeloperoxidase (MPO),
c-myc, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were
used as probes. All probes were labeled using a Rediprime kit (Amersham).
Hybridizations with phosphorus 32–labeled probes were done in ExpressHyb buffer (Clontech, Palo Alto, CA) according to the manufacturer’s
protocol. The membranes were washed in washing buffer of 2 ⫻ standard
saline citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) for 30 minutes
at room temperature, with several buffer changes, and this was followed by
2 washes in 0.1 ⫻ SSC and 0.1% SDS for 15 minutes at 42°C. The
membranes were exposed on XAR film (Kodak, Rochester, NY) at ⫺80°C
for 1 to 5 days.
Immunoprecipitation and Western blotting
Cells (1 ⫻ 107) were lysed in extraction buffer (10 mM Tris-hydrochloric
acid (HCl), 50 mM sodium chloride (NaCl), 5 mM EDTA, 50 mM sodium
fluoride, 30 mM sodium pyrophosphate, 100 ␮M sodium orthovanadate,
1% Triton-X 100, and 1 mM phenylmethylsulfonyl fluoride). Lysates were
centrifuged at 12 000g for 15 minutes at 4°C to remove debris, and protein
concentrations were measured using the bicinchoninic acid method (Pierce,
Rockford, IL). For immunoprecipitation, lysates were incubated with
primary antibodies and protein A–Sepharose beads for several hours at 4°C.
The beads were washed extensively with lysis buffer, and recovered
proteins were eluted with sample buffer (50 mM Tris [pH 6.8], 2% SDS,
10% glycerol, 1 mM dithiothreitol, and 0.1% bromophenol blue). The
immune complexes or 50 ␮g of each cell extract were/was resolved on 4%
to 20% SDS-polyacrylamide gel and transferred to Hybond ECL (Amersham). The membrane was blocked in 5% nonfat milk in TBS-T (20 mM
Tris-HCl, 137 mM NaCl, and 0.1% Tween 20) hybridized sequentially with
primary antibodies and horseradish peroxidase–conjugated anti-immunoglobulin secondary antibody (Amersham). Bound antibodies were detected
by an enhanced-chemiluminescence Western blotting kit (Amersham).
Rabbit anti-CRP1 (C/EBP⑀) polyclonal antibody was purchased from Santa
Cruz Biotechnology (Santa Cruz, CA).
Morphologic analyses, immunohistochemical studies, and
nitroblue tetrazolium reduction assays
Samples of 32Dcl3 and 32D/⑀ cells treated with IL-3 or G-CSF
were prepared on glass slides using the cytospin method. Morphologic features were evaluated with use of Wright-Giemsa staining.
Immunohistochemical staining of MPO was done with an MPO
detection kit (Sigma, St Louis, MO). For NBT assays, 1 ⫻ 106 cells
were centrifuged, suspended in 500 ␮L phosphate-buffered saline
(PBS), and incubated at 37°C for 30 minutes with 1 mg/mL NBT
and 30 ng/mL 12-O-tetradecanoylphorbol-13-acetate. Cytospin
preparations were made and stained with Wright staining. The
percentage of cells with formazan deposits in the cytoplasm was
determined by microscopical examination.
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BLOOD, 15 AUGUST 2001 䡠 VOLUME 98, NUMBER 4
G-CSF REGULATES C/EBP⑀
899
Flow cytometry
Cells were resuspended in PBS containing 1% FBS and 0.1% sodium azide.
Nonspecific antibody binding to surface Fc receptors was blocked by
incubating cells with Fc Block (Pharmingen, San Diego, CA) for 15
minutes at 4°C. Cells were then stained with phycoerythrin-conjugated
anti–Mac-1␣ antibody (Pharmingen) for 30 minutes at 4°C. Analysis was
done with a FACS Calibur flow cytometer using CellQuest software
(Becton Dickinson, Franklin Lakes, NJ).
Results
G-CSF induces C/EBP⑀ in 32Dcl3 cells, and its expression
highly correlates with myeloid differentiation
G-CSF induces granulocytic differentiation in 32Dcl3 cells (murine myeloid cell line). Because G-CSF is a key cytokine in
myeloid differentiation, we wondered whether G-CSF induces
C/EBP⑀ in 32Dcl3 cells. As illustrated in Figure 1A, we found that
G-CSF clearly induced C/EBP⑀ expression in 32Dcl3 cells after
one day of stimulation. Expression reached the maximum level in 3
days and remained unchanged thereafter for up to 5 days. Upregulation of C/EBP⑀ protein was also demonstrated by Western
blot analysis (Figure 2A). This induction cannot have been due to
Figure 2. Protein expression and growth characteristics of 32Dcl3 cells
expressing C/EBP⑀. (A) Parental 32Dcl3 cells were washed and transferred from
medium containing IL-3 to G-CSF, and cell lysates were prepared at the times
indicated. Lysates of 32D/⑀ cells were obtained from cells growing in IL-3. Each
protein extract (50 ␮g) was resolved on 4% to 20% gradient SDS-polyacrylamide gel
and transferred to a nitrocellulose membrane. The membrane was probed with
polyclonal antibody against C/EBP⑀. (B) Parental 32Dcl3 cells (solid symbols) and
32D/⑀ cells (open symbols) maintained in IL-3 were washed with PBS and transferred
to medium containing 25 U/mL IL-3 (circle), 50 ng/mL G-CSF (square), or no
cytokines (triangle). Viable cells were counted using the trypan blue dye exclusion
method. Cells were diluted with each medium to keep cell density within 2 to
10 ⫻ 105/mL.
Figure 1. G-CSF regulates C/EBP⑀. (A) Induction of C/EBP⑀ by G-CSF. Total RNA
extracted from 32Dcl3 cells (20 ␮g) treated with 50 ng/mL recombinant human
G-CSF for the indicated times was subjected to Northern blot analysis. (B) Growth
curve for 32Dcl3 cells cultured in various cytokines. The 32Dcl3 cells maintained in
the presence of IL-3 were washed extensively with PBS and transferred to medium
containing 25 U/mL IL-3 (F), 50 ng/mL G-CSF (f), IL-3 and G-CSF (Œ), or medium
without cytokines (E). Viable cells were counted using the trypan blue dye exclusion
method at the times indicated. Cells were diluted with each medium to keep cell
density within 2 to 10 ⫻ 105/mL. (C) Expression of C/EBP⑀ and C/EBP␣ in 32Dcl3
cells treated with IL-3, G-CSF, or both. Cells growing in IL-3 were washed twice with
PBS and transferred to medium containing the indicated cytokines. Northern blot
analysis was done as described above. (D) Comparison of expression of C/EBP⑀ and
C/EBP␣ in 32Dcl3 cells and NFS60 cells. Cells were starved in RPMI 1640 with 10%
FBS lacking cytokines for 14 hours and then stimulated with 50 ng/mL G-CSF for the
times indicated. Each total RNA (20 ␮g) was then analyzed by Northern blot analysis.
withdrawal of IL-3 because such withdrawal itself did not induce
C/EBP⑀ expression (data not shown), and examination of other cell
lines expressing various truncation mutants of G-CSF receptor
showed that a signal from G-CSF receptor is required for induction
of C/EBP⑀ (see below). Taken together, these results indicate that
C/EBP⑀ is a downstream target of G-CSF signaling.
We next examined the correlation between C/EBP⑀ expression
and granulocytic differentiation. In medium containing IL-3,
32Dcl3 cells grow and maintain blast-like morphologic characteristics. They also grow in medium containing both IL-3 and G-CSF
(Figure 1B). If C/EBP⑀ is crucial in G-CSF–induced granulocytic
differentiation, C/EBP⑀ should not be expressed in cells stimulated
with both IL-3 and G-CSF. As expected, we found that induction of
C/EBP⑀ was completely suppressed by costimulation with IL-3 and
G-CSF (Figure 1C). Likewise, IL-3 alone did not induce C/EBP⑀.
We also examined another C/EBP family member, C/EBP␣, which
is expressed at high levels in 32D cells, regardless of added
cytokines. Expression of C/EBP␣ was unchanged after G-CSF
stimulation, whereas it was slightly suppressed by IL-3 alone or
IL-3 and G-CSF. This finding suggests that C/EBP⑀, rather than
C/EBP␣, is an important regulator in G-CSF–induced granulocytic
differentiation and that IL-3 may prevent differentiation by inhibiting the induction signal for C/EBP⑀ from G-CSF receptor.
We also examined NFS60 cells, which do not differentiate but
rather proliferate in response to G-CSF. As shown in Figure 1D,
G-CSF did not induce C/EBP⑀ in these cells. In contrast to our
findings with C/EBP⑀, C/EBP␣ was highly expressed in NFS60
cells, as it was in 32D cells. This observation confirms the
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BLOOD, 15 AUGUST 2001 䡠 VOLUME 98, NUMBER 4
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Table 2. Myeloperoxidase and nitroblue tetrazolium reducing activity in
32Dcl3 and 32D/⑀ cells
Cells
MPO (%)
NBT (%)
IL-3
0⫾0
10.3 ⫾ 2.1
G-CSF
0⫾0
50.3 ⫾ 2.5
32Dcl3
32D/⑀
IL-3
46.0 ⫾ 4.6
60.0 ⫾ 7.2
G-CSF
86.7 ⫾ 4.2
95.3 ⫾ 2.5
Values are the mean ⫾ SD percentages of cells positive for MPO or NBT from 3
independent experiments. Cells were treated with G-CSF for 3 days.
MPO indicates myeloperoxidase; NBT, nitroblue tetrazolium. See Table 1 for
other abbreviations.
Figure 3. Priming of 32D/⑀ cells for granulocyte differentiation. Shown are the
morphologic characteristics of parental 32Dcl3 cells and 32D/⑀ cells maintained in
IL-3, treated with 50 ng/mL G-CSF for the period indicated (d4 indicates 4 days; and
d7, 7 days), and cultured in medium without cytokines (no factor) for 4 days. The
morphologic features of the cells were visualized using Wright-Giemsa staining
(original magnification, ⫻400).
correlation between expression of C/EBP⑀ and G-CSF–regulated
differentiation.
C/EBP⑀ facilitates morphologic and functional
differentiation to granulocytes
To investigate the role of C/EBP⑀ in granulocytic differentiation,
we stably expressed C/EBP⑀ in 32Dcl3 cells. As shown in Figure
2A, these cells (32D/⑀) expressed C/EBP⑀ protein at a level
comparable to that in a parental cell line treated with G-CSF for 2
days. Interestingly, 32D/⑀ cells grew at a slower rate than parental
32Dcl3 cells in IL-3 (Figure 2B). In addition, they showed
spontaneous differentiation to granulocytes in IL-3, at a significantly higher rate than parental cells (Figure 3 and Table 1).
Surprisingly, when 32D/⑀ cells were transferred to medium containing G-CSF, they immediately started to differentiate to granulo-
cytes, and 4 days later, almost all cells looked like segmented
neutrophils (Table 1 and Figure 3). In contrast, parental 32Dcl3
cells still showed immature blast–like morphologic features after 4
days of treatment with G-CSF and they took 14 days to fully
differentiate (Table 1 and Figure 3). Moreover, even though both
these cell lines underwent apoptosis in a few days after removal of
IL-3, few surviving 32D/⑀ cells showed spontaneous differentiation to granulocytes, whereas parental 32Dcl3 cells maintained
their immature blast–like morphologic characteristics under the
same conditions (Figure 3).
We then examined whether the morphologic change observed in
32D/⑀ cells was accompanied by functional maturation. It was
previously reported that C/EBP⑀ knockout mice have a defective
oxidative burst, a finding that suggests that C/EBP⑀ is critical in the
generation of reactive oxygen. To test whether C/EBP⑀ is sufficient
to elicit an oxidative burst, we conducted an NBT reduction test in
32D/⑀ cells. The number of NBT-positive cells in medium containing either IL-3 or G-CSF was much higher among 32D/⑀ cells than
parental 32Dcl3 cells (Table 2). We also examined expression of
MPO as a differentiation marker. In parental 32D cells, neither IL-3
alone nor IL-3 and G-CSF induced MPO expression (Figure 4A).
In contrast, G-CSF strongly induced MPO in these cells in 2 days.
Surprisingly, we observed constitutive MPO expression in 32D/⑀
cells even in the presence of IL-3, and G-CSF treatment marginally
increased this expression (Figure 4B). We confirmed this observation by immunohistochemical staining of MPO in vivo (Figure 4C
and Table 2). In this setting, G-CSF significantly increased the
percentage of MPO-positive 32D/⑀ cells (Table 2). MPO is a
primary granule protein known to be induced by C/EBP␣, but this
observation suggests that C/EBP⑀ can also induce MPO. We did
not observe any MPO activity in G-CSF–treated parental 32D cells
on immunohistochemical examination, whereas Northern blotting
analysis found that G-CSF induced MPO messenger RNA (mRNA)
in 2 days. This discrepancy could be due to either delayed protein
synthesis of MPO or the different sensitivity of the assays.
Table 1. Differential counts of 32Dcl3 and 32D/⑀ cells treated with interleukin 3, granulocyte colony-stimulating factor, or both
IL-3 ⫹ G-CSF
G-CSF
Cells
IL-3
Day 4
Day 7
Day 10
Day 14
Day 4
Day 7
32Dcl3
Myeloblasts
96.6 ⫾ 1.5
87.0 ⫾ 3.6
3.7 ⫾ 4.0
3.3 ⫾ 1.5
0.3 ⫾ 0.6
92.0 ⫾ 2.6
93.7 ⫾ 3.1
Myelocytes
5.0 ⫾ 2.0
11.3 ⫾ 3.5
72.6 ⫾ 8.0
29.0 ⫾ 3.6
21.0 ⫾ 5.6
8.0 ⫾ 2.6
6.3 ⫾ 3.1
Band (seg)
0⫾0
2.3 ⫾ 1.2
23.7 ⫾ 11.1
67.7 ⫾ 4.6
78.3 ⫾ 5.5
0⫾0
0⫾0
32D/⑀
Myeloblasts
85.0 ⫾ 7.0
1.0 ⫾ 1.0
0⫾0
—
—
83.7 ⫾ 3.2
83.7 ⫾ 6.0
Myelocytes
11.0 ⫾ 5.3
25.7 ⫾ 4.0
21.0 ⫾ 3.6
—
—
9.3 ⫾ 2.1
11.3 ⫾ 3.2
Band (seg)
4.0 ⫾ 2.6
73.3 ⫾ 3.8
79.0 ⫾ 3.6
—
—
7.0 ⫾ 3.6
5.0 ⫾ 3.5
Values are the mean ⫾ SD percentages of cells from 3 independent experiments. Myelocytes includes promyelocytes, myelocytes, and metamyelocytes.
G-CSF, granulocyte colony-stimulating factor; IL-3, interleukin-3; band (seg), band and segmented neutrophils.
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Tyr 703 of G-CSF receptor is important for C/EBP⑀ induction
Tyr residues on the cytoplasmic domain of cytokine receptors are
phosphorylated by Janus kinase and serve as docking sites for
various signaling molecules that contain the Src homology 2
domain. Yoshikawa et al24 showed that the first Tyr residue (Tyr
703) of G-CSF receptor is important for a myeloid differentiation
signal that leads to morphologic change, induction of MPO, and
growth arrest. On the basis of this observation, we speculated that
Tyr 703 might be important for induction of C/EBP⑀ by G-CSF. We
addressed this question by using FDCP1 cells expressing various
truncation mutants of G-CSF receptor (Figure 5A).23 As shown in
Figure 5B, C/EBP⑀ was induced by G-CSF in FDCP1–G-CSF
receptor wild-type (WT) cells. Induction of C/EBP⑀ was reduced
but was significant in A and E mutant cells, both of which retain Tyr
703. However, T mutant cells, which lack all Tyr residues on the
receptor, showed no C/EBP⑀ induction. A densitometric analysis of
relative C/EBP⑀ mRNA expression compared with GAPDH is
shown in Figure 5C. Although the differences in C/EBP⑀ induction
among mutants were subtle, the data were highly reproducible.
This result suggests that the region surrounding Tyr 703 is
sufficient to generate the induction signal for C/EBP⑀ and that a
Figure 4. Constitutive MPO and enhanced Mac-1␣ expression in 32D/⑀ cells. (A)
MPO mRNA expression in 32Dcl3 cells. The blot shown in Figure 1C was reprobed
with MPO. (B,C) 32D/⑀ cells cultured in IL-3 were washed twice with PBS and then
stimulated with 50 ng/mL G-CSF. Cells were harvested at the times indicated and
subjected to Northern blot analysis (B). Expression of MPO was confirmed by
immunohistochemical staining of 32Dcl3 and 32D/⑀ cells cultured in IL-3 or treated
with 50 ng/mL G-CSF for 3 days (C; original magnification, ⫻400). (D) FACS analysis
of Mac-1␣ in 32D and 32D/⑀ cells cultured in IL-3 or treated with 50 ng/mL G-CSF for
3 days.
The enhanced differentiation capacity of 32D/⑀ cells was also
confirmed by flow cytometric analysis of Mac-1␣ expression. As
shown in Figure 4D, 32D/⑀ cells expressed about 3 times more
Mac-1␣ than parental cells when cultured in IL-3. Moreover,
compared with parental cells, 32D/⑀ cells had enhanced Mac-1␣
expression in response to G-CSF. All these data strongly indicate
that C/EBP⑀ is the principal downstream target of G-CSF, whose
expression alone is sufficient to support terminal granulocyte
differentiation.
Figure 5. Induction of C/EBP⑀ by mutant G-CSF receptor. (A,B) FDCP1 cells
expressing various truncation mutants of G-CSF receptor described in panel A were
analyzed for expression of C/EBP⑀ (B). Cells growing in IL-3 were washed twice with
PBS and transferred to medium containing 50 ng/mL G-CSF. Cells were harvested at
the times indicated and subjected to Northern blot analysis. (C) Relative mRNA
induction of C/EBP⑀. Northern blotting film shown in panel B in the linear exposure
was subjected to densitometric analysis. The graph is plotted as the signal ratio of
C/EBP⑀ compared with GAPDH. WT indicates wild-type G-CSF receptor; detailed
constructions of A, E, and T mutants are described in Fukunaga et al.23
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C-terminal portion of the receptor including Tyr 763 contributes to
this induction.
STAT3 is critical in G-CSF–induced myeloid differentiation but
acts in a pathway different from that of C/EBP⑀
Previously, it was shown that Tyr 703 is a major docking site for
STAT3.25 This raised the possibility that C/EBP⑀ could be regulated
by STAT3. To explore this possibility, we created 32D cells
expressing a carboxyl-truncated STAT3 that lacked 55 amino acids
including the transactivation domain, which acts in a dominantnegative fashion with respect to endogenous STAT3 (Figure 6A).26
In contrast to parental cells, 32D cells expressing dominantnegative STAT3 (32D/DN-STAT3) proliferated in G-CSF (Figure
6B), and G-CSF could support their long-term growth over 2
months (data not shown). Morphologic analysis showed that
32D/DN-STAT3 cells maintained immature morphologic characteristics in G-CSF, without evidence of differentiation (Figure 6C).
These observations are consistent with previous reports showing
that dominant-negative STAT3 inhibits G-CSF– or IL-6–induced
differentiation and growth arrest in other hematopoietic cell
lines26-28; and this indicates that the dominant-negative construct is
working properly.
We then examined expression of C/EBP⑀ and MPO in 32D/DNSTAT3 cells and found that C/EBP⑀ and MPO were both normally
induced in response to G-CSF (Figure 6D). This result indicates
that STAT3 and C/EBP⑀ work in different pathways and that
C/EBP⑀ is regulated by a signaling molecule other than STAT3.
c-myc blocks myeloid differentiation by inhibiting expression
of C/EBP⑀ but not C/EBP␣
Aberrant proliferation control leads to altered differentiation and
ultimately to malignant transformation. c-myc is one of the critical
regulators of cellular proliferation, and it is known that overexpression of c-myc blocks differentiation in many cell types.19-21 In
32Dcl3 cells, overexpression of c-myc also blocks differentiation
by G-CSF, but the underlying molecular mechanism of this effect is
not well understood. We speculated that c-myc may block differentiation by inhibiting genes critical for differentiation, and this led us
to examine expression of C/EBP⑀ and C/EBP␣ in 32Dcl3 cells
stably expressing c-myc (32D/myc).29
As expected, basal expression of C/EBP⑀ in cultures containing
IL-3 was completely abrogated in 32D/myc cells on Northern blot
analysis, whereas expression of C/EBP␣ was unaffected. Moreover, G-CSF treatment did not cause any measurable increase in
C/EBP⑀ expression, whereas expression of C/EBP␣ was unchanged (Figure 7A). We also observed no induction of MPO by
G-CSF (Figure 7A). These data suggest that c-myc inhibits
G-CSF–induced differentiation by inhibiting transcription of
C/EBP⑀ in 32D/myc cells.
We hypothesized that if c-myc blocks granulocytic differentiation by suppressing expression of C/EBP⑀, introduction of C/EBP⑀
into 32D/myc cells would overcome this differentiation block. To
test this hypothesis, we transiently overexpressed C/EBP⑀ in
32D/myc cells, selected transfected cells by means of drug
resistance, and examined their morphologic features. As shown in
Figure 7B, enforced expression of C/EBP⑀ induced a change in
nuclear morphologic characteristics and the appearance of cytoplasmic granules in 32D/myc cells (ii-iv), whereas the control vector
did not (i). Expression of C/EBP⑀ in transfected cells was
confirmed by immunofluorescence analysis (data not shown).
These data indicate that c-myc suppresses C/EBP⑀ expression in
myeloid cells, thereby blocking differentiation in response to
G-CSF.
Discussion
Figure 6. The 32D cells expressing dominant-negative STAT3 show differentiation block but normal C/EBP⑀ induction. (A) Construction of dominant-negative
STAT3. (B) Growth curve of 32D/DN-STAT3. Cells were cultured in 25 U/mL IL-3 (E),
50 ng/mL G-CSF (Œ), or without cytokines (f). (C) Morphologic features of
32D/DN-STAT3 cells cultured in IL-3 or G-CSF and stained with Wright-Giemsa stain
(original magnification, ⫻400). (D) Expression of C/EBP⑀ and MPO mRNA in
32D/DN-STAT3 cells. Cells were transferred from IL-3 to G-CSF and subjected to
Northern blot analysis at the times indicated.
Unlike expression of other members of the C/EBP family, expression of C/EBP⑀ was found to be highly restricted to myeloid cells
and lymphoid cells.3,4 Although human primary CD34⫹ cells do not
express substantial levels of C/EBP⑀, expression of C/EBP⑀ was
induced dramatically when such cells were induced to differentiate
to granulocytes by G-CSF.4 However, it was not clear whether
expression of C/EBP⑀ is regulated directly by G-CSF. In this study,
we showed that G-CSF induces C/EBP⑀ in myeloid progenitor
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BLOOD, 15 AUGUST 2001 䡠 VOLUME 98, NUMBER 4
G-CSF REGULATES C/EBP⑀
903
replaced by Epo signaling and thus should be a common pathway
used by both Epo and G-CSF. Our data showing that STAT3 is not
involved in this pathway also fit into this model, because Epo and
G-CSF activate different STAT proteins.
Although our results place C/EBP⑀ downstream of G-CSF
signaling, the phenotypes of G-CSF and C/EBP⑀ knockout mice
were apparently different, suggesting that C/EBP⑀ could also be
regulated by other pathways. In fact, C/EBP⑀ can be induced by
other growth factors or differentiation inducers, such as retinoic
acid, stem cell factor (SCF), PIXY321 (an engineered recombinant
fusion protein of IL-3 and granulocyte-macrophage colonystimulating factor) and dimethyl sulfoxide (DMSO), in leukemic
cell lines or immature myeloid cells, thereby suggesting that
C/EBP⑀ is a common integrator of various differentiation signals
(Figure 8A).3,31,32 In addition, Park et al32 showed that promoter of
C/EBP⑀ is regulated directly by retinoic acid through the retinoic
acid responsive element. It is not clear whether other cytokines or
chemicals such as SCF or DMSO directly affect the promoter
activity of C/EBP⑀. In the case of G-CSF, induction of C/EBP⑀
does not occur in an immediate-early fashion, indicating that
multiple signaling steps are involved in this process. In fact, new
protein synthesis is required for induction of C/EBP⑀ by G-CSF,
because such induction is blocked by cycloheximide (H. N., J. N. I.,
unpublished data, November 1998).
Figure 7. C/EBP⑀ is critical in myc-induced differentiation block in 32D/myc
cells. (A) Expression of C/EBP⑀, C/EBP␣, and MPO in 32D/myc cells. Cells
maintained in IL-3 were washed and transferred to medium containing 50 ng/mL
G-CSF. The cells were harvested at the times indicated and subjected to Northern
blot analysis. (B) Ectopic expression of C/EBP⑀ induces differentiation in 32D/myc
cells. C/EBP⑀ expression vector (20 ␮g) with a cistronic zeomycin-resistance marker
(pcDNA3Zeo-C/EBP⑀) was electroporated into 32D/myc cells, and transfected cells
were selected with 750 ␮g/mL zeomycin for 6 days. The empty vector was used as a
negative control (i indicates negative control; and ii-iv, C/EBP⑀-transfected cells).
Morphologic features of cells were visualized by using Wright-Giemsa staining
(original magnification, ⫻400).
cells and that expression of C/EBP⑀ alone is sufficient to support
both morphologic and functional differentiation. Because of these
data and the phenotype of C/EBP⑀-deficient mice that revealed the
essential role of C/EBP⑀ in myeloid development, we conclude that
C/EBP⑀ is the key downstream effector in G-CSF–induced granulocyte differentiation.
There is controversy about whether the only role of cytokines in
hematopoiesis is to support survival of the progenitors that express
its cognate receptor on their surface and receptor-expressing cells
are destined to execute an intrinsically preset differentiation
program (stochastic model). In contrast, an instructive model
suggests active roles for lineage-specific cytokines in directing
various differentiation processes. Our data show that G-CSF
actively regulates the myeloid differentiation program by inducing
C/EBP⑀. Previously, mutant mice whose cytoplasmic domain of
G-CSF receptor was replaced with the cytoplasmic domain of the
Epo receptor were generated, and surprisingly, the mice had no
apparent defect in lineage commitment.30 This finding suggests that
signaling to induce C/EBP⑀ generated from G-CSF receptor can be
Figure 8. C/EBP⑀ is an integrator of multiple signaling pathways that induce
myeloid differentiation. (A) Expression of C/EBP⑀ is up-regulated not only by
G-CSF but also by retinoic acid and other differentiation-inducing chemicals. Retinoic
acid (RA) acts through the retinoic acid responsive element (RARE) on the promoter
of C/EBP⑀. IL-3 and c-myc suppress C/EBP⑀ expression at the transcriptional level.
(B) A model of G-CSF–induced granulocytic differentiation. C/EBP␣ up-regulates
G-CSF receptor and enhances signals to induce STAT3 and C/EBP⑀. Expression of
G-CSF receptor can also be induced by C/EBP⑀ as a positive feedback regulation.
C/EBP⑀ induces morphologic and functional differentiation to granulocytes and may
be involved in cell-cycle arrest. STAT3 is critical for cell-cycle arrest, cooperatively or
independently with C/EBP⑀. STAT3 may also be essential for differentiation, and
C/EBP␣ is one possible downstream target of the STAT3-dependent pathway.
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904
BLOOD, 15 AUGUST 2001 䡠 VOLUME 98, NUMBER 4
NAKAJIMA and IHLE
MPO was previously shown to be the target gene of C/EBP␣33;
however, the existence of constitutive expression of MPO in 32D/⑀
cells indicates that MPO can also be induced by C/EBP⑀. The
previous observation that MPO is present in C/EBP⑀ knockout
mice34 but not in C/EBP␣ knockout mice7 seems to indicate that
C/EBP␣ is the only regulator of MPO. However, because of the
lack of C/EBP⑀ expression in C/EBP␣ knockout mice that results
from differentiation block at the myeloid progenitor stage, it is
possible that MPO could also be regulated by C/EBP⑀. Hence, the
best interpretation of our data is that both C/EBP␣ and C/EBP⑀ can
regulate MPO during myeloid differentiation.
Previous investigations found that C/EBP␣ is critical for early
granulocyte differentiation.33,35 In those studies, induction of
C/EBP␣ induced morphologic differentiation and expression of
granulocyte-specific genes, such as G-CSF receptor, lactoferrin,
neutrophil collagenase, and C/EBP⑀.35 Our study showed that
C/EBP⑀ is also capable of facilitating granulocytic differentiation.
Taken together, the data from all these studies suggest that both
C/EBP␣ and C/EBP⑀ regulate granulocytic differentiation, cooperatively or independently, in vivo. Because C/EBP␣ induces expression of G-CSF receptor,35 it is intriguing to speculate that C/EBP␣
not only directs granulocytic differentiation by itself but also by
enhancing C/EBP⑀ expression by up-regulating G-CSF receptor. In
addition, C/EBP⑀ also enhances expression of G-CSF receptor,36
which then creates a positive feedback regulation on G-CSF
signaling. On the other hand, our study showed that STAT3 plays
an essential role in G-CSF–induced differentiation by means of a
pathway distinct from that of C/EBP⑀. The molecular mechanism
by which STAT3 regulates granulocyte differentiation is not
currently understood, but one possibility is regulation through
C/EBP␣. A proposed model for G-CSF signaling pathways to
granulocytic differentiation is shown in Figure 8B.
A previous study showed the importance of Tyr 703 of G-CSF
receptor in differentiation signaling.24 In that investigation, mutation of Tyr 703 abolished the ability of G-CSF to induce morphologic differentiation, MPO expression, and cell-cycle arrest. The
downstream molecule responsible for these effects was not identified, but our study suggests that C/EBP⑀ and STAT3 are the 2
principal candidates. With regard to cell-cycle arrest, we observed a
compromised growth rate in 32D/⑀ cells compared with parental
cells, indicating that C/EBP⑀ may be involved in growth inhibition.
In fact, C/EBP␣, another C/EBP family member, was previously
found to inhibit proliferation of adipocytes and hepatocytes by
stabilizing protein levels of p21/WAF1/CIP1.37,38 Therefore, an
intriguing possibility is that C/EBP⑀ may also inhibit progression
of the cell cycle by means of p21 or other CDK inhibitors.
However, because IL-3 can support long-term growth (⬎ 5 months;
unpublished data) of 32D/⑀ cells, though at a slower rate, it is
possible that some additional mechanisms are involved in cellcycle arrest. Data showing involvement of STAT3 in growth arrest
of M1 cells by IL-6, G-CSF receptor–transfected LGM-1 cells by
G-CSF, and macrophages by IL-10 suggest that STAT3 has a role in
cell-cycle arrest associated with differentiation.26,27,39 Similarly, we
observed G-CSF–dependent proliferation in 32Dcl3 cells expressing dominant-negative STAT3, a finding that also suggests that
STAT3 sends a growth-inhibitory signal. All these data indicate that
both C/EBP⑀ and STAT3 are negative regulators of cell proliferation. Studies to address this issue are currently under way.
NF-M, the chicken homologue of C/EBP␤, plays an important
role in avian myelomonocytic and eosinophilic differentiation.40,41
NF-M was first discovered as a critical transcription factor required
for expression of chicken myelomonocytic growth factor, which is
distantly related to mammalian G-CSF and IL-6.42 Expression of
NF-M is restricted to the myelomonocytic lineage and is turned on
at some point before the myeloblast stage. Conditional expression
of NF-M in a hematopoietic progenitor cell line induced eosinophilic differentiation and apoptosis.41 Although NF-M is structurally most related to mammalian C/EBP␤, studies of its expression
pattern and function suggested that it is more closely related to
C/EBP⑀. In view of these findings on NF-M, it is intriguing to
speculate that C/EBP⑀ might activate transcription of the G-CSF
gene, which further facilitates the differentiation program to
granulocytes.
c-myc controls proliferation and differentiation of the cell
through its capacity as a transcription factor to promote the activity
of the cyclin E–CDK2 complex or to induce apoptosis.29 Expression of c-myc increases when quiescent cells are induced to
proliferate43 and decreases when the cells exit the cell cycle.15,16,44,45
In many cases, expression of c-myc overrides differentiation
signals generated by differentiation inducers,19-21 suggesting that
c-myc may negatively regulate differentiation by means of unknown mechanisms. We here observed that c-myc inhibited
expression of C/EBP⑀ in a myeloid differentiation system. This
finding raises the possibility that the overall balance of expression
of c-myc and differentiation-regulating genes such as C/EBP
determines the fate of cells, that is, whether they proliferate or
differentiate under various conditions. Because the reciprocal
pattern of regulation—inhibition of myc by differentiationpromoting genes—has not been reported, it can be speculated that
myc is the dominant regulator over differentiation; thus, explaining
the regulation of myc expression is essential for understanding the
complex network of growth and differentiation control.
In summary, this study revealed the essential, rate-limiting role
of C/EBP⑀ in G-CSF–induced granulocytic differentiation and
illuminated the role of C/EBP⑀ as a key integrator of various
differentiation signals during myeloid differentiation (Figure 8A).
Acknowledgments
We thank Dr Kleanthis Xanthopoulos for providing pcEpsilon32
vector, Dr Shigekazu Nagata for FDCP1 cells expressing G-CSF
receptor mutants, and Dr John Cleveland for 32D/myc cells.
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2001 98: 897-905
doi:10.1182/blood.V98.4.897
Granulocyte colony-stimulating factor regulates myeloid differentiation
through CCAAT/enhancer-binding protein ε
Hideaki Nakajima and James N. Ihle
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