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INTRODUCTION |
Granulocyte colony-stimulating factor
(G-CSF)1 is a cytokine that
is critical for supporting normal neutrophil production and maturation
(1, 2). G-CSF stimulates the proliferation, survival, and maturation of
cells committed to neutrophilic lineage (3, 4). The diverse biological
effects of G-CSF are mediated through a single class of cell-surface
receptor protein that forms homodimeric complexes on ligand binding
(5). The human G-CSF receptor is a member of the hematopoietin receptor
superfamily (6, 7). On the other hand, granulocyte-macrophage
colony-stimulating factor (GM-CSF) is also thought to play an important
role in the proliferation and maturation of neutrophil and macrophage
lineage cells (6, 7).
Promyelocytic leukemia HL-60 cells are known to differentiate into
macrophages or neutrophils in response to several stimuli. Dimethyl
sulfoxide (Me2SO), retinoic acid (RA), and
Bt2cAMP cause the neutrophilic differentiation of HL-60
cells, whereas interferon-
and phorbol ester cause the
differentiation of macrophages (8-10). Because of these advantages,
many studies have been performed on HL-60 differentiation as a model of
proliferation and maturation of myelogenic lineage cells. Several
reports have suggested that although G-CSF cannot itself induce
neutrophilic differentiation of HL-60 cells, it can potentiate the
neutrophilic differentiation of RA and Me2SO-treated HL-60
cells in terms of the O
2-generating ability (11-13). The
molecular mechanism of G-CSF role on the neutrophilic differentiation
of HL-60 cells has not been fully elucidated. Conversely, GM-CSF has
been shown to directly induce the monocytic differentiation of HL-60
cells (14), and also to induce the re-proliferation in
Me2SO-treated HL-60 cells in which growth has been
suppressed (15). The effect of GM-CSF on the
neutrophilic-differentiation of HL-60 cells remains unclear.
The addition of G-CSF causes the activation of several signaling
pathways in myelogenic cells, including the JAK and STAT pathway, as
well as the Ras-mitogen-activated protein (MAP) kinase cascade (16),
and in particular the rapid activation of STAT3 and STAT1 (17).
Activation of MAP kinase has been reported in the cells that
proliferate in response to G-CSF (18, 19). Nicholson et al.
(19) have suggested that phosphorylation of MAP kinase was correlated
with both proliferative response and JAK2 activation. On the other
hand, the importance of p70 S6 kinase in cell cycle progression of
numerous cells has been reported (20). However, it has not yet been
studied whether or not G-CSF signaling cascade involves p70 S6 kinase.
Furthermore, it also remains unclear how these signaling pathways
coincide with the proliferation and differentiation of neutrophil
lineage cells.
In the present study, we analyzed the G-CSF-dependent
enhancement of differentiation of Me2SO-treated HL-60
cells, and observed the cross-talk of G-CSF and GM-CSF on this
differentiation. Furthermore, to clarify the molecular mechanism of the
role of G-CSF on neutrophilic differentiation,
G-CSF-dependent activations of STAT3, MAP kinase, and p70
S6 kinase were studied in Me2SO-treated HL-60 cells.
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EXPERIMENTAL PROCEDURES |
Materials--
Human recombinant G-CSF (Chugai Pharmaceutical
Co., Tokyo Japan) and human recombinant GM-CSF (Dainippon
Pharmaceutical Co., Osaka Japan) were obtained. Anti-STAT3 (monoclonal
antibody (mAb), Transduction Lab., S21320), and
anti-tyrosine-phosphorylated STAT3 (polyclonal antibody (pAb), New
England Biolab Inc., 9131S), anti-serine 727-phosphorylated STAT3 (pAb,
New England Biolab Inc., 9134S), and anti-phosphorylated MAP kinase
(New England Biolab Inc., 9100) antibodies were purchased from the
aforementioned companies. Anti-human C3bi receptor mAb was purchased
from Nichirei Co. (Tokyo, Japan) and fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse IgG and anti-rabbit IgG were obtained
from Jackson Immuno Research Lab., Inc. (West Grove, PA).
FITC-N-formyl-Met-Leu-Phe-Lys (fMLP) was purchased from
Peninsula Lab. Inc. (Belmont, CA). Horse heart ferricytochrome
c, fMLP, zymosan A, and bovine erythrocyte superoxide dismutase were obtained from Sigma. Zymosan particles were opsonized by
gently mixing with freshly prepared guinea pig serum (50 mg of zymosan
to 5 ml of serum) for 30 min at 37 °C. The zymosan particles were
washed several times with HEPES-buffered saline (HBS, 122 mM NaCl, 4.9 mM KCl, 1 mM
MgCl2, 16.7 mM HEPES, adjusted to pH 7.4 with
NaOH). The opsonized zymosan particles were suspended in HBS (50 mg/ml)
and used as a stimulant for O
2 generation at a final
concentration of 2.5 mg of zymosan equivalent/ml.
Cell Culture--
HL-60 cells were kindly supplied by the
Japanese Cancer Research Resources Bank (Tokyo, Japan). Cells were
maintained in a RPMI 1640 culture medium containing 10%
heat-inactivated fetal bovine serum and 30 µg/ml kanamycin at
37 °C in moisturized air containing 5% CO2. To
standardize the quality of cells throughout this series, frozen cells
of the same lot were thawed every three months and used for
experiments. Differentiation was initiated by dispersing the cells in a
fresh culture medium containing 1.25% (v/v) Me2SO to make
a final cell density of about 2.5 × 105 cells/ml.
G-CSF and/or GM-CSF were added to the cell suspension 2 days after the
addition of Me2SO. After culturing for 5 days, the cells
were collected by centrifugation at 600 × g for 5 min and were washed several times with HBS. The resultant cells were resuspended in HBS and kept in an ice-cold bath until used for analyses. The viability of the cells was evaluated by the trypan blue
dye-staining exclusion method, and more than 95% viability was
confirmed in HL-60 cells treated with or without differentiating agents.
O2-generating Activity--
O2
generation by differentiated cells was measured by ferricytochrome
c reduction assay (21). The cells (1.0 × 106) were preincubated in a substrate solution (50 µM ferricytochrome c, 5 mM
D-glucose, and 0.5 mM CaCl2 in HBS)
for 10 min at 37 °C, and the assay was initiated by the addition of
a stimulant. The rate of O
2 generation was measured by
continuously recording the increase in absorbance at 550-540 nm using
a Hitachi 557 double-beam spectrophotometer. In the assay with
insoluble stimulants, such as opsonized zymosan particles, the assay
mixture was constantly stirred by a windmill mixer as described
previously (22).
Expression of C3bi and fMLP Receptors on the Cell
Surface--
The expression of stimulant-receptors on the cell surface
was examined by flow cytometry with a Cyto-ACE-15 Auto Cell Screener (JASCO Co., Tokyo, Japan). For the detection of C3bi receptor, cells
(106 cells/100 µl of phosphate-buffered saline, PBS) were
incubated with anti-human-Mac-1 monoclonal antibody for 1 h at
4 °C. The cells were then washed several times in PBS, and incubated
with FITC-conjugated goat anti-mouse IgG in 100 µl of PBS for 1 h at 4 °C. The cells were again washed several times with PBS and
were finally suspended in an aliquot of PBS and subjected to flow cytometry.
The FITC-labeled chemotactic peptide (FITC-fMLP) was utilized to
estimate the expression of the fMLP receptor on the cell surface (23,
24). Approximately 106 cells were incubated with 0.1 µM FITC-fMLP in HBS containing 2% (w/v) bovine serum
albumin (BSA) for 30 min at 4 °C. For the evaluation of the specific
binding of the peptide to the receptor, the cells were preincubated
with 10 µM fMLP for 15 min at 4 °C, and then 0.1 µM FITC-fMLP was added. After the incubation with the
FITC-labeled peptide, the cells were collected by centrifugation at
600 × g for 5 min and washed twice with HBS containing
0.2% (w/v) BSA. The washed cells were suspended in 1 ml of HBS
containing 0.2% (w/v) BSA and subjected to flow cytometry.
Immunoblot Analysis--
Fifty nanograms/ml G-CSF and/or GM-CSF
were added to Me2SO-treated HL-60 cells, and then three
cells were incubated for 5 to 60 min at 37 °C. After the appropriate
incubation time, the cells were mixed with chilled PBS containing a
mixture of phosphatase and protease inhibitors and were transferred
into an ice bath. Then, the cells were sedimented and dissolved with
radioimmune precipitation assay buffer to 107 cells/0.1 ml
(25). The extract of the cells was prepared by short time sonication
and subjected to Western blot analysis of MAP kinase, p70S6 kinase, and
STAT3. 10 to 30 µg of the extracted protein was separated onto an
SDS-PAGE gel and transferred to a nitrocellulose membrane (Highbond
ECLTM film; Amersham Pharmacia Biotech). Membranes were
incubated with the primary antibodies anti-ERK (1:2000, pAb prepared in
rabbit), anti-p70 S6 kinase (1:2000, pAb prepared in rabbit),
anti-STAT3 (1:2000), anti-tyrosine-phosphorylated STAT3 (1:1000) and
anti-serine 727-phosphorylated STAT3 and then visualized with secondary
antibodies coupled to peroxidase and an ECL reagent (Amersham Pharmacia Biotech).
In order to analyze nuclear translocation of tyrosine-phosphorylated
STAT3, after the incubation with G-CSF and/or GM-CSF, HL-60 cells were
collected in a hypotonic buffer containing phosphatase and protease
inhibitors by differential centrifugation as previously reported (30).
After vortexing for 10 s, nuclei were sedimented by centrifugation
at 10,000 rpm for 5 min. Nuclear fractions were dissolved in SDS-sample
buffer containing Benzon nuclease. The nuclear extracts were subjected
to Western blotting analysis with anti-tyrosine-phosphorylated STAT3.
Observations of Confocal Laser-scanning Microscopy--
The
analysis of the alteration of STAT3 localization in
Me2SO-treated HL-60 cells by G-CSF and/or GM-CSF was
examined by confocal laser-activated microscopy (ACAS ULTIMA, Meridian
Instruments and Zeiss LSM410, Carl Zeiss Inc.). The cells were treated
with 50 ng/ml G-CSF and/or GM-CSF for 30 or 60 min. After the
incubation, the cells were fixed with an equal volume of 3.8%
paraformaldehyde in PBS and then washed with PBS. After treatment with
ethanol for 5 min, the fixed cells were incubated with anti-STAT3
(1:100, mAb) or anti-tyrosine-phosphorylated STAT3 antibodies for
1 h and visualized with secondary antibody coupled to FITC. Cells were examined using an ACAS ULTIMA or Zeiss LSM410 microscope, with an
extinction wavelength of 488 nm, emission of 530/30 nm.
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RESULTS |
Effects of G-CSF and GM-CSF on the O2-generating
Ability of HL-60 Cells Differentiating to Neutrophil-like
Cells--
The neutrophilic differentiation of HL-60 cells was
initiated by incubation with 1.25% (v/v) Me2SO. Two days
after the addition of Me2SO, 50 ng/ml G-CSF and/or 50 ng/ml
GM-CSF were added to Me2SO-treated cells, and then the
cells were incubated for 5 days. Fig. 1
shows the O
2-generating activities of the differentiated HL-60
cells. The O
2 generation from the G-CSF-treated HL-60 cells in
response to fMLP or OZ was found to be much higher than that of the
cells treated with Me2SO alone, as reported previously (13). On the other hand, the incubation with GM-CSF reduced the fMLP or
OZ-induced O
2-generating ability of HL-60 cells. It was noted
that GM-CSF markedly reduced the G-CSF-dependent enhancement of neutrophilic differentiation of HL-60 cells in terms of
O
2-generating ability. As previously reported (13), nondifferentiated HL-60 cells did not produce O
2 in the
presence fMLP or OZ (data not shown).

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Fig. 1.
Superoxide generation by differentiated HL-60
cells. Differentiation of HL-60 cells was initiated by dispersing
the cells in a fresh culture medium containing 1.25% (v/v)
Me2SO to establish a final cell density of about 2.5 × 105 cells/ml. Two days after the addition of
Me2SO, G-CSF and/or GM-CSF were added to the cell
suspension and then the cells were incubated for 5 days. The
O 2 generation from the cells was measured by means of a
ferricytochrome c reduction assay. The cells (1 × 106) were preincubated in a substrate solution (50 µM ferricytochrome c, 5 mM
D-glucose, and 0.5 mM CaCl2 in HBS)
for 10 min at 37 °C, and the assay was initiated by the addition of
either 500 nM fMLP (a) or 1.25 mg/ml opsonized
zymosan (b). Data represent the mean ± S.D. from four
experiments. *, p < 0.01 versus
control.
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G-CSF but Not GM-CSF Enhances the Expression of the fMLP Receptor
in HL-60 Cells during Neutrophilic Differentiation--
Expressions of
the fMLP and OZ (C3bi) receptors on the surface of HL-60 cells during
the neutrophilic differentiation were analyzed by flow cytometry. As
previously reported (13), the fluorescent signal representing the C3bi
receptor remarkably increased in HL-60 cells from the treatment of
Me2SO (data not shown). The expression of the C3bi receptor
in the Me2SO-treated HL-60 cells, however, was not altered
by incubation with either G-CSF or GM-CSF. On the other hand, the
expression of the fMLP receptor in these cells markedly changed after
incubation with either G-CSF and/or GM-CSF (Fig.
2). The expression of fMLP-receptor
(fMLP-R) in Me2SO-differentiated HL-60 cells was
characterized by both a high and a low level population, whereas
the nondifferentiated cells expressed only a low level of fMLP-R, as
previously reported (13). The population of cells showing a high level
of fMLP-R expression increased markedly after incubation with G-CSF.
The enhancement of fMLP-R expression by G-CSF may coincide with
G-CSF-dependent enhancement of O
2-generating ability. On the other hand, GM-CSF decreased the population of high
level fMLP-R-expressing cells that were treated with Me2SO alone. Furthermore, in the presence of GM-CSF, G-CSF failed to enhance
high level fMLP-R-expressing cells. These results, therefore, suggest
that G-CSF augments the neutrophilic differentiation of Me2SO HL-60 cells, whereas GM-CSF inhibits neutrophilic
differentiation of HL-60 cells.

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Fig. 2.
Expression of fMLP receptor and C3bi on the
differentiated HL-60 cells. Differentiation of HL-60 cells was
induced in the same manner as in Fig. 1. Approximately106
cells were incubated with 0.1 µM fMLP-FITC in HBS
containing 2% (w/v) bovine serum albumin for 30 min at 4 °C. For
the evaluation of the specific binding of the peptide to the receptor,
the cells were preincubated with 10 µM fMLP for 15 min at
4 °C prior to adding 0.1 µM fMLP-FITC. After the
incubation with the FITC-labeled peptide, the cells were collected by
centrifugation at 600 × g for 5 min and then washed
twice with HBS containing 0.2% (w/v) bovine serum albumin. The washed
cells were suspended in 1 ml of HBS containing 0.2% (w/v) bovine serum
albumin and subjected to flow cytometry. For the detection of a C3bi
receptor, the cells (106 cells/100 µl of PBS) were
incubated with 5 µl of diluted mouse ascitic fluid containing
anti-human-C3bi receptor monoclonal antibody for 1 h at 4 °C.
Washing with PBS several times, the cells were incubated with
FITC-conjugated goat anti-mouse IgG in 100 µl of PBS for 1 h at
4 °C.
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G-CSF and GM-CSF Enhance the Growth of Me2SO-treated
HL-60 Cells--
Myelogenic cytokines, such as G-CSF or GM-CSF, are
thought to modulate not only the differentiation but also the
proliferation of myelogenic cells. We, therefore, examined the effects
of G-CSF and GM-CSF on the growth of Me2SO-treated HL-60
cells. As shown in Fig. 3, both G-CSF and
GM-CSF accelerated the proliferation of Me2SO-treated HL-60
cells. Whereas the proliferation curve representing the presence of
GM-CSF was much higher than that for the presence of G-CSF, the
proliferation curve showing in the presence of both cytokines was
similar to that for the presence of GM-CSF alone, indicating that the
growth-promoting effects of either cytokine on
Me2SO-treated HL-60 cells were not cumulative.

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Fig. 3.
G-CSF and/or GM-CSF induce cell proliferation
in Me2SO-treated HL-60 cells. The
differentiation of HL-60 cells was induced in the same manner as in
Fig. 1. After 2 days of incubation, 50 ng/ml G-CSF and/or GM-CSF were
added to HL-60 cell suspension, and the cells were subsequently
cultured for various periods. The number of cells were determined at
each indicated time. Open circle, Me2SO alone;
closed circle, with G-CSF; open triangle, with
GM-CSF; closed triangle, with G-CSF and GM-CSF. Data
represent the mean ± S.D. from three experiments. *,
p < 0.01 versus control; **,
p < 0.01 versus G-CSF.
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Effect of G-CSF and GM-CSF on Signal Transduction--
The
previously mentioned results indicate that G-CSF acts as an accelerator
on the maturation of HL-60 cells to neutrophils, whereas this action is
inhibited by GM-CSF. Accordingly, we examined the cross-talk of these
cytokines in terms of the signal transduction pathways.
Fig. 4 shows the effects of G-CSF and/or
GM-CSF on the activation of MAP kinase and p70 S6 kinase. Within 5 min,
GM-CSF clearly induced a mobility shift of MAP kinase, indicating the
phosphorylation of MAP kinase. The GM-CSF-dependent MAP
kinase activation gradually ceased within 30 min (Fig. 4a).
The phosphorylation of MAP kinase by GM-CSF was confirmed by Western
blot using anti-phosphorylated MAP kinase antibody (Fig.
4b). An SDS-PAGE mobility shift assay indicated that GM-CSF
also induced a delayed phosphorylation of p70 S6 kinase relative to
that of MAP kinase (Fig. 4c). G-CSF, on the other hand, did
not induce the activation of MAP kinase but caused a similar delayed
activation of p70 S6 kinase. The addition of both G-CSF and GM-CSF
caused the rapid activation of MAP kinase and the delayed activation of
p70 S6 kinase.

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Fig. 4.
Activation of MAP kinase and p70 S6 kinase
induced by G-CSF and/or GM-CSF. The differentiation of HL-60 cells
was initiated by dispersing the cells in a fresh culture medium
containing 1.25% Me2SO in RPMI 1640 containing 10% fetal
bovine serum. G-CSF and/or GM-CSF were added to the cell suspension 2 days after the addition of Me2SO. After incubation for the
indicated time, the cells were mixed with ice-chilled PBS and
transferred into an ice bath. Then, the cells were sedimented and
dissolved with radioimmune precipitation assay buffer. Ten to 30 µg
of the extracted protein was separated on an SDS-PAGE gel and
transferred onto a nitrocellulose membrane. All lanes in
each panel have the same total protein. Next the membranes
were incubated with anti-ERK (MAP kinase, pAb), anti-phosphorylated ERK
(pAb), and anti-p70 S6 kinase (pAb) antibodies and visualized with
secondary antibodies coupled to peroxidase. The arrowhead
indicates the mobility shift of ERK by phosphorylation.
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Next, we examined whether STAT3 is activated upon the addition of G-CSF
and/or GM-CSF. As shown in Fig.
5b, G-CSF induced a tyrosine
phosphorylation of STAT3 in Me2SO-treated HL-60 cells, whereas GM-CSF alone did not. G-CSF-dependent tyrosine
phosphorylation of STAT3 continued for 30 min. These results are
consistent with those of the G-CSF- and GM-CSF-coupled JAK-STAT
pathways (16, 26). On the other hand, Sengupta et al. (27)
reported that tyrosine phosphorylation of STAT3, which was induced by
IL-6, was partially inhibited by GM-CSF. In the present experiment, G-CSF-induced tyrosine phosphorylation of STAT3 was not affected by
GM-CSF in the Me2SO-treated HL-60 cells, as shown in Fig.
5. Several investigators have reported that maximal activation of transcription by STAT3 requires both tyrosine and serine
phosphorylation (28-31). Serine-phosphorylated STAT3 shows a mobility
shift on SDS-PAGE (32). GM-CSF induced the rapid SDS-PAGE mobility
shift of STAT3. However, G-CSF caused a delayed SDS-PAGE mobility shift of STAT3. As shown in Fig. 5c, the experiment using
anti-serine 727-phosphorylated STAT3 antibody showed that GM-CSF caused
rapid serine 727 phosphorylation in STAT3, which corresponded to the SDS-PAGE mobility-dependent assay; however, G-CSF did not induce the serine 727 phosphorylation of STAT3.

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Fig. 5.
Phosphorylation of STAT3 induced by G-CSF
and/or GM-CSF. Differentiation of HL-60 cells was initiated in the
same manner as in Fig. 1. Two days after the addition of
Me2SO, G-CSF and/or GM-CSF were added to the cell
suspension, and the extraction of cells was performed as in Fig. 4. Ten
to 30 µg of the extracted protein was separated on an SDS-PAGE gel
and transferred onto a nitrocellulose membrane (ECL bond film, Amersham
Pharmacia Biotech). All lanes in each panel have
the same total protein. The membranes were incubated with anti-STAT3
(mAb), anti-tyrosine-phosphorylated STAT3 (pAb), and anti-serine
727-phosphorylated STAT3 (pAb) antibodies and were visualized with
secondary antibodies coupled to peroxidase.
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STAT3 is thought to be activated at the plasma membrane by tyrosine
phosphorylation and translocated into the nucleus to induce the
transcription of target genes. We, therefore, analyzed the cytokine-dependent nuclear translocation of STAT3 in
Me2SO-treated HL-60 cells by using confocal microscopy. As
shown in Fig. 6a (G-CSF), G-CSF caused the translocation of STAT3 into nuclei
within 60 min. It was, however, demonstrated that in the presence of GM-CSF, G-CSF failed to cause the nuclear translocation of STAT3, even
when the STAT3 was tyrosine-phosphorylated (Fig. 5b).
Furthermore, GM-CSF alone did not alter the cellular location of STAT3.
We therefore hypothesized that the activation of the GM-CSF-specific signal cascade disturbs the nuclear translocation of STAT3, and therefore examined the effect of MEK1 inhibitor, PD98059, on STAT3 translocation. As shown in Fig. 6
(PD-98059+G/GM-CSF), in the presence of PD98059,
the addition of both G-CSF and GM-CSF caused the nuclear translocation
of STAT3.

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Fig. 6.
Localization of STAT3 after the activation of
G-CSF and/or GM-CSF. The differentiation of HL-60 cells was
initiated as in Fig. 1. Two days after the addition of
Me2SO, G-CSF and/or GM-CSF were added to the cell
suspension, and then the cells were incubated for 60 min. After the
incubation, cells were fixed with 1.9% paraformaldehyde in PBS. After
a 1-h fixation, the cells were washed with PBS. After the treatment
with ethanol for 5 min, fixed cells were incubated with anti-STAT3
(1:100) antibody for 1 h (a) or
anti-tyrosine-phosphorylated STAT3 (1:100) antibody
(b) and then visualized with a secondary antibody coupled to
FITC. In some experiments, the cells were preincubated for 30 min with
100 µM PD98059 before the addition of G-CSF and/or
GM-CSF. a, immunostaining for STAT3 using anti-STAT3
mAb. Cells were observed using a Zeiss LCM410 microscope.
Inset indicates Nomarsky photograph of each cell.
b, immunostaining for tyrosine-phosphorylated STAT3 using
anti-tyrosine-phosphorylated STAT3 pAb. Arrows indicate the
localization of cells. Cells were observed with an ACAS ULTIMA. For
each experiment, data were collected at identical black level and gain
settings. In some experiments, cells were processed using a
nonimmuno-mouse or -rabbit IgG and subsequently stained irrelevant of
the secondary antibody. Cells stained with nonimmuno-IgG exhibited a
nonfluorescence signal.
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G-CSF-dependent nuclear translocation of STAT3 was analyzed
using anti-tyrosine 705-phosphorylated STAT3 pAb. As shown in Fig.
6b, whereas control cells and GM-CSF-treated cells did not show any response to anti-tyrosine-phosphorylated STAT3 antibody, G-CSF resulted in translocation of tyrosine-phosphorylated STAT3. In
the presence of GM-CSF, G-CSF prompted the tyrosine phosphorylation of
STAT3 but failed to induce the translocation of tyrosine-phosphorylated STAT3 into a nuclear portion. In the presence of PD98059, upon the
addition of both cytokines, tyrosine-phosphorylated STAT3 was
translocated into a nuclear portion.
On the other hand, a rapid mobility shift of STAT3 induced by GM-CSF
was clearly inhibited by PD98059 (Fig.
7a). In the presence of either
G-CSF alone or both cytokines, tyrosine-phosphorylated STAT3 exhibited
an SDS-PAGE mobility shift as compared with that in the presence of
PD98059. In the presence of PD98059, the addition of G-CSF or both
cytokines caused a delayed SDS-PAGE mobility-shift in
tyrosine-phosphorylated STAT3, suggesting that the
G-CSF-dependent delayed serine (or threonine)
phosphorylation may be catalyzed by kinases other than MAP kinase, or
its downstream kinase. Furthermore, the SDS-PAGE mobility shift pattern
of G-CSF-dependent tyrosine-phosphorylated STAT3 differed
from that of STAT3 with G-CSF, suggesting that a small amount of STAT3
was phosphorylated to tyrosine residue upon the addition of G-CSF.
Using an anti-serine 727-phosphorylated STAT3 antibody, GM-CSF induced
a rapid serine 727 phosphorylation of STAT3, which was markedly
inhibited by the addition of PD98059, whereas G-CSF alone did not
induce the serine 727 phosphorylation, suggesting that the delayed
serine (or threonine) phosphorylation of STAT3 induced by G-CSF may be
a result other than from the serine 727 residue. In the presence of
PD98059, the addition of GM-CSF did not induce the activation of MAPK
(data not shown).

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Fig. 7.
Effect of PD98059 on the phosphorylation of
STAT3 induced by G-CSF and/or GM-CSF. The differentiation of HL-60
cells was initiated as in Fig. 1. Two days after the addition of
Me2SO, 100 µM PD98059 was added to the cell
suspension. After the incubation with or without PD98059 for 30 min,
G-CSF and/or GM-CSF were added to the cell suspension, and then the
extraction of cells was performed as in Fig. 5. Panels a,
b, and c indicate the Western blots of whole cell
extracts. For the analysis of nuclear translocation of
tyrosine-phosphorylated STAT3, cells were disrupted with a hypotonic
solution, and a nuclear fraction was prepared as described under
"Experimental Procedures" (d).
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To confirm the nuclear translocation of STAT3, we prepared a nuclear
fraction from HL-60 cells and determined the content of
tyrosine-phosphorylated STAT3. As shown in Fig. 7d,
tyrosine-phosphorylated STAT3 was detected in the nuclear fraction in
G-CSF-treated cells but not that in both cytokine-treated cells.
Furthermore, in the presence of PD98059, tyrosine-phosphorylated STAT3
was detected in nuclear fractions in G-CSF-treated cells and both
cytokine-treated cells. These results indicate that the GM-CSF inhibits
G-CSF-dependent nuclear translocation and PD98059 blocks
the inhibition.
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DISCUSSION |
It is known that HL-60 cells possess the ability to differentiate
into neutrophilic cells by treatment with Me2SO (8) or RA
(9). Several reports have suggested that G-CSF cannot itself induce
neutrophilic differentiation in nondifferentiated HL-60 cells, but it
can potentiate the differentiation into neutrophils initiated by
Me2SO or RA (11-13). However, the role of G-CSF in the
development of functional maturation during the differentiation of
HL-60 cells into neutrophilic cells has not been extensively elucidated. We confirmed that G-CSF enhanced the neutrophilic differentiation of Me2SO-treated HL-60 cells (Figs. 1 and
2). However, whereas GM-CSF has been shown to be a potential inducer of
monocytic differentiation of human myeloid leukemia cells, including
HL-60 cells (33), the effect of GM-CSF on neutrophilic differentiation
of HL-60 cells remains unclear. In this paper, we showed that GM-CSF
inhibited the neutrophilic differentiation of Me2SO-treated
HL-60 cells in terms of O
2- generating ability and fMLP-R
expression. Furthermore, the G-CSF-dependent enhancement of
neutrophilic differentiation in Me2SO-treated HL-60 cells
was markedly inhibited by GM-CSF, suggesting that GM-CSF acts as an antagonist of G-CSF in regard to the neutrophilic differentiation of
HL-60 cells.
As a result of this data, we postulated that a G-CSF-specific signal
transduction pathway may be implicated in the enhancement of
neutrophilic differentiation of HL-60 cells. It has been reported that
G-CSF activates STAT3 through the activation of JAK2, whereas GM-CSF
activates STAT5 through the activation of JAK2 (17, 19, 26, 34-37). In
a study using dominant negative STAT3, Minami et al. (37)
speculated that STAT3 activation is a critical step in gp130-mediated
terminal differentiation and growth arrest in a myeloid cell line. In
this paper, we showed that G-CSF activated STAT3 by phosphorylation of
tyrosine residue, whereas GM-CSF alone did not. Sengupta et
al. (27) reported that GM-CSF partially inhibited interleukin-6
(IL-6)-induced tyrosine phosphorylation of STAT3. In this study, the
addition of GM-CSF did not inhibit the tyrosine phosphorylation of
STAT3 in Me2SO-treated HL-60 cells. This discrepancy may be
because of a difference of cell type-specific activation of STAT3.
In the presence of GM-CSF, however, G-CSF failed to induce the nuclear
translocation of STAT3, even though G-CSF induced the tyrosine
phosphorylation of STAT3. The inhibitory effect of GM-CSF on the
nuclear translocation of tyrosine-phosphorylated STAT3 was restored by
the addition of PD98059 (Fig. 6 and 7). Through the activation of MAP
kinase, GM-CSF induces the serine phosphorylation of STAT3, which
interrupts the nuclear translocation of STAT3 in HL-60 cells. It
remains unclear why the serine-phosphorylated STAT3 does not
translocate into the nucleus in HL-60 cells, as it can in other cells
(34, 38). Further research is required to clarify the molecular
mechanism in the inhibition of nuclear-translocation of STAT3.
GM-CSF inhibited the G-CSF-dependent enhancement of
neutrophilic differentiation in Me2SO-treated HL-60 cells
(Figs. 1 and 2). We postulated that G-CSF-dependent
tyrosine phosphorylation and nuclear-translocation of STAT3 may be
associated with the promotion of neutrophilic differentiation in
Me2SO-treated HL-60 cells and that the inhibition of
G-CSF-dependent neutrophilic differentiation by GM-CSF may
be because of the interruption of nuclear translocation of STAT3, as
discussed above. In regard to the effect of PD98059 on the
differentiation of Me2SO-treated HL-60 cells, our
preliminary results indicated that the decrease in fMLP receptor
expression by GM-CSF was restored by the addition of 100 µM PD98059. However, cell growth and
O
2-generating activity were markedly inhibited by PD98059,
both in the presence and absence of G-CSF and/or GM-CSF (data not
shown). Thus, a different approach is necessary to clarify the role of
MAP kinase on the inhibitory effect of GM-CSF.
It was reported that the maximal activation of the transcription of
STAT3 requires both serine and tyrosine phosphorylation; however,
serine kinase(s) that is responsible for the phosphorylation of STAT3
has not yet been clarified (28-31). The proposed MAP kinase phosphorylation site, PXSP, is conserved among members of the STAT
family, suggesting a role for MAP kinase in serine phosphorylation (28,
29). Other groups have suggested that H-7-sensitive serine kinase is
responsible for the phosphorylation of STAT3 (30, 31). Whereas the
addition of GM-CSF rapidly induced an SDS-PAGE mobility shift and
serine 727 phosphorylation of STAT3 in either the presence or absence
of G-CSF, G-CSF alone did not show the above reaction. On the other
hand, G-CSF induced the tyrosine phosphorylation of STAT3 but not
serine 727 (Figs. 5 and 7). The addition of PD98059 inhibited serine
727 phosphorylation induced by GM-CSF. In the presence of PD98059,
G-CSF caused a delayed SDS-PAGE mobility shift of
tyrosine-phosphorylated STAT3. These results suggest that G-CSF induces
serine (or threonine) phosphorylation, other than serine 727, in STAT3.
Furthermore, both GM-CSF-dependent MAP kinase activation
and serine 727 phosphorylation of STAT3 were observed by as early as 5 min. The GM-CSF-dependent mobility shift of STAT3 was
markedly reduced by the addition of PD98059, a MEK1 inhibitor (Figs. 4
and 7). Therefore, it was suggested that GM-CSF-dependent
serine phosphorylation of STAT3 is induced by the activation of MAP
kinase in HL-60 cells.
GM-CSF and G-CSF are thought to play an important role in the growth of
myelogenic cells. As previously reported, we observed that G-CSF and
GM-CSF augmented the cell number of Me2SO-treated HL-60
cells (15, 39). GM-CSF-dependent cell growth of
Me2SO-treated HL-60 cells was much higher than that of
G-CSF. The enhancement of GM-CSF and G-CSF on the growth of
Me2SO-treated HL-60 cells was not cumulative. Therefore, it
appears that the signaling pathway of GM-CSF-dependent cell
growth in Me2SO-treated HL-60 cells may also include the
pathway of G-CSF.
A number of hematopoietic growth factors, including GM-CSF, IL-3, and
steel factor have been shown to activate the Ras MAP kinase pathway
(40, 41). Conversely, the phosphatidylinositol 3-kinase-p70 S6 kinase
pathway in several cells has also been shown to be associated with the
growth signals of many cytokines (20). In the present study, we
observed that GM-CSF induced a rapid electromobility shift in
immunoreactive MAP kinase (ERK1 and ERK2) and a time-delayed
electromobility shift in immunoreactive p70 S6 kinase. On the other
hand, G-CSF was shown to induce a time-delayed activation of p70 S6
kinase but not MAP kinase. When both GM-CSF and G-CSF were added to
Me2SO-treated HL-60 cells, the activation pattern of both
MAP kinase and p70 S6 kinase were similar to that of GM-CSF alone. In
conclusion, we postulate that the activation of both kinases provides
maximum activation of cell growth in Me2SO-treated HL-60
cells. Furthermore, it appears that the activation of p70 S6 kinase in
Me2SO-treated HL-60 cells may be a mutual pathway for the
G-CSF- and GM-CSF-dependent enhancement of cell proliferation.