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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1142-1149
RAPID COMMUNICATION
Synergistic Activation of MAP Kinase (ERK1/2) by Erythropoietin and
Stem Cell Factor Is Essential for Expanded Erythropoiesis
By
Xingwei Sui,
Sanford B. Krantz,
Min You, and
Zhizhuang Zhao
From the Hematology Division, Department of Medicine, Department of
Veterans Affairs Medical Center, and Vanderbilt Cancer Center,
Vanderbilt University, Nashville, TN.
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ABSTRACT |
Stem cell factor (SCF) and erythropoietin (EPO) work synergistically
to support erythropoiesis, but the mechanism for this synergism is
unknown. By using purified human erythroid colony-forming cells (ECFC),
we have found that SCF and EPO synergistically activate MAP kinase
(MAPK, ERK1/2), which correlates with the cell growth and thus may be
responsible for the synergistic effects. Treatment of the cells with
PD98059 and wortmannin, inhibitors of MEK and PI-3 kinase,
respectively, inhibited the synergistic activation of MAPK and also the
cell growth, further supporting this conclusion. Wortmannin only
inhibits MAPK activation induced by EPO but not that by SCF, suggesting
that SCF and EPO may activate MAPK through different pathways, which
would facilitate synergy. Furthermore, EPO, but not SCF, led to
activation of STAT5, whereas SCF and wortmannin had no effect on the
EPO-induced STAT5 activation, suggesting that STAT5 is not involved in
the synergistic action of SCF and EPO. Together, the data suggest that
synergistic activation of MAPK by SCF and EPO is essential for expanded
erythropoiesis.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ERYTHROPOIESIS IS REGULATED by a number
of growth factors and cytokines, among which stem cell factor (SCF) and
erythropoietin (EPO) play an essential role both in vivo and in
vitro.1-3 Mice with mutations in the SCF gene, Steel
(Sl), or its receptor gene c-kit (W) develop severe
anemia characterized by depressed erythropoiesis4-6; EPO
and EPO-receptor (EPO-R) knockout mice die of failure of fetal liver
erythrocyte generation.7 In vitro, whereas EPO alone is
able to produce human erythroid cell development and maturation from
progenitor cells, a markedly enhanced, synergistic proliferation and
expansion of developing erythroid cells is supported by the combination
of EPO and SCF.1,8-10
Although the synergistic effects of EPO and SCF on the production of
erythroid cells have been well documented, the intracellular mechanism
by which SCF and EPO coordinate the synergistic effect is poorly
understood. In a recent study, Wu et al11 reported that, in
HCD57 cells, SCF rapidly induces tyrosine phosphorylation of EPO-R and
c-Kit physically associates with the cytoplasmic domain of EPO-R. They
proposed that c-Kit activates EPO-R by tyrosine phosphorylation to
induce further proliferation and maturation of erythroid progenitor
cells. This could provide an explanation for the synergistic effect of
EPO and SCF on erythropoiesis. However, HCD57 is an immortal cell line
that does not exhibit synergistic cell growth upon combined treatment
with SCF and EPO. For this reason, we performed a study using primary
erythroid progenitor cells. We demonstrate that, although SCF can
induce phosphorylation of EPO-R, it is not sufficient to transduce
signals via EPO-R and support cell growth. Instead, our data show that
SCF and EPO synergistically activate MAP kinase (MAPK, ERK1/2),
which correlates with the cell growth and thus may be responsible for
the synergistic effects.
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MATERIALS AND METHODS |
Reagents.
Recombinant human (rh) SCF and rhEPO were kindly provided by Amgen, Inc
(Thousand Oaks, CA) and Ortho Biotech, Inc (Raritan, NJ), respectively.
Rabbit phospho-specific MAPK and MEK1/2 (Ser217/221) antibodies were
purchased from New England Biolabs, Inc (Beverly, MA). Phospho-specific
MAPK antibody detects p42 and p44 (Erk1 and Erk2) only when
catalytically activated by phosphorylation at Tyr 204. Rabbit anti-MAPK
polyclonal antibody (7884) was raised in rabbits against a 22-amino
acid peptide derived from the subdomain X1 of MAPK and recognized both
Erk1 and Erk2.12 Rabbit polyclonal EPO receptor (EPOR),
c-Kit, and STAT5b (C-17; catalogue no. sc-835; recognizes both STAT5a
and 5b) antibodies were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Mouse antiphosphotyrosine antibody 4G-10 was from Upstate
Biotechnology, Inc (Lake Placid, NY). MEK1 inhibitor (PD98059) was from
New England Biolabs, Inc, and phosphatidylinositol 3-kinase
(PI3-kinase) inhibitor Wortmannin from Calbiochem (La Jolla, CA).
[ -32P]-dCTP and [-32P]-dTTP,
[-32P]-ATP, and horseradish peroxidase-conjugated
secondary antibodies were from Amersham Corp (Arlington Heights, IL).
Two buffers were used for extraction of proteins from ECFC. Buffer A
consisted of 50 mmol/L  -glycerophosphate (pH 7.3), 2 mmol/L EDTA, 1 mmol/L EGTA, 5 mmol/L -mercaptoethanol, 1% Triton X-100, and 0.1 mol/L NaCl. Buffer B consisted of 20 mmol/L HEPES (pH 7.9), 20 mmol/L NaF, 1 mmol/L EDTA, and 1 mmol/L dithiothreitol. Both buffers were also
supplemented with 0.2 mmol/L Na3VO4, 0.1 µmol/L microcystin, 1.0 mmol/L benzamidine, 0.1 mmol/L
phenylmethysulfonyl fluoride, 2 µg/mL leupeptin, 1 µmol/L pepstain
A, and 1 µg/mL aprotinin.
Preparation of erythroid colony-forming cells (ECFC).
Purification of human ECFC was performed as previously reported, with
minor modification.13 Four hundred milliliters of heparinized peripheral blood was obtained from healthy donor, after
informed consent was obtained, which was approved by the Vanderbilt
Committee for the Protection of Human Subjects. The light-density
mononuclear cells were separated on Ficoll-Hypaque (F-H; 1.077 g/mL) at
400g at 24°C for 25 minutes by centrifugation. The
interface mononuclear cells were collected, washed twice with Dulbecco's phosphate-buffered saline (D-PBS), and then
overlaid on 30 mL of 10% bovine serum albumin (BSA) in D-PBS at
180g at 24°C for 10 minutes to remove platelets. This
step was repeated once and then the cells were incubated with one-half
volume of neuraminidase-treated and one-half volume of IgG-coated sheep red blood cells (RBCs). After centrifugation at 130g at
24°C for 10 minutes, the cells were placed in ice for 60 minutes
and then separated over F-H at 400g at 24°C for 10 minutes
to remove the rosetted lymphocytes. The interface cells were then
incubated overnight at 37°C in Iscove's modified Dulbecco's
medium (IMDM) containing 20% fetal calf serum
(FCS), 1% BSA, and 4% giant cell tumor
(GCT)-conditioned medium to remove adherent cells. The
nonadherent cells were collected the next day (day 1) and incubated
with mouse monoclonal antibodies to CD11b, CD2, CD45R, and CD16 for 60 minutes at 4°C on a gently rocking platform. The cells were then
washed three times with IMDM containing 2% FCS and resuspended in IMDM containing 5% FCS. Five-milliliter aliquots were incubated at 4°C
for 90 minutes in 100-mm plastic tissue culture dishes (100 × 200) coated with affinity-purified goat antimurine IgG to remove the
antibody-positive cells.13 The antibody-negative
nonadherent cells were then collected, washed, and are refered to as
day-1 cells. The collected day-1 cells were cultured in a suspension mixture containing 30% FCS, 1% deionized BSA, 2 U/mL of rhEPO, 100 ng/mL of rhSCF, 50 U/mL of rhIL-3, 10 µg/mL of insulin,
10 4 mol/L of 2-mercaptoethanol, 500 U/mL of
penicillin, and 40 µg/mL of streptomycin in IMDM at 37°C in a
high humidity 5% CO2, 95% air incubator. At day 6 of the
suspension culture, equal volumes of fresh IMDM containing 20% FCS and
1% BSA and additional doses of SCF and EPO were added to the culture.
The cultured cells were collected at day 8 of suspension culture, and
these cells are referred to as day-8 ECFC. Compared with the day-6 ECFC
used in our previous study,15 the yield and purity of day-8
ECFC were significantly increased with this modified method (yield: 3 to 5 times more than that at day 6; purity: 80% ± 4.6% v
63% ± 7.9%). The purity of the ECFC refers to the percentage of
cells that can form erythroid colonies in the plasma
assay.13 The cells that cannot form colonies are mainly
proerythroblasts and erythroblasts, as shown by cytospin
mophological studies. The day-8 ECFC were washed twice with PBS
and used for all further studies.
Cell starvation and stimulation.
After washing with PBS, day-8 ECFC were resuspended at a cell density
of 2 to 5 × 106/mL in IMDM containing 0.1% BSA and
incubated at 37°C for 1 hour. The cells were then washed once again
with PBS and reincubated at 37°C for an additional 2 hours. After
the serum-deprived starvation, the cells were exposed to EPO and SCF at
their optimal concentrations for various periods of time. Optimal
concentrations of SCF at 100 ng/mL and EPO at 40 U/mL were obtained
from dose-response experiments. Activation of MAPK induced by SCF and
EPO in human ECFC reached plateaus at these concentrations,
respectively. These are concentrations at which the receptors (EPO,
c-Kit) on the ECFC cell surface are completely
saturated.13,17 For studies involving kinase inhibitors
(PD98059 and Wortmanin), the ECFC were preincubated with the inhibitors
at varying concentrations for 1 hour before stimulation or further cell
culture. The stimulation was stopped by adding 10 vol of cold PBS and
the cells were washed and lysed in buffer A for whole cell extraction.
For preparation of nuclear extracts, the pelleted cells were
resuspended in three packed cell volume of hypotonic buffer B, swollen
for 10 minutes, and lysed by repeated passage through a 25-gauge
needle. Nuclei were collected and washed by centrifugation at
16,000g for 10 minutes and then extracted in buffer A
supplemented with 0.42 mol/L NaCl and 20% glycerol on ice for 30 minutes. The supernatants (referred to as the nuclear extracts) were
cleared by centrifugation at 16,000g for 1 minute and used for
STAT activity analyses.
Gel mobility shift assays of STATs.
The activities of STATs were determined by analyzing the ability of the
protein to form complexes with specific DNA probes in gel shift assays.
The DNA probes used were PIE-derived from a prolactin-inducible element
and hSIE, a high-affinity sequence for the sis-inducible element of the
fos promotor. PIE is specifically for detection of STAT5, whereas hSIE
contains high affinity binding sites for STAT1 and STAT3.
PIE and SIE were made by annealing 5 -AGATTTCTAGGAATTCAA-3
with 5-GGATTGAATTCCTAGAAA-3 and
5-GTCGACATTTCCCGTAAATC-3 with
5-TCGACGATTTACGGGAAATG3, respectively.14
These double-stranded DNA probes with staggered ends were labeled by
Klenow fragment in the presence of [-32P]dCTP and
[-32P]dTTP for PIE and [-32P]dCTP
alone for SIE at room temperature for 30 minutes. Free nucleotides were
removed by using a nucleotide removing kit from Qiagen
(Chatsworth, CA). Nuclear extracts containing 10 µg protein were
preincubated with 2 µg of poly(dI-dC).poly(dI-dC) (Pharmacia, Uppsala, Sweden) for 10 minutes, which was followed by the
addition of 2 mL 32P-labeled DNA probes and further
incubated at room temperature for an additional 15 minutes. The
protein-DNA complexes were resolved on 5% polyacrylamide gels
(acrylamide:bisacrylamide = 39:1) containing 2.5% glycerol made in
0.5× TBE buffer. Gels were dried and exposed on x-ray film.
MAPK activity assay.
The whole cell extracts made in buffer A were immunoprecipitated with
phospho-specific MAPK antibody, and the MAPK activity in the
immunoprecipitates was assayed with myelin basic protein (MBP) as a
substrate in the presence of [-32P]-ATP, as described
previously.12
Suspension and colony assays.
Suspension and plasma-clot assays of ECFC were performed as we
previously reported.10,15 Cell numbers in the
serum-containing suspension culture were determined every other day. To
examine the effects of kinase inhibitor on cell growth, the ECFC were cultured in the presence of varying concentration of inhibitors. Because both PD98059 and Wortmannin were reconstituted in dimethyl sulfoxide (DMSO) and DMSO might have toxic effects on the
cells, the final concentration of DMSO in all culture was limited to less than 0.2%, which has been shown to have no effects on ECFC growth
in our studies. Plasma clot culture was maintained for 7 days and the
colonies formed were fixed and stained with 3,3 dimethoxybenzidine and hematoxylin for enumeration of erythroid colonies.
Sodium dodecyl sulfate (SDS)-gels and Western blotting.
Protein extracts were separated by 10% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membranes (Amersham). The membranes were probed with
various primary antibodies that were detected by using the enhanced
chemiluminescence (ECL) system with horseradish peroxidase-conjugated secondary antibodies (Amersham) according to the manufacturer's protocol.
| |
RESULTS |
SCF induces transient phosphorylation of EPO-R, but cannot replace EPO
to support erythropoiesis in vitro.
Over the past years, we have established a method to purify large
amounts of human ECFC from human peripheral blood.13 Our previous studies have shown that these cells at day 6 to 8 express about 1,000 EPO-R and 23,000 c-Kit receptors per cell and are highly
sensitive to treatment with SCF and EPO. A majority of these cells are
able to form RBC colonies in semisolid cultures.13,16,17 These highly purified ECFC thus provide a good primary cell system to
study cell signaling. To explore the mechanisms for the synergism of
SCF and EPO, we first examined tyrosine phosphorylation of EPO-R and
c-Kit after stimulation by their ligands. As shown in Fig 1A and B, both SCF and EPO stimulated
rapid and transient tyrosine phosphorylation of their own receptors,
respectively. Stimulation of cells with SCF alone also induced tyrosine
phosphorylation of EPO-R, although the degree of phosphorylation was
less than that observed with EPO and disappeared after 10 minutes.
However, in the presence of EPO, SCF appeared to show no enhancing
effect on EPO-R phosphorylation (Fig 1B). Whereas SCF alone possessed some effect on EPOR phosphorylation, EPO alone had no effect on c-Kit
phosphorylation, and a combination of SCF with EPO did not enhance
SCF-induced c-Kit phosphorylation (Fig 1A). Phosphorylation of EPO-R
upon treatment of the cells with SCF confirms the results reported by
others.11,18 However, no physical association between these
two receptors was observed, because Western blotting of EPO-R
immunoprecipitates with c-Kit antibody or c-Kit immunoprecipitates with
EPO-R antibody did not show any coimmunoprecipitation of one receptor
with the other (data not shown).
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| Fig 1.
SCF causes tyrosine phosphorylation of EPO-R, but cannot
replace EPO to support human erythropoiesis in vitro. Phosphorylation of c-Kit (A) and EPO-R (B) after stimulation of ECFC with EPO, SCF, or
EPO + SCF. Replicate day-8 ECFC (~5 × 106) were
starved and then treated with SCF and EPO separately or in combination
for 5 or 10 minutes as described in Materials and Methods. Cell
extracts were subject to immunoprecipitation with anti-c-Kit and
anti-EPO-R antibodies in the presence of protein A-Sepharose beads.
Immunoprecipitates (corresponding to whole cell extracts containing 50 µg of total protein) were subjected to SDS-PAGE and Western blot
analysis with antiphosphotyrosine antibody 4G-10. (C) To determine the
requirement of SCF and EPO for growth of ECFC, day-6 ECFC (5 × 103) were incubated in 20% FBS, 1% BSA-containing medium
supplemented with SCF, EPO, or SCF + EPO. Cell numbers were
determined after 7 days in culture. Data represent the mean values from
three independent experiments.
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We next determined whether SCF could stimulate erythroid cell growth
while inducing EPO-R phosphorylation. Figure 1C shows the growth of
erythroid cells in suspension culture. Compared with the
synergistically stimulated cell growth induced by the combination of
EPO and SCF, EPO alone manifested only 20% to 25% of this erythroid
cell production from ECFC over a period of 7 days, whereas SCF alone
did not support cell growth at all. Similar results were observed in
the erythroid colony-forming assays with semisolid culture medium. In
this case, the addition of SCF to EPO significantly increased both the
number and the size of RBC colonies. These results indicate that,
although SCF can induce transient phosphorylation of EPO-R on tyrosine,
it cannot replace EPO to support erythroid cell growth. Thus,
SCF-induced EPO-R phosphorylation may not be sufficient to activate
EPO-R and transduce signals through it. The synergistic effects of SCF
and EPO on erythropoiesis are most likely mediated by intracellular
signaling events downstream of the receptors.
SCF and EPO synergistically activate MAPK in human erythroid
progenitor cells.
MAP kinase (MAPK, ERK1/2) is a major transducer of extracellular
mitogenic signals that promote cell proliferation and
differentiation,19-22 and we thought that it might be
involved in the synergistic effects of EPO and SCF on erythropoiesis.
Because phosphorylation of MAPK is absolutely required for its
activation, we used a phospho-specific MAPK antibody that only
recognizes the phosphorylated form of the enzyme to determine its
activation. Figure 2A illustrates the time
course of the activation. When serum-starved ECFCs were treated with
EPO or SCF alone, marginal MAPK phosphorylation was observed. The
phosphorylation induced by SCF or EPO appeared after 5 minutes, reached
a peak at 10 minutes, and then declined slowly thereafter. In contrast
to the minimal phosphorylation of MAPK induced by EPO or SCF alone, a
combination of SCF and EPO gave rise to markedly enhanced MAPK
phosphorylation. The phosphorylation appeared within 5 minutes, reached
a maximum at 10 minutes, and then decreased gradually with detectable
phosphorylation remaining at 60 minutes. Densitometric analyses
indicated that the level of MAPK phosphorylation induced by the
combination of SCF and EPO was at least 10-fold higher than the
combined value obtained when cells were treated with SCF and EPO
separately. In all cases, Western blot analyses with antibodies to
regular anti-MAPK antibody showed that the protein levels of MAPK in
the cells were not altered by the treatment (data not shown). These
data demonstrate a synergistic activation of MAPK by SCF and EPO.
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| Fig 2.
Synergistic effects of SCF and EPO on phosphorylation of
MAPK (ERK1 and ERK2) (A) and MEK (B) and on stimulation of MAPK
activity (C). Serum-starved ECFC were treated with SCF, EPO, or SCF + EPO for the indicated periods of time. Whole cell extracts (20 µg protein) were subject to SDS-PAGE and Western blot analyses with anti-phospho-MAPK (A) or anti-phospho-MEK (B) antibodies. (C) For
determination of MAPK activity, cells were stimulated for 10 minutes
with SCF, EPO, or SCF + EPO and whole cell extracts containing about
60 µg of total protein were subjected to immunoprecipitation with
anti-phospho-MAPK. The MAPK activities in the immunoprecipitates were
analyzed by using myelin basic protein as a substrate in the presence
of [-32P]-ATP in buffer A containing PKI peptide and
calmidizolium, inhibitors of PKA and PKC, respectively. A
representative figure from four experiments with similar results is
shown. Each experiment was performed with blood samples from different
normal volunteers.
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We further measured the activation of MEK, a protein kinase responsible
for activation of MAPK. Because MEK also requires phosphorylation to be
activated, we used a phospho-MEK-specific antibody. As expected, EPO
or SCF alone induced very low levels of MEK phosphorylation, whereas a
combination of SCF and EPO induced synergistic MEK phosphorylation (Fig
2B). This result indicates that the synergistic activation of MAPK was
mediated through the synergistic activation of MEK.
To confirm the results obtained with phospho-specific antibodies, we
determined the activity of MAPK by using myelin basic protein as a
substrate.12 For this purpose, MAPK was first
immunoprecipitated with anti-phospho-MAPK and its kinase activity was
assayed in liquid-solid reaction. Consistent with the results obtained
with MAPK phosphorylation, the combined treatment of ECFCs with SCF and
EPO gave rise to a markedly enhanced MAPK activity, fourfold to sixfold
greater than that observed when cells were treated with SCF or EPO
alone (Fig 2C). The degree of synergy is less than that observed with
anti-phospho-MAPK antibodies, but this could be due to a lesser
efficiency of the liquid-solid phase reaction for activity assays.
Collectively, the data indicate that SCF and EPO synergistically
stimulate MAPK activation by inducing significant,
greater-than-additive increases in both MAPK phosphorylation and kinase
activity. Because the synergistic activation of MAPK correlates with
the synergistic effect on cell growth, we conclude that the synergistic
effect of SCF and EPO on erythropoiesis is mediated by the synergistic
activation of MAPK.
PD98059 (MEK1/2 inhibitor) and Wortmannin (PI-3 kinase inhibitor)
affect MAPK activation and cell growth induced by SCF and EPO.
To prove further the importance of MAPK activation in erythropoiesis,
we examined the effect of PD98059, which acts as a highly selected
inhibitor of MEK, on MAPK activation and cell growth induced by SCF and
EPO. Pretreatment of cells with 100 µmol/L PD98059 completely
inhibited activation of MAPK induced by EPO and SCF
(Fig 3A). Concomitantly, PD98059 treatment
also dose-dependently inhibited growth of ECFC in suspension culture.
The inhibitory effect was observed at a concentration of 50 µmol/L,
and at 200 µmol/L, greater than 70% inhibition was observed (Fig
3B). In an attempt to show the mechanism controlling MAPK activation by SCF and EPO, we also treated the cells with wortmannin, a PI-3 kinase
inhibitor. Wortmannin markedly inhibited the activation of MAPK induced
by EPO but had no inhibitory effect on that induced by SCF alone (Fig
3A). Furthermore, it reduced the synergistic activation of MAPK
phosphorylation by SCF and EPO to a level similar to that attained with
SCF alone (lanes 2, 10, and 12, Fig 3A). It should be noted that a
nonspecific band right below phospho-Erk2 was equally distributed in
all lanes. Accordingly, wortmannin also markedly inhibited EPO, but not
SCF-induced MEK activation (data not shown). These results suggest that
PI-3 kinase is involved in the EPO- but not in SCF-mediated MAPK
activation and that SCF and EPO may activate MAPK through different
pathways. The insensitivity of SCF-induced MAPK activation to
wortmannin also suggests that SCF does not transduce EPO-R-mediated
MAPK activation, although it causes tyrosine phosphorylation of EPO-R.
As expected, pretreatment of ECFC with wortmannin also inhibited the
growth of ECFC in liquid medium cultures, although the degree of
inhibition was slightly lower compared with that exhibited by PD98059
(Fig 3C). While Fig 3 demonstrated the inhibition of ECFC growth in
suspension culture, similar results were also obtained with erythroid
colony formation assays in semisolid medium. Together, these data
indicate that the synergistic activation of MAPK by SCF and EPO plays
an essential role in the proliferation and maturation of human
erythroid progenitors.
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| Fig 3.
MEK inhibitor PD98059 and PI-3 kinase inhibitor
wortmannin inhibit synergistic activation of MAPK (A) and cell growth
(B and C). Serum-starved cells were left untreated (control) or
pretreated with 100 µmol/L PD98059 or 0.5 µmol/L wortmannin for 1 hour and then stimulated with SCF and EPO separately or jointly for 10 minutes. Whole cell extracts were subject to SDS-PAGE and Western blotting with phospho-specific MAPK antibody or anti-MAPK
antibody. The latter detects both phosphorylated and nonphosphorylated
MAPK. A representative figure from four experiments with similar
results is shown. Each experiment was performed with blood samples from different normal volunteers. It should be noted that a nonspecific band
right below phospho-Erk2 was equally distributed in all lanes. To
analyze the effects of the inhibitors on erythroid cell growth, day-6
ECFC (5 × 103) were allowed to grow for another 7 days in
liquid medium supplemented with EPO ( ) or SCF + EPO ( ) in the
presence of varying concentrations of PD98059 (B) or wortmannin (C).
Cell numbers were determined after 7 days in culture. Results represent
data from three independent experiments.
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SCF has no effects on EPO-induced STAT activation in human erythroid
progenitor cells.
Activation of STAT family transcription factors is another major signal
transduction pathway involved in the action of growth factor and
cytokines.23,24 It has been reported that EPO activates STAT1 and STAT5,25-27 whereas SCF may be involved in STAT3
or STAT5 activation.28,29 To examine whether activation of
STATs is involved in the synergistic action of SCF and EPO in
erythropoiesis, we analyzed STAT activity by using electrophoretic
mobility shift assays with specific DNA probes.14 With
hSIE, a high-affinity mutant form of the serum-inducible element widely
used to detect STAT1 and STAT3,30 no binding was observed
after stimulation of cells with SCF and EPO. With PIE, a
prolactin-inducible element that can specifically bind STAT5a and
STAT5b,25 stimulation of ECFCs with EPO induced marked DNA
binding activity (Fig 4A). The presence of
STAT5 in the DNA-protein complex is evident in the super-shift analyses
with anti-STAT5 antibody (Fig 4A, lane 12). EPO-induced STAT5
activation was detected after 5 minutes of incubation and remained at
30 minutes. SCF alone failed to stimulate any STAT5 activity and
addition of SCF to EPO had no significant effect on EPO-induced STAT5
activation. This again suggests that, although SCF causes tyrosine
phosphorylation of EPO-R, it cannot transduce a signal through the
receptor. PD98059 and wortmannin, which displayed marked effects on
cell growth and MAPK activation, did not affect the EPO-induced STAT5
activation, suggesting that MAPK and PI-3 kinase may not be involved in
the EPO-dependent STAT5 activation. These experiments indicate that activation of STAT5 does not appear to be involved in the synergistic effect of SCF and EPO. The fact that both STAT5a and STAT5b knock-outs did not show any abnormalities of erythropoiesis suggests that STAT5
may not be important for erythropoiesis in vivo.31,32 However, because of the possible redundancy between STAT5a and STAT5b,
double knock-out mice of STAT5a and STAT5b need to be generated to test
the role of STAT5 in erythropoiesis.
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| Fig 4.
SCF and inhibitors of MEK and PI-3 kinase have no effects
on EPO-induced activation of STAT5. (A) Serum-starved day-8 ECFC were
treated with SCF, EPO, or SCF + EPO for 5, 10, and 30 minutes. Nuclear extracts were extracted and analyzed for STAT activity with a
32P-labeled PIE oligonucleotide probe. EPO* (lane 12, A)
denotes a supershift analysis with the nuclear extracts preincubated
with anti-STAT5b antibody (0.2 µg) for 10 minutes at room temperature before the addition of the PIE probe. (B) Serum-starved cells were
pretreated with 100 µmol/L PD98059 or 0.5 µmol/L wortmannin for 1 hour before stimulation with EPO for 10 and 60 minutes. STAT5 activity
was determined as described above.
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| |
DISCUSSION |
In the present study, we demonstrated that SCF and EPO synergistically
activate MAPK, which correlates with their effects on erythroid cell
growth, thus providing an intracellular mechanism for the synergistic
action of SCF and EPO in erythropoiesis. Our data also suggest that
tyrosine phosphorylation of EPO-R produced by SCF is not sufficient to
transduce the EPO signal, which is evident in the failure of SCF to
induce wortmannin-sensitive MAPK activation and activation of STAT5.
SCF may cause phosphorylation of EPO-R at sites that are not essential
for signal transduction.
MAPK lies at a convergent point for many signaling events. It plays an
important role in promoting cell proliferation and differentiation19-22 and preventing
apoptosis.33 The inhibition of erythroid cell growth and
colony formation by inhibitors of MEK and PI-3 kinase demonstrate the
crucial role of MAPK in the SCF- and EPO-mediated erythropoiesis. Both
the magnitude and duration of the activation are crucial, and studies
suggest that transient activation of MAPK may trigger proliferation,
whereas sustained activation allows differentiation.34 The
synergistic effects of SCF and EPO observed in our current study not
only greatly enhanced the magnitude, but also enhanced the
duration of the activation, which may be important for both
proliferation and differentiation of erythroid cells. Prolonged
activation of MAPK might also play a role in preventing apoptosis, as
reported in rat pheochromocytoma PC12 cells.33 Prevention
of apoptosis is believed to be the primary role for EPO.35
Although synergistic activation of MAPK has been previously observed in
various cell lines when treated with insulin and phorbol
ester,36 integrins and growth factors,37
granulocyte-macrophage colony-stimulating factor (GM-CSF) and
SCF,38 or interleukin-3 (IL-3) and SCF,39 the
degree of the synergistic effects was not as significant as we observed
with SCF and EPO in the primary erythroid progenitor cells. Moreover,
in this study, we were able to correlate directly the synergy in signal
transduction with the synergy in cell growth. Considering that
hematopoiesis requires the presence of multiple growth factors or
cytokines, this study begins to show how these growth factors and
cytokines interact to produce balanced growth and differentiation. It
also suggests that the synergistic effects of other growth factors and
cytokines in other tissue systems may be mediated through synergistic
activation of MAPK.
The general scheme of the MAPK activation pathway includes a series of
protein phosphorylations and protein-protein interactions involving,
sequentially, Grb2, mSOS, Ras, Raf-1, MEK, and MAPK. SCF transduces its
signal by binding to its receptor c-Kit, a PDGF family receptor
tyrosine kinase, and, thus, presumably, it activates MAPK via the
Ras/Raf pathway.40 However, for EPO, direct recruitment of
Grb2 to EPO-R has not been reported. Although phosphorylation of SHC
and insulin receptor substrate-2 after EPO stimulation might provide
docking sites for Grb2 binding,41-43 recent studies
suggests the existence of a SHC/Grb2-independent pathway.44,45 The fact that the PI-3 kinase inhibitor
wortmannin inhibited EPO-induced MAPK activation, but not that of SCF,
suggested that SCF and EPO may lead to activation of MAPK through
different pathways. Because SCF and EPO synergistically activate MEK,
the convergent point at which these two pathways interact should be at
the level of MEK or above MEK. Presumably, these two pathways would
transduce signals that act on different sites of the target protein to
facilitate the synergistic effects. How EPO transduces its signal
through PI-3 kinase to MAPK is still unknown. It has been postulated
that protein kinase C might be one of the mediators.44
| |
FOOTNOTES |
Submitted March 17, 1998;
accepted June 1, 1998.
Supported by a Veterans Health Administration Merit Review Grant (to
S.B.K.), Grants No. DK-15555 and 2 T32-DK07186 (to S.B.K.) and HL 57393 and P30ES00267 (to Z.Z.) from the National Institutes of Health, and
Markey Trust Funds (to Z.Z.). X.S. is an Ortho Biotech Hematology
Fellow.
Address reprint requests to Sanford B. Krantz, MD, or Zhizhuang Zhao,
PhD, Department of Medicine/Hematology, Vanderbilt University Medical
Center, MRBII, Nashville, TN 37232-6305.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Ella Stitt, Judy Luna, and Amanda Hodges for their
excellent technical assistance.
| |
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Abstract
Introduction
Abstract