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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 462-471
A Novel Function of Stat1 and Stat3 Proteins in
Erythropoietin-Induced Erythroid Differentiation of a Human Leukemia
Cell Line
By
Keita Kirito,
Mie Uchida,
Masaaki Takatoku,
Koichi Nakajima,
Toshio Hirano,
Yasusada Miura, and
Norio Komatsu
From the Division of Hematology, Department of Medicine, Jichi
Medical School, Tochigi, Japan; and the Department of Molecular
Oncology, Biomedical Research Center, Osaka University Medical School,
Osaka, Japan.
 |
ABSTRACT |
We recently determined that erythropoietin (EPO) activates 3 members
of the signal transducer and activator of transcription (STAT) family,
Stat1 , Stat3, and Stat5, in the human EPO-dependent cell lines, UT-7
and UT-7/EPO (Kirito et al, J Biol Chem 272:16507, 1997). In
addition, we have shown that Stat1, but not Stat3, is involved in
EPO-induced cellular proliferation. In this study, we examined the
roles of Stat1 and Stat3 in EPO-induced erythroid differentiation.
UT-7/GM was used as a model system, because this cell line can
differentiate into erythroid-lineage cells with EPO treatment (Komatsu
et al, Blood 89:4021, 1997). We found that EPO did not activate
Stat1 or Stat3 in UT-7/GM cells. Transfection experiments showed
that both Stat1 and Stat3 inhibited the induction by EPO of
 -globin and erythroid-specific 5-aminolevulinate synthetase transcripts, resulting in a reduction of the percentage of
hemoglobin-positive cells. Dominant negative forms of Stat1 or Stat3
promoted the EPO-induced erythroid differentiation of UT-7/GM cells,
even in the presence of granulocyte-macrophage colony-stimulating
factor, although this cytokine never induced erythroid differentiation of the parent UT-7/GM cells with or without EPO. A cell cycle analysis
showed that the constitutive activation of Stat1, but not Stat3,
shortened the period of G0/G1 prolongation caused by EPO stimulation.
Taken together, our data suggest that Stat1 and Stat3 act as
negative regulators in EPO-induced erythroid differentiation.
Specifically, Stat1 may activate a cell cycle-associated gene(s),
leading to the entry of cells into the cell cycle.
| |
INTRODUCTION |
ERYTHROPOIETIN (EPO) plays an important
role in the proliferation and differentiation of erythroid progenitor
cells.1 The binding of EPO to its specific receptor on the
cell surface induces tyrosine phosphorylation and the activation of
several proteins, including Janus kinase 2 (JAK2),2 signal
transducer and activator of transcription 5 (Stat5),3-5
mitogen-associated protein kinases,6 and phospholipase
C-1.7
We recently demonstrated that EPO induces the activation
of Stat1 and Stat3 in the EPO-dependent cell lines UT-7, its subline UT-7/EPO, and F36E.8-13 By contrast, EPO did not activate
Stat1 and Stat3 in UT-7/GM, another subline of UT-7, or in TF-1
cells, which grow only minimally in the presence of
EPO.13-15 Transfection experiments with Stat1
and/or Stat3 showed that the overexpression of Stat1, but
not Stat3, promoted the EPO-induced proliferation of UT-7/GM cells. In
addition, the introduction of EPO receptor (EPOR) cDNA into UT-7/GM
cells restored not only the growth response to EPO, but also the
activation of Stat1 and Stat3.13 These results suggested
that Stat1 is at least partly involved in EPO-induced cell growth.
In the present study, we examined the roles of Stat1 and Stat3 in
EPO-induced erythroid differentiation, because STAT proteins appear to
play some role in hematopoiesis.16-18 For example, Stat3 plays a critical role in interleukin-6 (IL-6)-induced macrophage differentiation and granulocyte colony-stimulating factor
(G-CSF)-induced granulocytic differentiation.16-18
Although the concept is controversial at present, Stat5 may be involved
in EPO-induced erythroid differentiation.19,20 Thus, STAT
proteins appear to be involved in the differentiation of hematopoietic
cells. Because UT-7/GM and TF-1 cells can differentiate into erythroid
cells after exposure to EPO,14,15 the loss of activation of
Stat1 and Stat3 by EPO may be involved in the EPO-induced erythroid
differentiation of these cell lines. To test this possibility, we used
UT-7/GM transfectant cells exogenously expressing Stat1 and/or Stat3 proteins13 and examined the biological
functions of these Stat proteins in EPO-induced erythroid
differentiation. We show here that both Stat1 and Stat3 proteins
inhibit the EPO-induced erythroid differentiation of UT-7/GM cells.
| |
MATERIALS AND METHODS |
Cells.
The UT-7 cell line was derived from the bone marrow of a patient with
megakaryoblastic leukemia.10 UT-7 and UT-7/GM cells were
maintained in liquid culture with Iscove's modified Dulbecco's medium
(IMDM; GIBCO Laboratories, Grand Island, NY) containing 10% fetal calf
serum (FCS; Hyclone Laboratories, Logan, UT) and 1 ng/mL
granulocyte-macrophage colony-stimulating factor (GM-CSF).
Hematopoietic growth factors and reagents.
Recombinant human EPO was a gift from the Life Science Research
Institute of the Snow Brand Milk Company (Tochigi, Japan). Recombinant
human GM-CSF was provided by Sumitomo Pharmaceutical Co (Osaka, Japan).
Neomycin (G418) was purchased from GIBCO BRL Life Technologies
(Gaithersburg, MD). Human erythroid-specific 5-aminolevulinate
synthetase (ALAS-E) cDNA was provided by Dr M. Yamamoto (Tsukuba
University, Tsukuba, Japan). Human -globin cDNA and a human
ribosomal DNA probe were provided by the Japanese Cancer Research
Resources Bank (Tokyo, Japan). The human EPOR cDNA was a gift from Dr
H. Nakauchi (Tsukuba University, Tsukuba, Japan).
Dianisidine staining.
Hemoglobin concentrations were examined by incubating cells in
serum-free IMDM medium containing 0.2% 3,3 -dimethoxybenzidine, fast blue B (dianisidine; Sigma Chemicals, St Louis, MO), 0.3% acetic
acid, and 0.3% H2O2 for 30 minutes at room
temperature (RT).21
Preparation of nuclear and cytoplasmic extracts.
After stimulation, cells were washed with ice-cold phosphate-buffered
saline (PBS) containing 2 mmol/L Na3VO4,
resuspended in a hypotonic buffer (20 mmol/L HEPES [pH 7.9], 10 mmol/L KCl, 1 mmol/L MgCl2, 10% glycerol, 0.5 mmol/L
dithiothreitol [DTT], 1 mmol/L phenylmethylsulphonyl fluoride
[PMSF], 15 µg/mL aprotinin, 3 µg/mL leupeptin, 3 µg/mL
pepstatin, and 2 mmol/L Na3VO4)
with 0.2% Nonidet P-40 (NP-40) and then homogenized. After
centrifugation at 1,000g for 5 minutes, the supernatant was
separated from the nuclear pellet and then centrifuged at
14,000g for 20 minutes at 4°C. The debris was removed, and
the supernatants were collected as cytoplasmic extracts. The nuclear
pellets were resuspended in hypotonic buffer with 300 mmol/L NaCl,
debris was removed by centrifugation (14,000g for 20 minutes),
and the supernatants were collected as nuclear extracts.
Electromobility shift assay (EMSA).
Nuclear or cytoplasmic extracts (5 µg of protein) were incubated for
15 minutes at 4°C in 10 mmol/L Tris-HCl, pH 7.5, 75 mmol/L KCl, 1 mmol/L DTT, 1.5 µg of poly dIC/dAT, 1 mmol/L EDTA, and 4% Ficoll
type 400, with 2 ng of a 32 P-labeled oligonucleotide.
Samples were loaded on a 5% nondenaturating polyacrylamide gel, run
for 1.5 hours at 150 V, vacuum-dried, and exposed to x-ray film. For
the supershift study, nuclear extracts were incubated with antibodies
at RT for 10 minutes before the above reaction. The probes were
double-stranded oligonucleotides corresponding to the sis-inducible
element (SIE; upper strand, 5-GTCGACATTTCCCGTAAATC-3)22 and the
 -casein promoter region (-CAP; upper strand,
5-AGATTTCTAGGAATTCAAATC-3).23 In the study of
competition, extracts were incubated with a 150 molar excess of the
unlabeled probe.
Generation of stable transfectants.
UT-7/GM cells were transfected with mammalian expression vector
(pCAGGSneo) alone or pCAGGSneo containing human Stat1 cDNA, murine
Stat3 cDNA,14 or human EPOR cDNA by conventional
electroporation (1,000 V, 25 µFD). We selected 3 independent clones
resistant to neomycin (0.8 mg/mL). To make UT-7/GM cells that
overexpressed both Stat1 and Stat3, we constructed a pCAGGS-BSD
vector containing a blasticidin-resistant gene.24
The tyrosine residues at 701 of Stat1 and at 705 of Stat3 were replaced
with phenylalanine to make Stat1F and Stat3F,
respectively.16 Stat1F or Stat3F cDNA also was inserted
into pCAGGS vector and introduced into UT-7/GM cells under the above
conditions.
Luciferase assay.
UT-7/GM cells were transfected with DNA by the lipofectin method
according to the manufacturer's instructions (Promega, Madison, WI).
Typically, 1.0 µg of one of the reporter plasmids containing the
firefly luciferase gene and 1 µg of pSV--galactosidase (Promega), an expression vector containing the LacZ gene encoding
-galactosidase as an internal control for transfection efficiency
were used. Two micrograms of the pCAGGS-Neo expression vector system
with a cDNA encoding HA-Stat1, HA-Stat3, or their derivatives were cotransfected. After transfection, cells were starved for 24 hours and
stimulated with 10 ng/mL of GM-CSF for 6 hours. The cells were then
collected in 100 µL lysis buffer and subjected to the assay for
luciferase and -galactosidase activity. The reporter genes contain 4 copies of the acute-phase response element (APRE), which is
transactivated by Stat1 and Stat3, in front of the minimal junB promoter linked to the luciferase gene
(4XAPREluc).16
Colorimetric MTT assay for cell proliferation.
Cell growth was also examined by a colorimetric assay according to
Mosmann,25 with some modification. Briefly, cells were incubated at a density of 1 × 104/0.1 mL in 96-well
plates in IMDM containing 10% FCS in the absence or presence of
various concentrations of GM-CSF or EPO. After 72 hours of culture at
37°C, 20 µL of sterilized 5 mg/mL MTT
[3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide; Sigma]
was added to each well. After 2 hours of incubation at 37°C, 100 µL of 10% sodium dodecyl sulfate (SDS) was added to each well to
dissolve the dark-blue crystal product. The optical density (OD) was
measured at a wavelength of 595 nm using a microplate reader (model
3550; Bio-Rad, Richmond, CA).
RNA extraction and Northern blotting.
Total RNA was isolated from cells according to the method of
Chomczynski and Sacchi.26 RNA was resolved by
electrophoresis on agarose formaldehyde gels, transferred to nylon
membranes (Zeta-probe; Bio-Rad) in 10× standard sodium citrate
(SSC), and hybridized to human cDNA fragments for ALAS-E or -globin.
The fragment was labeled with 32P-CTP by random-priming.
After an overnight incubation at 43°C in the presence of 50%
formamide, blots were washed 3 times with 2× SSC, 0.5× SSC,
or 0.1× SSC containing 0.1% SDS for 15 minutes each. The
membranes were autoradiographed using Kodak XAR-5 film (Eastman Kodak,
Rochester, NY) with an intensifying screen at  70°C.
Cell cycle analysis.
A cell cycle analysis was performed by staining DNA with propidium
iodide in preparation for flow cytometry with the FACScan/ModFit LT system (Becton Dickinson, San Jose, CA).
| |
RESULTS |
Correlation between the GM-CSF-induced activation of
Stat1 and Stat3 and the GM-CSF-induced inhibition of
EPO-induced erythroid differentiation.
We previously reported that Stat1, Stat3, and Stat5 were commonly
activated by EPO in UT-7 cells and that EPO induced the activation of
Stat5 but not of Stat1 or Stat3 in UT-7/GM cells.13 In
addition, we demonstrated that UT-7/GM cells can differentiate along
the erythroid lineage in the presence of EPO and that GM-CSF inhibited
the EPO-induced erythroid differentiation of UT-7/GM cells in a
dose-dependent manner.14 Collectively, the activation of
Stat1 and Stat3 may be closely involved in the inhibition by GM-CSF
of EPO-induced erythroid differentiation.
To test this notion, we examined whether SIE-binding complexes formed
after stimulation with EPO (10 U/mL) plus GM-CSF (10 ng/mL). About 80%
of the cells became dianisidine-positive after 7 days of exposure to
EPO alone (Fig 1A). By contrast, GM-CSF strongly reduced the percentage of dianisidine-positive cells, even in
the presence of high-dose EPO (Fig 1A). GM-CSF activated Stat1 and
Stat3 at 10 ng/mL, at which concentration the dianisidine-positive cells completely disappeared (Fig 1B). However, Stat5 was commonly activated by GM-CSF and EPO (Fig 1B). Thus, there seems to be a
positive correlation between the GM-CSF-induced activation of Stat1
and Stat3 and the GM-CSF-induced inhibition of EPO-dependent erythroid
differentiation.
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| Fig 1.
Activation by GM-CSF of Stat1 and Stat3 and
suppression by GM-CSF of EPO-induced erythroid differentiation. (A)
UT-7/GM cells were cultured with GM-CSF (10 ng/mL) and/or EPO
(10 U/mL). Seven days later, cells were harvested for dianisidine
staining. The data are the means ± standard deviation (SD) of
triplicate cultures. (B) Growth factor-starved UT-7/GM cells were
treated with EPO (10 U/mL) and/or GM-CSF (10 ng/mL) for 15 minutes. Nuclear extracts were then prepared for an EMSA with
32P-labeled SIE or -CAP probes. Arrows A, B, and C
indicate a homodimer of Stat3, a heterodimer of Stat1 and Stat3, and
a homodimer of Stat1, respectively (see Kirito et al13
and text).
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Overexpression of Stat1 or Stat3 proteins suppressed
the EPO-induced erythroid differentiation.
To examine whether the activation of Stat1 and Stat3 inhibits the
EPO-induced erythroid differentiation, we used Stat1- or
Stat3-transfected UT-7/GM cells that we generated
previously.13 We confirmed that Stat1 and Stat3 proteins
are overexpressed and constitutively activated in each type of
transfectant cells (Kirito et al13 and data not shown).
UT-7/GM clones transfected with vector alone were used as controls.
These transfectant cells were cultured with 10 U/mL EPO for 7 days and
then harvested for dianisidine staining. Dianisidine-positive cells
were not observed before exposure to EPO, but 70% to 80% of the
control cells were positive for dianisidine after 7 days of exposure to
EPO (Fig 2A). The percentage of
dianisidine-positive cells decreased to 40% to 45% in the Stat1 or
Stat3 transfectant clones (Fig 2A). We also evaluated the expression
levels of -globin or ALAS-E mRNA, both of which are involved in
hemoglobin synthesis. EPO enhanced the expression level of both genes
in the control cells. Although the expression levels of these two genes
were elevated by the EPO treatment of Stat1 or Stat3 transfectants,
the degree of elevation was lower than that in the control cells (Fig
2B). This finding is consistent with dianisidine-staining.
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| Fig 2.
Effects of overexpression of Stat1 and/or
Stat3 on the EPO-induced differentiation of UT-7/GM cells. (A) UT-7/GM
clones transfected with vector alone (lane 1; n = 10),
Stat1-transfected clones (lane 2; n = 10), Stat3-transfected
clones (lane 3; n = 10), and Stat1 and Stat3-cotransfected clones
(lane 4; n = 3) were cultured with EPO (10 U/mL). Seven days later,
cells were harvested for dianisidine-staining. The data are the mean ± SD. (B) Parent UT-7/GM cells, Stat1-transfected clones (clones
29, 32, and 47), and Stat3-transfected clones (clones 5, 12, and 29)
were cultured with EPO (10 U/mL) and harvested for the isolation of
total cellular RNA. -Globin and ALAS-E transcripts were detected by
Northern blotting. The membrane was rehybridized with a
32P-labeled human ribosomal DNA probe to show the amounts
of RNA loaded.
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Stat1 suppressed the prolongation of the G0/G1 phase of the cell
cycle after EPO stimulation.
Cellular proliferation and differentiation are closely associated with
cell cycle events. For example, G0/G1 prolongation is a critical event
for erythroid differentiation.27,28 We previously reported
that prolongation of the G0/G1 phase of the cell cycle was required for
the EPO-induced erythroid differentiation of UT-7/GM
cells.14 This observation prompted us to examine whether
Stat proteins inhibited the EPO-induced erythroid differentiation through an effect on cell cycle progression. After the 24-hour deprivation of growth factors, parent UT-7/GM or transfectant cells
were exposed to EPO for various periods, and a cell cycle analysis was
performed using flow cytometry. The percentage of G0/G1 cells at the
start time was 50% to 55% in all clones. The G0/G1 percentage
decreased after 12 to 24 hours of exposure to GM-CSF in both the
parental and transfectant cells (Fig 3A and B). By contrast, there was a large difference in the cell cycle patterns after EPO treatment. In the parental UT-7/GM and Stat3 transfectant cells, the percentage of G0/G1 cells was unchanged even
after 48 hours of culture with EPO (Fig 3B). However, when Stat1-transfected cells were cultured with EPO, the percentage of
G0/G1 cells was reduced to 35% to 40% after 24 to 48 hours of
exposure to EPO (Fig 3A). These kinetics were almost identical to those
produced by GM-CSF treatment (Fig 3A). These results may explain our
previous observation that the cells transfected with Stat1 grew
better than did the parent or Stat3-transfected cells.13
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| Fig 3.
Cell cycle analysis. Parental UT-7/GM cells,
Stat1-transfected clones (A; clones 29 and 47), or Stat3-transfected
clones (B; clones 5 and 29) were starved for 24 hours. The cells were subsequently stimulated with GM-CSF (10 ng/mL, left panel) or EPO (10 U/mL, right panel) and harvested at the indicated times for cell cycle
analysis. The percentage of cells in G0/G1 was determined by a ModFit
LT program. The data are the means of three independent
experiments.
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Activation of both Stat1 and Stat3 additively
inhibited the EPO-induced erythroid differentiation.
Shortening of the G0/G1 phase after EPO stimulation occurred in the
cells transfected with Stat1 but not in those transfected with Stat3
cDNA. These results imply that Stat1 and Stat3 inhibit EPO-induced
erythroid differentiation through distinct pathways. If so, the
activation of both Stat proteins could be expected to reduce the
erythroid differentiation additively or synergistically. To test this
notion, we introduced Stat3 cDNA into a Stat1-transfected clone
(clone 29) and selected 3 independent clones overexpressing both
Stat1 and Stat3 by Western blotting analysis (data not shown). The
constitutive activation of Stat1 and Stat3 was observed in these
clones (Fig 4). However, the growth
patterns in these clones were unchanged; the clone cells required
GM-CSF or EPO for growth and survival (data not shown). Only 20% to
30% of these cells became dianisidine-positive after EPO treatment
(Fig 2A). However, cell cycle patterns of the cells doubly transfected
with Stat1 and Stat3 were similar to those of the cells transfected
with Stat1 alone (data not shown).
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| Fig 4.
Establishment of UT-7/GM cells coexpressing Stat1 and
Stat3. UT-7/GM cells were cotransfected with Stat1 and Stat3 cDNA. UT-7/GM and transfectant cells were deprived of growth factors for 24 hours and then incubated with GM-CSF (10 ng/mL) or EPO (10 U/mL) for 15 minutes. Nuclear extracts were then prepared from the cells, and an
EMSA was performed using 32P-labeled SIE probes.
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Overexpression of the full-length EPOR also decreased the EPO-induced
erythroid differentiation.
Overexpression of the full-length EPOR (UT-7/GM-EPOR) restored the
growth response to EPO in UT-7/GM cells (Kirito et al13 and
data not shown). Moreover, we confirmed by EMSA that Stat1 and Stat3
proteins were activated by EPO in these transfectant cells (Kirito et
al13 and data not shown).
We examined whether EPOR overexpression affects the EPO-induced
erythroid differentiation of UT-7/GM cells. We confirmed by Northern
blotting that the parental UT-7/GM cells expressed EPOR at low levels,
whereas the transfectant cells abundantly expressed EPORs
(Fig 5A). Transfectant cells were cultured
with EPO for 7 days and then harvested for dianisidine staining and
Northern blot analysis. The percentages of dianisidine-positive cells
in the EPOR-transfected cells were much lower than in the parental UT-7/GM cells (Fig 5B; 0% v 80%). EPO induced EPOR, ALAS-E,
and -globin genes at the mRNA level in the parental UT-7/GM cells, whereas neither ALAS-E nor -globin gene was induced in the
EPO-treated UT-7/GM-EPOR cells (Fig 5A). This result was consistent
with dianisidine staining. The percentage of cells in G0/G1 was clearly
reduced after EPO treatment in the UT-7/GM-EPOR cells but not in the
parental UT-7/GM cells (Fig 5C), suggesting that G0/G1 prolongation did not occur in the UT-7/GM-EPOR cells.
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| Fig 5.
Effects of overexpression of EPOR on the EPO-induced
erythroid differentiation of UT-7/GM cells. (A) Parent UT-7/GM and
UT-7/GM-EPOR cells were treated with EPO (10 U/mL) for 7 days, and
total cellular RNA was isolated. The transcripts of EPOR, ALAS-E, and
-globin were examined by Northern blotting. The membrane was
rehybridized with a 32P-labeled human ribosomal DNA probe
to show the amounts of RNA loaded. (B) Dianisidine-staining.
UT-7/GM-EPOR cells were cultured with 10 U/mL of EPO for 7 days. The
cells were harvested for dianisidine staining. The data are the mean ± SD of triplicate cultures. (C) Parent UT-7/GM cells and
UT-7/GM-EPOR cells were starved for 24 hours. The cells were stimulated
with GM-CSF (10 ng/mL, left panel) or EPO (10 U/mL, right panel) and
harvested for cell cycle analysis. The percentage of cells in G0/G1 was
determined. The data are the means of three independent experiments.
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Effect of dominant negative forms of Stat1 and Stat3
on the inhibition by GM-CSF of EPO-induced erythroid differentiation.
To confirm that Stat1F and Stat3F function as the dominant negative
forms in our system,16,29,30 we cotransfected these cDNAs
and 4XAPREluc into the parent UT-7/GM cells. After 24 hours of
starvation, these transfectant cells were stimulated with GM-CSF, and
the cells were harvested 6 hours later for a luciferase assay. As shown
in Fig 6A, the luciferase activity was
clearly reduced in the Stat1F and Stat3F transfectant cells, indicating
that these dominant-negative forms work well in our system. In
addition, we confirmed by EMSA that the dominant-negative Stats
actually inhibited the DNA binding activities of the wild-type Stats in UT-7/GM cells (data not shown).
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| Fig 6.
Effects of dominant-negative forms of Stat1 and Stat3
on the inhibition by GM-CSF of EPO-induced erythroid differentiation. (A) UT-7/GM cells were transfected with a plasmid DNA mixture (1 µg
of reporter genes containing 4 copies of APRE in front of the minimal
junB promoter linked to the luciferase gene, 2 µg of either
expression vector pCAGGS-Neo, with no insert or with an insert of Stat
cDNA encoding either HA-Stat3F or HA-Stat1F, and 1 µg of
pSV--galactosidase. After transfection, the cells were cultured
without growth factors and then stimulated with GM-CSF (10 ng/mL) for 6 hours. Luciferase values were normalized for -galactosidase activity
and expressed relative to the normalized luciferase activity in the
extracts from unstimulated cells transfected with the reporter plasmids
and a control expression plasmid. The data are the mean ± SD of more
than four independent experiments. (B) MTT reduction assay. Cells were
plated at a density of 104/well in IMDM supplemented with
5% FCS and cultured with GM-CSF (10 ng/mL). MTT reduction was measured
after 3 days of culture. The data are the mean ± SD of triplicate
cultures. (C) Dianidisine staining. Cells were cultured in the presence
of GM-CSF (10 ng/mL) or a combination of EPO (10 U/mL) and GM-CSF (10 ng/mL). Seven days later, cells were harvested for dianisidine
staining. The data are the mean ± SD from three independent clones.
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To investigate the functional roles of Stat1 and Stat3 in erythroid
differentiation, we generated stable transfectant cells exogenously
expressing Stat1F or Stat3F. An MTT assay showed that Stat1F but not
Stat3F partially inhibited the GM-CSF-induced cell growth of UT-7/GM
cells (Fig 6B). Then, these transfectant cells were cultured with
GM-CSF alone (10 ng/mL), EPO (10 U/mL), or a combination of GM-CSF (10 ng/mL) and EPO (10 U/mL) for 7 days and then harvested for dianisidine
staining. GM-CSF completely blocked the EPO-induced erythroid
differentiation in the parental UT-7/GM cells. However, unlike the
parental UT-7/GM cells, about 20% of the cells transfected with Stat1F
or Stat3F showed the positivity for dianisidine staining after exposure
to EPO, even in the presence of GM-CSF (Fig 6C). Approximately 80% of
the cells treated with EPO became positive for dianidisine staining in
these transfectant cells (Stat1F, 75.0% ± 2.7%; Stat3F, 75.4% ± 5.0%), as in the case of the parental UT-7/GM cells (76.5% ± 8.7%).
| |
DISCUSSION |
In this study, we established the functional role of Stat proteins in
EPO-induced erythroid differentiation. The forced activation of
Stat1 and/or Stat3 suppressed the induction of -globin
and the erythroid-specific ALAS gene, resulting in decreased hemoglobin synthesis. In addition, UT-7/GM cells transfected with EPOR cDNA restored the EPO-induced activation of Stat1 and Stat3 and acquired high responsiveness to EPO for cell proliferation. However, these cells
lost their capacity to become hemoglobin-positive in the presence of
EPO. Dominant negative forms of Stat1 or Stat3 promoted EPO-induced
erythroid differentiation even in the presence of GM-CSF. These results
indicate that both Stat1 and Stat 3 act as negative regulators of
EPO-induced erythroid differentiation.
The regulation of cell cycle progression appears to be involved in the
cellular switch to the proliferation and differentiation of erythroid
progenitor cells.27,28 Carroll et al27 reported that prolongation of the G0/G1 phase was required for EPO-induced erythroid differentiation. We recently found that the addition of
GM-CSF reduced the G0/G1 prolongation, accompanied by a reduction in
the ratio of dianisidine-positive cells.14 In addition, we showed here that the inhibition by GM-CSF of EPO-induced erythroid differentiation correlated with the activation of Stat1 and Stat3 (Fig 1A and B) and that the activation of Stat1 promoted the entry
of UT-7/GM cells into the cell cycle. This notion was also supported by
the finding that Stat1F but not Stat3F inhibited the GM-CSF-induced
cell growth of UT-7/GM cells. Taken together with reports that Stat1
is also involved in the proliferative effects of platelet-derived
growth factor (PDGF) and epidermal growth factor (EGF),31
our data raise the possibility that Stat1 is directly or indirectly
involved in the activation of cell cycle-associated gene transcription.
There are several lines of evidence that the activation of Stat
proteins is involved in the cell cycle. It was demonstrated that a
dominant-negative form of Stat3 inhibited growth arrest at the G0/G1
phase induced by IL-6 in a murine leukemic cell line, M1.16
In addition, another group reported the EGF- or interferon- (IFN-)-induced growth arrest of A431 or HT29 cells by the
upregulation of the cyclin-dependent kinase inhibitor, p21; this was
mediated through the binding of activated Stat1 to the promoter of
the p21 gene.32 Thus, Stat1 and Stat3 proteins appear to
play a role in the blockade of cell cycle progression. However, this notion is inconsistent with our present finding that Stat1 proteins promoted entry into the cell cycle. This discrepancy may be explained partly by the evidence that p21 acts not only as a negative regulator, but also as a positive regulator of cell cycle
progression.33,34
It is apparent that the inhibition by Stat3 of the EPO-induced
erythroid differentiation of UT-7/GM cells is independent on the cell
cycle. Indeed, the forced overexpression of Stat3 had no effect on the
EPO-induced proliferation of UT-7/GM cells.13 Moreover,
Stat3 promotes the differentiation of M1 cells to macrophages. Very
recently, it was found that Stat3 inhibits the neural differentiation of PC12 cells.29 Whereas all of these data were obtained
using immortalized cell lines, it was recently shown that Stat3 is
involved in the maintenance of embryonic stem (ES) cells by leukemia
inhibitory factor (LIF); the Stat3 dominant-negative form induced
morphological differentiation of ES cells even in the presence of
LIF.35 Thus, it is unlikely that the inhibitory effects of
Stat3 on differentiation are limited to immortalized cells. Stat3 could
also be expected to play an important role in normal development,
presumably as an inhibitor of differentiation.
Although the dominant-negative forms of Stat1 and Stat3 certainly
inhibited the GM-CSF-induced activation of Stat1 and Stat3, the
majority of the transfectant cells did not become dianisidine-positive in the presence of GM-CSF alone, as was the case for the parental UT-7/GM cells. However, unlike the parent cells, about 20% of the
transfectant cells became dianidisine-positive after exposure to EPO,
even in the presence of GM-CSF (Fig 6C). These observations strongly
suggest that the loss of activation of Stat1 and Stat3 is required,
but is not sufficient by itself, for EPO-induced erythroid
differentiation, and that the EPO signal(s) is a prerequisite for
erythroid differentiation. In addition, the finding that the constitutive activation of Stat1 and Stat3 proteins partially but
not completely inhibited EPO-induced erythroid differentiation of
UT-7/GM cells suggests that other molecules such as Lyn36 and SHP-137 may be in part involved in the suppression by
GM-CSF of the EPO-induced erythroid differentiation of UT-7/GM cells.
The constitutive activation of Stat1 or Stat3 is frequently observed
in leukemia cells.38-40 In addition, Stat1 and Stat3 are
activated by Bcr-Abl products41 and
src-kinase,42,43 respectively. The infection of Friend
spleen focus-forming virus in the EPO-dependent murine cell line HCD 57 causes cytokine-independent cell growth and the constitutive activation
of Stat1 and Stat3.44 A mutation in the Drosophilia
homolog of the JAK kinase gene causes the hyperactivation of this
signal transduction pathway and subsequent leukemia-like hematopoietic
defects.45-47 Thus, aberrations in the JAK-STAT pathway
could cause the development of hematologic malignancy. Although the
constitutive activation of Stat1 and/or Stat3 blocked the
EPO-induced erythroid differentiation of these transfectant cells, we
did not observe autonomous cell growth (Kirito et al13 and
data not shown). These results indicate that the constitutive
activation of Stat1 and/or Stat3 is insufficient for the
autonomous growth of leukemia cells in vitro. The mechanism by which
exogenous expression of Stat1 and Stat3 in UT-7/GM cells resulted in
constitutive activation of these molecules is unknown (Fig 4). Because
mere overexpression of Stat molecules, even in COS cells, usually does
not give rise to constitutive activation, the overexpressed Stat1
and Stat3 proteins might be showing the action of normally subthreshold
amounts of an autocrine factor.
Because Stat3 knockout mice have a lethal defect, it is difficult to
estimate the function of Stat3 on erythropoiesis.48 Stat1 knockout mice studies demonstrated that the elimination of the
Stat genes does not affect erythropoiesis.49,50 This seems
to contradict our observation. This discrepancy may be explained by two
possibilities. One is that other Stat proteins such as Stat3 compensate
for the lack of Stat1. The other is that Stat1 is involved in
abnormal erythropoiesis, as shown by the results obtained using
leukemic cell line cells.
It is also interesting that most of the UT-7/GM cells lost the capacity
to differentiate into erythroid cells by overexpression of the
full-length EPOR. By contrast, these transfectant cells had a restored
growth response to EPO and activation of Stat1 and Stat3 by
EPO.13 This indicates that Stat1 and Stat3 lie downstream of the EPO receptor in the EPO signaling pathway. However, Stat3-Stat3 homodimers were almost undetectable in the EPO-treated UT-7/GM-EPOR cells (data not shown). This may be explained by one
possibility that the ratio of activated Stat3 to activated Stat1 by
EPO is relatively small, resulting in the increased formation of
Stat1-Stat3 heterodimers and the decreased formation of Stat3-Stat3
homodimers.
It seems likely that the inability of UT-7/GM cells to proliferate in
response to EPO is due to low levels of endogenous EPO receptor
expression. Although we cannot completely exclude the possibility that
decreased sensitivity to EPO is due to the presence of mutations in the
endogenous EPO receptor, we and another group demonstrated that the
original UT-7 cells expressed structurally normal EPOR
mRNA.51,52
Whether Stat5 plays a critical role in EPO-induced erythroid
differentiation is still controversial. Chretien et al19
reported that the activation of Stat5 promotes cell growth and inhibits erythroid differentiation in a human erythroleukemia cell line. By
contrast, Iwatsuki et al20 found that the activation of
Stat5 leads to erythroid differentiation in a murine erythroleukemia cell line. We observed that EPO induced erythroid differentiation in
UT-7/GM cells but not in UT-7 cells. However, there was no significant
difference in the degree of Stat5 activation by EPO between these cell
lines (Kirito et al13 and data not shown). Although the
possibility that this discrepancy is caused by differences in the cell
lines cannot be excluded completely, our data suggest that Stat5
activation is insufficient for EPO-induced erythroid differentiation. A
separate factor(s) is required for the terminal maturation of erythroid
cells.
It is still controversial as to whether EPO induces the activation of
Stat1 and/or Stat3 as well as Stat5. Some investigators reported that Stat5, but not Stat1 or Stat3, is activated by EPO in
Ba/F3 cells transfected with EPOR cDNA.53,54 To test this,
we also generated Ba/F3 cells exogenously expressing human EPOR and
examined whether EPO induces the activation of Stat1 and Stat3
proteins in these transfectant cells. As reported previously, neither
of these Stat proteins was activated by EPO stimulation, although Stat5
protein was strongly activated by EPO treatment (data not shown). Thus,
the data obtained from Ba/F3 transfectant cells were in contrast with
those from the UT-7 cells expressing endogenous human EPOR. Although we
cannot completely exclude the possibility that this discrepancy may be
due to the different cell lines used in these experiments or the
different EPOR number on the surface of the cells, it is likely that
EPO induces the activation of Stat1 and/or Stat3 proteins in
primary erythroid cells and cell lines abundantly expressing endogenous
EPOR.13,44,55,56
In summary, we propose a novel function of Stat1 and Stat3 in
EPO-induced erythroid differentiation of a leukemia cell line; Stat1
may inhibit EPO-induced erythroid differentiation, presumably mediated
through the activation of cell cycle-associated gene(s). Although the
roles of Stat1 and Stat3 in normal erythropoiesis remains to be
undetermined, the constitutive activation of these Stat proteins may be
involved in the erythroid differentiation arrest in erythroleukemia.
| |
FOOTNOTES |
Submitted December 30, 1997;
accepted March 18, 1998.
Supported by Grants-in-Aid for Cancer Research and Scientific Research
from the Ministry of Education, Science and Culture of Japan and by a
grant from the Yamanouchi Foundation for Research on Metabolic
Disorders.
Address reprint requests to Norio Komatsu, MD, PhD, Division of
Hematology, Department of Medicine, Jichi Medical School
Minamikawachi-machi, Tochigi-ken 329-04, Japan; e-mail:
nkomatsu{at}ms.jichi.ac.jp.
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 the Life Science Research Institute of Snow Brand
Milk Co for providing recombinant human EPO. We are also grateful to
Tomoko Ando for technical assistance and Motoko Yoshida for manuscript
preparation.
| |
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Abstract
Introduction
Abstract