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RAPID COMMUNICATION
Induction of Sequence-Specific DNA-Binding Factors by Erythropoietin and
the Spleen Focus-Forming Virus
By Takashi Ohashi, Michiaki Masuda, and Sandra K. Ruscetti
The signal transduction mechanism of
erythropoietin (Epo),
whichregulates growth and differentiation of erythroid
cells, is still unclear. Recent studies showing the activation
by various ligandsof a group of proteins called Stat (signal
transducers and activators of transcription) proteins raised
the possibility that such proteins may also be involved in
the Epo signal transduction
pathway. In this report,we show
that Epo induces factors that specifically bind to the sisinducible element and the gamma response region of the
Fcyreceptorfactor I gene in the Epo-dependentmouse
erythroleukemiacell line HCD-57.Thesefactorscontain
phosphotyrosineandantibodiesagainst
Statl and Stat3
proteins reacted with them. In HCD-57 cells infected with
Friend spleen focus-forming virus, which now grow in an
Epo-independentmanner, the DNA-bindingfactors were
constitutively activated even in the absence of Epo. These
results suggestthat thefactors induced
by Epo contain components identicalor related to known Stat proteins. It is also
suggested that continuous activationof these DNA-binding
factors may be responsible for the ability of spleen focusforming virus to abrogate the Epo-dependence of HCD-57
cells and cause erythroleukemia in susceptible mice.
0 1995 by The American Society of Hematology.
E
RYTHROPOIETIN (Epo) is a growth factor that plays
a major role in the regulation of the growth and differentiation of hematopoietic cells in the erythroid lineage. The
genes encoding Epo and the Epo receptor (EpoR) have been
isolated, and the relationship between their structure and
function has been studied extensively.”6However, the actual
mechanism by which binding of Epo to the EpoR triggers
the signal for erythroid cell growth and differentiation is
largely unclear. Previous studies showed that stimulation of
the EpoR by Epo induces tyrosine phosphorylation of various cellular substrates, such as the EpoR itself:-9 the 85-kD
regulatory subunit of phosphatidylinositol 3-kinase,IoShc,”
R&-1,” and an unidentified 97-kD protein.I3 Recent studies
also showed that Janus kinase 2 (JAK2) is associated with
a membrane-proximal region of the EpoR and phosphorylated at its tyrosine residue in response to Epo stimulati~n.’~.’’
Activation of JAK2 kinase appears to be essential
for Epo-induced signal transduction.16
There are a family of proteins called Stat (signal transducers and activators of transcription) proteins, which include
Statl and Stat2, that have been identified through the study
of transcriptional regulation in response to stimulation by
interferon-a (IFN-a) and IFN-y.’”*’ There are an increasing
number of studies showing that a variety of other cytokines,
hormones, and growth factors can activate a certain set of
Stat proteins, including Statl through Stat5 and interleukin4 (IL-4) Stat.**-**
After ligand stimulation, specific Stat proteins appear to be phosphorylated at their tyrosine residues
in the cytoplasm, to be transported into the nucleus, and to
bind to certain target sequences, such as the sis-inducible
element (SIE) and the gamma response region (GRR) of the
FCy receptor factor I gene.
Because some of the protein substrates phosphorylated by
Epo stimulation have molecular weights similar to those of
Stat proteins and activation of JAK2 kinase was shown to
be associated with phosphorylation of Stat protein^:^*^' the
possibility was raised that these proteins might be involved
in the Epo signal transduction pathway. Recently, itwas
shown that a sequence-specific DNA-binding activity that
interacts with the GRR sequence was induced in the human
erythroleukemia cell line TF1 in response to Epo stimulation.
However the relationship of the DNA-binding factor to Stat
proteins was unclear.” In this study, we show that DNAbinding factors that bind specifically to the SIE and GRR
sequences are rapidly and transiently induced after Epo stimulationin the Epo-dependent mouse erythroleukemia cell
line HCD-57 and that these factors contain components detected by antiphosphotyrosine antibodies and antibodies
against Statl or Stat3 proteins. We also demonstrate that
infection of HCD-57 cells with the polycythemic strain of
Friend spleen focus-forming virus (SFFV,), which causes
acute erythroblastosis and erythroleukemia in susceptible
mice and abrogates Epo-dependent growth of HCD-57
cells,3*can activate both the SIE-binding and the GRR-binding factors constitutively. Our results suggest that DNAbinding factors identical or related to the known Stat proteins
are involved in the Epo signal transduction pathway and that
constitutive activation of these factors can cause autonomous
cell growth that leads to hematopoietic hyperplasia and leukemia.
Fromthe Laboratory of Molecular Oncology, National Cancer
Institute, Frederick, MD.
Submitted November 2, 1994; accepted December 28, 1994.
Address reprint requests to Sandra K. Ruscetti, PhD, Bldg 469,
Room 140, NCI-FCRDC, Frederick, MD 21702-1201.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1995 by The American Sociev of Hematology.
OOO6-4971/95/8506-0036$3.00/0
Cells. HCD-57 cells are Epo-dependent mouse erythroleukemia
cells derived from an NIH Swiss mouse infected with Friend murine
leukemia virus (F-MuLV) at birth.”.” HCD-SFFV cells are HCD57 cells chronically infected with SFFV,, which abrogated the requirement of Epo for cell growth.’* HCD-57 cells were maintained
in Epo-containing conditioned medium as previously described.”
HCD-SFFV cells were maintained in the same medium without Epo.
For Epo stimulation, cells grown in the regular medium to the
density of 1 x 10‘ cells/mL were collected by centrifugation at
1,OOOg for 5 minutes at 4”C, resuspended in medium lacking serum
1454
MATERIALS AND METHODS
Blood, Vol85, No 6 (March 15). 1995: pp 1454-1462
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EPO- AND SFFV-INDUCED DNA-BINDING FACTORS
and Epo, and incubated in 5% CO2 at 37°C for 2 hours. After
starvation, the cells were collected again, resuspended in regular
medium with or without 100 U/mL of recombinant human Epo
(Ortho Biologic Inc, Raritan, PA; a kind gift from the R.W. Johnson
Pharmaceutical Research Institute), and incubated at 37°C. In some
experiments, 100 pg/mL of genistein (GIBCO-BRL, Gaithersburg,
MD) was added to the media during the starvation period. EL-4
mouse lymphoma cells used as a control for the RNA analysis experiment were grown in Dulbecco's modified Eagle's medium with
10% fetal calf serum (FCS).'4
Cell extracts. Cells were rinsed with ice-cold phosphate-buffered saline (PBS) containing 1 mmol/L Na3V04, resuspended in
ice-cold hypotonic buffer (20 mmol/L HEPES [pH 7.91, 10 mmoY
L KCl, 1 mmol/L MgCl,, 10% glycerol, 0.5 mmol/L dithiothreitol
[D'IT], 0.5 mmol/L phenylmethylsulphonyl fluoride [PMSFJ, 1 pg/
mL aprotinin, 1 pg/mL leupeptin, and 1 mmol/L Na3V04)with 0.2%
Nonidet P-40 (NP-40), and homogenized by a Dounce homogenizer.
After centrifugation at 1,OOOg for 5 minutes, the supernatant was
separated from the nuclear pellet. Cytoplasmic extracts were prepared by removing debris from the supernatants by centrifugation
(14,OOOg for 20 minutes) at 4°C. The nuclear pellets were rinsed with
hypotonic buffer containing 0.2% NP-40, resuspended in hypotonic
buffer with 300 mmol/L NaCl, and gently rocked for 30 minutes.
Residual nuclei were removed by centrifugation (14,OOOg for 20
minutes) at 4°C and the supernatants were collected as nuclear extracts. For whole cell extracts, cells were resuspended in ice-cold
extraction buffer (20 mmol/L HEPES [pH 7.91, 10 mmol/L KCl, 1
mmol/L MgC12, 150 mmol/L NaCl, 1% Triton X-100, 0.5 mmoyL
DIT, 0.5 mmol/L PMSF, 1 pg/mL aprotinin, 1 pg/mL leupeptin,
and 1 mmol/L Na3V04) and gently rocked for 30 minutes. After
centrifugation at 14,OOOg for 20 minutes at P C , the supernatant was
collected as a whole cell extract. The protein concentration of each
sample was determined using a Bio-Rad protein assay kit (Bio-Rad,
Hercules, CA).
The electrophoretic mobility shifr assay (EMSA). EMSAs were
performed as previously d e ~ c r i b e d .A~ ~cytoplasmic extract containing 30 pg of protein or a nuclear extract containing 10 pg of
protein was mixed with 5 pg of poly(dI)(dC) (Pharmacia, Piscataway, NJ) with or without an unlabeled competitor oligonucleotide
(100-fold molar excess over labeled probe) and incubated on ice for
10 minutes. Probe DNA labeled with Klenow fragment and [a"PIdCTP was then added so that the binding reaction mixture had
a total volume of 30 pL and contained a final concentration of 13
mmol/L HEPES (pH 7.9), 65 mmol/L NaCl, 1 mmol/L D=, 0.15
mmovL EDTA, and 8% glycerol. Then, the mixture was incubated
at room temperature for 30 minutes. In some experiments, antibodies
were added to the mixture of the extract and poly(dI)(dC) and incubated at 4°C for 16 hours before the labeled probe was added. After
the binding reaction, samples were subjected to electrophoresis in a
5% polyacrylamide gel containing 5% glycerol with 0% TBE
buffer for 2 hours at 200 V. The gels were then dried and exposed
to Kodak XR-5 film at -80°C. The probes used in this study were
double-stranded oligonucleotides corresponding to the high-affinity
S E (m67) ~equence'~
(5'-GTCGACATTTCCCGTAAATC-3')
and
the GRR sequence (5'-AAGGATGTATITCCCAGAAAAG-3').
Competitors were unlabeled SIE probe, GRR probe, and an oligonucleotide (5'-CGAAGCCTGATTCCGTAGAGCC-3')
corresponding
to a part of the P-globin gene promoter sequence.
Antibodies. Antiphosphotyrosinemonoclonal antibodies (MoAbs)
1G2, 25.2G4, 4G10, and PY-20 were purchased from Oncogene
Science (Uniondale, NY), Wako Chemicals (Osaka, Japan), Upstate Biotechnology Inc (UBI; Lake Placid, NY), and Transduction
Laboratories (Lexington, KY), respectively. The anti-Stat1 MoAb
raised against the N-terminal 194 amino acids of human Stat1 was
1455
purchased from Transduction Laboratories and is designated as
anti-StatlN antibody in this report. Another anti-Stat1 MoAb raised
against the aminoacid residues 613-739 of human Statl (designated
as anti-StatlC antibody) was purchased from Santa CNZ Biotechnology (Santa CNZ, CA).Anti-Stat2 antibody raised against amino
acids 813-831 of human Stat2 was purchased from UBI. Anti-Stat3
polyclonal antibody raised against amino acids 626-640 of mouse
Stat3 was purchased from Santa CNZ Biotechnology. Anti-p53
MoAb and antirabbit IgG polyclonal antibody were purchased from
Oncogene Science and Amersham Corp (Arlington Heights, IL),
respectively.
Protein analysis. Whole cell extracts were prepared as described
above and incubated with agarose-conjugated anti-StatlN or antiStatlC antibody for 2 hours at 4°C. After extensive washing with
the extraction buffer, samples were eluted from the agarose and
affinity-purified proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) using a 7.5% gel
and transferred electrophoretically to nitrocellulose filters. The filters
were incubated at room temperature for 1 hour in blocking buffer
(2% bovine serum albumin in 10 mmovL Tris-HC1 [pH 7.51 and
100 mmol/L NaCl), followed by the addition of antiphosphotyrosine
antibodies (PY20 and 4G10, each diluted 1:2,500). After incubation
at 4°C for 16 hours, the filters were rinsed in washing buffer (10
mmol/L Tris-HC1 [pH 7.51, 10 mmom NaCl, and 0.2% [voVvol]
Tween 20), incubated in horseradish peroxidase-conjugated antimouse IgG antibody (Amersham) at room temperature for 40
minutes, and rinsed in the washing buffer. Antibodies bound to the
filter were detected by the enhanced chemiluminescence method
(Amersham).
Reverse transcription-polymerasechain reaction (RT-PCR). Total
RNAs were obtainedfrom HCD-57 and E L 4 cells by using R N h l
(Tel-test Inc, Friendswood, TX) and reverse transcribed to cDNA by
using acDNA synthesis kit (Invitmgen, San Diego, CA). Downstream
primers used for cDNA synthesis were 5'"KGATGGAA'ITCAGCAAC"CTC"GA3', 5"'ITCTG'ITCTAGATCCTGCACTCGCI'-3',
and 5'-AGCr'ClTCTAGAAGCATGCTG'ITCA3'for Statl, Stat3,
and Stat4, respectively. For the PCR amplification, each of the upstream primers(5'-GGGGTACCGCAGGATGTCACAGTGGTTC-3'
for Statl, S'-GGGGTACCGCAGGATGGCTCAGTGGAAC-3'for
Stat3,5'-CiGGGTACCGCAGCATGTCTCAGTGGAAT-3'
for Stat4)
was added and30 cycles of denaturation (94°Cfor 1 minutes), annealing (55°C for 1 minutes),andpolymerization(72°C for 3minutes)
were performed according to the manufacturer's instructions (PerkinElmer/Cetus, Norwalk, CT). Some of the primers include additional
sequences providing restriction enzyme
sites for further cloning procedures. PCR products were electrophoresed in a 2%
agarose gel and
stained by ethidium bromide (Sigma, St Louis, MO). A I-kb DNA
ladder (GIBCO-BRL) was used as a molecular size standard.PCR
products were clonedin pGE"7Zf(+) and their nucleotide sequences
were determined by a Taq dye primer cycle sequencing system and a
DNA sequencer (373A; Applied Biosystems, Inc. Foster City, CA).
RESULTS
Epo stimulation induces transient activation of sequencespecific DNA-binding factors in HCD-57 cells. To determine if stimulation of HCD-57 cells by Epo induces DNAbinding factors that interact with the S E and GRR sequences
known to be recognized by Stat proteins, EMSAs were performed by using 32P-labeledoligonucleotide probes corresponding to these two sequences. Cells were starved for 2
hours in the medium depleted of serum and Epo, and then
the medium was changed to fresh regular medium with or
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1456
OHASHI, MASUDA,AND
A. S I E
B. GRR
+ + + +
- + + + +
-
- S
G C
- -
S
G
C
2
3
5
2
4
5
6
EPO
- -
Competitor
1
RUSCElTI
4
6
3
Fig 1. Induction of the SIEand the GRR-binding factors by
Epo in HCD-57 cells. Nuclear extracts were prepared from HCD57 cells after starvation (lane l),
15 minutes after incubation in
complete medium without (lane
2) or with (lane 3) Epo and incubated with a 3ZP-IabeledSIE (A)
or GRR (B) probe. Competition
assays were performed by adding a 100-foldmolar excess of an
unlabeled oligonucleotide corresponding to SI€ (lanes 4). GRR
(lanes 5). or p-globin promoter
sequences (lanes 6). Samples
were subjected to 5% polyacrylamide gel electrophoresis. Positions of the t w o SIE-binding
factor complexes and the GRRbinding complex are indicated
by arrows.
without Epo. No DNA-bindingactivity was detected by these
two probes before stimulation (Fig
1A and B. lanes 1 ) or
after the addition of the medium without Epo (Fig IA and
B, lanes 2). However, when the medium supplemented with
Epo was used, two mobility shift signals were detected by
the SIE probe (Fig I A , lane 3) and one signal was detected
by the GRR probe (Fig IB,
lane 3). The mobilityofthe
signal detected by the GRR probe was slower than that of
either signal detected by the SIE probe. The shifted bands
detected by the SIE probe disappeared when an excess of
unlabeled SIE or GRR probe was
included in the binding
reaction (Fig IA, lanes 4 and S). whereas the same amount
of an unrelated oligonucleotide had no effect (Fig IA, lane
6). The shifted band detected by the GRR probe was completely competed by the homologous GRR competitor (Fig
1B, lane S) and partially by the SIE probe (Fig 1 B, lane 4).
but not by the unrelated oligonucleotide (Fig 1B. lane 6 ) .
Toanalyzethe kineticsoftheinduction
of the DNAbindingfactors, we prepared cytoplasmicand nuclear extracts from HCD-S7 cells stimulated by Epo for different
periods of time and examined the extracts for the presence
of factors that bind to the SIE or GRR probes. As shown in
Fig 2A, a significant level of SIE-binding factors was detected in the cytoplasm 1 minute after Epo stimulation and
in the nucleus 5 minutes after stimulation. In contrast, the
GRR-binding factor was detected in the cytoplasm as early
as 30 seconds after Epo stimulation and in the nucleus by 1
minute (Fig 2B). High levelsof both the SIE- and GRRbinding factors could be detected in the cytoplasm and nucleusforupto
30 minutes after Epo stimulation but the
levelsdecreasedafter
60 minutes.HCD-S7 cells continuously maintained in the presence of Epo showed neither the
SIE- nor GRR-binding factors (data not shown).
The SIE- andthe GRR-binding fnctors renct with antipho.sphol?.rf~sineantibodies. To characterize the DNAbinding factorsinduced by Epo stimulation. we analyzed
them for the presence of phosphotyrosine by examining the
effect of antiphosphotyrosine antibodies on the mobility shift
complex formation.Asshown in Fig 3A. additionof IG2
antiphosphotyrosine antibody resulted in a supershifted SIE
signal but it did not show a significant effect on the complex
formation by the GRR-binding factor.However, another
antiphosphotyrosine antibody, 25.264. strongly inhibited the
formation of the GRR-protein complex and caused a slight
enhancement of the signal correspondingto the faster migrating SIE complex.Otherantiphosphotyrosine
antibodies,
such as PY-20 and 4G10, failed to exert a significant effect
on the complex formation by either the SIE- or the GRRbinding factors induced by Epo (data not shown). When the
cellswere treatedbefore Epo stimulation with atyrosine
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EPO- AND SFFV-INDUCED DNA-BINDING FACTORS
1457
A. SIE
Nucleus
Cytosol
Time (min) 0 0.5 1
15 30 60 0 0.5 1
5
5
15 30 60
8. GRR
Nucleus
Cytosol
-
Time (min) 0 0.5 1
Fig 2. Kinetics of the DNAbinding factor induction by Epo.
After starvation, HCD-57 cells
were treated with
Epo for the indicated periods (0. 0.5, 1, 5, 15,
30, or 60 minutes) and the cytoplasmic and nuclear extracts
were prepared. Samples were
then incubated with a "P-labeled SIE (A) or GRR (B)probe
and subjected to 596 polyacrylamide gel electrophoresis.
kinase inhibitor, genistein, the SIE- and the GRR-binding
factors could not be detected either in the cytoplasm or the
nucleus (Fig 3B).
Anti-Stat protein antibodies react with the SIE- and the
GRR-bindingfactors induced by Epo stimulation. To determine whether the DNA-binding factors induced by Epo stimulation are related to Stat family proteins, wetested the
reactivity of anti-Stat protein antibodies with these factors.
As shown in Fig 4A, both of the signals corresponding to
the SIE complexes were supershifted by antiStat1N antibody. After addition of anti-StatlC antibody, formation of
the SIE-complexes was partially inhibited. Anti-Stat3 antibody altered the ratio of the two SIE complexes indicated
by a slight decrease in the relative intensity of the band
corresponding to the slower migrating complex and an increase in that corresponding to the faster migrating complex.
Effects of the same antibodies on the GRR complex formation were also examined (Fig 4B). Although anti-StatlN
antibody did not have any effect, both anti-StatlC and antiStat3 antibodies showed inhibitory effects on the formation
of the GRR complex. We failed to detect any effect of antiStat2 antibody on the complex formation by any of the SIEor GRR-binding factors, and unrelated antibodies, such as
anti-p53 and antirabbit IgG antibodies, also failed to have
5
-'
15 30 60 0 0.5 1
'-
.
.
5
15 30 60
"
an effect (data not shown). Because we found that anti-Stat 1
antibodies reacted with the DNA-binding factors induced by
Epo, we investigated whether any of the proteins recognized
by the anti-Stat1 antibodies are phosphorylated in response
to Epo stimulation. Whole cell extracts prepared from HCD57 cells stimulated with or without Epo were immunoprecipitated with anti-StatlN or anti-StatlC antibody. Precipitates
were then analyzed by immunoblotting with antiphosphotyrosine antibodies (Fig 4C). A signal corresponding to a 92kD tyrosine-phosphorylated protein was clearly detected by
anti-StatlN and anti-StatlC antibodies only after Epo stimulation.
Statl and Stat3 mRNA.7 are expressed in HCD-57 cells.
Because the involvement of Stat family proteins in the Epo
signal transduction pathwaywas suggested, we examined
the mRNA expression of known Stat genes in HCD-57 cells
by an RT-PCR analysis. Because the mouse nucleotide sequences were available only for Statl, Stat3, and Stat4,2'.23
we analyzed the expression of these three genes. The given
sets of primers described in the Materials and Methods are
expected to generate amplifiedDNA products of 687 bp,
495 bp,and 720 bp in length for Statl, Stat3, and Stat4
mRNA, respectively. As shown in Fig 5, the expected size
of signals for Statl and Stat3 mRNA were detected in both
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OHASHI, MASUDA, AND RUSCETI
1458
A.
B.
Probe
25.
Ab
GRR
SIE
"
"" '
-
25.
IG2 2G4
-
Probe
SIE
Fraction
Cyt.
Nuc.
GRR
Cyt.
Nuc.
fig 3. Tyrosine phosphorylstion of the Epo-inducedDNAbindingfactors. (A). HCD-57
cells
were treated for 15 minutes with
Epo and the cytoplasmic
extracts were prepared and incubated without (-1 or with an
antiphosphotyrosine antibody
(10 p g of 1G2 or 5 p g of 25.264)
for 16 hours at 4°C. The samples
were then incubated with a 32Plabeled SIE or GRR probe and
subjected to 5% polyacrylamide
gel electrophoresis.The position
of the complex supershifted by
l G 2 antibody is indicated by an
were
arrow. (B). HCD-57cells
treated without 1-1 or with (+)
genistein (l00 pg/mL) for 2
hours and then stimulated with
Epofor 15 minutes. The cytoplasmic (Cyt) and nuclear (Nuc)
extracts were then prepared, in-
"
"
162 2G4
=,
-
Genistein
.
m
HCD-57 and EL-4 cells. In contrast, the DNA product for
Stat4 mRNA was detected in E L 4 cells, but not in HCD57 cells, indicating the lack of Stat4 mRNA expression in the
latter cell line. Nucleotide sequence analysis of the cloned
fragment corresponding to these signals confirmed that the
amplified DNA was actually derived from Stat1 and Stat3
mRNA (data not shown).
Epo-induced DNA binding factors are constitutively activated in SFFV-infected HCD-57 cells. To investigate the
effects of SFFV,, which abrogates Epo dependence of HCD57 cells, on activation of the S E - and the GRR-binding
factors, we performed EMSAs by using cell extracts prepared from HCD-SFFV cells that were treated with or without Epo (Fig 6). As previously shown, HCD-57 cells expressed SIE- and GRR-binding activities only after Epo
stimulation (Fig 6A and B, lanes 3). In contrast, two distinct
SIE-binding activities and one GRR-binding activity were
detected in SFFV-infected HCD-57 cells after starvation in
serum-free medium (Fig 6A and B, lanes 4) or after incubation in complete medium without Epo (Fig 6A and B, lanes
5). When HCD-SFFV cells were treated with Epo, an increase in all DNA-binding activities was observed (Fig 6A
and B, lanes 6). The DNA-protein complexes formed by the
HCD-SFFV cell extracts showed the same mobilities as
those formed by the Epo-stimulated HCD-57 cell extracts.
To determine whether the DNA-binding factors expressed
in SFFV-infected HCD-57 cells are also related to known
Stat family proteins, we tested the reactivity of anti-Stat
protein antibodies with these DNA-binding factors. As
shown in Fig 6C, both of the signals corresponding to the
SIE complexes were supershifted by anti-StatlN antibody,
- + ~-- +. -. +. - . - i-.
..
.
and the complex formation was partially inhibited by antiStatlC antibody. Anti-Stat3 antibody altered the ratio of the
two SE-complexes so that the faster migrating complex was
more abundant than the slower migrating complex. AntiStatlN antibody did not have any effects on the GRR complex formation, whereas the complex formation was partially
inhibited by both anti-StatlC and anti-Stat3 antibodies (Fig
6D). These results observed in SFFV-infected HCD-57 cells
were very similar to those observed in uninfected HCD-57
cells stimulated with Epo.
DISCUSSION
In this study, we showed that DNA-binding factors that
specifically recognize the S E and GRR sequences are induced by Epo stimulation in the Epo-dependent mouse erythroleukemia cell line HCD-57. These DNA-binding factors
are activated in the cytoplasm immediately after Epo stimulation and, after a brief delay, the same factors are detected
in the nucleus, suggesting that the activated factors are transported from the cytoplasm to the nucleus. Activated factors
were detected at a significant level only transiently both in
the cytoplasm and the nucleus. They, therefore, might be
involved in the initial rapid phase of the signal transduction
pathway required for resting cells to resume their ability to
proliferate.
The two SE-binding factors and the GRR-binding factor
appear to contain different components because the DNAprotein complexes formed by these factors exhibited distinct
mobilities and the kinetics of their activation were different.
However, both unlabeled S E and GRR oligonucleotides
could inhibit (at least partially) SIE- and GRR-binding fac-
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€PO-
AND SFFV-INDUCED DNA-BINDING FACTORS
A. S I E
1459
B. GRR
C.
a-Stat
a-Stat
Antibody
-
1N 1C 3
-
a-Stat
LP.
1N 1C 3
EPO
1N
- + - +
KD
20097 68 43-
1C
1
i
-
92 KD
"W
Fig 4. ReMonship of theEpo-induced DNA-binding factorst o Stat proteins. Cytoplasmic extracts wereprepared from HCD-57 cells stimulated with Epo for 15 minutes and incubated without
(-1 or with 0.25 p g of anti-StatlN UN), 10 p g of anti-StatlC (1C). or 10 p g of anti-Stat3
(3)antibody for16 hours at 4°C. The samples were then incubated with
a '2P-labeled SIE (A) orGRR (B) probe and subjected t o 5% polyacrylamide
gel electrophoresis. The position of the SIE complex supershifted by anti-StatlN antibody is indicated by an arrow. (C) Whole cell extracts
were prepared from HCD-57 cells treated without (-1 or with (+) Epo for 15 minutes and incubatedwith agarose-conjugated anti-StatlN IlN)
or anti-StatlC (1C) antibody for 2 hours at 4°C. After extensive washing, the immunoprecipitated proteins wereseparated by 7.5% SDS-PAGE
and transferred t o a nitrocellulose filter. The filter was incubated with a mixture of antiphosphotyrosine antibodies IPY20 and 4G10, each
diluted 1:2,500) and then with an antimouse
lgG antibody conjugatedt o horseradish peroxidase. Antibodies boundto the filter were
detected
by theenhanced chemiluminescence method. The position of the immunoprecipitated 92-kD phosphotyrosyl protein is indicated by arrow.
an
tors from binding to the labeled probes. This cross-competition might be caused by limited homology between the SIE
and GRR sequences or may suggest that the SIE- and GRRbinding factors share a common component(s) thatis depleted by addition of an excess of either oligonucleotide.
The DNA-binding factors induced by Epo appear to contain phosphotyrosine because antiphosphotyrosine antibodies affected complex formation by these factors. Because
treatment of the cells with a tyrosine kinase inhibitor prevented induction of the DNA-binding factors by Epo, tyrosine phosphorylation of these factors may be crucial for their
activity. Interestingly, different effects on complex formation were observed for different antiphosphotyrosine antibodies. This finding suggests that the conformation surrounding the phosphotyrosines in the SIE-binding factors
and the GRR-binding factor is different and that each antiphosphotyrosine antibody can react with phosphotyrosine
when it is present only in a certain context.
DNA-binding proteins activated by a number of growth
factors and cytokines have been shown to be members of
the Stat family of transcription factors. Stat1 is activated
by IFN-a, IFN-y, epidermal growth factor (EGF), IL-10,
platelet-derived growth factor, and ciliary neurotrophic factor,18-21.374i
whereas Stat3 has been shown to be activated in
response to IL-6, EGF, and granulocyte colony-stimulating
factor (G-CSF).23.24.42
Several observations from our studies
suggest that the DNA-binding factors induced by Epo stimulation belong to the Stat family of DNA-binding proteins.
The factors specifically bind tosequences known to be recognized by Stat proteins and the kinetics of their induction are
rapid and transient like those of known Stat proteins. The
proteins are phosphorylated on tyrosine and tyrosine phosphorylation appears to be required for their DNA binding
activity. Finally, antibodies raised against certain known Stat
proteins react with these factors. Both of the SIE-binding
factors contain a component thatisrecognized
by antiStatlN and anti-StatlC antibodies and the molecular weight
of the phosphotyrosyl protein detected by immunoprecipita-
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1460
OHASHI. MASUDA,AND
EL-4HCD-57 Cell
I
Primer
51 7/506 -1
396
344
298
method.We are currently attempting to affinity-purify the
DNA-binding proteins using agarose-conjugated probes so that we canfurther characterize their molecular nature. Because there is a possibility that a novel Stat
protein antigenically related to Statl or Stat3 is contributing
to the SIE- or the GRR-binding factor formation, attempts
are being made to search for a novel. Stat-related gene expressed in HCD-S7 cells. We are also testing other Eporesponsive hematopoietic cells, including various myeloid
cell lines thathavebeen
transfected withtheEpoR gene,
mouse erythroleukemia cell lines, and human leukemic cell
lines, for activation of Stat-related DNA-binding factors.
Studies with the erythroleukemia-inducing SFFVhave
clearly shown that viruses can affect the proliferation and
differentiation of hematopoietic cells without carrying an
oncogene. So far, it has been shown that the primary effect
of SFFV on erythroid cells is induced by the product of its
unique envelope gene, and the interaction of the envelope
glycoprotein withtheEpoR
appears to be crucial for its
effect on erythroid cellgrowth.'2.J""hHowever.
little is
known about how this interaction leads to autonomous cell
growth. In this study, we showed thatSFFV,infection of
HCD-S7 cells results in the constitutive activation of DNAbinding factors that are transiently induced by Epo in uninfected cells. This result suggests that SFFV, can activate the
Epo signal transduction pathway in a prolongedmanner.
This continuous activation of the DNA-binding factors by
SFFV, might be triggering the signal that enables the erythroid cells to proliferate autonomously without Epo. It is
not known how SFFV, activates these DNA-binding factors
without Epo, but because neither the EpoR nor the SFFV,
envelope glycoprotein have an effector regionsuch as a
tyrosine kinase domain, it is likely that they are activating
these DNA-binding factors indirectly. Recently, it has been
reported that JAK2 tyrosine kinase is associated withthe
cytoplasmic domain of theEpoRand
activated after Epo
Because JAK2 kinase was shown to be responsible for the tyrosine phosphorylation of Stat proteins,'".'" it is possible that the SFFV, envelope glycoprotein
can generate a functionally active form of the EpoR complex
more stably than Epo and that the associated JAK2 is activated constitutively, which in turn activates the DNA-binding factors. A recent report demonstrating the constitutive
tyrosine phosphorylation of JAK2 in an SFFV-induced
erythroleukemia cell line" is in agreement with this hypothesis. Alternatively, theSFFV, envelope glycoprotein might
be negatively affecting the mechanism that inactivates the
JAK2 kinase or the DNA-binding factors (eg, phosphatases).
Further studies using the anemia-inducing strain of SFFV
that does not induce Epo-independence, other recombinant
SFFVs, and mutant Epo receptors will allow us to determine
the structures of the viral envelope glycoprotein andthe
EpoR required for the constitutive activation of the DNAbinding factors. Identification of the cellular target genes
that these DNA-binding factors can regulate wouldalso give
us further insights into the functional significance of these
factors in the physiologic mechanism for erythropoiesis as
well as the pathogenic mechanism for leukemia induction.
$
7
1
Epo-induced
Stat
-
7
Fig 5. Expression of Stat family mRNAs in HCD-57 cells. Total
RNAs obtained from HCD-57 and EL-4 cellswere reverse transcribed
to cDNA andamplified by PCR by usingprimer pairs specific to Statl,
Stat3, and Stat4. PCR productswere electrophoresed in a 2% agarose
gel and stained by ethidium bromide. Molecular sizes of the markers
(MI are indicated on the left.
tionwith anti-Stat1 antibodies coincides withthat of the
phosphorylated form of Stat la. In addition, Statl mRNA
was detected by RT-PCR using specific primers in HCD-S7
cells. Therefore, it is likely that Stat la is a component of
the SIE-binding factors. although involvement of a novel
Stat protein antigenically relatedto Statl is also possible.
The complex formation by the GRR-binding factor was inhibited by anti-StatlC antibody, whichwasraised against
amino acid residues 6 13-739 in the C-terminus of Stat l , but
notby anti-StatlN antibody, which was raised against the
amino terminal194 amino acids. In addition, antibodies
raisedagainst amino acids 607-647 or the C-terminal 39
amino acids of Stat la were recently shown to have no effect
on complex formation by the GRR-binding factor induced
by Epo in human erythroleukemia TFI cells." Therefore, it
is possible thatthe GRR-binding factor does not include
Stat I but another protein whose antigenicity is related to that
of the epitope recognized by anti-StatIC antibody. Alternatively, the anti-StatlN antibody used in this study and the
other anti-Stat I antibodies used in the previous study" may
notbe able to react with the cognate epitopes of the Statl
protein when it is incorporated in the GRR-binding complex.
The SIE-binding factor complex with a slower mobility contains a component recognized by anti-Stat3 antibody. and
the activity of the GRR-binding factor was also affected by
the same antibody. This combined with data showing that
Stat.? mRNA can be detected in HCD-S7 cells by RT-PCR
suggests that Stat3 may also be activated by Epoand included in the SIE- and the GRR-binding factors, although it
is also possible that a distinct proteinrelatedto
Stat3 is
involved. Lack of reactivity of anti-Stat2 antibody with any
of the DNA-binding factors suggests that Stat2 is not a component of Epo-induced DNA-binding complexes. Likewise,
involvement of Stat4 in the Epo signal transduction pathway
in HCD-S7 cells is unlikely because Stat4 mRNA was not
detected in these cells usingthehighly sensitive RT-PCR
RUSCElTI
DUCED
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EPO- AND
DNA-BINDING FACTORS
1461
c. S I E
A. S I E
SFFV
(+)
SFFV
(-)
EPO
- 1
2
a-Stat
+ - - +
3
4
D. G R R
5
Antibody
-
1N 1C 3
a-Stat
- 1N 1C 3
6
6. GRR
EPO
SFFV
SFFV
(-)
(+l
- -
+ -
- +
1
3
5
2
4
6
Fig 6. Constitutive activation of the SIE- and the GRR-binding factors by SFFV,. Cytoplasmic extracts prepared from HCD-57 cells (lanes 1
t o 3) or HCD-SFFV cells (lanes 4 t o 6) after starvation (lanes 1 and 4). 15 minutes after incubationin complete medium without (lanes 2 and
51 or with (lanes 3 and 6) Epo were incubatedwith a 32P-labeledSIE (A) or GRR (B) probe andsubjected t o 5% polyacrylamide gel electrophoresis.
For supershift and inhibition analysis with anti-Stat antibodies, cytoplasmic extracts were prepared from HCD-SFFV cells after 15 minutes of
(-) or with 0.25 p g of anti-StatlN (1N). 10 p g of antiincubation in complete medium withoutEpo. The extracts were then incubated without
StatlC (1CI. or 10 p g of anti-Stat3 (3) antibody for 16 hours at 4°C. The samples were incubated with a 32P-labeledSIE (C) or GRR ID) probe
and subjected t o 5% polyacrylamide electrophoresis.
ACKNOWLEDGMENT
We are grateful to Drs Rosemarie E. Aurigemma, Lin Sun-Hoffman, andKyoichi Shibuya for helpful discussions. We also thank
Charlotte Hanson for technical assistance and Denise Rubens for
facilitating DNA sequencing.
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1995 85: 1454-1462
Induction of sequence-specific DNA-binding factors by erythropoietin
and the spleen focus-forming virus
T Ohashi, M Masuda and SK Ruscetti
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