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Oncogene (2000) 19, 2296 ± 2304
2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00
www.nature.com/onc
Epo regulates erythroid proliferation and di€erentiation through distinct
signaling pathways: implication for erythropoiesis and Friend virus-induced
erythroleukemia
Barry Zochodne1,4, Amandine HL Truong1,4, Kendra Stetler1, Rachel R Higgins1, Je€ Howard3,
Dan Dumont1, Stuart A Berger2 and Yaacov Ben-David*,1
1
Department of Medical Biophysics, University of Toronto, Cancer Biology Research, Sunnybrook and Women's College Health
Sciences Centre, 2075 Bayview Avenue, S-Wing, Toronto, Ontario, M4N 3M5 Canada; 2Department of Immunology, University of
Toronto, Arthritis and Immune Disorder Network, University Health Network, 620 University Avenue, Suite 700, Toronto, Ontario
M5G 2M9 Canada
We have recently isolated the erythroleukemic cell line,
HB60-5, that proliferates in the presence of erythropoietin (Epo) and stem cell factor (SCF), but undergoes
terminal di€erentiation in the presence of Epo alone.
Ectopic expression of the ets related transcription factor
Fli-1 in these cells resulted in the establishment of the
Epo-dependent cell line HB60-ED that proliferates in the
presence of Epo. In this study, we utilized these two cell
lines to examine the signal transduction pathways that
are activated in response to Epo and SCF stimulation.
We demonstrate that Epo, but not SCF, phosphorylates
STAT-5 in both HB60-5 and HB60-ED cells. Interestingly, SCF activates the Shc/ras pathway in HB60-5
cells while Epo does not. However, both Epo and SCF
are capable of activating the Shc/ras pathway in HB60ED cells. Furthermore, enforced expression of gp55 in
HB60-5 cells by means of infection with the Spleen
Focus Forming virus-P (SFFV-P), confers Epo independent growth, which is associated with the up-regulation
of Fli-1. Activation of the Shc/ras pathway is readily
detected in gp55 expressing cells in response to both Epo
and SCF, and is associated with a block in STAT-5B
tyrosine phosphorylation. These results suggest that
STAT-5 activation, in the absence of Shc/ras activation,
plays a role in erythroid di€erentiation. Moreover, Fli-1
is capable of switching Epo-induced di€erentiation to
Epo-induced proliferation, suggesting that this ets factor
regulated genes whose products modulate the Epo-Epo-R
signal transduction pathway. Oncogene (2000) 19,
2296 ± 2304.
Keywords: erythropoiesis; Epo; Fli-1; signal transduction; Friend erythroleukemia
Introduction
In the past decade a number of important intracellular
and extracellular factors have been indenti®ed that
alter the fate of erythroid progenitor cells, namely
whether to divide (self-renew), die (apoptosis), or
*Correspondence: Y Ben-David
3
Current Address: HULC Cell & Molecular Biology Laboratory,
Lawson Research Institute, St. Joseph's Health Centre, 268
Grosvenor St. London, Ontario N6A 4V2 Canada
4
Equal contribution made by ®rst two authors
Received 20 October 1999; revised 20 January 2000; accepted 27
March 2000
di€erentiate. Of these, perhaps hematopoietic growth
factors (HGF) have been the most thoroughly
characterized. Erythropoietin (Epo) and stem cell
factor (SCF) are two HGFs that have been identi®ed
as important regulators of erythropoiesis. Recognition
of a role for SCF and its receptor, the tyrosine kinase
c-Kit, in erythroid development stems largely from the
study of W (White spotting) and Sl (Steel) mice that
exhibit erythroid and other lineage-speci®c defects due
to inherited mutations within the c-Kit and SCF genes
(Pawson and Bernstein, 1990). Studies examining the
pleiotropic e€ects of c-Kit signaling have identi®ed a
number of important downstream e€ectors that are
believed to be responsible for its potent mitogenic
e€ects, including phosphatidylinositol 3'-kinase (PI3kinase), the SH2 adaptor protein (Shc), p21ras and
mitogen-activated protein kinase (MAPK) (Tauchi et
al., 1994; Timokhina et al., 1998; Sattler et al., 1997;
Lev et al., 1992; Kinashi et al., 1995).
Arguably the most important erythroid-speci®c HGF
is Epo. The signaling events triggered by the binding of
Epo to the Epo-receptor (Epo-R), like many other
HGFs, is rather complex. However, it is clear that Epo
possesses anti-apoptotic activity (Koury and Bondurant, 1990), a function that is somehow tightly coupled
to its proliferative and di€erentiation-inducing e€ects
on erythroid progenitor cells (Youssou®an et al., 1993;
Liboi et al., 1993). Although the exact molecular
mechanisms underlying these contrasting cellular responses are not well understood, a number of downstream signaling e€ectors have been identi®ed,
including Shc/ras, PI3-kinase, SHIP, as well as the
JAK2/STAT5 pathways (Lecoq-Lafon et al., 1999;
Damen et al., 1993, 1995a,b; He et al., 1995; Miura et
al., 1994; Verdier et al., 1997; Gouilleux et al., 1995 ID:
1002; Pallard et al., 1995; Wakao et al., 1995). Epoinduced phosphorylation of the Epo-R at Tyrosine 343
promotes proliferation and an increase in STAT-5
activity (Damen et al., 1995b). However, recent studies
link STAT-5 phosphorylation to Epo-induced di€erentiation of erythroid progenitor cells. Iwatsuki et al.
(1997), showed that tyrosine phosphorylation of the
Epo-R at position Y343 and Y401 is required for
STAT-5 activation and erythroid di€erentiation. These
con¯icting results underscore the need to clarify the
exact role of STAT-5 phosphorylation during Epoinduced signaling events.
Friend erythroleukemia is a well known multi-staged
tumor model that is aptly suited for studying the
Epo signaling in erythropoiesis and Friend leukemia
B Zochodne et al
mechanisms governing the proliferation and maturation of erythroid progenitor cells (Ben-David and
Bernstein, 1991). In the past decade alone researchers
have discovered that the signaling pathway de®ned by
Epo-Epo-R is a common target of the transforming
strains of Friend virus. The genomic integrity of both
Epo and the Epo-R are often speci®cally disrupted
during the course of F-MuLV and FV-P-induced
erythroleukemias (Li et al., 1990; McDonald et al.,
1987; Hankins et al., 1989; Howard et al., 1996).
Moreover, gp55p, the glycoprotein of the env of the
polycythemia strain of spleen focus-forming virus
(SFFVp), has been shown to bind to and activate the
Epo-R (Li et al., 1990). The Epo mimicry of gp55p is
thought to be responsible for the early polyclonal
proliferation of erythroid progenitor cells during the
preleukemic stage of FV-P induced disease (Kabat,
1990). Overall, Friend virus has also been found to
suppress the di€erentiation signals controlled by the
Epo/Epo-R pathway, through retroviral insertional
mutagenesis (Ben-David and Bernstein, 1991).
Genes speci®cally targeted by retroviral insertional
mutagenesis have been identi®ed for each strain of
Friend virus. These genes include the p53 tumor
suppressor gene and two members of the ets family
of transcription factors, Spi-1 and Fli-1 (Ben-David et
al., 1988, 1991; Hicks and Mowat, 1988; MoreauGachelin et al., 1988). Activation of Fli-1 has been
identi®ed as an early transformation event associated
with F-MuLV induced erythroleukemias (Ben-David et
al., 1990). Examination of its role during the
transformation of erythroid progenitor cells suggest
that constitutive expression of Fli-1 dramatically
increases the self-renewal potential of proerythroblasts.
This block in erythroid di€erentiation appears to stem,
in part, from the ability of Fli-1 to transcriptionally
repress the retinoblastoma (Rb) tumor suppressor gene,
a central component of the cell cycle machinery (Tamir
et al., 1999). Disruption of the cell cycle in this manner
would be advantageous to the virus since exit from the
cell cycle is a prerequisite for terminal erythroid
di€erentiation but detrimental to further viral replication.
In this study, we have investigated the role of Epo and
SCF in regulating the self-renewal and di€erentiation
potential of erythroid progenitor cells. Toward this goal,
we used a novel erythroleukemia cell line, designated
HB60-5, that proliferates in the presence of SCF and
Epo, but undergoes terminal erythroid di€erentiation in
response to Epo alone (Tamir et al., 1999). We previously
demonstrated that a transient but dramatic decrease in
the expression levels of Fli-1 is associated with cell cycle
arrest and terminal di€erentiation of this cell line (Tamir
et al., 1999). Ectopic expression of Fli-1 in HB60-5 or
expression as a result of insertional mutagenesis
conferred Epo-dependent proliferation, suggesting that
constitutive high levels of Fli-1 expression constitutes an
important molecular `switch' that disables the di€erentiation signaling of Epo. By using HB60-5 and its Epodependent derivative HB60-ED cells, we provide
evidence that the proliferation of HB60-5 cells is
speci®cally associated with the activation of the PI3K
and Shc/ras pathways, while Epo-induced terminal
di€erentiation appears to be mainly associated with
STAT-5 activation. Interestingly, expression of gp55 in
HB60-5 cells resulted in Epo and SCF independent
growth. The block in Epo-induced di€erentiation elicited
by gp55p was found to be associated with a block in
STAT-5 phosphorylation and activation of the Shc/ras
pathway, which correlated with high levels of Fli-1
expression.
2297
Results
Epo-induced differentiation of HB60-5 cells is associated
with the phosphorylation of STAT-5
Previous studies have demonstrated that Epo stimulates the phosphorylation of both Shc and STAT-5b
protein whereas SCF activates Shc/ras signaling (Penta
and Sawyer, 1995; Damen et al., 1993, 1995b;
Gouilleux et al., 1995; Wakao et al., 1995). To verify
the status of these phosphorylation events in HB60-5
cells, we brie¯y starved and then incubated these cells
with Epo, SCF or both growth factors (10 min) and
then immunoprecipitated with STAT-5b and Shc
antibodies. Hybridization of these blots with anti-Shc
and anti-STAT-5b antibodies clearly showed the
presence of Shc (52 and 46 kDa) and STAT-5b
(93 kDa) in these IPs. Western blot analysis with
anti-PY antibodies also revealed that STAT-5b became
phosphorylated in response to Epo or Epo and SCF
together, but not in response to SCF alone in HB60-5
cells (Figure 1B). However, Shc phosphorylation was
only detected in cells after stimulation with SCF or
SCF and Epo, but not Epo alone (Figure 1A). These
results demonstrate that Epo, but not SCF, can
activate STAT-5b in HB60-5 and that Shc phosphorylation correlates with proliferation.
Since phosphorylated STAT-5b enters the nucleus
(Mui et al., 1995), we next examined its localization in
HB60-5 cells following growth factor stimulation. After
10 minutes of stimulation with Epo or Epo and SCF,
the majority of the phosphorylated STAT-5b band was
found in the nucleus (Figure 1C). As expected, SCF
alone did not alter the phosphorylation status of
STAT-5b in either cytoplasmic or nuclear fractions. It
has recently been reported that the Src homology 2containing inositol phosphate (SHIP) is associated with
Shc and is phosphorylated in response to Epo and SCF
(Damen et al., 1996). Indeed, we also detected the
140 kDa phosphorylated SHIP proteins in IPs with Shc
antibody in extracts from SCF or SCF+Epo stimulated HB60-5 cells, but not with Epo alone (Figure
1A). These experiments, therefore, demonstrated that
in HB60-5 cells Epo but not SCF activated STAT-5
while the Shc/ras pathway was activated by SCF.
Activation of Shc/ras pathway by Epo in HB60-ED cells
Unlike HB60-5 cells, stimulation of the Fli-1 overexpressed HB60-ED cells with Epo resulted in the
phosphorylation and activation of Shc (Figure 2A). A
similar phosphorylation pattern was also detected in the
Epo-dependent cell line HB9.1-ED, a F-MuLV induced
erythroleukemic cell line which also expresses high levels
of Fli-1 due to retroviral insertional activation of this
gene (Figure 2B). Activation of Shc was also detected in
HB60-ED cells following SCF stimulation, although the
extent of phosphorylation was signi®cantly reduced in
these cells. Similar to HB60-5 cells, stimulation of
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Epo signaling in erythropoiesis and Friend leukemia
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2298
Figure 1 SCF and Epo activate Shc and STAT-5 in HB60-5 cells, respectively. Lysates (100 mg) from HB60-5 cells, stimulated with
the indicated growth factors were immunoprecipitated with either Shc (A) of STAT-5b (B) antibodies and subjected to Western blot
analysis using the indicated antibodies. (C) Cytoplasmic and nuclear proteins were prepared from HB60-5 cells after stimulation
with the indicated growth factor. 100 mg of proteins from each preparation was immunoprecipitated with STAT-5b antibody,
subjected to SDS ± PAGE and Western blotted with the STAT-5b antibody
Figure 2 Mitogenic activation of Shc/ras pathway by Epo and SCF correlates with proliferation. Lysates prepared from HB60-ED
cells (A) or the Epo-dependent HB9.1-ED cells (B) after stimulation with the indicated growth factors, were subjected to IP-Western
blot analysis with the indicated antibodies (left panel), as described in Figure 2. Arrows (right panel) show the positions of the
detected proteins. The symbol (*) indicates the position of a phosphorylated protein that co-immunoprecipitated with the antiSTAT-5b antibody but whose identity is yet to be determined
HB60-ED and HB9.1-ED with Epo also activated
STAT-5b phosphorylation (Figure 2A,B). Interestingly,
a 180 kDa protein that was also phosphorylated in
response to Epo, co-immunoprecipitated with STAT-5b
Oncogene
in HB60-ED cells (Figure 2A). This band was not
detected in the STAT-5b IP of HB60-5 cells (Figure 1B).
Together these results suggest that di€erentiation of
HB60-5 cells is associated with STAT-5 phosphoryla-
Epo signaling in erythropoiesis and Friend leukemia
B Zochodne et al
tion, whereas their proliferation may require Shc/ras
pathway activation by Epo.
SCF activated the Epo-R through a direct interaction of cKit with the Epo-R
Previously we showed that HB60-5 cells proliferate in
response to SCF, and that the addition of Epo
dramatically increases this proliferative response (Tamir et al., 1999). The activation of Epo-R through
direct interaction with the phosphorylated c-Kit
receptor has been suggested to be responsible for the
synergistic e€ect of Epo and SCF during erythroid
proliferation (Wu et al., 1995). However, a recent
con¯icting study showed that in erythroid cells c-Kit,
although critical to the phosphorylation of Epo-R by
SCF, does not directly interact with the Epo-R
(Jacobs-Helber et al., 1997). To verify the role of
SCF in erythroid proliferation, HB60-5 cells were
starved for 12 h and subsequently stimulated with
either Epo or SCF. SCF stimulation followed by
immunoprecipitation with the Epo-R antibody revealed
a 145 kDa phosphorylated band, as well as three
additional phosphorylated bands of 68, 66 and 95 kDa
(Figure 3A). Phosphorylation of the latter three bands
was also stimulated by Epo with their precipitation
being blocked by the addition of competitor Epo-R
peptide (Figure 3A). A similar pattern of cytokine
mediated-phosphorylation was also seen in HB60-ED
cells (Figure 3B) and the Epo-dependent HB9.1-ED
cell line (Figure 3C), which was derived from a FMuLV induced-erythroleukemia (Howard et al., 1993).
Based on their molecular weight and interaction with
speci®c antibodies, the 68 and 95 kDa proteins appear
to be identical to the Epo-R and STAT-5, respectively
(data not shown here). Interestingly, the phosphorylated 145 kDa band that immunoprecipitated with the
Epo-R also weakly hybridized with the c-Kit antibody
(Figure 3A,B). To con®rm that the 145 kDa band was
indeed c-Kit, SCF stimulated cell extracts were ®rst
immunoprecipitated with Epo-R, separated from the
beads, re-immunoprecipitated with the c-Kit antibody
and Western blotted with anti-phospho-tyrosine (PY)
antibody. As shown in Figure 3D, a 145 kDa band
migrates with the phosphorylated c-Kit band was
precipitated by the double IP procedure. These data
show that the phosphorylated c-Kit and the Epo-R are
found in the same complex. The identity of the 66 kDa
band that was also precipitated with the Epo-R from
HB60-5 and HB60-ED cell extracts is yet to be
determined.
Phosphatidylinositol 3-kinase (PI3K) has been previously shown to be phosphorylated in response to Epo
and SCF stimulation and has been shown to bind to
both receptors (Verdier et al., 1997; Damen et al.,
1995a; Sattler et al., 1997; Lev et al., 1992). Using
HB60-5 and HB60-ED cells, we also examined the
binding of the p85 subunit of PI3K to the Epo-R and
c-Kit. Western blot analysis revealed the PI3K was
associated with both the Epo-R and c-Kit in IP with
extracts from SCF stimulated HB60-5 and HB60-ED
cells (Figure 3A,B). However, the Epo-induced association of p85 with the Epo-R was only seen in the IP
with extracts from HB60-ED cells, and not with
extracts from the parental HB60-5 cells, demonstrating
that PI3K activation is connected with the Epodependent proliferation observed in this cell line.
2299
gp55 induces Epo and SCF independent growth HB60-5
cells
The cell line HB60-5 is similar to FV-P inducederythroleukemia cell lines in that it has undergone
insertional activation of the Spi-1 gene and a p53
mutation (Johnson et al., 1993; Tamir et al., 1999).
However, since this cell line was derived from a F-MuLV
infected tumor, it is negative for gp55 expression (Figure
4). Given that gp55p mimics the proliferative e€ects of
Epo in erythroblasts, we examined the e€ect of gp55p
expression in HB60-5 cells. The resulting SFFV-P
Figure 3 Epo-R is phosphorylated by SCF stimulation through a direct interaction with the activated c-Kit. Cell extracts (100 mg)
prepared from either SCF or Epo stimulated HB60-ED (A), HB60-5 (B) and HB9.1-ED (C) were immunoprecipitated with either an
Epo-R antibody (with or without a competitive Epo-peptide), or a c-Kit antibody, as indicated. The precipitates were then subjected
to SDS ± PAGE, transferred to PVDF membrane, and immunoblotted with the anti-phosphotyrosine antibody (anti-PY). These
same blots were then sequentially hybridized with the anti-c-Kit antibody and an anti-p85 antibody. (D) Extracts (2 mg) prepared
from HB60-5 cells, that were stimulated with SCF (lane 4) or without SCF (lane 3), were immunoprecipitated with an anti-Epo-R
antibody and then re-immunoprecipitated with an anti-c-Kit antibody, as described in the methods. The blotted membrane was then
immunoblotted with the anti-phosphotyrosine antibody (a-PY). Extracts (200 mg) from the starved HB60-5 cells (lane 1) or SCF
stimulated cells (lane 2) that were only immunoprecipitated with the c-Kit antibody, were used as controls
Oncogene
Epo signaling in erythropoiesis and Friend leukemia
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2300
infected HB60-5 cell line, HB60-SSFV, was similar to the
FV-P induced-erythroleukemia cell line DP28-9 (BenDavid et al., 1988) in expressing gp70 (encoded by FMuLV) and gp55 (encoded by SFFV) (Figure 4). In
contrast to HB60-5 cells that undergo cell growth arrest
when cultured in the presence of Epo, expression of gp55
in these cells conferred SCF and Epo independent
growth in culture (Figure 5A,B). We have previously
shown that the expression of globin genes (di€erentiation
marker for erythroid cells) is elevated in response to Epo
in HB60-5 cell (Tamir et al., 1999). Since the expression
of globin in HB60-SSFV cells was not a€ected by the
addition of Epo (Figure 5C), this data suggests that the
expression of gp55 in HB60-5 cells blocks di€erentiation.
Moreover, gp55 appears to stabilize the levels of Fli-1
expression in HB60-SSFV cells, since previously we have
shown that Fli-1 levels undergo a transient but dramatic
decrease in response to Epo (Figure 5c) (Tamir et al.,
1999). These results suggest that gp55p prevents the
transient but dramatic decrease in Fli-1 expression upon
Epo stimulation.
(Figure 6A). Moreover, Shc activation by these growth
factors was also associated with SHIP phosphorylation
which co-immunoprecipitated with the anti-Shc antibody.
Discussion
We have shown here, using the unique erythroleukemia
cell lines HB60-5 and HB60-ED, that Epo-induced
di€erentiation correlates with distinct signaling events.
STAT-5 phosphorylation correlates with the di€eren-
Expression of gp55 in HB60-5 cells overrides STAT-5b
phosphorylation by Epo
Since gp55 expression confers SCF and Epo independent growth to HB60-5 cells, we examined the
phosphorylation status of Shc and STAT-5b in
HB60-SFFV cells. In contrast to HB60-5, incubation
of HB60-SSFVcells with Epo (1 units/ml), was not able
to activate STAT-5b phosphorylation (Figure 6B). As
shown in Figure 6C, regardless of the concentration of
Epo used for cell stimulation, STAT-5b remained
unphosphorylated in HB60-SFFV cells, whereas maximum STAT-5b phosphorylation levels were readily
detected in HB60-5 cells stimulated with only 0.5 units
of Epo. A similar block in STAT-5b activation by Epo
was also seen in the FV-P induced erythroleukemia cell
line DP28-9 (Figure 6C). However, a low but
constitutive level of STAT-5b phosphorylation was
seen in these cells.
With regard to the status of Shc phosphorylation,
the addition of both Epo and SCF resulted in an
increase in Shc phosphorylation in HB60-SFFV cells
Figure 4 Expression of gp55 in HB60-5 cells. Total protein
extracts (20 mg) prepared from the indicated cells were resolved
on a 12% SDS-polyacrylamide gel, transferred to a PVDF
membrane and hybridized with a gp55 speci®c antibody. Equal
loading was determined by hybridizing the same membrane to
Erk-2 antibody. The locations of gp70 and gp55 bands are shown
by arrows
Oncogene
Figure 5 Expression of gp55 in HB60-5 cells confers growth
factor independence. Triplicate culture (56104) of HB60-5 (A)
and HB60-SSFV (B) cells were grown in absence of no growth
factor, Epo (0.5 units/ml), SCF (100 ng/ml) or both Epo and
SCF, and the number of viable cells was determined at days 1 ± 4.
(C) Total RNA was isolated from HB60-SSFV cells after 12 and
24 h growth with or without Epo and separated on a 1% agarose
gel, transferred to the nitrocellulose membrane and sequentially
hybridized with Fli-1, a-globin and GAPDH probes
Epo signaling in erythropoiesis and Friend leukemia
B Zochodne et al
2301
Figure 6 Expression of gp55 in HB60-5 cells promotes the activation of Shc while inhibiting STAT-5b phosphorylation. HB60SSFV, HB60-5 and DP28-9 erythroleukemic cells were starved in a-MEM+0.5% FBS, stimulated with Epo (1 units/ml for A and
0.5 units/ml for C) or SCF (100 ng/ml for B) for 10 min, washed with PBS and lysed. The lysates (100 mg) were immunoprecipitated
with either Shc (A) or STAT-5b antibodies (B and C), separated on SDS-polyacrylamide gels and subjected to Western blot analysis
using the indicated antibodies
tiation of HB60-5 cells but not with the proliferation of
these cells by Epo. Conversely, activation of the Shc/
ras pathway by Epo was only detected under
proliferative conditions but not during di€erentiation
of these cells. As well, since constitutive expression of
Fli-1 in these cells can switch di€erentiation to
proliferation in response to Epo stimulation, these
results demonstrate the importance of this transcription
factor in the regulation of Epo/Epo-R signaling and
erythroid transformation.
Analysis of Shc activation by Epo had revealed that
this SH2-domain containing adaptor was mainly
phosphorylated in the proliferative HB60-ED cells.
Moreover, activation of SHIP and p85/PI3K was also
detected in proliferating HB60-ED cells. Our results are
consistent with published studies that have shown
increased phosphorylation of Shc and other SH2
containing protein such as P85/PI3K, SHIP and
Grb2 during Epo-induced proliferation of erythroblasts
(Damen et al., 1993, 1995a, 1996; He et al., 1995;
Verdier et al., 1997). Interestingly, Epo-induced
di€erentiation of the parental HB60-5 cells requires
very distinct signaling events, namely the tyrosine
phosphorylation of STAT-5b. The ability of gp55p to
block STAT-5b tyrosine phosphorylation in response
to Epo supports the notion that it may be an
important di€erentiation signaling event. This is
further supported by the lack of STAT-5b phosphorylation by SCF, which is a known mitogenic factor for
erythroid cells. Several reports (Sui et al., 1998;
Gregory et al., 1998; Pallard et al., 1995), with the
exception of one (Brizzi et al., 1999), also suggest that
SCF does not activate STAT-5b. However, SCF
phosphorylated Shc and its associated protein SHIP
in both HB60-5 and HB60-ED cells resulting in
proliferation. Interestingly, during proliferation of
HB60-5 cells with Epo and SCF, phosphorylation of
STAT5 is also detected in these cells. Based on the
results presented here, it is likely that activation of Shc/
ras pathway by SCF may block STAT-5 mediated
di€erentiation signal which can also be induced by Epo
in HB60-5 cells.
Further support for the involvement of STAT-5b in
erythroid di€erentiation comes from recent knock-out
studies. Double knock-out STAT-5a/5b mice are viable
with no apparent defect in erythropoiesis (Teglund et
al., 1998). However, these mice eventually developed
splenomegaly which is associated with a dramatic
increase in the number of Ter 119 erythroid positive
spleen cells (Moriggl et al., 1999). While the authors
attributed this altered morphology to spleen hyperplasia, there is a reasonable possibility that the lack of
STAT-5 in the erythroid progenitor cells of the double
knockout mice may enhance their proliferation.
Interestingly, Socolovsky et al. (1999) have recently
shown that the STAT-5 double knockout embryos are
anemic. The low number of erythroid progenitor cells
seen in these mice was shown to be associated with a
higher apoptotic rate due to lower Bcl-xl expression.
These data strongly suggest that STAT-5 is essential
for high erythropoietic rate during fetal development.
Additional support for the role of STAT-5 in erythroid
development has emerged from a study using a
dominant negative mutant of STAT-5 that inhibited
the di€erentiation of erythroblasts and erythroleukemic
cells (Iwatsuki et al., 1997). It will therefore be
intriguing to determine whether STAT-57/7 mice
are more susceptible to Friend virus induced-erythroleukemias.
Perhaps the most striking result that supports the
above observation is the ability of gp55 to block
Epo-induced phosphorylation in HB60-5 cells. In
addition, these gp55 expressing cells are Epo and
SCF independent with an intact Epo-activated Shc/
ras response. Interestingly, Yamamura et al. (1998)
have recently reported that normal erythroblasts and
erythroleukemic cells expressing gp55 have constitutive STAT-5 phosphorylation. However, they reported that the phosphorylated STAT-5 was unable
to bind to a speci®c STAT-5 consensus DNA
sequence and did not translocate to the nucleus.
Indeed, we also see constitutive phosphorylation of
STAT-5b in a FV-P induced erythroleukemia cell
line, but at very low levels. Nevertheless, their
results are consistent with ours in that gp55/Epo-R
signaling in erythroblasts is distinct from Epo-R
signaling. Although our results also suggest that
gp55/Epo-R signaling could also be identical to Epo/
Oncogene
Epo signaling in erythropoiesis and Friend leukemia
B Zochodne et al
2302
Oncogene
Epo-R only in transformed erythroblasts that
proliferate in response to Epo.
Our previous studies have demonstrated that in
HB60-5 cells Fli-1 ¯uctuates during Epo-induced
di€erentiation while constitutive expression of this
transcription factor results in Epo-induced proliferation (Tamir et al., 1999). Recently, a similar result was
also reported in chicken erythroblasts transfected with
Fli-1 (Pereira et al., 1999). Similarly, gp55 expression in
HB60-5 cells conferred a growth advantage that was
associated with elevated Fli-1 expression levels. Given
that gp55p activates the Shc/ras pathway in HB60-5
cells, it may be that Fli-1 is a downstream component
of this pathway. In support of this notion we have
recently identi®ed Fli-1 as a downstream target of the
SCF/c-Kit signaling which also activates the Shc/ras
pathway (Tamir et al., 1999). Fli-1 may therefore
belong to a growing list of ets related transcription
factors whose expression is thought to be regulated by
the ras pathway (Janknecht, 1996; O'Hagan et al.,
1996; Wasylyk et al., 1998; Fowles et al., 1998).
Our data demonstrates that Fli-1 over-expression in
HB60-5 cells converts Epo-induced di€erentiation to
Epo-induced proliferation. Furthermore, this di€erentiation is correlated with the activation of Shc/ras
through the Epo-R. While the mechanism by which
Fli-1 alters Epo signaling has not yet been identi®ed,
these results suggest that some of the downstream
target genes of this transcription factor may be
involved in the modulation of the Epo/Epo-R signal
transduction pathway. Hence, identi®cation of these
target genes should provide important insights into the
molecular events that regulate normal erythropoiesis
and malignant transformation by Fli-1.
The nature of the synergy between Epo and SCF
with respect to the proliferation of HB60-5 cells
appears to be closely linked to the ability of c-Kit to
phosphorylate the Epo-R. Previously, Wu et al. (1995)
demonstrated that c-Kit was capable of interacting
with and rapidly phosphorylating the Epo-R. Although
a recent report (Jacobs-Helber et al., 1997) suggested
an indirect mechanism of Epo-R phosphorylation by cKit, our data certainly provides evidence that c-Kit
binds to the Epo-R and induces its phosphorylation.
Since the phosphorylation of either c-Kit or Epo-R
activates the Shc/ras pathway in HB60-ED cells, both
SCF and Epo likely promote the growth of these cells
through their accumulative e€ects on multiple overlapping and distinct pathways that are also undoubtedly targeted by gp55.
In summary, by utilizing the cell lines HB60-5 and
HB60-ED, we have shown that activation of Shc/ras
pathway was strongly associated with SCF and Epoinduced cell growth. In contrast, Epo-induced activation of the STAT-5 pathway was associated with
terminal di€erentiation of HB60-5 cells. Constitutive
expression of Fli-1 dramatically modi®ed the e€ect of
Epo on these cells by blocking their di€erentiation and
promoting their proliferation, which was associated
with Shc activation. These same responses to Epo were
produced in cells ectopically expressing gp55p. The
viral glycoprotein activated Fli-1 expression and
blocked STAT-5 activation by Epo. Together, these
results shed further light on the signaling events that
discriminate between Epo-induced proliferation and
di€erentiation.
Materials and methods
Tumors and cell lines
The establishment of the erythroleukemic cell line HB60-5
cells was previously described (Tamir et al., 1999). HB60-ED
cells are polyclonal derivative of HB60-5 cells transfected
with a Fli-1 expression vector (Tamir et al., 1999). The Epodependent erythroleukemia cell line HB9.1-ED, was derived
from a culture of greatly enlarged spleens of BALB/c mice
injected at birth with F-MuLV (Howard et al., 1993). The
erythroleukemia cell lines DP28-9 and CB7 were induced by
FV-P and F-MuLV, respectively as described elsewhere (BenDavid et al., 1988). Cells were maintained in alpha minimum
essential medium (a-MEM) with 10% fetal bovine serum
(FBS).
HB60-5 and HB60-ED cells were cultured in a-MEM
medium supplemented with 15% heat inactivated FBS,
0.5 unit/ml of Epo (Boehringer Mannheim) and 100 ng/ml
of SCF. To induce di€erentiation, HB60-5 cells were washed
twice with PBS and incubated in the presence of 15% FBS
and 1 unit/ml of Epo.
Viral infection
HB60-5 cells were infected with SFFV-P virus by co-culturing
these cells with an SSFV producing amphotrophic GP+
envAM12 helper free packaging cell line (Markowitz et al.,
1988) for 48 h in the presence of Epo and SCF. The
unattached HB60-5 cells were separated from the viral
producer packaging cell lines, washed with PBS and grown
in the presence of Epo and SCF for two additional days. The
polyclonal HB60-SFFV cell line was then established by
growing these cells in the absence of growth factors.
RNA extraction and Northern blotting
Total cellular RNA from erythroleukemia cell lines was
isolated by using TRIzol reagent as described by the supplier
(Gibco BRL). Twenty mg of total RNA was dissolved in
2.2 M formaldehyde, denatured at 658C for 5 min and
electrophoresed in a 1% agarose gel containing 0.66 M
formaldehyde. After transfer to Zetaprobe membranes, the
®lters were hybridized with 26106 c.p.m./ml of a-32P-dCTP
labeled probe.
Western blotting and immunoprecipitations
Erythroleukemia cell lines were lysed with RIPA bu€er (0.5%
NP-40, 50 mM Tris HCl, pH 8.0, 120 mM NaCl, 50 mM NaF,
10 mg/ml aprotinin, 100 mg/ml leupeptin and 10 mM PMSF).
For Western blotting, 50 mg of cell lysates were resolved on a
12% SDS-polyacrylamid gel and immunoblotted as described
(Howard et al., 1996). Antibody to gp55 was obtained from
Quality Biotech Inc. (New Jersey) and antibody against Erk-2
from Santa Cruz, CA, USA.
For immunoprecipitations (IPs), cells were starved for 9 h
in a a-MEM+0.5% FBS. Subsequent to induction with Epo
(1 units/ml) or SCF (100 ng/ml) for 10 min at 378C, the cells
were collected by centrifugation, washed with cold PBS and
lysed in RIPA bu€er as previously described (Tamir et al.,
1999). Protein concentration was determined using the
BioRad protein assay (Bio-Rad Laboratories, Munchen,
Germany). After pre-clearing with Protein-G and -A,
200 mg of lysates were used in IPs and subjected to Western
blotting, as described (Tamir et al., 1999). For double IP
procedure, the lysates (2 mg) from starved or SCF stimulated
cells were ®rst immunoprecipitated with antibody to Epo-R,
bound to sepharose-A beads and the bound proteins were
washed three times with the IP lysis bu€er. The proteins were
released by boiling the beads in the same lysis bu€er
containing 1% SDS for 1 min. Supernatant was then diluted
10-fold with the IP bu€er and immunoprecipitated for c-Kit
Epo signaling in erythropoiesis and Friend leukemia
B Zochodne et al
using the c-Kit antibody. The Shc antibody was a gift from
Dr Jane McGlade (University of Toronto). STAT-5b, p85
subunit of PI3K and cKit antibodies were purchases from
Santa-Cruz, CA, USA.
After a brief vortexing and a further incubation on ice for
5 min, the suspension was spun at 14000 r.p.m. for 5 min at
48C. Supernatants, containing the nuclear extract, were
aliquoted and stored at 7808C.
2303
Preparation of cytoplasmic and nuclear extracts
Approximately 16106 cells, cultured as described above,
were pelleted, washed once with PBS and once with
hypotonic bu€er (10 mM HEPES, pH 7.5, 1.5 mM MgCl2,
10 mM KCl, 0.5 mM DTT). Cell pellets were resuspended in
400 uL of hypotonic bu€er and incubated on ice for 10 min.
After a brief vortexing (10 s) and further incubation on ice
for 5 min, cells were pelleted at 8000 r.p.m. for 10 min. The
supernatant, which contains the cytoplasmic material, was
aliquoted and stored at 7808C. Pellets, which contain nuclei,
were resuspended in 200 uL bu€er C (20 mM HEPES, pH 7.5
1.5 mM, MgCl2, 0.5 mM DTT 0.5 mM PMSF 25% glycerol)
containing 420 mM NaCl and incubated on ice for 20 min.
Acknowledgments
We thank Dr Jane McGlade for the generous gift of Shc
antibody. We also thank Drs Kirill Rosen for his comment
on the manuscript and Lynda Woodcock for help in
preparation of the manuscript. This work was supported
by grants from the National Cancer Institute of Canada to
Y Ben-David and SA Berger and Medical Research
Council of Canada to D Dumont. RR Higgins was
supported by a fellowship from the Leukemia Research
Fund of Canada.
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