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INTRODUCTION |
The survival, proliferation, and differentiation of erythroid
progenitor cells depends to a large extent on the signals emanating from c-Kit, a receptor tyrosine kinase, and the erythropoietin (Epo)1 receptor (Epo-R), a
member of the hematopoietin receptor family (1, 2). Mutant mice that
lack the expression of c-Kit (dominant white spotting, or W,
mutants) or Epo-R demonstrate severe deficiencies in erythroid cell
development (1, 2). c-Kit-deficient mice exhibit a severe reduction of
colony-forming unit-erythroid (CFU-E) progenitors in the fetal liver
and die of anemia around day 16 of gestation (1, 3). Epo-R-deficient
mice also demonstrate a significant decrease in CFU-E progenitors and
die of anemia between days 13 and 15 of gestation (2). The data suggest
that committed erythroid progenitors cannot survive, proliferate, or differentiate unless both the c-Kit and the Epo-R signal transduction pathways are functional. Recent studies have suggested that Epo-R may
contribute to this process by preventing committed erythroid progenitors from undergoing apoptosis (4, 5). The role of c-Kit in the
process of erythroid cell development alone or in combination with
Epo-R remains unclear. Recent studies have demonstrated that erythroid
precursors from mice deficient in the expression of Epo-R can be
rescued by in vitro infection with an Epo-R-containing retrovirus but only in the presence of stem cell factor (SCF) (6). From
these data, it has been inferred that an interaction between Epo-R and
c-Kit, which was previously demonstrated in a cultured cell line (7),
is required for erythroid colony formation, presumably at a stage prior
to that at which Epo-R-deficient precursors are arrested.
Bcl-2 family proteins are likely to be key effectors of growth factor
receptor-mediated survival signals (8). The balance of antiapoptotic
(Bcl-2, Bcl-xL, A1, and MCL1) and proapoptotic (Bax, Bad,
Bak, and Bcl-xs) Bcl-2 family proteins in the cell is
critical in determining its ability to survive and subsequently proliferate and differentiate (9). Consistent with these observations, both Bcl-xL and Bcl-2 are involved in regulating erythroid
progenitor cell survival (10-15). However, it is uncertain during
which phase of erythroid cell maturation Bcl-xL and Bcl-2
function. Many cytokines induce the expression of antiapoptotic Bcl-2
family proteins, although the mechanisms involved are not clear
(16-18). When and how c-Kit and Epo-R induce the expression of Bcl-2
and/or Bcl-xL in erythroid cells remains poorly defined. In
particular, the role of c-Kit in regulating the expression of Bcl-2 and
Bcl-xL during erythroid cell development remains unknown.
Recent studies have shown that Stat5 may in part be responsible for
regulating the expression of Bcl-xL in erythroid progenitor cells (19). Mutants of mice that lack both Stat5a and Stat5b demonstrate embryonic anemia (19). In vivo deficiency of
Stat5 does not result in embryonic lethality (19, 20), suggesting that
other factors may regulate the expression of Bcl-xL, either independently or cooperatively with Stat5 during embryogenesis. Stat5
is activated by Epo-R (21) and other cytokine receptors including those
for thrombopoietin; granulocyte-macrophage colony-stimulating factor;
granulocyte colony-stimulating factor; interleukin-2, -3, and -5;
prolactin; and growth hormone.
In addition to a role for growth factors in inducing the expression of
antiapoptotic genes, hematopoietic cells also use internal programs to
ensure their survival. The transcription factor GATA-1, which is
abundant in erythroid progenitor cells, is essential for maturation of
erythroblasts (22-24). Although GATA-1 is expressed in multipotential
progenitor cells prior to the commitment to a single lineage,
disruption of the GATA-1 gene produces maturation arrest relatively
late in erythroid development (22-24). GATA-1-deficient embryos are
embryonic lethal at the yolk sac stage (24). They appear colorless;
proerythroblast-like cells are present in the yolk sac and circulation.
Upon in vitro differentiation of GATA-1-deficient embryonic
stem (ES) cells, definitive erythroid precursors are arrested at the
proerythroblast stage and undergo apoptosis (25, 26).
The generation of an erythroid cell line (G1E-ER2) from
GATA-1-deficient ES cells provides an excellent model to study the function of c-Kit and Epo-R in erythroblasts in the context of GATA-1
function (27). G1E-ER2 cells express erythroid but not myeloid genes
(27). These cells proliferate continuously in culture as
developmentally arrested erythroid precursors. G1E-ER2 cells stably
express a conditional (estrogen-responsive) form of GATA-1, and upon
exposure to
-estradiol they undergo synchronous erythroid maturation
(15). Utilizing G1E-ER2 cells, recent studies have shown that GATA-1
can induce the expression of Bcl-xL and cooperate with Epo
to promote the survival of these cells, although the mechanism of
Bcl-xL expression remains to be determined (15). In the
present study, we demonstrate an essential role for c-Kit in
proliferation and survival of erythroid progenitors via the induction
of Bcl-2 expression. We also demonstrate that c-Kit synergizes with
Epo-R in enhancing proliferation and survival of erythroid progenitors
in the absence of GATA-1 function by inducing the expression of Epo-R,
Stat5, and Bcl-xL. Restoration of GATA-1 function results
in terminal erythroid differentiation, down-regulation of c-Kit and
Bcl-2, and up-regulation of Epo-R and Bcl-xL, leading to
significantly enhanced survival of differentiating erythroid
progenitors. These results suggest that c-Kit and Epo-R have unique
role(s) during distinct phases of erythroid cell maturation and
describe an essential role for c-Kit in proliferation and in inducing
the expression of critical genes involved in regulating survival of
erythroid progenitors by Epo, including Epo-R, Stat5, and
Bcl-xL.
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EXPERIMENTAL PROCEDURES |
Cell Line, Antibodies, and Flow Cytometric Analysis--
G1E-ER2
cells have been described previously (15, 28). Unless otherwise
specified, G1E-ER2 cells were grown in Iscove's modified Dulbecco's
medium (Life Technologies, Inc.) with 15% heat-inactivated embryonic
stem cell serum (Hyclone, Logan, UT), recombinant erythropoietin (Epo)
(2 units/ml) (Amgen, Thousand Oaks, CA), and recombinant rat SCF (50 ng/ml) (Amgen, Thousand Oaks, CA).
Phycoerythrin-conjugated monoclonal antibodies were directed against
Ter119. All of the phycoerythrin-conjugated monoclonal antibodies,
including the isotype control antibodies, were purchased from
Pharmingen (San Diego, CA). G1E-ER2 cells (1 × 106)
were incubated at 4 °C for 30 min with 1 µg of the primary
monoclonal antibody. Cells were washed three times with
phosphate-buffered saline containing 0.1% bovine serum albumin (Sigma)
and analyzed by a fluorescence-activated cell sorter (Becton Dickinson,
San Jose, CA).
Effects of SCF and Epo on Proliferation and Survival of G1E-ER2
Cells--
The effect of SCF and Epo on proliferation of G1E-ER2 cells
was assayed using thymidine incorporation. 96-Well tissue culture plates were utilized for these studies. G1E-ER2 cells were plated at
5 × 104 cells/well for 48 h, either in the
presence or absence of recombinant rat SCF and/or Epo. Subsequently,
1.0 µCi of [3H]thymidine (Amersham Pharmacia Biotech)
was added to each well for 6-8 h at 37 °C. Cells were then
harvested using an automated cell harvester (96-well harvester;
Brandel, Gaithersburg, MD), and thymidine incorporation was determined
in a scintillation counter. The effect of SCF and/or Epo on cell death
(apoptosis and necrosis) of G1E-ER2 cells was assayed by staining the
cells with annexin-fluorescein isothiocyanate-conjugated and propidium iodide according to the manufacturer's instructions (Pharmingen, San
Diego, CA). 24-Well tissue culture plates were utilized for these
studies. G1E-ER2 cells were plated at 5 × 105
cells/well for 48 h, either in the presence or absence of SCF and/or Epo. Subsequently, cells were harvested and stained with annexin-fluorescein isothiocyanate and propidium iodide and analyzed by
flow cytometry.
Western Blotting--
Western blotting was performed according
to standard protocols. 1-2 × 106 G1E-ER2 cells were
plated in duplicate six-well tissue culture plates for 48 h at
37 °C in the presence or absence of SCF and/or Epo. Thereafter,
cells were harvested and lysed in lysis buffer (10 mmol/liter
K2HPO4, 1 mmol/liter EDTA, 5 mmol/liter EGTA,
10 mmol/liter MgCl2, 1 mmol/liter
Na2VO4, 50 mmol/liter
-glycerol phosphate,
10 µg/ml leupeptin, 1 µg/ml pepstatin, and 10 µg/ml aprotinin) at
4 °C for 30 min. Cell lysates were clarified by centrifugation for
30 min at 10,000 × g at 4 °C. An equal amount of
protein was fractionated on 10% polyacrylamide/SDS gel and electrophoretically transferred to nitrocellulose membrane. Expression of c-Kit was determined by using a 1:1000 dilution of an anti-c-Kit antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Expression of Epo-R was determined by using a 1:1000 dilution of an anti-Epo-R antibody (Santa Cruz Biotechnology). Expression of Bcl-xL
and Bcl-2 was determined by using a 1:1000 dilution of anti-Bcl-2 (Santa Cruz Biotechnology) and anti-Bcl-xL (Pharmingen, San
Diego, CA) antibody. The expression of Erk and p38 was determined by using anti-Erk and p38 antibodies (all purchased from New England Biolabs, Beverly, MA).
Activation of Stat5 was determined by utilizing a phosphospecific Stat5
antibody (Tyr694; New England Biolabs, Beverly, MA).
This antibody detects Stat5 only when it is catalytically activated by
phosphorylation at Tyr694. Briefly, G1E-ER2 cells
were factor-starved for 6-8 h in Iscove's modified Dulbecco's
medium. Subsequently, 6-8 × 106 cells were
stimulated with SCF and/or Epo for the indicated time points.
Thereafter, cells were harvested and lysed in lysis buffer as described
above. Western blot analysis was performed according to the
manufacturer's instructions (New England Biolabs, Beverly, MA).
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RESULTS |
Stimulation of c-Kit by SCF Results in Proliferation of G1E-ER2
Cells but Impairment of GATA-1-induced Differentiation--
Previous
work, including the analysis of mice deficient in the expression of
c-Kit and Epo-R, has suggested an essential function for c-Kit and
Epo-R in erythroid cell development (1, 2). To determine the role(s) of
c-Kit and Epo-R in erythroid cell development, we first examined
proliferation in G1E-ER2 cells stimulated with either SCF or Epo or
with the combination of these two cytokines in the absence or presence
of GATA-1 function. As expected, in cultures in which no cytokines were
added, G1E-ER2 cells demonstrated no proliferation after 48 h.
Stimulation of G1E-ER2 cells with SCF alone, in the absence of GATA-1,
resulted in a significant increase in DNA synthesis in comparison with unstimulated cells (68,144 ± 13,765 versus 455 ± 194 cpm (mean ± S.D.), respectively, p < 0.0001)
(Fig. 1A). Interestingly,
stimulation of these cells with Epo alone resulted in only a modest
increase in DNA synthesis in comparison with unstimulated cells
(5144 ± 2711 versus 455 ± 194 cpm, mean ± S.D., respectively, p < 0.0001) (Fig. 1A).
DNA synthesis in response to Epo could not be augmented by higher Epo
concentration (data not shown). Stimulation of G1E-ER2 cells with
combined Epo and SCF resulted in significantly greater proliferation in
comparison with cells stimulated with SCF alone (92,013 ± 17,923 versus 68,144 ± 13,765 cpm (mean ± S.D.),
respectively, p < 0.0001) (Fig. 1A). These
results demonstrate an essential and nonredundant role for c-Kit in
promoting G1E-ER2 cell proliferation in the absence of GATA-1
function.

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Fig. 1.
Stimulation of c-Kit by SCF results in
proliferation of G1E-ER2 cells. G1E-ER2 cells were cultured in the
presence of SCF (50 ng/ml) or Epo (25 units/ml) or both SCF (25 ng/ml)
and Epo (12.5 units/ml) either in the absence (A) or
presence of GATA-1 expression (B) for 48 h.
Proliferation was measured by thymidine incorporation assay.
Bars denote the mean thymidine incorporation (cpm ± S.D.) of two independent experiments performed in replicates of six. *,
p < 0.01 for SCF versus Epo and no
growth factor. **, p < 0.01 for Epo versus
no growth factor. ***, p < 0.01 for SCF + Epo
versus SCF, Epo, and no growth factor.
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We next examined the effect of c-Kit and Epo-R activation on
proliferation of G1E-ER2 cells in the presence of GATA-1 expression.
-Estradiol-induced GATA-1-expressing G1E-ER2 cells were left unstimulated or were stimulated with SCF or Epo or with the combination of these two cytokines as described above. Stimulation with SCF alone
resulted in a modest but statistically significant increase in DNA
synthesis (1181 ± 478 versus 356 ± 170 cpm
(mean ± S.D.), respectively, p < 0.01) (Fig.
1B). A modest but statistically significant increase in DNA
synthesis over cytokine-unstimulated cells was also apparent following
stimulation with Epo alone (780 ± 133 versus 356 ± 170 cpm (mean ± S.D.), respectively, p < 0.01) (Fig. 1B). Combined stimulation of GATA-1-expressing
cells with SCF and Epo also demonstrated significantly greater
proliferation in comparison with cells stimulated with SCF alone
(6006 ± 1356 versus 1181 ± 478 cpm, mean ± S.D., respectively, p < 0.01) (Fig. 1B).
Previous studies have shown that the expression of GATA-1 by adding
-estradiol in the presence of Epo triggers synchronous erythroid
maturation in G1E-ER2 cells, G1 phase cell cycle arrest, followed by the appearance of benzidine-staining cells (15). Consistent
with these observations, in our hands, the expression of GATA-1 in the
presence of Epo also resulted in the emergence of benzidine-positive
cells (data not shown) and induction of cell surface protein, Ter119
(Fig. 2). However, GATA-1-induced differentiation, as measured by the induction of Ter119, was
significantly reduced in the presence of SCF compared with Epo
stimulation (20.3 ± 3 versus 72.6 ± 7.5% Ter119
expression (mean ± S.D.), respectively, p < 0.005) (Fig. 2). These results demonstrate that in contrast to the
effect of c-Kit activation on G1E-ER2 cell proliferation in the absence
of GATA-1, in the presence of GATA-1 expression, SCF significantly
impairs erythroid differentiation.

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Fig. 2.
Restoration of GATA-1 function results in
terminal differentiation of G1E-ER2 cells. G1E-ER2 cells were
cultured in the presence of SCF (50 ng/ml) or Epo (25 units/ml) or both
SCF (25 ng/ml) and Epo (12.5 units/ml) either in the absence or
presence of GATA-1 expression for 48 h. Differentiation was
measured by examining the induction of Ter119 following -estradiol
treatment of G1E-ER2 cells as described under "Experimental
Procedures." Bars denote the mean ± S.D. Ter119
expression of three independent experiments. *, p < 0.05 for SCF versus no growth factor. **, p < 0.05 for Epo and Epo + SCF versus SCF and no growth
factor.
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Stimulation of c-Kit by SCF Maintains the Survival of G1E-ER2 Cells
in the Absence of GATA-1 but Not in the Presence of GATA-1
Expression--
A reasonable hypothesis to explain the differential
effects of Epo and SCF on proliferation and differentiation of G1E-ER2 cells would be that these two cytokines maintain the survival of
G1E-ER2 cells during distinct stages of maturation, which may be
regulated by GATA-1 (Fig. 7). To test this, we examined apoptosis in
G1E-ER2 cells following stimulation with either SCF or Epo or with the
combination of these two cytokines in the absence or presence of GATA-1
function. Following no cytokine treatment, factor-starved G1E-ER2 cells
lacking the expression of GATA-1 demonstrated significantly greater
cell death in comparison with cells cultured in the presence of either
SCF or Epo or with the combination of these two after 48 h (Fig.
3A). Cell death of G1E-ER2 cells in the presence of SCF alone was significantly less relative to
cultures stimulated with Epo alone (28 ± 3.4 versus
49 ± 3.7% (mean ± S.D.), respectively, p < 0.001) (Fig. 3A). Further, combined stimulation of these
cells with both SCF and Epo resulted in significantly less cell death
relative to cultures stimulated with SCF alone (13 ± 3.5 versus 28 ± 3.4% (mean ± S.D.), respectively,
p < 0.001) (Fig. 3A).

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Fig. 3.
Differential effects of c-Kit and Epo-R
stimulation on the survival of G1E-ER2 cells in the absence and
presence of GATA-1 expression. G1E-ER2 cells were cultured in the
presence of SCF (50 ng/ml) or Epo (25 units/ml) or both SCF (25 ng/ml)
and Epo (12.5 units/ml) either in the absence (A) or
presence of GATA-1 expression (B) for 48 h. Cell death
was quantitated by performing annexin V and propidium iodide staining
as described under "Experimental Procedures." Bars
denote the percentage of total cell death ± S.D. of three
independent experiments performed in replicates of three. A,
*, p < 0.001 for SCF versus Epo and no
growth factor; **, p < 0.001 for Epo versus
no growth factor; ***, p < 0.001 for SCF + Epo
versus Epo, SCF, and no growth factor. B, *,
p < 0.01 for SCF versus no growth factor;
**, p < 0.01 for Epo versus SCF and no
growth factor; ***, p < 0.01 for SCF + Epo
versus Epo, SCF, and no growth factor.
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In contrast to significantly less cell death of SCF-treated cells in
the absence of GATA-1, only a modest decrease in cell death was
observed in SCF treated cultures in the presence of GATA-1 (92 ± 2.2 versus 96 ± 2.4% (mean ± S.D.),
respectively, p < 0.007) (Fig. 3B). In
contrast, the addition of Epo to GATA-1-expressing cultures was
associated with significantly less cell death of G1E-ER2 cells relative
to SCF-stimulated cultures (61 ± 9.1 versus 92 ± 2.2% (mean ± S.D.), respectively, p < 0.01)
(Fig. 3B). Further, analogous to the results observed in the
absence of GATA-1 (see Fig. 3A), stimulation of
GATA-1-expressing cells with SCF and Epo resulted in least cell death
(Fig. 3B). These results demonstrate that in the absence of
GATA-1, c-Kit-mediated antiapoptotic signals appear to be essential in
maintaining the survival of G1E-ER2 cells. In contrast, Epo-R-mediated
antiapoptotic signals play a dominant role in maintaining the survival
of GATA-1-induced terminally differentiating G1E-ER2 cells. Combined
stimulation of c-Kit and Epo-R is essential for maximum survival of
G1E-ER2 cells, both in the absence and presence of GATA-1 function
(Fig. 7).
Stimulation of c-Kit by SCF Induces Bcl-2 but Not
Bcl-xL Expression in G1E-ER2 Cells--
To determine the
mechanism of differential survival of G1E-ER2 cells in response to SCF
and Epo stimulation during distinct stages of erythroid cell
maturation, we examined the expression of antiapoptotic gene family
members Bcl-2 and Bcl-xL in the absence and presence of
GATA-1 function. G1E-ER2 cells grown in log phase in the presence of
SCF and Epo were factor-starved for 5 h and stimulated with
cytokines (Fig. 4). 48 h later,
cells were harvested and lysed, and an equal amount of protein was
subjected to immunoblot analysis using an anti-Bcl-2 or
anti-Bcl-xL antibody. In the absence of GATA-1, stimulation
of G1E-ER2 cells with SCF resulted in significant expression of Bcl-2
(Fig. 4, lane 2). In contrast, stimulation of
these cells with Epo was associated with complete loss of Bcl-2 protein
(Fig. 4, lane 3). Combined stimulation with SCF
and Epo was associated with significant levels of Bcl-2 protein after 48 h of culture (Fig. 4, lane 4). However,
stimulation of G1E-ER2 cells with Epo resulted in significant
Bcl-xL expression (Fig. 4, lane 3).
Surprisingly, stimulation of c-Kit with SCF resulted in a significant
decrease in the expression of Bcl-xL after 48 h (Fig.
4, lane 2). Consistent with the survival data
noted above, combined stimulation of c-Kit and Epo-R significantly
increased the expression of Bcl-xL relative to Epo
stimulation alone (Fig. 4, lane 4). These data
demonstrate that in the absence of GATA-1 expression, survival of
G1E-ER2 cells by SCF is associated with the expression of Bcl-2. In
contrast, Epo-mediated survival of these cells is associated with the
induction of Bcl-xL. Further, maximum survival in cultures
stimulated concurrently with SCF and Epo is associated with the
combined antiapoptotic effects of both Bcl-2 and Bcl-xL
(Fig. 7).

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Fig. 4.
Stimulation of c-Kit and Epo-R results in
differential expression of Bcl-2 and Bcl-xL in G1E-ER2
cells. G1E-ER2 cells were cultured in the presence of SCF (50 ng/ml) or Epo (25 units/ml) or both SCF (25 ng/ml) and Epo (12.5 units/ml) in the absence or presence of GATA-1 expression for 48 h. For Western analysis, 5 µg of protein from whole cell lysates was
fractionated on a 10% SDS-polyacrylamide gel and probed with
anti-Bcl-2 or anti-Bcl-xL antibody. The position of Bcl-2
and Bcl-xL is indicated. Lanes 2-4
show expression of Bcl-2 and Bcl-xL in the absence of
GATA-1 expression. Lanes 5-7 show expression of
Bcl-2 and Bcl-xL in the presence of GATA-1
expression.
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We next examined the antiapoptotic effects of Bcl-2 and
Bcl-xL in GATA-1-induced terminally differentiating G1E-ER2
cells in the presence of SCF or Epo or the combination of these two cytokines. Stimulation of these cells with SCF resulted in neither Bcl-2 nor Bcl-xL expression (Fig. 4, lane
5). Further, the lack of Bcl-2 induction noted earlier in
response to Epo in the absence of GATA-1 was also apparent in the
presence of GATA-1 expression (Fig. 4, lane 6).
These data suggest that GATA-1, either directly or indirectly,
down-regulates the expression of Bcl-2, which is associated with
complete loss of survival in G1E-ER2 cells stimulated with SCF (Fig.
7).
In contrast to the apparent lack of Bcl-2 effects on the survival of
terminally differentiating G1E-ER2 cells in the presence of SCF or Epo,
expression of GATA-1 significantly enhanced the expression of
Bcl-xL in presence of Epo but not SCF (Fig. 4,
lane 6). Epo-mediated increase in
Bcl-xL expression in GATA-1-expressing cells was
significantly greater than that observed in cells stimulated with Epo
in the absence of GATA-1 (Fig. 4, lane 3). The
increase in Bcl-xL expression under these conditions
correlated with enhanced survival of G1E-ER2 cells in the presence of
Epo but not SCF noted earlier (see Fig. 3B). Analogous to
our previous observation in the absence of GATA-1, combined stimulation
of GATA-1-expressing G1E-ER2 cells with SCF and Epo also resulted in
enhanced survival via the induction of both Bcl-xL and
Bcl-2 (Fig. 4, lane 7), although the level of
Bcl-2 was less than that observed in the absence of GATA-1. These
results demonstrate that the expression of Bcl-xL is
associated with the survival of terminally differentiating G1E-ER2
cells. Combined expression of Bcl-2 and Bcl-xL is
associated with further augmenting the survival of terminally
differentiating G1E-ER2 cells. Further, unlike the lack of cooperation
noted between GATA-1 and SCF in inducing the expression of Bcl-2, Epo
in collaboration with GATA-1 significantly enhances the expression of
Bcl-xL and survival of terminally differentiating G1E-ER2
cells (Fig. 7).
Stimulation of c-Kit by SCF Does Not Activate Stat5 but Maintains
Its Protein Expression--
Stat5 binds to the Bcl-xL
promoter and induces the expression of Bcl-xL in response
to Epo stimulation in HCD57 cells (19). To explain the observation that
c-Kit stimulation by SCF in GATA-1-nonexpressing cells does not
maintain the expression of Bcl-xL but stimulation with Epo-R significantly enhances the expression of Bcl-xL,
we hypothesized that activation of Stat5 in G1E-ER2 cells is mediated differentially via Epo-R but not c-Kit stimulation. To test this hypothesis, c-Kit-expressing G1E-ER2 cells were factor-starved for 5-6
h and stimulated with SCF or Epo or with the combination of these two
cytokines (Fig. 5A).
Thereafter, cells were lysed, and immunoblot analysis was performed
with an antiphospho-Stat5 antibody. Consistent with previously
published observations in HCD57 cells (19), stimulation of G1E-ER2
cells with Epo resulted in Stat5 activation (Fig. 5A,
lanes 5 and 6). Consistent with our
hypothesis and with the lack of Bcl-xL induction noted in response to c-Kit activation, stimulation of G1E-ER2 cells with SCF did
not activate Stat5 (Fig. 5A, lanes 2 and 3), despite the presence of Stat5 protein in these cells
(Fig. 5A, lower panel). Combined
stimulation of Epo-R and c-Kit did not appear to enhance the activation
of Stat5 (Fig. 5A, lanes 8 and
9). These data demonstrate that activation of Stat5 in
G1E-ER2 cells is mediated via the activation of Epo-R but not c-Kit
(Fig. 7).

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Fig. 5.
Stimulation of c-Kit by SCF does not activate
Stat5 but maintains its protein expression. A,
factor-starved G1E-ER2 cells were stimulated for indicated time points
in the presence of SCF (50 ng/ml) (lanes 2 and
3) or Epo (25 units/ml) (lanes 5 and
6) or with the combination of SCF (25 ng/ml) and Epo (12.5 units/ml) (lanes 8 and 9). Cell
lysates were collected and subjected to Western blot analysis with an
antiphospho-Stat5 antibody that specifically detects phosphorylated
Tyr694. The bottom panel shows
total Stat5 protein levels in each lane as a loading control. The
position of phosphorylated Stat5 and total Stat5 is indicated.
B, G1E-ER2 cells were cultured in the presence of SCF
(lane 2) or Epo (lane 3) or
both SCF and Epo (lane 4) in the absence of
GATA-1 expression for 48 h. For Western analysis, 5 µg of
protein from whole cell lysates was fractionated on a 10%
SDS-polyacrylamide gel and probed with anti-Stat5 (top
panel), anti-Erk (middle panel), and
anti-p38 (bottom panel) antibodies. The position
of Stat5, Erk2, and p38 is indicated.
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We next hypothesized that the expression of Bcl-xL after
stimulation of both c-Kit and Epo-R and the survival of G1E-ER2 cells in the absence of GATA-1 and these cytokines might in part be due to
the regulation of the Stat5 protein by SCF. In this model (Fig. 7), we
propose that regulation of Stat5 occurs in two steps. First,
stimulation of c-Kit by SCF induces the expression of Stat5 protein in
G1E-ER2 cells, followed by phosphorylation and subsequent activation of
Stat5 protein via the activation of Epo-R by Epo. To test if c-Kit
stimulation by SCF was associated with the induction of expression of
Stat5, G1E-ER2 cells grown in the presence of SCF and Epo were washed
three times in medium containing no growth factors and stimulated with
either SCF or Epo or with the combination of these two cytokines (Fig.
5B). 48 h later, cells were harvested and lysed, and an
equal amount of protein was subjected to immunoblot analysis.
Consistent with our hypothesis, activation of c-Kit by SCF is essential
for maintaining the expression of Stat5 in G1E-ER2 cells (Fig.
5B, lane 2). Stimulation of these
cells with Epo alone lead to a significant decrease in the expression
of Stat5 protein after 48 h of culture (Fig. 5B,
lane 3). As a loading control, the expression of
mitogen-activated protein kinase family members Erk and p38 was
examined. The expression of these proteins remained similar in cultures
stimulated with either SCF or Epo (Fig. 5B,
middle and lower panels). These
results demonstrate a novel mechanism of cooperation between c-Kit and
Epo-R in erythroid cells, whereby stimulation of c-Kit by SCF is an
essential prerequisite for activation of Stat5 via Epo stimulation and
resulting enhanced Bcl-xL expression and survival (Fig.
7).
GATA-1 Modulates the Expression of c-Kit and Epo-R during Terminal
Stages of Erythroid Maturation--
To further explore the
differential responsiveness of G1E-ER2 cells to SCF and Epo in the
absence and presence of GATA-1 function, we examined the consequence(s)
of GATA-1 in modulating the expression of c-Kit and Epo-R. We
hypothesized that GATA-1 either directly or indirectly modulates the
expression of c-Kit and Epo-R during terminal stages of erythroid
differentiation, rendering these cells nonresponsive to SCF but not
Epo. To test this, we examined the expression of c-Kit and Epo-R in
G1E-ER2 cells by immunoblot analysis in the absence and presence of
GATA-1 expression. G1E-ER2 cells grown in the presence of SCF and Epo
were starved for 5 h and stimulated with either SCF or Epo or with
the combination of these two cytokines in the absence or presence of
-estradiol. 48 h later, cells were harvested and lysed, and an
equal amount of protein was subjected to immunoblot analysis.
Stimulation of G1E-ER2 cells by SCF in the absence of GATA-1 resulted
in significant c-Kit expression after 48 h of culture (Fig.
6, lane 2).
However, stimulation of these cells in the presence of Epo
down-regulated the expression of c-Kit after 48 h (Fig. 6,
lane 3). In contrast, induction of GATA-1
significantly down-regulated the expression of c-Kit in the presence of
SCF (Fig. 6, lane 5). These data demonstrate that
restoring the function of GATA-1, either directly or indirectly down-regulates the expression of c-Kit in G1E-ER2 cells. Therefore, loss of Bcl-2 expression and reduced survival noted earlier in G1E-ER2
cells in the presence of SCF may in part be due to the loss of c-Kit
protein in terminally differentiating G1E-ER2 cells (Fig.
7).

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Fig. 6.
Expression of GATA-1 results in modulation of
c-Kit and Epo-R expression. G1E-ER2 cells were cultured in the
presence of SCF (50 ng/ml) or Epo (25 units/ml) or both SCF (25 ng/ml)
and Epo (12.5 units/ml) in the absence or presence of GATA-1 expression
for 48 h. For Western analysis, 5 µg of protein from whole cell
lysates was fractionated on a 10% SDS-polyacrylamide gel and probed
with anti-c-Kit or anti-Epo-R antibody. The position of c-Kit and Epo-R
is indicated. Lanes 2-4 show expression of c-Kit
and Epo-R in the absence of GATA-1. Lanes 5-7
show expression of c-Kit and Epo-R in the presence of GATA-1
expression.
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Fig. 7.
Survival pathways during erythroid cell
development. In immature erythroid cells, c-Kit signaling induces
Bcl-2, Epo-R, and Stat5 expression. Epo-R signaling induces
phosphorylation of Stat5 and expression of Bcl-xL.
Expression of GATA-1 results in terminal erythroid differentiation and
down-regulation of c-Kit and Bcl-2 and up-regulation of Epo-R and
Bcl-xL. Combined expression of Bcl-2 and Bcl-xL
is associated with maximum survival of these cells.
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We next examined the effect of GATA-1 induction on Epo-R expression. As
seen in Fig. 6, stimulation of G1E-ER2 cells with SCF in the absence of
GATA-1 resulted in significant expression of Epo-R after 48 h of
culture (Fig. 6, lane 2). In contrast, a
significant loss of Epo-R was observed in cells stimulated with Epo
alone (Fig. 6, lane 3). However, restoration of
GATA-1 in the presence of Epo significantly enhanced the level of Epo-R in these cells (Fig. 6, lane 6). Thus, analogous
to described previously role for GATA-1 and Epo in enhancing the
expression of Bcl-xL in terminally differentiating G1E-ER2
cells (see Fig. 4B, lane 6), these
data also demonstrate cooperativity between GATA-1 and Epo in inducing
the expression of Epo-R (Fig. 7).
 |
DISCUSSION |
SCF is crucial for the development of CFU-E, since mice lacking
SCF or its receptor c-Kit exhibit significant reduction of fetal liver
CFU-E progenitors and suffer severe anemia (1, 3). The survival of
CFU-E progenitors also depends on Epo, suggesting that committed
erythroid progenitors cannot proliferate or mature further unless both
c-Kit and the Epo-R signal transduction pathways are functional. Our
results demonstrate an essential and nonredundant function for
SCF/c-Kit in proliferation and survival of proerythroblasts. In
contrast, we demonstrate that stimulation of Epo-R signaling does not
induce proliferation, although it maintains the survival of
proerythroblasts. These observations are consistent with a
previously reported role for Epo in the survival of CFU-Es without
affecting their cell cycle status (29-31). Interestingly, the combined
stimulation of G1E-ER2 cells with SCF and Epo significantly enhanced
the proliferation of these cells. In this regard, we and others have
previously shown that SCF can replace Epo in supporting the growth of
HCD57 cells, an Epo-dependent erythroid cell line (7, 32).
In these cells, SCF rapidly induced tyrosine phosphorylation of both
c-Kit and Epo-R. These data suggest that c-Kit may activate the
Epo/Epo-R signal transduction pathway via tyrosine phosphorylation of
the Epo-R and that, in turn, the activated Epo-R may induce further proliferation and maturation of committed erythroid progenitors. We
believe that this may in part be the mechanism of increased proliferation noted in G1E-ER2 cells in response to simultaneous activation of both c-Kit and Epo-R.
The mechanism of Epo and SCF regulation of red cell production is not
clear. The maintenance of cell survival may constitute one such
mechanism, since we demonstrate that the maximum number of erythroid
progenitors rescued from apoptosis depends on simultaneous activation of both c-Kit and Epo-R. The graded response of G1E-ER2 cells to Epo and SCF could in part be due to variation in the antiapoptotic signaling that progenitors require; a number of distinct
antiapoptotic pathways may interact additively or synergistically to
rescue maximum numbers of progenitors stimulated with Epo and SCF.
Support for this model comes from our studies demonstrating induction
of two separate antiapoptotic proteins, Bcl-2 and Bcl-xL, by c-Kit and Epo-R, respectively (Fig. 7). We show that the combined induction of both Bcl-2 and Bcl-xL by two completely
separate cytokines is associated with maximum survival of G1E-ER2 cells both at the proerythroblast stage (in the absence of GATA-1) as well as
in terminally differentiated erythroid cells (in the presence of
GATA-1). Our results also demonstrate that SCF-induced Bcl-2 expression
is predominantly associated with the survival of proerythroblasts. In
contrast, Epo-induced Bcl-xL is associated with the
survival of terminally differentiated erythroid cells.
The production of definitive erythroid lineage cells to a large extent
is controlled by Epo (33). Epo prevents the apoptotic cell death of
definitive erythroid progenitors. The antiapoptotic effect of Epo on
definitive erythroid progenitors has been observed from late erythroid
progenitors (CFU-E) up to the onset of hemoglobinization (4, 34-36).
Epo-deprived apoptotic cell death is observed less at the end of
maturation when maximum hemoglobin synthesis occurs. On the other hand,
studies performed in mice lacking Bcl-xL have clearly
demonstrated that apoptotic cell death in Bcl-xL mice occurs only at the end of terminal differentiation in both primitive and definitive erythropoiesis (37). Therefore, it is reasonable to
propose that accumulation of Bcl-xL resulting from Epo
stimulation probably prevents the apoptotic cell death of terminally
differentiated erythroid cells. Consistent with this hypothesis, our
results in G1E-ER2 cells demonstrate maximum induction of
Bcl-xL in terminally differentiated erythroid cells.
Further, our data show that the lack of Bcl-xL induction,
noted in response to SCF stimulation of GATA-1-expressing G1E-ER2 cells
resulted in significantly reduced differentiation. The data demonstrate
an essential role for Bcl-xL in maintaining the survival of
differentiated erythroid cells. However, the accumulation of
Bcl-xL in terminally differentiated cells cannot be the
only mechanism for the antiapoptotic effect of Epo, since some
Bcl-x
/
colonies contain a significant proportion of viable cells
that partially hemoglobinize (15, 37). Therefore, it is possible that
other death-antagonizing genes also play a role in the survival of
terminally differentiated erythroid cells. Our data would suggest that
c-Kit-induced Bcl-2 expression may in part be responsible for partial
survival of hemoglobinized colonies seen in Bcl-x
/
mice (Fig.
7).
A significant increase in the expression of Bcl-xL during
terminal differentiation is intriguing. Clearly, this increase is mediated either directly or indirectly via the induction of GATA-1. How
GATA-1 up-regulates the expression of Bcl-xL and
down-regulates the expression of Bcl-2 remains to be determined. One
possible explanation might involve the modulation of c-Kit and Epo-R.
GATA-1 has previously been shown to directly bind the Epo-R promoter and induce its expression (38-40). It is conceivable that the enhanced expression of Bcl-xL during terminal stages of erythroid
maturation may be an indirect effect of increased Epo-R expression by
GATA-1. Further, during terminal stages of erythroid maturation GATA-1 alone is not sufficient to induce the expression of Bcl-xL;
rather, GATA-1 cooperates with Epo in this process. Consistent with
these observations, our results demonstrate a significant increase in the expression of Epo-R in GATA-1-expressing cells compared with GATA-1-nonexpressing cells. This increase in the expression of Epo-R
may partly account for the increase in the expression of Bcl-xL in these cells.
In addition to the previously described mechanism of cross-talk between
c-Kit and Epo-R in inducing proliferation of erythroid progenitors (7,
32), we describe a novel mechanism via which c-Kit and Epo-R may
augment the proliferation and survival of proerythroblasts. We
demonstrate that the expression of Epo-R and Stat5 is dependent upon
the activation of c-Kit by SCF. Although some survival of G1E-ER2 cells
is observed in the presence of Epo stimulation alone, stimulation by
both SCF and Epo significantly enhances the survival of these cells. We
demonstrate that the increase in the survival of proerythroblasts in
the presence of both SCF and Epo is partly due to the increase in the
expression of Epo-R and Stat5 by SCF. We propose that c-Kit stimulation
by SCF is essential for the expression of Epo-R and Stat5, resulting in
optimal activation of Epo-R and Stat5 by Epo, followed by
Bcl-xL induction and enhanced survival of these cells (Fig.
7). We hypothesize that the lack of this process may in part be
responsible for reduced numbers of CFU-Es noted in mutants of SCF and
c-Kit. Further support for this mechanism of cooperation between c-Kit
and Epo-R comes from studies performed in a human leukemic cell line,
HML/SE (41). These studies demonstrated an increase in the Epo-R
mRNA in response to stimulation by SCF (41). These studies also
showed that SCF can activate the human Epo-R promoter containing the
GATA and Sp1 binding sites, and mutations in the Sp1 binding site
resulted in abrogation of Epo-R mRNA in response to SCF
stimulation. Together, these results may also help explain an absolute
requirement for both Epo and SCF in the formation of CFU-E colonies
from fetal livers of Epo-R-deficient mice infected in vitro
with a retrovirus expressing the wild-type Epo-R (6). Taken together,
we demonstrate a novel mechanism for controlling the
Epo-R-Stat5-Bcl-xL signaling pathway in erythroid cells by
regulating the expression of Epo-R and Stat5 via the activation of
c-Kit.