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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 87-96
GATA-1 and Erythropoietin Cooperate to Promote Erythroid Cell
Survival by Regulating bcl-xL Expression
By
Todd Gregory,
Channing Yu,
Averil Ma,
Stuart H. Orkin,
Gerd A. Blobel, and
Mitchell J. Weiss
From Ontogeny, Inc, Cambridge, MA; the Division of
Hematology-Oncology, Children's Hospital, Dana-Farber Cancer Institute
and the Department of Pediatrics, Harvard Medical School, Boston, MA;
the Department of Medicine, Committee on Immunology, University
of Chicago, Chicago, IL; the Division of Hematology,
Children's Hospital of Philadelphia, University of Pennsylvania School
of Medicine, Philadelphia, PA; and the Howard Hughes Medical Institute,
Boston, MA.
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ABSTRACT |
The transcription factor GATA-1 is essential for normal
erythropoiesis. By examining in vitro-differentiated embryonic stem cells, we showed previously that in the absence of GATA-1, committed erythroid precursors fail to complete maturation and instead undergo apoptosis. The mechanisms by which GATA-1 controls cell survival are
unknown. Here we report that in erythroid cells, GATA-1 strongly induces the expression of the anti-apoptotic protein
bcl-xL, but not the related proteins bcl-2 and mcl-1.
Consistent with a role for bcl-xL in mediating
GATA-1-induced erythroid cell survival, in vitro-differentiated
bcl-xL / embryonic stem cells fail to
generate viable mature definitive erythroid cells, a phenotype
resembling that of GATA-1 gene disruption. In addition, we show that
erythropoietin, which is also required for erythroid cell survival,
cooperates with GATA-1 to stimulate bcl-xL gene expression
and to maintain erythroid cell viability during terminal maturation.
Together, our data show that bcl-xL is essential for normal
erythroid development and suggest a regulatory hierarchy in which
bcl-xL is a critical downstream effector of GATA-1 and
erythropoietin-mediated signals.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE GENERATION of mature hematopoietic
cells relies upon the establishment of lineage-specific developmental
programs and the maintenance of precursor viability during cellular
maturation. Both of these requirements are achieved during erythroid
development in embryos and adults by the essential X-linked
transcription factor GATA-1. The first erythrocytes, termed primitive
(or embryonic), arise in the extraembryonic yolk sac. Later,
erythropoiesis occurs in the fetal liver where adult (or definitive)
erythrocytes are produced. Finally, at birth, erythrocyte production
shifts to the bone marrow (and spleen in mice). Although primitive and
definitive erythrocytes exhibit unique patterns of gene expression and
growth factor requirements,1-7 GATA-1 is required for both
lineages.8,9 Gene knockout studies in mice have shown that
in the absence of GATA-1, committed erythroid precursors undergo
maturation arrest and apoptosis.9-11 Moreover, inhibition
of GATA-1 in erythroleukemia cells causes apoptosis.12 In
addition to its functions in erythroid development, GATA-1 is required
for normal megakaryocytic differentiation.13,14
GATA-1 recognizes conserved GATA-motifs found in the regulatory regions
of virtually all erythroid-expressed genes including globins, heme
biosynthetic enzymes, membrane proteins, and red blood cell
transcription factors.15-17 It is believed that GATA-1 helps to establish and maintain the erythroid phenotype by activating these genes. How GATA-1 sustains erythroid precursor viability is unknown.
Erythropoietin (Epo) promotes erythroid development by maintaining the
survival of committed precursors at the late colony-forming unit-erythroid (CFU-E) stage.4,5 Depriving
primary definitive erythrocyte precursors and erythroid cell lines of
Epo leads to apoptosis.18-20 The gene encoding the Epo
receptor (EpoR) contains functional GATA-binding sites in its promoter
and enhancer, suggesting that it is a downstream target of
GATA-1.21,22 However, GATA-1
proerythroblasts express a normal level of EpoR mRNA.10
Transcription of the EpoR gene and several other GATA target genes
presumably occurs through the action of GATA-2, a related transcription
factor whose expression is elevated approximately 50-fold in the
absence of GATA-1. The formation of GATA-1 erythroid
colonies is Epo-dependent, demonstrating that the EpoR signaling
pathway is functionally intact in the mutant cells. These observations
exclude the simple model that apoptosis of GATA-1
proerythroblasts is due to absence of the Epo receptor, and compelled us to search for additional GATA-1-induced survival pathways in erythroid cells.
The bcl-2 gene family consists of various members with either pro- or
anti-apoptotic activity.23 One member, bcl-x,24 appears to be especially important for erythropoiesis. bcl-x is upregulated during terminal erythroid maturation,25 and
bcl-x/ embryos exhibit apoptosis of fetal
liver hematopoietic cells and pallor.26 However, the
erythropoietic defect resulting from loss of bcl-x function has not
been well characterized.
bcl-x has been implicated in mediating some effects of Epo signaling.
For example, forced bcl-x expression inhibits apoptosis of Epo-deprived
erythroid cells, and bcl-x expression in erythroid cells is
Epo-dependent.20,27 The latter observation positions bcl-x
downstream of the EpoR in a regulatory pathway that supports erythroid
development by preventing apoptosis. How EpoR signaling regulates bcl-x
and how GATA-1 fits into this scheme are not known.
GATA-1-regulated cellular maturation and viability occur through
distinct pathways that have been uncoupled in the
GATA-1 erythroid cell line G1E.28 G1E
cells were derived from in vitro-differentiated GATA-1 embryonic stem (ES) cells and represent
primary GATA-1 erythroblasts as determined by the
expression of erythroid, but not myeloid, genes.28 A
distinguishing property of G1E cells is that instead of undergoing
apoptosis, they proliferate continuously in culture as developmentally
arrested erythroid precursors. This feature makes G1E cells ideally
suited for genetic and cell biological studies. Restoration of GATA-1
function in G1E cells triggers terminal erythroid maturation, providing
a physiologically meaningful assay to determine structure-function
relationships within GATA-1, to evaluate protein interactions
surrounding GATA-1, and to identify novel genes activated or repressed
by GATA-1 during terminal maturation.28,29 To facilitate
these studies, we created a conditional (estrogen-responsive) form of
GATA-1 by fusing its coding region to the ligand-binding domain of the
human estrogen receptor (ER). G1E cells stably expressing the GATA-1/ER
fusion protein (termed G1E-ER cells) undergo synchronous erythroid
maturation after exposure to  -estradiol.
Here we show that bcl-xL (a splice variant which
counteracts apoptotic signals) mRNA and protein are strongly induced
following GATA-1 activation in G1E-ER cells. In vitro differentiation
of bcl-x/ ES cells shows maturation arrest
and apoptosis of definitive erythroid precursors, similar to what we
have observed upon loss of GATA-1 function. Although GATA-1 and Epo
stimulate bcl-x expression independently in G1E-ER cells, their
combined action is required for maximal bcl-x induction and complete
maturation. Our data extend previous work which implicates a role for
bcl-x in erythroid development by identifying the stage of erythroid
maturation at which bcl-x becomes essential and by establishing a
regulatory hierarchy in which GATA-1 and Epo cooperate to stimulate
bcl-x expression and prevent apoptosis of incipient erythrocytes.
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MATERIALS AND METHODS |
Cytokines.
All cytokines used were purchased from R & D Systems (Minneapolis, MN),
except for Epo (Amgen, Thousand Oaks, CA).
Cell lines.
G1E-ER cells are G1E cells that stably express murine GATA-1 fused to
the ligand-binding domain of the human estrogen
receptor.30,31 For the experiments described here, we used
the clone G1E-ER2, which expresses a high level of GATA-1/estradiol
receptor fusion protein and undergoes nearly complete
estradiol-dependent differentiation (>80% benzidine positive cells
at 72 hours). Unless otherwise specified, G1E-ER2 cells were grown in
IMDM (GIBCO-BRL, Grand Island, NY) with 15% heat-inactivated fetal
calf serum (Hyclone, Logan, UT), Epo 2 U/mL, and kit ligand (KL, stem
cell factor), 50 ng/mL. For Epo-starvation experiments, cells were
washed three times in phosphate-buffered saline, then resuspended in
the same medium without Epo. To induce activation of the conditional
GATA-1, cells were cultured in the presence of 107
mol/L -estradiol (Sigma, St Louis, MO). Benzidine staining
was performed according to standard protocol.32 Cell
viability was determined using the Live/Dead Viability/Cytotoxicity Kit
(Molecular Probes, Eugene, OR). bcl-x+/ and
bcl-x/ gene-targeted ES cells were
generated as described.33
Cell-cycle analysis.
Cells were fixed in 50% ethanol and stained for DNA content with 7 × 105 mol/L propidium iodide. Flow cytometry
was performed using a FACScan flow cytometer (Becton Dickinson, San
Jose, CA). Propidium iodide fluorescence was recorded using linear
amplification and LYSYS II software, and analyzed using Cellfit
software (Becton Dickinson). Data were acquired from intact single
cells by using a doublet discriminator. Ten thousand cells were
analyzed from each sample.
Plasmids and probes.
Probes for murine bcl-x and bcl-2 were supplied by Stanley Korsmeyer
(Dana
Farber Cancer Institute, Boston, MA). Probes for  - and -globins,
GATA-2, c-myb, and -actin were generated by reverse-transcribed
polymerase chain reaction (RT-PCR).10 Plasmids encoding
murine bcl-xL and bcl-x for
transcription-coupled translation were supplied by Harvey
Cantor and Xiao-Feng Yang (Dana-Farber Cancer Institute).34
RT-PCR for bcl-x mRNA splice variants.
Total RNA was isolated using Trizol reagent (GIBCO-BRL). Complementary
DNA was synthesized using Superscript II reverse transcriptase (GIBCO-BRL) per the supplier's instructions. The following primers, indicated schematically in Fig 2B, were used for PCR:
bcl-xL and bcl-xs, LX8
(5'-CTCTCCTACAAGCTTTCCCAG-3') and LX10
(5'-CCAGCGGTTGAAGCGCTCC-3'); bcl-x 1
(5'-AAATGTCTCAGAGCAACCG-3') and 3
(5'-CACAGAGAAGAGAGACACAAGC-3'). Plasmids containing the
bcl-xL coding region and mouse thymus cDNA were used as
controls. PCR was performed using Taq DNA polymerase (Boehringer
Mannheim, Indianapolis, IN) for 30 cycles under the following
conditions: 94°C, 30 seconds; 50°C, 30 seconds; and 72°C,
90 seconds. PCR products were resolved by electrophoresis on 1.6%
agarose/Tris acetate gels.
Northern blotting.
Total RNA was isolated using Trizol reagent and fractionated on 1.2%
agarose-formaldehyde gels according to standard conditions. Northern
blot analysis was performed using Hybond C+ (Amersham, Arlington
Heights, IL), according to the manufacturer's instructions. Blots were
washed at a final stringency of 0.5X SSC at 65°C.
Western blotting.
Western blotting was performed according to standard protocols. G1E-ER2
cells were lysed in 1X SDS sample buffer (Sigma) and passed through a
21-gauge needle several times to shear DNA. Fifty micrograms of protein
was fractionated on 14% polyacrylamide/sodium dodecyl sulfate (SDS)
gel and electrophoretically transferred to nitrocellulose membrane.
Equal loading of proteins between samples was verified by Coomassie
Blue staining of SDS-polyacrylamide gels in parallel experiments. Two
polyclonal rabbit anti-human bcl-x antibodies were used in independent
experiments: no. B22630 (Transduction Laboratories, Lexington, KY),
1:1,000 dilution; and bcl-xS/L S-18 (Santa Cruz
Biotechnology, Santa Cruz, CA) at 1:100 dilution. The secondary
antibody was horseradish peroxidase-conjugated donkey anti-rabbit
(Amersham) at 1:5,000 dilution. Bound antibody was detected by
chemiluminescence using the ECL Western blot kit (Amersham). In vitro
transcription and translation of bcl-xL and bcl-x was performed using the TNT coupled reticulocyte
lysate system (Promega, Madison, WI).
In vitro differentiation of ES cells.
Generation of hematopoietic colonies from ES cells was performed as
described.10,35 The specific conditions used to assess various hematopoietic lineages are described in the text and figure legends. Cytokine concentrations used for ES cell replating are as
follows: Epo, 2 U/mL; KL, 50 ng/mL; granulocyte colony stimulating factor (G-CSF), 1,000 U/mL; granulocyte macrophage colony-stimulating factor (GM-CSF), 15 U/mL; M-CSF, 100 U/mL.
In situ detection of apoptosis.
Cells from erythroid colonies were deposited onto glass slides by
cytocentrifugation. The TUNEL (terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling) assay for
apoptosis was performed using the ApopTag Apoptosis Detection Kit
(Oncor, Gaithersburg, MD) according to the manufacturer's instructions.
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RESULTS |
G1E-ER2 cells undergo estradiol-dependent terminal erythroid
maturation.
To achieve synchronous activation of GATA-1 in G1E cells, we generated
a conditional form of murine GATA-1 in which its coding region was
fused to that of the ligand-binding domain of the human ER.29-31 This chimeric protein activates GATA-dependent
promoter-reporter constructs in an estradiol-dependent fashion (not
shown). After exposure to -estradiol, G1E cells stably expressing
the GATA-1/ER fusion product induced hemoglobin, as measured by
benzidine staining. The degree of benzidine staining varied among
individual clones and correlated with the level of GATA-1/ER protein
expressed (not shown). These observations are consistent with
concentration-dependent requirements for GATA-1 observed during
erythroid differentiation in vivo.36 A clone expressing a
relatively high level of the GATA-1/ER fusion protein, referred to as
G1E-ER2, exhibited nearly complete estradiol-dependent maturation
(benzidine staining observed in more than 80% of cells after 72 hours); this clone was used in the experiments described below.
Addition of estradiol to G1E-ER2 cultures triggered synchronous
erythroid maturation (Fig 1, see page 91). Adult-type
globin mRNAs were induced by 6 hours, and within 12 to 24 hours, cell division ceased with concomitant G1-phase cell-cycle arrest, followed by the appearance of benzidine-staining cells with a late normoblast morphology at 24 to 72 hours (Fig 1A and B). Concurrently, mRNA levels
of c-myb and GATA-2, markers for less differentiated states associated
with higher proliferative capacity,31,37 decreased (Fig
1C). Estradiol had no apparent effect on the parental G1E line, which
does not express the GATA-1/ER fusion protein. Of note, the partial ER
antagonist tamoxifen induced maturation of G1E-ER2 cells (not shown).
Tamoxifen binds to the ER moiety of chimeric fusion proteins leading to
activation, but in contrast to estradiol, fails to induce interaction
of the ER ligand-binding domain with its cellular
coactivators.38,39 This indicates that the biological
effects of GATA-1/ER are attributable to the GATA-1 moiety of the
fusion protein, which is consistent with our previous finding that
wild-type GATA-1 introduced into G1E cells via retroviral transfer
triggers terminal differentiation.28
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| Fig 1.
Activation of conditional GATA-1 in G1E-ER2 cells
triggers terminal erythroid maturation. Conditional GATA-1 was created
by fusing the full-length murine cDNA to the ligand-binding domain of
the human ER. G1E-ER2 cells are a clone derived from stable expression
of the GATA-1/ER chimeric protein in the GATA-1
erythroid cell line, G1E. (A) Kinetics of G1 cell cycle arrest (left)
and hemoglobin accumulation (benzidine staining, right) after addition
of estradiol. Note that estradiol has no cell-cycle-arresting or
hemoglobin-inducing effects on the G1E parental line, which does not
express estradiol-inducible GATA-1. (B) G1E-ER2 cell morphology
(May-Grünwald-Giemsa staining, top panels) and benzidine staining
(bottom panels) before and 72 hours after addition of estradiol.
Original magnification: 1,000× top panels; 400×, bottom panels. (C)
Northern blot analysis of erythroid-expressed genes. Each lane contains
15 µg of total RNA.
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bcl-x mRNA and bcl-xL protein are upregulated during
G1E-ER2 cell maturation.
We examined expression patterns of various anti-apoptotic bcl-2 gene
family members during estradiol-induced G1E-ER2 cell maturation. Among
several candidates tested including bcl-2, mcl-1, and bcl-xonly
bcl-x was strongly upregulated upon GATA-1/ER activation (Fig 2 and not
shown). After addition of estradiol, bcl-x mRNA induction was detected
at 24 hours and became more substantial by 48 hours (Fig 2A). In
addition, bcl-x mRNA was upregulated by tamoxifen in G1E-ER2 cells, but
not by estradiol in parental G1E cells, excluding the possibility that
bcl-x is induced by estradiol in a GATA-1-independent manner (see
above).
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| Fig 2.
Induction of bcl-xL mRNA and protein by
GATA-1 in G1E-ER2 cells. G1E-ER2 and parental G1E cells were treated
with estradiol (E2) or tamoxifen (Tamox) and sampled for bcl-x mRNA and
protein at the indicated times. (A) Northern blot analysis using a
bcl-xL cDNA probe. Each lane contains 20 µg of total RNA.
(B) Expression of splice variants bcl-xL,
bcl-xs, and bcl-x in maturing G1E-ER2 cells.
The top panel shows a schematic of the various bcl-x mRNAs examined and
the primers used for detection by RT-PCR. The bottom panels show the
RT-PCR reactions for bcl-xL and bcl-xs (left)
and bcl-x (right). The positive controls are
bcl-xL cDNA plasmid (C) and mouse thymus cDNA (T). (C)
bcl-x protein expression in G1E-ER2 cells. For Western analysis (left
panel), 50 µg of protein from whole-cell lysates was fractionated on
a 14% SDS polyacrylamide gel. 35S methionine-labeled in
vitro transcribed/translated (TNT) bcl-xL and
bcl-x isoforms were fractionated in adjacent lanes
(right panel).
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Differential splicing of primary bcl-x transcripts leads to the
generation of proteins with varying effects on
apoptosis.24,40,41 RT-PCR analysis showed that
bcl-xL, the anti-apoptotic form, and bcl-x,
which can be either pro- or anti-apoptotic depending on cellular
context, were induced during G1E-ER2 cell maturation (Fig 2B). mRNA
encoding the short isoform, bcl-xs, which antagonizes anti-apoptotic effects of bcl-2 or bcl-xL, was
undetectable. A similar pattern of bcl-x mRNA expression is observed in
day 15.5 fetal liver, which consists primarily of erythroid
cells.41
Western blots using two different polyclonal antibodies against bcl-x
detected the induction of a single protein of approximately 29 kD,
paralleling the increase of bcl-x mRNA (Fig 2C and not shown). This
bcl-x protein migrated at a position identical to in vitro
transcribed/translated bcl-xL, and more slowly than
bcl-x. Therefore, bcl-xL appears to be the
predominant form expressed in G1ER-E2 cells. These data show that
bcl-xL is a direct or indirect downstream target of GATA-1,
suggesting a mechanism by which GATA-1 maintains the viability of
developing erythrocytes.
bcl-x is essential at a late stage of definitive erythroid
maturation.
bcl-x knockout mice die at embryonic day 13 with apoptosis of fetal
liver hematopoietic cells and pallor.26 To investigate whether failure to produce viable definitive erythroid cells is caused
by a cell-autonomous defect of bcl-x-null precursor cells, and to
pinpoint the stage at which loss of bcl-x function affects erythropoiesis, we analyzed the developmental potential of heterozygous mutant (bcl-x+/) and homozygous null
(bcl-x/) ES cells using a two-step in vitro
differentiation protocol.35 Upon differentiation in vitro,
ES cells form aggregates, termed embryoid bodies (EBs), which contain
numerous differentiated cell types, including hematopoietic precursors
of multiple lineages, which appear in a well-defined temporal sequence.
Pure hematopoietic colonies are generated by disaggregating EBs and
replating the single-cell suspension into cytokine-containing
methylcellulose cultures. This technique allowed us to quantitate and
evaluate primitive and definitive erythroid precursors from the bcl-x
gene-targeted ES cells. To minimize the possibility that any
differences between homozygous and heterozygous mutant cells are caused
by a mutation unrelated to bcl-x acquired during cell passage, all
experiments were performed using two independently derived clones of
bcl-x/ ES cells (nos. 48 and 99). The results
obtained from these clones were indistinguishable (not shown).
To generate primitive erythroid (EryP) colonies, 6-day-old EBs were
replated into cultures with Epo. Under these conditions, the majority
of colonies formed were EryP as determined by histological staining of
cytocentrifugation preparations (not shown).
bcl-x/ EryP progenitors formed in normal
numbers (Fig 3A), and generated normal-appearing,
hemoglobinized colonies when compared with the heterozygous mutant
cells (Fig 3B). There were no marked differences in size or extent of
hemoglobinization between bclx/,
bcl-x+/, and bcl-x+/+ EryP colonies (Fig
3 and not shown). These observations are consistent with the phenotype
of bcl-x/ embryos, in which primitive
erythropoiesis is not drastically impaired.26
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| Fig 3.
Bcl-x is dispensable for normal development of
primitive erythroid colonies. (A) Primitive erythroid (EryP) precursors
were enumerated after secondary replating of 6-day-old embryoid bodies
derived from in vitro differentiation of bcl-x+/ and
bcl-x/ ES cells. EryP colonies were generated in
methylcellulose cultures containing Epo. Similar results were obtained
using two independently derived bcl-x/ ES cell clones
(nos. 48 and 99). Bcl-x/ EryP colonies shown in B are
from clone no. 48. Error bars represent standard error of the mean from
three separate cultures. (B) bcl-x+/ and
bcl-x/ EryP colonies exhibit similar morphologies.
Five-day-old colonies are shown. Original magnification × 400.
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Definitive erythroid (EryD) bcl-x/ colonies,
which were generated from 10-day-old EBs replated into cultures with
Epo and KL, were also normal in number. However, in contrast to their
EryP counterparts, bcl-x/ EryD precursors
were significantly impaired in their maturation as reflected by their
failure to form fully hemoglobinized colonies (Fig 4B), and by their abnormal cellular
morphology (Fig 4C). In addition, the delay in maturation was
accompanied by excessive apoptosis as shown by TUNEL staining of the
bcl-x/ erythroid cells (Fig 4D). These
results are consistent with the observation that loss of bcl-x function
impairs viability of fetal liver erythrocytes in mice,26
and localize the defect to between the proerythroblast and early
normoblast stage of development. Therefore, bcl-x is required for the
survival and normal maturation of definitive erythroid cells. The
phenotype resulting from bcl-x deficiency in EryD cells is
similar to that observed with GATA-1 loss, albeit less severe.
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| Fig 4.
Bcl-x/ definitive erythroid cells
hemoglobinize poorly and undergo excessive apoptosis. (A)
bcl-x+/ and bcl-x/ definitive
erythroid (EryD) precursors were enumerated after replating 10-day-old
embryoid bodies into cultures containing Epo and Kit ligand (KL, stem
cell factor). Similar results were obtained using two independently
derived bcl-x/ ES cell clones (nos. 48 and 99).
Analyses in B through D were performed using bcl-x/
EryD colonies from clone no. 48. Error bars represent standard error of
the mean from three separate cultures. (B) bcl-x/
EryD colonies are normal in size, but exhibit defective
hemoglobinization. Colonies shown are 6 days old. Original
magnification × 200. (C) May-Grünwald-Giemsa staining of
erythroid cells from 6-day-old bcl-x+/ and
bcl-x/ EryD colonies. Note the larger blast-like
cells and cells with pyknotic nuclei within bcl-x/
colonies. Original magnification × 600. (D) Excessive apoptosis
within cells derived from bcl-x/ EryD
colonies. Upper triangles show DAPI staining (blue), which displays the
nuclei of all cells. The same cells were stained for apoptosis using
the TUNEL stain (lower triangles, green).
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Nonerythroid colonies derived from in vitro differentiation of
bcl-x/ ES cells, including macrophage,
granulocyte-macrophage, and mast, were indistinguishable from wild-type
and heterozygous mutant colonies in number and appearance (not shown).
GATA-1 and Epo are both required for optimal bcl-x expression.
Because both GATA-1 and Epo are required for erythroid cell survival
and optimal bcl-x expression4,5,20,25 (and this manuscript), we determined the effects of Epo deprivation on viability and bcl-x expression in G1E-ER2 cells. G1E-ER2 cells are typically grown in Epo and KL. After Epo removal in the absence of estradiol, G1E-ER2 proliferation decreased slightly (not shown), but more than
90% of the cells remained viable ( Epo, E2;
Fig 5A). In contrast, Epo starvation during
estradiol-induced maturation reduced viability by approximately 50% at
48 hours (Epo,+E2; Fig 5A), although induction of globin RNAs
(Fig 5B) and benzidine-positive cells still occurred (not shown).
Therefore, Epo deficiency impairs survival of G1E-ER2 cells only during
GATA-1-induced terminal maturation, but induction of terminal
differentiation markers still occurs, consistent with models that
assign a supportive, rather than instructive, role for Epo in erythroid
differentiation.18,42,43
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| Fig 5.
Epo cooperates with GATA-1 to maximize bcl-x induction
and G1E-ER2 cell viability during terminal maturation. (A) Viability of
G1E-ER2 cells cultured with various combinations of Epo and E2. Live
and dead cells were quantitated using calcein AM and ethidium bromide,
respectively. Note that Epo is required for cell survival only during
estradiol-induced terminal maturation. (B) Effects of Epo starvation on
bcl-x and -globin mRNA expression in G1E-ER2 cells. Cells were
cultured with the indicated combinations of Epo and/or estradiol (E2),
sampled at various times and analyzed for gene expression by Northern
blotting. Note that Epo and GATA-1 can induce bcl-xL mRNA
independently, although both are required for maximal expression. Each
lane contains 20 µg of total RNA.
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Next, we tested whether Epo stimulates bcl-x expression in G1E-ER2
cells (Fig 5B). In the absence of estradiol, removal of Epo reduced
bcl-x mRNA levels (Epo, E2; Fig 5B) with little or no
effect on cell viability (see above) or -globin expression; readdition of Epo restored bcl-x mRNA level to baseline within 24 hours
(not shown). During estradiol-induced maturation, Epo starvation
markedly reduced but did not completely block the ability of GATA-1 to
induce bcl-x (Epo,+E2; Fig 5B). Similar results were obtained
when G1E-ER2 cells were cultured in serum-free defined medium (not
shown), excluding the possibility that trace amounts of Epo present in
serum are required for induction of bcl-x expression by GATA-1.
Therefore, Epo and GATA-1 exert independent, cooperative effects on
bcl-x mRNA induction in erythroid cells. In this cellular model for
erythroid development, a strict requirement for Epo becomes manifest
mainly during the later stages of maturation.
| |
DISCUSSION |
For mature blood cells to form properly, it is essential that committed
precursors are protected from cell death. In most cell types, apoptosis
is tightly regulated by interacting networks of cytokine signaling
pathways, nuclear factors, and other effectors such as members of the
caspase and bcl-2 families. Increased anti-apoptotic activity may be
required in developing erythrocytes to counter stress caused by
accumulation of intracellular iron and consequent production of toxic
reactive oxygen species.25,44 Here we establish a
regulatory pathway in which EpoR and GATA-1 cooperate to induce bcl-xL expression, which in turn is critical for the
survival of late proerythroblasts and early normoblasts
(Fig 6). Regulated expression of bcl-2 gene
family members is one mechanism by which apoptosis is
controlled.45,46 However, it remains possible that bcl-x
activity in erythroid cells might also be regulated through
posttranslational mechanisms such as phosphorylation and availability
of dimerization partners.46
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| Fig 6.
Anti-apoptosis pathways during terminal erythropoiesis.
GATA-1 and Epo receptor signaling both induce bcl-x mRNA, and all three
genes are required for survival of maturing definitive erythroid
cells.
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Knockout studies in mice can demonstrate the requirement of certain
genes for the formation of a given cell type. One limitation of such
studies is that phenotypic analysis of homozygous gene-targeted animals
cannot distinguish between cell-autonomous versus indirect effects.
Here we have used in vitro differentiation of
bcl-x/ ES cells to make this distinction. Our
observation that bcl-x/ definitive erythroid
colonies fail to mature properly indicates that bcl-x is required in a
cell-autonomous fashion for normal erythroid development.
The similarity between the phenotypes observed in
GATA-1 and bcl-x/
definitive erythroid cells is consistent with a model which places bcl-xL downstream of GATA-1 in an erythroid cell survival
pathway. However, several notable differences exist between the two
mutant phenotypes. First, while GATA-1 is required for normal
development of both primitive and definitive erythrocytes, bcl-x
appears to be essential predominantly for the definitive lineage, as
evidenced by our results and the phenotype of homozygous null
embryos.26 It is possible that GATA-1 maintains viability
of yolk sac erythrocytes by regulating the expression of other bcl-2
family members, although none as yet have been shown to be limiting for
primitive erythropoiesis. Second, while GATA-1 EryD
colonies exhibit complete maturation arrest with failure to
hemoglobinize, some age-matched bcl-x/
colonies contain a higher proportion of viable cells and hemoglobinize partially. Therefore, it is possible that GATA-1 activates other death-antagonizing genes (or represses death-promoting ones) in definitive erythrocytes, in addition to bcl-x.
An open question remains as to whether bcl-x is a direct
transcriptional target of GATA-1. Two GATA consensus motifs reside in
the 5' region of the human and mouse bcl-x genes, upstream of a
major transcription start site mapped by primer extension using several
hematopoietic cell lines,47 including G1E-ER2 (unpublished
data, April 1998). DNA segments from this region activate transcription
of reporter genes in several erythroid lines including MEL, G1E-ER2
(unpublished data, April 1998), and K562,47 with the
majority of promoter activity residing in a 57-bp fragment that
contains the proximal GATA motif.47 Mutations in this motif
which prevent GATA-1 binding have little effect on promoter activity in
MEL, K562, or G1E cells (unpublished data, August 1998). It is possible
that GATA-1 regulates bcl-xL expression through other
elements present elsewhere in the gene. Alternatively, bcl-x might be
an indirect target of GATA-1. This latter possibility is supported by
the delayed kinetics of bcl-xL induction after GATA-1
activation (Fig 2A, above).
Loss of Epo signaling through gene targeting results in apoptosis of
committed definitive erythroid precursors at the late CFU-E
stage,4,5 and Epo is required for optimal expression of
bcl-x in erythroid cells.20,25 Therefore, it is possible that Epo exerts some of its anti-apoptotic effects by inducing bcl-x
expression. Epo and several other hematopoietic cytokines are believed
to induce bcl-x through a JAK-dependent pathway.48,49 In
addition, Epo may upregulate bcl-x by inhibiting the expression of
JunB, a member of the AP-1 transcription factor family.50 While GATA-1 can induce bcl-xL expression independent of
Epo, we do not exclude the possibility that crosstalk also exists. For
example, GATA-1 might regulate the expression of genes that modulate
EpoR signaling. However, in G1E-ER2 cells, it is unlikely that GATA-1
influences bcl-xL expression by directly stimulating transcription of the EpoR gene, as EpoR mRNA level decreases slightly during the period of maximal bcl-x induction (unpublished data, October
1998), and the half-life of EpoR protein in hematopoietic cells is only
about 60 minutes.51,52
Epo requirements vary in G1E cells according to their state of
maturation, which in turn is controlled by GATA-1. In the absence of
GATA-1 activity, the cells exhibit an immature erythroid phenotype, are
relatively Epo insensitive, but require KL for proliferation and
survival. Based on these cytokine requirements,4 G1E cells most closely approximate the late BFU-E or early CFU-E stage of erythroid development. Because primary GATA-1
erythroid progenitors die at the proerythroblast stage, it appears that
G1E cells were immortalized at a level of differentiation upstream of
where the antiapoptotic effect of GATA-1 is exercised.10,11 Estradiol-induced GATA-1 activation in G1E-ER2 cells triggers terminal
maturation accompanied by transition into a state of Epo-dependency,
consistent with the in vivo requirement for Epo during the late CFU-E
and proerythroblast stages.4,5,18 GATA-1 activation without
Epo causes cellular maturation, but bcl-xL induction is
reduced and the cells die. We speculate that Epo is required along with
GATA-1 to achieve a critical level of bcl-xL that is
required for terminal differentiation. The apparently normal erythroid
phenotype in bcl-x heterozygous mutant states implies that either 50%
bcl-x protein is sufficient, or that there is a compensatory increase
in expression of the intact allele. In contrast to Epo, KL is not
required for viability or bcl-xL mRNA induction during
GATA-1-stimulated maturation of G1E-ER2 cells (unpublished data,
January 1999). The shift from KL to Epo-dependency during terminal
differentiation of G1E cells resembles the pattern of cytokine response
exhibited during normal definitive erythropoeisis.4
Hematopoietic cytokines, including Epo, are believed to function as
survival factors that are supportive for terminal differentiation. Accordingly, forced expression of bcl-2 partially rescues the hematopoietic phenotypes of interleukin-7 (IL-7) receptor- and M-CSF
receptor-deficient mice.53-55 The EpoR probably has a
similar, supportive role,4,5 yet overexpression of bcl-2
does not rescue Epo-deprived erythroid precursors from
death.56 This may reflect a critical function for bcl-x in
erythroid cells that cannot be substituted for by related proteins. In
contrast to the role for Epo signaling in terminal erythropoiesis,
GATA-1 exerts more pleiotropic actions, fostering cell survival and
erythroid maturation. Therefore, while forced expression of bcl-x might rescue apoptosis of GATA-1 erythroid precursors in
vivo, it would not be expected to overcome the maturation arrest
associated with the mutant phenotype.
Induction of bcl-xL by GATA-1 shows a connection between an
essential anti-apoptosis pathway and a tissue-specific transcription factor required for erythroid differentiation. Gene targeting and
overexpression studies have identified numerous nuclear factors that
participate in the determination and differentiation of specific hematopoietic lineages.57 Enhancement of cell survival
through regulation of viability genes may be a common mechanism through which transcription factors participate in the developmental control of hematopoiesis.
| |
ACKNOWLEDGMENT |
We thank Craig Thompson, Tullia Lindsten, Haidi Yang, and Kay MacLeod
for providing bcl-x genomic DNA; Stanley Korsmeyer, Harvey Cantor, and
Xiao-Feng Yang for providing plasmid DNAs; and Christopher Guo for
performing site-directed mutagenesis on bcl-x promoter sequences. We
thank Merav Socolovsky and Gordon Keller for reviewing this manuscript
prior to publication.
| |
FOOTNOTES |
Submitted December 9, 1998; accepted February 24, 1999.
The first two authors contributed equally to this work.
Supported in part by a grant from the National Institutes of Health
(K08 HL03364, to M.J.W.)
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.
Address reprint requests to Mitchell J. Weiss, MD, PhD, Ontogeny, Inc,
45 Moulton St, Cambridge MA 02138-1118; e-mail:
mweiss{at}ontogeny.com.
| |
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TOP
ABSTRACT
INTRODUCTION