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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 450-457
Forced GATA-1 Expression in the Murine Myeloid Cell Line M1:
Induction of c-Mpl Expression and Megakaryocytic/Erythroid
Differentiation
By
Yuji Yamaguchi,
Leonard I. Zon,
Steven J. Ackerman,
Masayuki Yamamoto, and
Toshio Suda
From the Department of Cell Differentiation, Institute of Molecular
Embryology and Genetics, Kumamoto University School of Medicine,
Kumamoto, Japan; the Division of Hematology/Oncology, The Children's
Hospital, Harvard Medical School, Boston, MA; the Department of
Biochemistry, University of Illinois at Chicago, Chicago, IL; and the
Center of Tsukuba Advanced Research Alliance, University of Tsukuba,
Tsukuba, Japan.
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ABSTRACT |
The "zinc-finger" transcription factor GATA-1 was first shown
in cells of erythroid lineage. It is also expressed in cells of other
hematopoietic lineages including megakaryocytes, mast cells, and
eosinophils. GATA-1 is now considered to be one of the central
regulators in hematopoietic cell differentiation. To further analyze
the role of GATA-1 in controlling differentiation from hematopoietic
stem cells, we investigated the phenotypic changes induced by the
overexpression of murine GATA-1 in the murine myeloid leukemic cell
line, M1. Forced expression of GATA-1 induced the appearance of
erythroid cells and megakaryocytes as assessed by cellular morphology,
acetylcholinesterase activity, and expression of platelet factor 4 and
 -globin mRNA synthesis. Because the c-mpl ligand, thrombopoietin,
plays an important role in megakaryopoiesis, the expression of c-mpl
and c-mpl ligand (thrombopoietin) mRNA was analyzed by Northern blot
and reverse transcription-polymerase chain reaction (RT-PCR) in M1
cells overexpressing GATA-1. The c-mpl ligand mRNA was equally
expressed both in parental M1 cells and in those transfected with the
GATA-1 expression vector. In contrast, the mRNA expression of c-mpl was
increased only in GATA-1 expressing M1 cells differentiated towards
erythroid and megakaryocyte lineages. The increased expression of c-mpl
mRNA induced by GATA-1 raised the question as to whether or not GATA-1 transactivated the c-mpl promoter. The activity of the c-mpl promoter in the presence of cotransfected GATA-1 was significantly increased compared with that of the control. A plasmid with the mutated GATA-binding site did not show transactivation ability in the cotransfection with a GATA expression vector. These findings suggest that the upregulation of c-mpl induced by GATA-1 expression in M1 cells
is closely associated with erythroid and megakaryocytic differentiation.
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INTRODUCTION |
THE DIFFERENTIATION of hematopoietic stem
cells is regulated in part by the binding of hematopoietic growth
factors to their receptors.1 However, the molecular basis
for the lineage commitment of hematopoietic progenitors is still
unclear. In the erythroid lineage, GATA-1, Tal-1/SCL, or Rbtn2
transcription factors are involved in the differentiation of erythroid
cells.2,3 In the myeloid lineage, PU.1, first identified as
the Spi-1 oncogene activated in viral induced
erythroleukemias,4,5 may be one of the principal regulators
in myeloid differentiation because it activates numerous myeloid target
genes.6-13 It has been shown that p45 of the erythroid
transcription factor NF-E2 acts as an essential factor for
megakaryocyte maturation.14 Thus, studies of the role of
transcription factors in the commitment of hematopoietic stem cells
have suggested that various nuclear proteins are associated with
lineage-specific gene expression.
GATA-1 was originally isolated for its ability to bind a core consensus
(A/T)GATA(A/G) site present in the promoters or enhancers of many
erythroid genes. Studies of GATA-1 gene disruption have shown that
GATA-1 plays a pivotal role in the differentiation of the erythroid
lineage.15 However, GATA-1 is also expressed in cells of
other lineages, including mast cells, eosinophils, and
megakaryocytes.16-18 Forced GATA-1 expression in avian
Myb-Ets-transformed myeloblasts results in reprogramming of
myeloblasts towards the eosinophil and megakaryocyte
lineages.19 On the other hand, targeted disruption of the
mouse GATA-1 gene prevents primitive and definitive erythroid
development.15 To date, it is generally accepted that
GATA-1 plays an important role in hematopoietic stem cell
differentiation towards erythroid and myeloid lineages.
To further analyze the roles of GATA-1 in regulating the
differentiation of hematopoietic stem cells towards the myeloid
lineage, we investigated phenotypic changes in the murine myeloid
leukemia M1 cell line, which is a well-established in vitro model for
studying the differentiation pathway from immature blast cells to
mature macrophage, stably transfected with and expressing the murine GATA-1 gene.
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MATERIALS AND METHODS |
Cell culture.
The murine myeloid leukemia cell, M1, originally established by
Ichikawa,20 was maintained at 37°C under 5%
CO2 in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% heat-inactivated newborn calf serum (ICN
Biochemicals Japan Co, Osaka, Japan), 2 mmol/L L-glutamine, 100 U/mL
penicillin, and 100 µg/mL streptomycin. The cells were passaged twice
a week at 2 × 105 cells/mL.
Plasmids and electroporation.
We used a murine GATA-1 expression plasmid containing the SV40 enhancer
and adenovirus major late promoter (pMG-mGATA1), which provided
constitutive expression of GATA-1 mRNA in neomycin-resistant, stably
transfected cells. For electroporation, 1.5 × 107
cells were pelleted and resuspended in 0.5 mL of electroporation buffer, 21 mmol/L HEPES (pH 7.05), 137 mmol/L NaCl, 5 mmol/L KCl, 0.7 mmol/L Na2HPO4, and 6 mmol/L glucose containing
20 µg Nde I-linearized plasmid DNA and left at room temperature for 5 minutes before electroporation at 320 V, 960 µF, using a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, VA). The cells were kept on ice
for 15 minutes, then resuspended in 10 mL of complete DMEM. After 48 hours, the cells were seeded in Methocel containing 1 mg/mL of G418
(Geneticin; Sigma, St Louis, MO). Two to 3 weeks later, G418-resistant
colonies were transferred into liquid medium containing 0.4 mg/mL of
G418.
Cytochemical staining.
Cells on cytocentrifuge slides were visualized by means of
Wright-Giemsa and acetylcholine esterase staining.
Fluorescence-activated cell sorting (FACS) analysis.
M1 clonal lines transfected with the GATA-1 expression vector,
M1GATA-Y1, M1GATA-Y22, and M1GATA-Y25 clone, were stained with biotinylated TER-119 monoclonal antibody (MoAb; PharMingen, San Diego,
CA), followed by fluorescein isothiocyanate (FITC)-conjugated streptavidin (Becton Dickinson Immunocytometry System, San Jose, CA).
Stained cells were analyzed by FACSvantage (Becton Dickinson Immunocytometry System).
Northern blot analysis.
Total RNA was extracted from M1 cells and their subclones with
guanidine isothiocyanate.21 Poly(A)+ RNA was
separated by oligotex-dT30 (Takara Shuzo, Japan). The total RNA (20 µg/lane) was denatured in formamide-formaldehyde followed by
electrophoresis in a 1% agarose-formaldehyde gel. The RNA was then
transferred onto Hybond-N nylon membranes (Amersham International Plc,
Buckinghamshire, UK) and hybridized with radiolabeled cDNA probes
encoding murine (m) GATA-1,22 mGATA-2,23
m-globin,24 human (h) platelet factor 4 (PF4),25 mCD34,26 MafK,27 NF-E2 (p45),28 and 18S ribosomal RNA. The membranes were washed
twice in 2X SSC containing 0.2% sodium dodecyl sulfate (SDS) at
53°C for 30 minutes and then washed twice in 0.2X SSC containing
0.2% SDS at 55°C for 30 minutes.
Reverse transcription-polymerase chain reaction (RT-PCR).
For murine c-mpl ligand (thrombopoietin), the 5 primer sequence
used was 5-TGATGGCAGCACGAGGACAGTTGGAA-3 and the 3
primer sequence used was 5-GTGAGGTTCCAGCAAAGAGCCCATG-3,
corresponding to exon 4 and exon 5, respectively.29,30 For
murine -actin, the 5 primer sequence used was
5-CTAGACTTCGAGCAGGAGAT-3 and the 3 primer sequence
used was 5-GCTCAGTAACAGTCCGCCTAGA-3. The isolated total
RNA was reverse-transcribed in a 20 µL total buffer containing 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L dithiothreitol, a 10 mmol/L dNTP mixture, 100 pmol/L random
hexamer oligonucleotides (Takara Shuzo, Kyoto, Japan), and 200 U
Moloney murine leukemia virus reverse transcriptase (BRL, Gaithersburg,
MD). Double-stranded DNA was then synthesized from the single-stranded
DNA with 1 U Thermus aquaticus (Taq) polymerase (Takara Shuzo)
and two pairs of oligonucleotides (c-mpl ligand and -actin), using
25 PCR cycles on a thermocycler (Perkin-Elmer Cetus PCR 1000, Norwalk,
CT). Each cycle included denaturation at 94°C for 1 minute,
reannealing of the primer, fragmentation at 55°C for 2 minutes, and
polymerization at 72°C for 2 minutes.
Isolation of the murine c-mpl promoter.
The 5 sequence containing exon 1 of the murine c-mpl gene was
amplified and cloned from Sal I/BamHI-digested murine
genomic DNA by PCR based on the published sequence (31) with primers A (5-ACGCGTCGACCTGTTCTGACAGCCATATGC-3) and B
(5-CGCGGATCCCTTCTCCGGCACTGTGTGCCT-3). The synthesized
fragment was 324 bp long, extending from bp  312 to +12 with the
transcription start site designated +1.31 This PCR fragment
was digested with Sal I and BamHI and cloned upstream of the herpes simplex virus thymidine kinase (TK) promoter followed by
firefly luciferase cDNA, pTKLuc/m c-mpl.32 To generate the construct containing murine c-mpl promoter with a mutation in the
GATA-binding site (bp 85 to 80), pTKLuc/m c-mpl was used as the template. A 6-bp linker containing a Xho I site
(sequence 5-CTCGAG-3) was substituted for the wild-type
sequence of the 312 bp murine c-mpl promoter construct between
bp 85 and 80 using oligonucleotide-directed PCR
mutagenesis.33 The PCR fragment was cloned into the
luciferase vector pTKLuc, and the resulting construct was sequenced to
confirm the correct placement of the linker oligonucleotide.
Transient transfection.
Jurkat cells were electroporated at 250 V, 960 µF, using a Bio-Rad
Gene Pulser. The cells were harvested 24 hours posttransfection into
0.5 mL of lysis buffer, and 0.1 mL of this lysate was added to 0.3 mL
of assay buffer for luciferase assays. Details of the transfection and
luciferase assay procedures were as described.34 The
transfection efficiency was normalized to the levels of growth hormone
produced by 0.5 µg of cotransfected plasmid containing the
cytomegalovirus (CMV) promoter directing human growth hormone gene
expression (CMV-hGH). Growth hormone concentrations were measured by
radioimmunoassay (Nichols Institute, San Juan Capistrano, CA). Data are
expressed as the fold induction over that with the pTKLuc/m c-mpl.
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RESULTS |
GATA-1 induces the differentiation of the erythroid and megakaryocytic
lineages from a murine myeloid leukemic cell line, M1.
The M1 cell line was originally established in vitro from a spontaneous
myeloid leukemia of SL strain mice.20 This cell line
expresses abundant CD34, a marker of hematopoietic stem cells, and can
differentiate towards the macrophage lineage in response to
interleukin-6 (IL-6; Fig 1). As shown in
Fig 1B, M1 cells were capable of differentiating towards macrophages
that could phagocytize latex beads after stimulation with IL-6.
However, M1 cells have not been shown to differentiate towards
erythroid and/or megakaryocytic lineages. A linearized murine
GATA-1 expression vector containing a neomycin-resistance cassette was
electroporated into M1 cells. G418-resistant transfectants were
selected, and clonal lines were generated by in vitro colony formation.
Three G418-resistant colonies, M1GATA-Y1, M1GATA-Y22, and M1GATA-Y25,
were selected for further analysis. Wright-Giemsa staining showed that
the M1 clone transfected with a control vector
(Fig 2A) and the M1GATA-Y1 clone (Fig 2B) were similar to the parental M1 cells. In contrast, the M1GATA-Y22 and
-Y25 clones were distinctly different from the parental M1 cells,
consisting of blast cells, erythroblasts, and megakaryocytes (Fig 2C
and D). To confirm the presence of erythroblasts in M1 clonal lines
transfected with the GATA-1 expression vector, FACS analysis was
performed using TER-119 MoAb, which is specific for the erythroid
lineage.35 As shown in Fig 3,
TER-119 reacted with M1GATA-Y22 and M1GATA-Y25 clones, but not with
M1GATA-Y1. This result indicated the erythroid characteristics of
M1GATA-Y22 and M1GATA-Y25 clones. The megakaryocytic characteristics of
these GATA-1-expressing clones were confirmed by Wright-Giemsa and
acetylcholinesterase (ACh E)-specific (data not shown) staining.
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| Fig 1.
Differentiation of M1 cells to phagocytic cells induced
by IL-6. (A) Uninduced parental M1 cells, which are blast-like cells. (B) M1 cells were induced to differentiate to phagocytic cells (macrophages) by culturing the cells at an initial concentration of 2 × 105 cells/mL for up to 2 days in the presence of 50 ng/mL IL-6. These cells represent phagocytized latex beads.
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| Fig 2.
Morphological changes induced by electroporation of the
GATA-1 expression vector. (A) M1 clone transfected with the control vector (M1/pMG2), (B) M1GATA-Y1 clone, (C) M1GATA-Y22 clone, and (D)
M1GATA-Y25 clone. Arrows indicate the erythroblasts.
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| Fig 3.
Expression of the erythroid lineage in M1 clonal lines
transfected with the GATA-1 expression vector. Cells were incubated with biotinylated TER-119 and FITC-conjugated streptavidin. (A) M1
clone transfected with the control vector (M1/pMG2), (B) M1GATA-Y1 clone, (C) M1GATA-Y22 clone, (D) M1GATA-Y25 clone. (_____),
unstained cells; (----), stained cells.
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Northern blot analysis of erythroid and megakaryocytic lineage
markers.
Morphological studies indicated that the selected clones of M1 cells
transfected with mGATA-1, M1GATA-Y22, and M1GATA-Y25 possessed the
characteristics of erythroblasts and megakaryocytes. To determine
whether these clones expressed cellular markers of the erythroid or
megakaryocytic lineages, and whether or not the expression level of
other transcription factors were altered, we performed Northern blot
analyses for the expression of mRNAs for mGATA-1, mGATA-2, NF-E2 (p45),
Maf K, mCD34, human platelet factor 4 (hPF4), and m-globin
(Fig 4). In all of these clones, M1GATA-Y1,
-Y22, and -Y25, mGATA-1 mRNA was expressed. In contrast, the parental
M1 cells (M1) and M1 clone transfected with a control vector (M1/pMG2)
did not express GATA-1. The level of GATA-2 mRNA expression was lower
in the M1 clones expressing GATA-1 compared with parental M1 and
M1/pMG2 cells. The mRNAs encoding -globin, an erythroid marker, and
PF4, a megakaryocytic marker, were abundantly expressed in the
M1GATA-Y25 clone. In the M1GATA-Y22 clone, less -globin mRNA was
expressed and PF4 mRNA expression was minimal. However, neither
-globin nor PF4 were expressed in the M1GATA-Y1 clone. The level of
the mRNA encoding mCD34 was decreased in the M1 clones differentiated
towards the erythroid and megakaryocytic lineages. NF-E2 p45 and Maf K
form heterodimers with the NF-E2 site (TGCTGAGTCAT/C). Whereas the Maf
K mRNA was expressed equally in all these clones, M1GATA-Y1,
M1GATA-Y22, and M1GATA-Y25, as well as in the parental M1 cells and
M1/pMG2 clone, the mRNA encoding NF-E2 p45 was increased only in the M1
clones expressing GATA-1. These findings indicated that M1 clones
expressing GATA-1 possess the characteristics of both erythroid and
megakaryocytic cells.
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| Fig 4.
Northern blot analysis of M1 clonal lines transfected
with the GATA-1 expression vector. Lane 1, parental M1 cells; lane 2, M1 cells transfected with the control vector; lane 3, M1GATA-Y1 clone;
lane 4, M1GATA-Y22 clone; lane 5, M1GATA-Y25 clone; lane 6, EL-4 murine
thymoma cell line. The membrane containing 15 µg total RNA from each
cell line was sequentially hybridized with murine GATA-1, murine
GATA-2, NF-E2 (p45), Maf K, murine CD34, human platelet factor 4 (hPF
4), murine -globin, and 18S rRNA probes.
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c-mpl and c-mpl ligand (thrombopoietin) mRNA expression.
It has been reported that c-mpl and thrombopoietin play pivotal roles
in megakaryocyte development.36 Accordingly, to determine whether the c-mpl or c-mpl ligand are expressed in M1 clones that differentiate into megakaryocytes, we examined the expression of the
mRNA for the c-mpl and c-mpl ligand by Northern blot or RT-PCR followed
by Southern blot, respectively. Then each primer used for RT-PCR of
c-mpl ligand was derived from the different exons. Northern blot
analysis showed that the expression of mRNA for c-mpl was detected in
the M1GATA-Y22 and -25 clones but not M1, M1 cells transfected
with a control vector (M1/pMG), and M1GATA-Y1 clone
(Fig 5A). In contrast, the mRNA for
thrombopoietin was expressed in all clones of cells transfected with
the GATA-1 expression vector, as well as in M1 and M1/pMG cells (Fig
5B). These data suggested that c-mpl expression in stable
GATA-1-expressing M1 clones differentiated towards megakaryocytes
(M1GATA-Y22 and -Y25) is closely associated with megakaryocytic
differentiation.
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| Fig 5.
mRNA expression of murine c-mpl by Northern blot (A) and
that of murine c-mpl ligand (thrombopoietin) by RT-PCR followed by Southern blot (B) in the M1 clonal lines stably transfected with the
GATA-1 expression vector. The membrane containing 5 µg
poly(A)+ RNA from each cell was hybridized with murine
c-mpl and -actin cDNA (A). Murine -actin was the internal control
for RT-PCR (B). Lane 1, parental M1 cells; lane 2, M1 cels transfected
with control vector; lane 3, M1GATA-Y1 clone; lane 4, M1GATA-Y22 clone;
lane 5, M1GATA-Y25 clone.
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GATA-1 transactivates the c-mpl promoter.
The previously stated results suggested that c-mpl expression, as
regulated through GATA-1, may be important for megakaryocytic differentiation. Therefore, we addressed whether or not GATA-1 can
transactivate the c-mpl promoter in Jurkat cells that do not express
c-mpl or GATA-1. The promoter region and the first exon of murine c-mpl
gene (bp 312 to +12) were cloned upstream of the TK promoter
followed by firefly luciferase cDNA.32 Constructs containing the intact murine c-mpl promoter with the GATA motif (bp
85 to 80) and the mutated promoter with a 6-bp linker
substitution of the GATA-binding site (bp 85 to 80) were
cotransfected into Jurkat cells with the human GATA-1 expression vector
pEF-hGATA-1. GATA-1 was able to enhance the activity of a construct
that contained a GATA-binding site upstream of the minimal TK promoter
by an average of 2.3-fold (Fig 6). A
plasmid with the mutated GATA-binding site did not show transactivation
greater than that of the minimal TK promoter (Fig 6). These findings
support the functional importance of GATA-1 in the regulation of c-mpl
expression via binding to its recognition site.
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| Fig 6.
Transactivation of a minimal TK promoter containing the
murine c-mpl GATA-binding site. Jurkat cells were transfected with either pTKLuc/m c-mpl containing a wild-type GATA-binding site, or
pTKLuc/m c-mpl containing a mutated GATA-binding site, bp 85 to
80, with and without 10 µg of pEF-hGATA-1. The results are expressed as the average fold increase in ability on addition of
pEF-hGATA-1. The error bars represent the standard error of the mean of
at least four experiments. Luciferase assay was normalized to
-galactosidase produced by a cotransfected CMV- Gal plasmid.
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DISCUSSION |
We found that the overexpression of GATA-1 in the murine myeloid
leukemic cell line, M1, leads to erythroid and megakaryocytic differentiation. Erythroid cells were confirmed by the morphology, expression of TER-119, and -globin mRNA. Megakaryocytes were verified by the morphology, acetylcholinesterase staining and the
expression of PF4 mRNA. M1 clones differentiated by forced GATA-1
expression were heterogenous in nature and formed cells of both the
erythroid and megakaryocytic lineages. This finding suggests the
presence of bilineage precursor cells capable of differentiating
towards erythroid and megakaryocytic lineages in response to GATA-1
expression. Visvader et al37 have reported that introducing
a GATA-1 expression vector into the early myeloid cell line 416B, which
originally differentiated along the megakaryocytic and granulocytic
pathways, resulted in the most restricted differentiation to the
megakaryocytic lineage. Whereas 416B cells showed megakaryocytic characteristics before introducing a GATA-1 expression vector, the M1
cell line has been known for its potential for granulocyte-macrophage differentiation on stimulation with leukemia inhibitory factor (LIF) or
IL-6, but not for megakaryocytic differentiation. This indicates that
GATA-1 regulates the switching of the differentiation from bilineage
precursors to cells of either the myeloid or erythroid/megakaryocyte lineage.
M1GATA-Y22 and -Y25 clones which expressed abundant GATA-1 mRNA
differentiated towards erythroid and megakaryocyte lineages, but
M1GATA-Y1, which expressed a low level of GATA-1 mRNA, did not. This
result suggested that the high levels of GATA-1 expression are required
for the erythroblastic or megakaryocytic differentiation of the M1 cell
line by the GATA-1 transgene. Visvader et al37 have also
reported that clones expressing a low level of GATA-1 mRNA did not show
any megakaryocytic differentiation. In addition, it has been suggested
that high levels of GATA-1 correlate with the differentiation of
thromboblasts and erythroblasts, whereas intermediate levels induce
eosinophil development.19
The c-mpl ligand, thrombopoietin, has been cloned and shown to induce
progenitor cells to differentiate into platelet-producing megakaryocytes.29 In this study, mRNAs encoding c-mpl
ligand were expressed in parental M1 cells and in all the stably
transfected clones of M1 cells expressing GATA-1. In contrast, c-mpl
receptor mRNA was expressed only in the clones of M1 cells that
differentiated towards erythroblasts and megakaryocytes. In addition,
we showed that GATA-1 could significantly transactivate the c-mpl
promoter activity via the GATA-binding consensus site. In our study,
although the magnitude of transactivation of the c-mpl promoter by
GATA-1 was slight, the mutation at the GATA motif (bp 85 to
80) in the c-mpl promoter abolished the transactivation activity
by GATA-1 (Fig 6). This indicates that GATA-1 is one of the
transcription factors associated with the expression of the gene
encoding c-mpl. Alexander and Dunn31 have speculated that
Ets and GATA factors play an important role in expressing the c-mpl
gene. In the analysis of human c-mpl gene promoter, it has been shown
that GATA-1 binds with low affinity to a unique GATA motif at 70
bp in the human c-mpl promoter, and destruction of this site resulted
in a 15% decrease of the promoter activity.38,39 From our
data, although GATA-1 transactivated the murine mpl promoter activity,
it did not indicate the full promoter activity. Transcription factors other than GATA-1, such as Ets factor, which bound immediately downstream of the GATA motif in the murine c-mpl promoter, may act
synergistically with GATA-1 in the mpl gene expression. Deveaux et
al39 have shown that the c-mpl promoter can be bound by
GATA-1 and ETS proteins, including ETS-1, Fli-1, and Elf-1, and that the ETS motif adjacent to the GATA site is crucial for its expression. Also, it has been shown that the interaction between Sp1 and Ets factors play an important role in transcription of the
megakaryocyte-specific  IIb gene.40 It is important to
analyze which ets transcription factors are expressed during
megakaryocyte differentiation. Besides, GATA-1 might induce other
transcription factors that enhance c-mpl promoter activity. Further
studies are needed on the c-mpl promoter, including an examination of
the binding of GATA-1 in the murine c-mpl promoter by means of an
electrophoretic mobility shift assay (EMSA) or the detailed functional
promoter activity by linker-scanning mutants. Our findings suggested
that the stably transfected GATA-1-expressing M1 clones differentiate
towards megakaryocytes via an autocrine mechanism involving the
expression of both c-mpl ligand and c-mpl receptor. However, at
present, it is difficult to justify the autocrine mechanism via the
expression of c-mpl ligand and c-mpl receptor by GATA-1. It has been
reported that cells of the 32D promyelocytic line transfected with
murine c-mpl are morphologically similar to early megakaryocytes but
lack expression of the megakaryocyte markers, acetylcholinesterase and
PF4.41 These findings suggest that in 32D cells, signaling
through c-mpl and thrombopoietin may not be sufficient for
megakaryocytic differentiation. Recently, it has been shown that in
vivo overexpression via a retroviral vector of c-mpl leads to a fatal
myeloproliferative syndrome characterized by hyperproliferation of the
erythroblastic compartment.42 In addition, it has been
reported that mpl ligand enhances proliferation of erythroid
progenitors.43,44 This evidence may suggest that the c-mpl
induced by GATA-1 is involved in the proliferation and differentiation
of cells of not only the megakaryocytic but also the erythroid
lineages.
In all clones of M1 cells overexpressing GATA-1, expression of the mRNA
for mGATA-2 was downregulated (Fig 3). In mammalian hematopoietic
cells, GATA-2 mRNA is expressed at high levels in megakaryocytes and
mast cells but at very low or undetectable levels in murine
erythroleukemia cell lines, in which mGATA-1 mRNA expression is
increased (data not shown). In addition, the level of GATA-2 mRNA,
which is expressed in quiescent hematopoietic progenitor cells,
declines with erythroid and granulocytic differentiation, whereas
GATA-1 mRNA expression is induced during both erythroid and
megakaryocytic differentiation.45,46 These findings suggest that GATA-1 suppresses GATA-2 gene expression.
CD34 is expressed on pluripotential hematopoietic stem cells, and its
expression declines with differentiation.47 Expression of
mCD34 mRNA was also decreased in M1 cells overexpressing mGATA-1, which
were differentiated towards erythroid or megakaryocytic lineages (Fig
4). This evidence suggested that GATA-1 exerts a negative effect on
CD34 gene expression or perhaps induces transcription factors that bind
to a negatively acting cis element of the mCD34 promoter.
In conclusion, forced expression of GATA-1 in the myeloid leukemic cell
line, M1, induces the differentiation towards erythroid and
megakaryocytic lineage associated with the induction of c-mpl gene.
| |
FOOTNOTES |
Submitted February 3, 1997;
accepted September 12, 1997.
Supported by Grants-in-Aid from the Ministry of Education, Science and
Culture of Japan.
Address reprint requests to Yuji Yamaguchi, MD, Department of Cell
Differentiation, Institute of Molecular Embryology and Genetics,
Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto, 860 Japan.
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.
| |
ACKNOWLEDGMENT |
We are grateful to Dr M. Poncz for providing the human platelet factor
4 cDNA probe and Dr M. Hashiyama for the FACS analysis.
| |
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Abstract
Introduction
Abstract