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Prepublished online as a Blood First Edition Paper on July 12, 2002; DOI 10.1182/blood-2002-04-1177.
HEMATOPOIESIS
From the Department of Hematology/Oncology and
Molecular Oncology, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka, Japan; Center for TARA and Institute of Basic
Medical Institute, University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
Japan; Division of Cellular Therapy, Advanced Clinical Research Center,
Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo,
Japan; and Section of Gene Function and Regulation, Institute of Cancer
Research, Chester Beatty Laboratories, London, United Kingdom.
GATA-2 is considered to be essential for the development,
maintenance, and function of hematopoietic stem cells (HSCs). However, it was also reported that GATA-2 inhibits the growth of HSCs. To
examine the role of GATA-2 in the growth of hematopoietic cells, we
introduced an estradiol-inducible form of GATA-2 (GATA-2/estrogen receptor [ER]) into interleukin 3 (IL-3)-dependent cell lines, Ba/F3, 32D, and FDC-P1. Estradiol-induced GATA-2 suppressed
c-myc mRNA expression and inhibited IL-3-dependent growth
in these clones. As for this mechanism, GATA-2 was found to inhibit
ubiquitin/proteasome-dependent degradation of p21WAF1 and
p27Kip1 and to induce their accumulation by repressing the
expression of Skp2 and Cul1, both of which are components of the
ubiquitin ligase for p21WAF1 and p27Kip1.
Overexpression of c-myc restored the expression of Skp2 and Cul1 mRNA, reduced the amounts of p21WAF1 and
p27Kip1 proteins, and canceled GATA-2-induced growth
suppression, suggesting that down-regulation of c-myc
expression may be primarily responsible for GATA-2-induced growth
suppression. Next, we transduced retrovirus containing GATA-2/ER into
murine bone marrow mononuclear cells (MNCs) and stem/progenitor
(Sca-1+Lin GATA family transcription factors are composed of 6 members (GATA-1 through GATA-6) and play essential roles in the
development of diverse cell types according to their unique tissue
distribution (for a review, see Weiss and Orkin1). These
factors bind to a GATA motif (A/T)GATA(A/G), in the regulatory region
of their target genes through 2 highly conserved zinc finger domains.
The carboxyl (C) finger is absolutely required for the DNA binding, whereas the amino (N) finger stabilizes the DNA binding and confers full specificity.2,3 Among GATA family members, at least 3 GATA factors (GATA-1, GATA-2, and GATA-3) are critically involved in
different aspects of hematopoiesis. GATA-1 is highly expressed in
erythroid cells, mast cells, and megakaryocytes.4-7 GATA-1 is essential for primitive and definitive erythropoiesis, because targeted disruption of GATA-1 gene in mice causes fatal
embryonic anemia due to a block in erythroid maturation and
apoptosis.8-10 Furthermore, Shivdasani et
al11 showed that GATA-1 is also required for
megakaryopoiesis by generating lineage-selective GATA-1 knockout mice.
GATA-3 is exclusively expressed in T lymphocytes and indispensable for
the development of T lymphocytes.12,13 These results
suggest that both GATA-1 and GATA-3 are essential for the lineage
commitment or the subsequent maturation of committed hematopoietic
progenitor cells. In contrast, GATA-2 is highly expressed in
pluripotent hematopoietic stem cells (HSCs),14 whereas it
is also expressed in early stages of erythroid cells, mast cells, and
megakaryocytes.15-18 GATA-2 knockout mice die around
embryonic day 9.5 to 10.5 due to a pan-hematopoietic deficit,
suggesting that GATA-2 would be a key regulator of development,
maintenance, or function of HSCs.19 Furthermore, Tsai and
Orkin20 showed that GATA-2 was necessary for proliferation
and survival of early hematopoietic cells by analyzing
GATA-2 Cell growth is tightly regulated by cell cycle regulatory molecules,
including cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors
(CKIs).23 Cell cycle progression from G1 to S phase is regulated by CDK4, CDK6, and CDK2. The catalytic activities of
CDK4 and CDK6 are up-regulated in the mid G1 to late
G1 phases dependent on the formation of the complexes with
D-type cyclins, whereas they are also negatively regulated by INK4
family of CKIs included in these complexes (p16INK4A,
p15INK4B, p18INK4C, and p19INK4D).
CDK2 activities are induced at late G1 phase through its
interaction with cyclin D or cyclin E and are suppressed by KIP family
of CKIs (p21WAF1, p27Kip1, and
p57Kip2). At early G1 phase, a dephosphorylated
form of retinoblastoma protein (Rb) binds to E2F-1, thereby
inhibiting transcriptional activities of E2F-1. From the mid
G1 to late G1 phase, Rb is sequentially phosphorylated by CDK4, CDK6, or CDK2, and phosphorylated Rb, in turn,
releases E2F-1, which initiates transcription of its target genes such
as cyclin D, cyclin E, cyclin A, c-myc, c-myb, and DNA polymerase that are required for G1/S transition or
DNA synthesis. Expression levels of these cell cycle regulatory
molecules are regulated at both transcriptional and posttranscriptional levels, and the ubiquitin (Ub)/proteasome system plays central roles in
the later step.24,25 Ub/proteasome-mediated proteolysis is
executed through a series of enzymatic reactions. First, Ub is charged
with adenosine triphosphate (ATP) by a Ub-activating enzyme (E1), and
then transferred to the substrate by a Ub-conjugating enzyme (E2). An
element within the target protein termed a degron is recognized by E2
either alone or in combination with a Ub ligase (E3). After recognition
of the degron, the Ub target protein conjugates are formed via an
isopeptide bond between the carboxyl-terminal glysine of Ub and a
lysine residue on the target protein. Repeated rounds of
ubiquitination results in the highly ubiquitinated
target protein, which is recognized and degraded by 26S proteasome. At present, a number of cell cycle regulatory molecules such as E2Fs, cyclin D1, cyclin E, cyclin A, cyclin B, CDC25B, p21WAF1,
p27Kip1, and p53 are known to be the targets of the
Ub/proteasome system.
Pluripotent HSCs are characterized by their abilities for self-renewal
and multipotentiality.26,27 By using these abilities, HSCs
maintain their own population and keep supplying mature blood cells in
all lineages throughout the life period. Most of HSCs are kept dormant
under physiologically normal conditions, whereas these cells show
dramatic proliferative activities in response to hematopoietic injury
such as the treatment with myelotoxic reagents and irradiation. In a
previous study, Cheng et al28 reported that
p21WAF1 was required to keep HSCs in quiescence and to
prevent their exhaustion, indicating that cell cycle control would
affect biologic properties and fate of HSCs profoundly. However, the
precise mechanism governing the cell cycle in HSCs still remains to be determined.
In an attempt to clarify the roles of GATA-2 in the growth regulation
of HSCs, in this study we examined the effects of GATA-2 on the
expression and the function of cell cycle regulatory molecules. We
found here that enforced expression of GATA-2 inhibited
cytokine-dependent growth of normal hematopoietic stem/progenitor
cells, which was linked with the suppression of c-myc mRNA
expression and the accumulation of p21WAF1 and
p27Kip1 proteins. As for this mechanism, GATA-2 was found
to inhibit c-myc-dependent expressions of Skp2 and Cul1,
which act as components of E3 toward p21WAF1 and
p27Kip1 and participate in their degradation. In addition,
we found that the expression of GATA-2 protein was reduced in
hematopoietic stem/progenitor cells that were enforced to enter cell
cycle by the cytokine stimulation. These results suggest that GATA-2
may induce growth suppression of HSCs through up-regulated expressions of p21WAF1 and p27Kip1 proteins, and raise the
possibility that GATA-2 may contribute to the quiescence of HSCs.
Reagents and antibodies
Plasmid constructs and cDNAs
Cell lines and cultures Murine IL-3-dependent cells lines Ba/F3, 32D, and FDC-P1 were cultured in RPMI (Nakarai Tesq, Kyoto, Japan) supplemented with 10% fetal calf serum (FCS; Flow, North Ryde, Australia) and 1 ng/mL mIL-3. NIH3T3 cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FCS.Flow cytometry DNA content of cultured cells was quantitated by staining with propidium iodide (PI) and analyzed on FACSort (Beckon Dickinson, Oxnard, CA). Cell cycle analysis was performed with a program Modfit LT2.0 (Beckon Dickinson).Northern blot analysis The methods for isolation of total cellular RNA and Northern blotting were described previously.29Cell proliferation assays To quantitate proliferation of cultured cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma, St Louis, MO) rapid colorimetric assays were performed as previously reported.30 In brief, triplicate aliquots of 2.0 × 104 cells were suspended in 96-well flat-bottom microtiter plates and cultured for 72 hours in the presence or absence of 2 µM estradiol. MTT (10 µL of a 5-mg/mL solution in phosphate-buffered saline [PBS]) was added for the final 4 hours of the culture, and then 100 µL acid isopropanol (0.04 N HCL in isopropanol) was added and mixed. The optical density (OD) was measured on the Microelisa plate reader (Corona Electric, Ibaragi, Japan) with a test wavelength of 540 nm and a reference wavelength of 620 nm.Immunoprecipitation and immunoblotting Isolation of total cellular lysates, immunoprecipitation, gel electrophoresis, and immunoblotting were performed according to the methods described previously.29 Immunoreactive proteins were visualized with the enhanced chemiluminescence detection system (Dupont NEN, Boston, MA).Metabolic labeling and measurement of protein turnover To examine the half-lives of p21WAF1 and p27Kip1 proteins, a pulse chase experiment was performed according to the procedures described previously.31 In short, 1 × 107 cells for each sample were radiolabeled with 200 µCi (7.4 MBq) [35S]-methionine in 1 mL methionine-free DMEM supplemented with 10% dialyzed FCS for 60 minutes. Then, the cells were washed and resuspended in DMEM containing 2 mM unlabeled methionine. During the culture with or without estradiol, total cellular lysates were prepared at the time indicated. p21WAF1 and p27Kip1 were immunoprecipitated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were dried and subjected to the autoradiography.Preparation of stable transfectants from Ba/F3, 32D, and FDC-P1 To stably introduce an expression vector for GATA-2/ER into Ba/F3, 32D, and FDC-P1, we transfected 30 µg pEF-BOS-GATA-2/ER-puro by electroporation (250 V, 960 µFD; Bio-Rad Laboratory, Richmond, CA). After the culture with puromycin at a concentration of 1.2 µg/mL, several clones, in which GATA-2/ER was effectively expressed, were selected and subjected to analyses. We further introduced an expression vector for c-myc, CDK2, CDK4, or CDK6 into Ba/F3/GATA-2/ER, and these clones were selected by the culture with G418 (1.0 mg/mL).Luciferase assays Luciferase assays were performed with a Dual-Luciferase Reporter System (Promega, Madison, WI) as previously described.29 In short, NIH3T3 cells (2 × 105 cells seeded in a 60-mm dish) were transfected with 2 µg pEF-BOS-GATA-2/ER-puro or an empty mock vector together with 2 µg of a reporter gene for GATA (named 3 × M P-Luc) and 10 ng pRL-CMV-Rluc, an expression vector
of renilla luciferase, by the calcium phosphate
coprecipitation method. After 12 hours, the cells were washed,
serum-deprived for 12 hours, and then cultured with 2 µM estradiol
for 8 hours. Then, the cells were lysed in lysis buffer supplied by the
manufacturer, followed by the measurement of the firefly and
renilla luciferase activities on a luminometer LB96P
(Berthold Japan, Tokyo, Japan). The relative firefly luciferase
activities were calculated by normalizing transfection efficiency
according to the renilla luciferase activities. The
experiments were performed in triplicate and the similar results were
obtained from at least 3 independent experiments.
Immune complex kinase assays Immune complex kinase assays were performed according to the procedure described previously.32 Briefly, CDK2 was immunoprecipitated from 100 µg total cellular lysates. Kinase reactions were initiated by the addition of 20 µCi (0.74 MBq) -[32P]ATP (6000 Ci/mmol [222 GBq/mmol]) into
20 µL kinase buffer (50 mM HEPES
[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid] at pH 7.5, 10 mM MgCl2, 1 mM DTT
[dichlorodiphenyltrichloroethane], 1 mM EGTA
[ethyleneglycoltetraacetic acid], 0.4 mM NaVO4, 0.4 mM
NaF, and 40 µM of nonradioactive ATP) and performed at 30°C for 20 minutes (within a linear incorporation kinetics) by using 3 µg
Histone H1 (Boehringer Mannheim, Mannheim, Germany) as a substrate.
After addition of the protein-loading buffer, samples were boiled, and
subjected to SDS-PAGE. The gels were stained with Coomassie blue to
confirm the amounts of the immunoprecipitates, destained, dried, and
subjected to autoradiography.
Semiquantitative reverse transcription-PCR analyses Total cellular RNA was extracted from cultured cells (about 104 cells) and reverse transcribed (RT) into cDNA with oligo(dT) primers (Pharmacia, Piscataway, NJ) by using SuperScript reverse transcriptase according to the manufacturer's instruction (Gibco BRL, Gaithersburg MD). The cDNA product (1 µL) was resuspended in 20 µL of the PCR reaction buffer containing 0.5 U TaqGold DNA polymerase (Perkin Elmer, Foster City, CA), 2 mM MgCl2, 200 µM deoxynucleoside triphosphate (dNTP) mix, 30 µCi (1.11 MBq)-[32P] deoxycytidine
triphosphate (dCTP), and 15 pmol forward and reverse primers.
The primer sets to amplify c-myc, Cul1, and  -actin are as
follows: c-myc, a sense primer 5'-TCACCAGCACAACTACGCCG-3' and a reverse primer 5'-CAGGATGTAGGCGGTGGCTT-3'; Cul1, a sense primer
5'-GGACTGAAGCAGATCGGTCTT-3' and a reverse primer
5'-AATGTCTATTGAGGTAGGCA-3'; -actin, a sense primer
5'-CATCACTATTGGCAACGAGC-3' and a reverse primer
5'-ACGCAGCTCAGTAACAGTCC-3'. The samples were denatured at 94°C for 10 minutes, followed by 20 to 32 cycles of amplification (94°C, 30 seconds for denaturation; 55°C, 30 seconds for annealing; 72°C, 30 seconds for extension). At first, we adjusted the amounts of cDNA
products in several samples. Briefly, the aliquots (5 µL) of
-actin products obtained after 26, 28, 30, and 32 PCR cycles from
each sample were size fractionated on 12% polyacrylamide gels, dried,
and autoradiographed. Because -actin was amplified exponentially
during 28 to 34 cycles of PCR in all of the samples, we quantified the
amounts of cDNA by measuring the intensities of -actin products
obtained after 30 cycles of PCR with a densitometer. After adjustment
of the amounts of cDNA products, the equal amount of cDNA products were
subjected to the PCR. The amounts of c-myc and Cul1 mRNA
were evaluated after 28 to 34 cycles of PCR, during which PCR products
from all of the samples were exponentially amplified.
Preparation of the conditioned media containing high-titer virus particles The conditioned media containing high-titer virus particles were prepared as described previously.33 Briefly, the plasmid pMX-GATA-2/ER-neo, an empty pMX-neo or pMX-GFP (green fluorescent protein) was transfected into an ecotropic packaging cell line Plat-E by the calcium phosphate coprecipitation method. After 12 hours, the cells were washed, and then cultured for 48 hours in DMEM supplemented with 10% FCS. The supernatant containing virus particles was collected, centrifuged at 6900 rpm for 16 hours, and concentrated by 50-fold in volume.Retrovirus transfection into murine bone marrow cells Bone marrow cells were harvested from 9- to 12-week-old Balb/c mice pretreated with 150 mg/kg 5-fluoruracil (5-FU) for 2 days. Mononuclear cells were isolated by density gradient centrifugation and cultured in DMEM supplemented with 10% FCS in the presence of mIL-3 (10 ng/mL), mSCF (100 ng/mL), and hIL-6 (50 ng/mL) for 48 hours. Then, the cells were supplemented with 5% volume of conditioned media containing high-titer retrovirus and polybrene (3 µg/mL). After 24 hours, the cells were washed and cultured in DMEM supplemented with 10% FCS containing cytokines (as described above) and 1 mg/mL G418 for 72 hours. After the selection with G418, retrovirus-infected cells were further cultured with or without 2 µM estradiol for 72 hours, and subjected to MTT assays, RT-PCR analyses, and Western blot analyses. To assess the transfection efficiency, we infected retrovirus containing pMX-GFP and examined the proportion of GFP+ cells by flow cytometric analysis.Isolation of murine bone marrow stem/progenitor cells We isolated Lin cells by negative selection
of CD3+, CD20+, Ter-119+,
Mac-1+, and Gr-1+ cells with BioMAG
(Polysciences, Warrington, PA). LinSca-1+
cells were isolated from murine bone marrow mononuclear cells (MNCs) by
using MACS (Miltenyl Biotec, Bergisch Gladbach, Germany).
Effects of GATA-2 on the growth of Ba/F3, 32D, and FDC-P1 To examine the effects of GATA-2 on the growth of hematopoietic cells in a ligand-inducible system, we used GATA-2/ER, a chimeric molecule of GATA-2 consisting of full-length GATA-2 and the ligand-binding domain of ER. At first, we performed luciferase assays with a reporter gene for GATA, 3 × MP-Luc, in NIH3T3 cells. As
shown in Figure 1A, the estradiol
treatment stimulated 3 × MP-Luc by 4.7-fold in
GATA-2/ER-transfected cells, whereas it did not show any effect in
mock-transfected cells. These results indicate that GATA-2/ER reveals
its activities only when the cells are treated with estradiol. Next, we
stably introduced GATA-2/ER into mIL-3-dependent cell lines (a pro-B
cell line Ba/F3, a myeloid cell line 32D, and a hematopoietic
progenitor cell line FDC-P1); each clone was designated
Ba/F3/GATA-2/ER, 32D/GATA-2/ER, and FDC-P1/GATA-2/ER, respectively. At
first, we compared the expression levels of GATA-2/ER with those of
endogenous GATA-2 in these clones by immunoblot analysis. The
expression level of GATA-2/ER was almost equivalent to that of
endogenous GATA-2 in FDC-P1/GATA-2/ER and about 2-fold in
32D/GATA-2/ER, whereas Ba/F3/GATA-2/ER scarcely expressed endogenous GATA-2 protein (Figure 1B). Under the culture without estradiol, these
clones proliferated in response to IL-3 with the growth curves similar
to those of their parental clones (data not shown). In contrast, the
estradiol treatment inhibited IL-3-dependent growth of
Ba/F3/GATA-2/ER, 32D/GATA-2/ER, and FDC-P1/GATA-2/ER almost completely
(Figure 1C), whereas it did not affect the growth of mock (an empty
vector)-transfected Ba/F3, 32D, or FDC-P1 (data not shown). In DNA
content analysis, the estradiol treatment reduced the cells in S phase
in all of the clones (changes in the proportion of the cells in S phase
before and after the estradiol treatment: Ba/F3/GATA-2/ER, 59% to 5%;
32D/GATA-2/ER, 49% to 6%; FDC-P1/GATA-2/ER, 42% to 4%; Figure 1D).
Similar results were obtained from at least 3 GATA-2/ER-transfected
clones of Ba/F3, 32D, and FDC-P1 (data not shown).
Effects of GATA-2 on the expression of growth regulatory molecules To clarify the mechanism of GATA-2-induced growth suppression, we examined the expression levels of GATA-2/ER and growth-promoting molecules in Ba/F3/GATA-2/ER and FDC-P1/GATA-2/ER during the estradiol treatment by Northern blot analysis. As shown in Figure 2A, the expression of GATA-2/ER mRNA was kept constant in both clones during the test period. After the addition of estradiol into the culture medium, the expression of c-myc decreased as early as 4 hours in both clones (Figure 2A). Then, the expression of CDK4, cyclin A, and cyclin B declined gradually. In contrast, we did not detect an apparent change in the expression levels of CDK2 or CDK6 (Figure 2A) in addition to cyclin D2 or cyclin D3 (data not shown). Furthermore, neither cyclin D1 nor cyclin E was expressed in these clones during the test period (data not shown). We also examined the expression of growth inhibitory molecules during GATA-2-induced growth suppression. As shown in Figure 2A, the expression levels of p21WAF1 and p27Kip1 mRNA did not change for up to 120 hours. Moreover, we did not detect a significant change in the expression of other CDK inhibitors (p16INK4A, p15INK4B, p18INK4C, p19INK4D, and p57Kip2), p19ARF, or p53 (data not shown). Next, we examined the effects of GATA-2 on the expression levels of CKIs by Western blot analysis. Although the expression levels of p21WAF1 and p27Kip1 mRNA were kept constant during the estradiol treatment, the amount of p21WAF1 and p27Kip1 proteins increased in both Ba/F3/GATA-2/ER and FDC-P1/GATA-2/ER (Figure 2B). To investigate the significance of up-regulated expression of p21WAF1 and p27Kip1 proteins, we evaluated changes of CDK2 activities with immune complex kinase assays by using Histone H1 as a substrate. As shown in Figure 2C, CDK2 activities were reduced by the estradiol treatment in both Ba/F3/GATA-2/ER and FDC-P1/GATA-2/ER, probably due to the increased expression of p21WAF1 and p27Kip1 proteins. In contrast, the estradiol treatment did not affect the expression of these growth regulatory molecules at mRNA or protein levels in mock-transfected Ba/F3 or FDC-P1 (data not shown).
GATA-2 inhibits Ub/proteasome-dependent degradation of p21WAF1 and p27Kip1 through the suppression of Skp2 and Cul1 mRNA expression Next, we investigated the effects of GATA-2 on the half-lives of p21WAF1 and p27Kip1 proteins by a pulse chase experiment. [35S]-methionine-labeled FDC-P1/GATA-2/ER and Ba/F3/GATA-2/ER cells were cultured with or without estradiol for the time indicated, and changes in the expression levels of p21WAF1 and p27Kip1 proteins were examined in each clone. As shown in Figure 3A, both 35S-labeled p21WAF1 and p27Kip1 were degraded rapidly under the culture without estradiol (percent degradation assessed by the densitometric analysis: p21WAF1, 62% at 40 minutes and 93% at 80 minutes; p27Kip1, 32% at 40 minutes and 91% at 80 minutes). In contrast, the estradiol treatment maintained the expression levels of 35S-labeled p21WAF1 and p27Kip1 for up to 80 minutes (Figure 3A), whereas it did not retain their expression levels in mock-transfected FDC-P1 or Ba/F3 (data not shown). These results indicate that GATA-2 would enhance the protein stability of p21WAF1 and p27Kip1. Because degradation of p21WAF1 and p27Kip1 is executed by the Ub/proteasome system,34-36 we examined the effects of a proteasome inhibitor, lactacystin, on the amounts of p21WAF1 and p27Kip1 in FDC-P1/GATA-2/ER and Ba/F3/GATA-2/ER. Under the culture without estradiol, the treatment with lactacystin augmented the amounts of p21WAF1 and p27Kip1 in each clone (Figure 3B, lane 1 versus lane 2), implying that both p21WAF1 and p27Kip1 are constantly degraded by Ub/proteasome pathways under normal culture conditions. Meanwhile, the estradiol treatment augmented the amounts of p21WAF1 and p27Kip1 proteins as efficiently as lactacystin (Figure 3B, lane 2 versus lane 3), and lactacystin could not further augment their amounts in the estradiol-treated cells (Figure 3B, lane 3 versus lane 4). These results suggest that GATA-2 may inhibit Ub/proteasome-dependent degradation of p21WAF1 and p27Kip1. Next, we analyzed the mechanism through which GATA-2 inhibits Ub/proteasome-dependent degradation of p21WAF1 and p27Kip1. Because Skp1, Skp2, and Cul1 have been reported to form a complex and to act as a Ub ligase (E3) toward p21WAF1 and p27Kip1, we examined their expression in Ba/F3/GATA-2/ER and FDC-P1/GATA-2/ER during the estradiol treatment by Northern blot analysis.34-36 As shown in Figure 3C, the expression of Skp1 was retained in both Ba/F3/GATA-2/ER and FDC-P1/GATA-2/ER during the estradiol treatment. In contrast, the expression of Skp2 and Cul1 was gradually suppressed by the estradiol treatment in Ba/F3/GATA-2/ER. Also, in FDC-P1/GATA-2/ER, the expression of Skp2 and Cul1 was reduced by the estradiol treatment after 12 hours, whereas that of Skp2 was transiently up-regulated from 4 to 8 hours. Although the results shown in Figure 3 were obtained from a single cell clone, similar results were observed in 2 other clones of GATA-2/ER-transfected Ba/F3 and FDC-P1, respectively (data not shown). Together, these results suggest that GATA-2 might inhibit Ub/proteasome-dependent degradation of p21WAF1 and p27Kip1 through the suppression of Skp2 and Cul1 expression.
Reduced expression of c-myc mRNA is primarily responsible for GATA-2-induced growth suppression In this study, GATA-2 suppressed the expression of c-myc and CDK4 mRNA (Figure 2A). GATA-2 also inhibited the expression of Skp2 and Cul1 mRNA (Figure 3C), thereby inducing the accumulation of p21WAF1 and p27Kip1 proteins. In previous studies, c-myc was reported to induce the expression of CDK4 and Cul1 mRNA.37,38 Therefore, we speculated that down-regulation of c-myc expression might be primarily responsible for the reduced expression of CDK4 and Cul1 and for the subsequent accumulation of p21WAF1 and p27Kip1. To verify this hypothesis, we overexpressed c-myc, CDK2, CDK4, and CDK6 in GATA-2/ER-transfected Ba/F3 cells. As compared with the growth inhibition observed in the mock clone transfected with an empty vector, overexpression of c-myc, CDK2, and CDK4 canceled GATA-2-induced growth suppression by 90%, 40%, and 20%, respectively (Figure 4A). In contrast, overexpression of CDK6 showed little effect. Next, we examined the effects of overexpressed c-myc on the expression of CDK4, Skp2, and Cul1 mRNA in c-myc-introduced Ba/F3/GATA-2/ER. In contrast to the data obtained from Ba/F3/GATA-2/ER (Figures 2A and 3C), the expression of CDK4, Skp2, and Cul1 mRNA was maintained in Ba/F3/GATA-2/ER/c-myc even during the estradiol treatment (Figure 4B). In addition, the accumulation of p21WAF1 and p27Kip1 proteins and the suppression of CDK2 activities that are induced by the estradiol treatment in the mock clone were canceled in Ba/F3/GATA-2/ER/c-myc (Figure 4C-D). Although the results shown in Figure 4 were obtained from a single cell clone, similar results were observed in 2 other clones of each transfectant (data not shown). Together, these results suggest that the suppression of c-myc expression by GATA-2 may be primarily responsible for the accumulation of p21WAF1 and p27Kip1 and consequent growth inhibition.
GATA-2 inhibits cytokine-dependent growth of normal hematopoietic cells To examine the effects of GATA-2 on cytokine-dependent growth of normal hematopoietic cells, we isolated MNCs from bone marrow of mice treated with 5-FU. We also purified Sca-1+Lin
cells from MNCs. After culture with various cytokines, we infected retrovirus containing pMX-GFP into these cells and assessed the transfection efficiency after 72 hours. As shown in Figure
5A, about 55% to 65% of MNCs and
Sca-1+Lin cells were found to be
GFP+ by flow cytometric analysis. Next, we infected
retrovirus containing pMX-GATA-2/ER-neo or pMX-neo (a mock vector) into
these cells. After the selection with G418 for 3 days, we examined the
expression level of GATA-2/ER relative to endogenous GATA-2 in
Sca-1+Lin cells by immunoblot analysis. The
expression level of GATA-2/ER was almost equivalent to that of
endogenous GATA-2 in Sca-1+Lin cells (Figure
5B). We further cultured G418-resistant cells with or without estradiol
for 72 hours and subjected them to a proliferation assay, cell cycle
analysis, Western blot analysis, and RT-PCR analysis. As shown
in Figure 5C, GATA-2/ER inhibited the growth of MNCs by 75%
(P < .01) and that of Sca-1+Lin
cells by 65% (P < .01) in an MTT assay although the
estradiol treatment did not affect the growth of mock-infected MNCs or
Sca-1+Lin cells. In agreement with this
result, the estradiol treatment reduced the proportion of the cells in
S phase from 18% to 3% in GATA-2/ER-transfected MNCs (Figure 5D).
GATA-2 suppresses the expression of c-myc and Cul1 mRNA and induces the accumulation of p21WAF1 and p27Kip1 proteins in normal hematopoietic cells Next, we examined the effects of GATA-2 on the expression of c-myc, Skp2, and Cul1 in normal MNCs by semiquantitative RT-PCR analysis. After normalization of the amounts of cDNA according to the amounts of the-actin products, we evaluated their expression after 28 to 34 cycles of PCR, during which these PCR products were
amplified exponentially (Figure 5D). In agreement with the results
obtained from Ba/F3 and FDC-P1, the estradiol treatment severely
reduced the expression of c-myc, Skp2, and Cul1 mRNA in
normal hematopoietic cells (Figure 5E). Also, the estradiol treatment
induced the accumulation of p21WAF1 and p27Kip1
proteins in normal MNCs (Figure 5F).
The expression of GATA-2 protein decreases in cycling hematopoietic stem/progenitor cells To assess the roles of endogenous GATA-2 expressed in hematopoietic stem/progenitor cells, we compared expression levels of GATA-2 protein in quiescent and cycling stem cells. We isolated Lin cells from murine bone marrow cells and cultured with
mSCF, mIL-3, hIL-6, and mFLT3 ligand for 5 days because this
combination was reported to expand HSCs without spoiling their
mutipotentiality.39 Surface phenotypes of the cells were
essentially the same before and after the culture (data not shown).
However, the proportion of the cells in S phase increased from 2% to
25% after the culture (Figure 6A). The
expression of GATA-2 protein was found to decrease in the cultured
cells (Figure 6B), suggesting a possibility that down-regulated
expression of GATA-2 in hematopoietic stem/progenitor cells might be
prerequisite for their cell cycle entry.
In a previous study, Heyworth et al22 reported that
an estradiol-inducible form of GATA-2 (GATA-2/ER) inhibited
cytokine-dependent growth of normal hematopoietic progenitor cells in
vitro. Similarly, in this study, we found that GATA-2/ER inhibited the
growth of normal hematopoietic cells and various cell lines. However,
considering the fact that GATA-2/ER is an artificial form, we have to
pay special attention to the possibility that the ER portion of the conditional protein might reveal some artificial effects that are
different from those of wild-type GATA-2. Yet, supporting the data
obtained by GATA-2/ER, Persons et al21 showed that enforced expression of wild-type GATA-2 inhibited the expansion of HSCs
in vitro and in vivo. They introduced wild-type GATA-2 into murine
primitive Sca-1+Lin With regard to the mechanisms of GATA-2-induced growth suppression,
Persons et al21 could not detect an apparent effect of
GATA-2 on cell cycle profiles in normal hematopoietic progenitor cells,
whereas they found that GATA-2 inhibited their growth severely. On the
other hand, in the present study, we found that GATA-2 reduced the
proportion of the cells in S phase in normal hematopoietic cells as
well as in various hematopoietic cell lines. As for this mechanism, we
found that GATA-2 suppressed the expression of c-myc, CDK4,
and Cul1 mRNA and thereby induced the accumulation of
p21WAF1 and p27Kip1 proteins. Because
overexpression of c-myc restored these changes and
consequently canceled GATA-2-induced growth suppression, the suppression of c-myc expression was considered to be
primarily responsible for GATA-2-mediated growth inhibition. However,
it still remains unresolved how GATA-2 suppressed the expression of
c-myc mRNA. Up to the present, GATA-2 has been reported to act as a transcriptional repressor on promoters of erythropoietin and
peroxisome proliferator-activated receptor We found here that GATA-2 augments the amounts of p21WAF1 and p27Kip1 proteins. Because p21WAF1 and p27Kip1 were reported to play essential roles to keep HSCs and progenitor cells in quiescence, respectively,28,52 it was speculated that GATA-2 may play some role in their cell cycle regulation through modulating the expression levels of p21WAF1 and p27Kip1 proteins. In summary, we demonstrate here that enforced expression of GATA-2 inhibits cytokine-dependent growth of hematopoietic cells through the down-regulation of c-myc mRNA expression and the up-regulation of p21WAF1 and p27Kip1 protein expression.
After we had made a revision of this manuscript, Kitajima et al53 reported that the functions of GATA-2/ER were somewhat different from those of wild-type GATA-2.
We thank Drs E. Harlow, H. Matsushime, H. Kiyokawa, B. Vogelstein, J. Massague, T. Hama, K. Kataoka, C. Sherr, H. Zhang, and R. A. DePinho for providing the plasmids.
Submitted April 18, 2002; accepted June 27, 2002.
Prepublished online as Blood First Edition Paper, July 12, 2002; DOI 10.1182/blood-2002-04-1177.
Supported by grants from ministry of Education, Science and Culture of Japan, Mochida Foundation, Ichiro Kanehara Foundation, Uehara Memorial Foundation, Naito Foundation, and the Japan Medical Association.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Itaru Matsumura, Department of Hematology and Oncology (C9), Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan; e-mail: matumura{at}bldon.med.osaka-u.ac.jp
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Martin DI, Orkin SH.
Transcriptional activation and DNA-binding by the erythroid factor GF-1.
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1990;4:1886-1898 3. Yang H-Y, Evans-T. Distinct roles for the two cGATA-1 finger domains. Mol Cell Biol. 1992;12:4562-4570 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
) or without (
) 1 µM estradiol. Total number of viable cells was counted by trypan blue
dye exclusion method at the times indicated. The results are shown as
means ± SD of triplicate cultures. (D) Cells cultured as
described above were subjected to PI staining, and DNA content was
analyzed on FACSort. Cell cycle analysis was performed with a program
Modfit LT2.0. The results shown are representative of 3 independent
experiments.
