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Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1225-1234
Bcl-3 Expression and Nuclear Translocation Are Induced by
Granulocyte-Macrophage Colony-Stimulating Factor and Erythropoietin in
Proliferating Human Erythroid Precursors
By
Min-Ying Zhang,
Edward W. Harhaj,
Laurie Bell,
Shao-Cong Sun, and
Barbara A. Miller
From the Departments of Pediatrics and Microbiology and Immunology,
The Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, PA.
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ABSTRACT |
Bcl-3 is a proto-oncogene involved in the chromosomal
translocation t(14;19) found in some patients with chronic lymphocytic leukemia. It shares structural similarities with and is a member of the
I B family of proteins. In this report, involvement of Bcl-3
in hematopoietic growth factor-stimulated erythroid proliferation and
differentiation was examined. In TF-1 cells, an erythroleukemia cell
line, granulocyte-macrophage colony-stimulating factor (GM-CSF) and
erythropoietin (Epo) greatly enhanced Bcl-3 expression at both
the protein and mRNA levels in association with stimulation of
proliferation. Bcl-3 protein was also highly expressed in early burst-forming unit-erythroid (BFU-E)-derived erythroid
precursors (day 7) and decreased during maturation (days 10 and 14),
suggesting that Bcl-3 is involved in normal erythroid proliferation. In
these hematopoietic cells, Bcl-3 was hyperphosphorylated. GM-CSF and Epo modulated the subcellular localization of Bcl-3. Upon stimulation of TF-1 cells with GM-CSF or Epo, the nuclear translocation of Bcl-3 was dramatically enhanced. Overexpression of Bcl-3 in TF-1 cells by transient transfection along with the NF-B factors p50 or
p52 resulted in significant induction of an human immunodeficiency virus-type 1 (HIV-1) B-TATA-luceriferase reporter plasmid,
demonstrating that Bcl-3 has a positive role in transactivation of
B-containing genes in erythroid cells. Stimulation with GM-CSF
enhanced c-myb mRNA expression in these cells. Bcl-3 in nuclear
extracts of TF-1 cells bound to a B enhancer in the c-myb
promoter together with NF-B2/p52 and this binding activity was
enhanced by GM-CSF stimulation. Furthermore, cotransfection of Bcl-3
with p52 or p50 in TF-1 cells resulted in significant activation of a
c-myb B-TATA-luceriferase reporter plasmid. These findings
suggest that Bcl-3 may participate in the transcriptional regulation of
certain B-containing genes involved in hematopoiesis, including
c-myb.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
Bcl-3 WAS FIRST IDENTIFIED from a
chronic lymphocytic leukemia (CLL) patient with the chromosomal
translocation t(14;19) (q32; q13.1).1 This breakpoint
junction does not affect the structural integrity of Bcl-3, but
results in the overexpression of Bcl-3 mRNA in leukemic
cells.1 Bcl-3 overexpression is proposed to contribute to the development of CLL through dysregulation of a gene(s)
normally regulated by NF-B transcription factors and important in
cell proliferation and differentiation.1,2 The Bcl-3
protein has a distinct pattern of ankyrin-related repeats and is
structurally similar to other members of the IB family of proteins,
including IB , IB , and IB .3-5
NF-B/Rel designates a widely distributed family of transcription
factors that modulate the expression of genes involved in immune and
acute-phase responses as well as the response to signals for rapid gene
expression.5 The DNA-binding forms of NF-B transcription
factors are heterodimers or homodimers composed of different
combinations of 5 structurally related DNA-binding proteins, p50, p52,
RelA (p65), RelB, and c-Rel. These proteins share a highly conserved
amino-terminal sequence called the Rel homology region
(RHR).5 Specific heterodimers or homodimers of these
proteins bind to target enhancer elements (B) present in the
promoters of regulated genes. The activity of these proteins is
regulated through cytoplasmic retention by physical interaction with
cytoplasmic inhibitors termed IB.5-7 IB proteins
interact with the RHR of NF-B proteins through their ankyrin
repeats.8 Stimulation of cells with NF-B inducers leads
to rapid phosphorylation and degradation of IBs, allowing NF-B to
translocate to the nucleus to activate targets.5-8
Based on protein structural similarities and interaction with
NF-B/Rel proteins, Bcl-3 is considered to be a member of the IB
family. However, despite structural homology, Bcl-3 and IBs appear
to have different roles in the regulation of NF-B/Rel proteins.
Whereas IB is primarily cytoplasmic, transiently transfected Bcl-3 protein is predominantly located in the nucleus.2,3,9 IB inhibits nuclear translocation of NF-B/Rel, whereas Bcl-3 does not.2,5-8 Bcl-3 has been reported to form complexes
with p5010 or p529-11 homodimers that serve as
transcriptional coactivators of B-specific gene expression. Under
certain conditions, Bcl-3 may also function to antagonize the DNA
binding of nuclear p50 homodimers, which can be B-specific
repressors of active forms of NF-B/Rel.3,5,12 Thus,
these transient transfection studies suggest that Bcl-3 plays a
positive regulatory role in B-specific gene transcription. The in
vivo role of Bcl-3 in different cell types is less clear.
In this study, the involvement of Bcl-3 in erythroid
proliferation and differentiation was investigated using both an
erythroleukemia cell line, TF-1,13 and normal human
progenitor-derived erythroblasts. Proliferation of the TF-1 cells is
completely dependent on hematopoietic growth factors, including
granulocyte-macrophage colony-stimulating factor (GM-CSF),
erythropoietin (Epo), and interleukin-3 (IL-3).13 GM-CSF
and Epo stimulation of TF-1 proliferation resulted in marked induction
of Bcl-3 expression. Furthermore, Bcl-3 was highly expressed in
day-7 burst-forming unit-erythroid (BFU-E)-derived erythroid precursors and decreased with maturation, suggesting that Bcl-3 is
involved in normal erythroid proliferation. In contrast to previous
transfection studies,9-11 endogenous Bcl-3 in TF-1 cells and normal erythroblasts was located in both nucleus and cytoplasm. After growth factor stimulation of TF-1 cells, a gradual but dramatic translocation of Bcl-3 to the nucleus was observed. Transient transfection studies with TF-1 cells showed that overexpression of
Bcl-3 along with p50 or p52 resulted in significant transactivation of
a luciferase reporter gene driven by a human immunodeficiency virus-type 1 (HIV-1) B enhancer, suggesting that Bcl-3 can modulate expression of regulated genes in vivo in TF-1 cells. Induction of
c-myb mRNA was observed in GM-CSF-stimulated TF-1 cells.
Electrophoretic mobility shift assay (EMSA) using the NF-B binding
site of the c-myb promoter suggested that a nuclear complex of
Bcl-3 and p52 bound to the c-myb promoter and this complex was
induced by GM-CSF stimulation of TF-1 cells. Overexpression of Bcl-3
along with p52 or p50 was also able to significantly induce expression
of a luciferase reporter gene driven by a B site present in the c-myb promoter. These data indicate that Bcl-3 may participate in the regulation of B-containing genes involved in hematopoiesis including c-myb.
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MATERIALS AND METHODS |
Culture of BFU-E-derived erythroblasts and TF-1 cells.
Peripheral blood was obtained from normal volunteer donors at The
Milton S. Hershey Medical Center (Hershey, PA) under protocols approved
by the Institution's Clinical Investigation Committee. BFU-E-derived
erythroblasts were cultured as described previously.14 Briefly, peripheral blood mononuclear cells were separated on Ficoll-Paque (Pharmacia, Piscataway, NJ) and cultured in 0.9% methylcellulose media containing 30% fetal calf serum, 9.0 mg/mL deionized bovine serum albumin (Cohn fraction V; Sigma Chemical Co, St
Louis, MO), 1.4 × 10 4 mol/L
-mercaptoethanol, and 2 U/mL Epo (recombinant Epo >100,000 U/mg;
Amgen, Thousand Oaks, CA). Single BFU-E, when cultured in methylcellulose, proliferate and differentiate over 14 days to form
large colonies containing 1 to 5 × 104 mature
erythroblasts. These cells can be removed from culture at different
days to study a well-defined population of normal human cells at
distinct stages of maturation.14 Day-7 cells are poorly
hemoglobinized blasts with a large proliferative capacity, day-10 cells
are partially hemoglobinized proerythroblasts with decreased
proliferative capacity, and day-14 cells are terminally differentiating
polychromatophilic and orthochromatic normoblasts. Cells from maturing
BFU-E-derived colonies were plucked from culture on days 7, 10, and
14. Cytocentrifuge preparations of aliquots of BFU-E-derived cells
routinely identified greater than 99% as erythroid precursors.
TF-1 cells, a human erythroleukemia cell line,13 were
cultured in RPMI 1640 medium containing 10% fetal calf serum and 1 to
2 ng/mL human recombinant GM-CSF (R & D Systems, Minneapolis, MN) or 5 U/mL Epo. To examine Bcl-3 induction, TF-1 cells were removed
from growth factor for 24 hours and then stimulated with 2 ng/mL GM-CSF
or with 5 U/mL recombinant Epo. Samples were collected at intervals
over 0 to 24 hours. Viability of TF-1 cells after growth factor
stimulation was determined by trypan blue exclusion. The percentage in
apoptosis was determined with the ApoAlert Annexin V Apoptosis Kit
(Clontech, Palo Alto, CA) and analysis with a fluorescence-activated
cell sorter. The cell cycle status of TF-1 cells was determined by
propidium iodide staining.15
Cell lysate preparation and nuclear/cytoplasmic fractionation.
Whole cell lysates were prepared by suspending 1 × 106 TF-1 cells or BFU-E-derived erythroblasts in cell
lysate buffer (50 mmol/L Tris HCl, pH 8.0, 150 mmol/L NaCl, 0.05%
NP40, 100 mmol/L NaF, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.08 mmol/L
phenylmethyl sulfonyl fluoride [PMSF], 0.01 mg/mL of
leupeptin, and 0.01 mg/mL aprotinin). The suspension was vortexed and
centrifuged at 10,000 rpm for 10 minutes. The supernatant was saved for
Western blotting. Nuclear and cytoplasmic fractions were prepared as
previously described by Schreiber et al.16 TF-1 cells (1 × 107) or BFU-E-derived erythroblasts
(1.5 × 107) harvested at day 10 were washed twice
with cold phosphate-buffered saline (PBS). The cell pellet was
resuspended in 100 µL of cold buffer (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.4% NP40, 1 mmol/L dithiothreitol
[DTT], 0.5 mmol/L PMSF, and 1% volume protease
inhibitor cocktail) and pipetted several times. The lysates were spun,
and the supernatant was used for the cytosol preparation. The nuclear
pellet was extracted with 50 µL of ice-cold buffer (20 mmol/L HEPES,
pH 7.9, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, and 1 mmol/L PMSF)
and vigorously shaken for 15 minutes at 4°C. After centrifugation,
the nuclear extract was collected and kept at  70°C.
Human tissues frozen at 70°C were mixed 1:5 (wt/vol) with
fresh lysis buffer (10 mmol/L Tris HCl, pH 7.6, 5 mmol/L EDTA, 5 mmol/L
MgCl2, 0.8 mmol/L PMSF, 0.01 mg/mL leupeptin, and 0.01 mg/mL aprotinin), homogenized, and centrifuged at 10,000 rpm for 10 minutes. The supernatant was removed, quantitated, and used in Western
blot analysis.
Immunoblotting.
The whole cell lysate, nuclear, or cytoplasmic preparations were boiled
for 5 minutes in protein sample buffer and separated on 12% or 10%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins were electroblotted onto
Hybond-ECL nitrocellulose membrane (Amersham Life Sciences, Bucks, UK)
according to the recommended procedures of the manufacturer. After
blocking in 5% dry milk in TBST buffer,14 membranes then
were incubated with anti-Bcl-3 antibody (Santa Cruz Biotechnology,
Santa Cruz, CA; diluted 1:200). Donkey antirabbit antibody (1:2,000
dilution) was used as the secondary antibody and membranes were
detected with the ECL-Western blotting system (Amersham). Preincubation of anti-Bcl-3 antibody with Bcl-3 peptide (Santa Cruz Biotechnology) competitively removed recognition by the antibody of the Bcl-3 protein
band on Western blots (data not shown). As another control, an aliquot
of the protein sample was run on a 10% polyacrylamide gel and IB
protein levels were examined with anti-IB antibody17 (1:2,000 dilution) and ECL. In some experiments, anti-E47 antibody (Santa Cruz; diluted 1:250) was used to demonstrate subcellular localization of this basic helix-loop-helix transcription factor.
Northern blot analysis.
Total TF-1 RNA was isolated using TRI REAGENT-RNA/DNA/PROTEIN isolation
reagent (Molecular Research Center, Inc, Cincinnati, OH). RNA samples
(40 µg/lane) were separated on 1.2% agarose-formaldehyde gels and
alkaline transferred onto Zeta-Probe GT Genomic Tested Blotting
membrane (Bio-Rad, Hercules, CA). Membranes were
prehybridized for 5 minutes at 42°C in 50% formamide, 120 mmol/L
Na2HPO4, 250 mmol/L NaCl, and 7% SDS. The
32P-dCTP-labeled Bcl-3 cDNA probe was added and
the hybridization was continued for 18 hours, followed by standard
washing steps. Bcl-3 cDNA clone (kindly provided by Dr Timothy
McKeithan, University of Chicago, Chicago, IL) was
digested with EcoRI and HindIII to obtain the insert,
followed by Geno-Bind DNA purification (Clontech). The cDNA probe was
labeled by the random primer labeling method (Promega, Madison, WI).
Phosphatase treatment.
Whole cell lysates and nuclear/cytoplasmic extracts were prepared in
lysate buffer (50 mmol/L HEPES, 250 mmol/L NaCl, 0.1% NP40, 5 mmol/L
EDTA, 1 mmol/L DTT, 1 mmol/L PMSF, and 1% volume protease inhibitor
cocktail) without any phosphatase inhibitors. Thirty micrograms of
protein from normal human heart and TF-1 cells or 50 µg of protein
from BFU-E-derived erythroblasts was incubated with 26 U of calf
intestine phosphatase (CIP; GIBCO-BRL, Gaithersburg, MD) for 40 minutes
at 37°C and then separated on 10% SDS-PAGE gel for Western
analysis.3
TF-1 transfection and luciferase assay.
The HIV-1 B-TATA-luciferase reporter plasmid was generated by
transferring the insert, containing the HIV-1 B enhancer and TATA
box, from the B-TATA-CAT into the pGL2 plasmid 5 of the luciferase gene (Promega).18,19 The pGL2 basic vector
lacking the TATA box and the B enhancer was used as a negative
control. The cDNAs encoding Bcl-3, p50, and p52 have been described
previously.2,20,21 These cDNAs were cloned into the
expression plasmid pCMV4 as described.19,22 To construct
the c-myb B-luciferase reporter plasmid, 4 copies of a
14-nucleotide c-myb B element and 1 copy of the Herpes
simplex virus thymidine kinase (tk) minimal promoter were cloned in
front of a luciferase gene. In brief, the B oligonucleotide sequence present in the c-myb promoter (Harhaj and Sun, manuscript in
preparation) was cloned into the Kpn I/Mlu
I sites of the pGL2 basic vector (Promega). Subsequently, the tk
promoter was inserted between the B sites and luciferase gene at the
Bgl II/Nhe I sites. TF-1 cells were transfected at a
density of 1 × 106 cells/mL with Tfx-20 reagent
(Promega) at 3:1 ratio Tfx-20 to DNA in the presence of GM-CSF. The
quantity of reporter gene and effector plasmids used is present in the
Results. After 48 hours of culture, the transfectants were collected
and suspended in a lysis buffer (Reporter lysis buffer; Promega). Cell
extracts were normalized for protein recovery (Bio-Rad) and then
subjected to luciferase assay (Promega). Luciferase activity was
quantitated using a single photon channel of a scintillation counter
(Beckman, Fullerton, CA).
Electrophoretic mobility shift assay (EMSA).
EMSA was performed by the method described previously.23 A
double-stranded oligonucleotide covering a B site present in the
promoter of the human c-myb gene (Harhaj and Sun, manuscript in
preparation) was labeled as described by Ganchi et
al.20 Four microliters (6 µg) of nuclear extracts was
incubated with 0.5 to 2 µL of NF-B (anti-p50, anti-p52, anti-p65,
anti-RelB, and anti-c-Rel; from Dr Warner Greene, University of
California, San Francisco, CA and San Francisco General
Hospital) or anti-Bcl-3 antibodies in 12 µL of the
binding buffer (0.5 µL of 1 µg/µL polydI-dC, 1 µL of 0.1 mol/L
DTT, 3 µL of KCl-Dialysis buffer lacking KCl,24 12 µL
H2O) for 10 minutes at room temperature. Following this, 1 µL of 32P-radiolabeled c-myb-B probe (1 × 105 cpm) was added and incubated for another 25 minutes. The DNA-protein complex was resolved on a 5% native
polyacrylamide gel.
| |
RESULTS |
GM-CSF and Epo induce Bcl-3 expression in TF-1 cells.
To explore the potential role of the proto-oncogene Bcl-3 in
hematopoietic growth factor-stimulated proliferation, the expression of
Bcl-3 was investigated by Western blotting analysis in
GM-CSF-stimulated and Epo-stimulated TF-1 cells. The proliferation of
TF-1 cells is dependent on growth factors, including GM-CSF, Epo, and
IL-3, but the molecular mechanisms by which these hematopoietic growth factors induce the growth of TF-1 cells have only been partially identified. TF-1 cells were growth factor deprived for 24 hours. The
viability of TF-1 cells 24 hours after growth factor deprivation was
greater than 97%, and less than 5% of apoptotic cells were detected.
After growth factor deprivation, 2 ng/mL of GM-CSF or 5 U/mL of Epo was
added to the medium to induce cells to proliferate and the cells were
subsequently continuously stimulated by growth factor. At different
time points, stimulated TF-1 cells were collected for analysis of
Bcl-3 expression. Bcl-3 protein was markedly induced after
GM-CSF or Epo stimulation (Fig 1). At 2 hours (GM-CSF) or 4 hours (Epo) of stimulation, the level of Bcl-3
protein was approximately 3 times more than that in nonstimulated
cells. The level of Bcl-3 protein remained elevated for at least 24 hours after stimulation. Both higher and lower molecular weight
isoforms of Bcl-3 were induced. Because Bcl-3 has been proposed to be
an IB-like protein, the expression pattern of IB was also
analyzed in these stimulated TF-1 cells. However, in contrast to Bcl-3,
IB did not show increased expression in response to GM-CSF or Epo
stimulation (Fig 1). These data suggest that hematopoietic growth
factors participate in regulation of Bcl-3 expression in TF-1 cells.
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| Fig 1.
Bcl-3 expression in GM-CSF-stimulated and Epo-stimulated
TF-1 cells. Whole cell lysates were prepared from growth factor-induced TF-1 cells at the time points indicated. Thirty micrograms of protein
was loaded on each lane of a 12% polyacrylamide gel (Bcl-3) or 15 µg
of protein was loaded on each lane of a 10% polyacrylamide gel
(IB). The membranes were blotted with anti-Bcl-3
(1:200) or anti-IB (1:2,000) antibody as a control and then
detected with ECL. N.D. indicates time points that were not done. Three independent experiments showed induction of Bcl-3 expression.
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To examine the mechanism underlying the induction of Bcl-3 by
hematopoietic growth factors, total RNA was extracted from TF-1 cells
after stimulation by GM-CSF or Epo. Northern blotting showed that
Bcl-3 transcripts increased significantly after 30 minutes of
TF-1 stimulation by GM-CSF and continued to be elevated for 24 hours
(Fig 2). This expression pattern is
consistent with results of Western blotting. With Epo, a similar
increase in Bcl-3 mRNA was observed. As a control, 18S rRNA
showed no significant change (Fig 2). Taken together, these data
suggest that growth factor stimulation of TF-1 proliferation results in
enhanced Bcl-3 expression and that induced Bcl-3
expression is mediated at the level of transcription.
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| Fig 2.
GM-CSF and Epo induce Bcl-3 mRNA expression in
TF-1 cells. Total RNA was isolated from GM-CSF-induced or Epo-induced
TF-1 cells and Northern blotting analysis was performed using 40 µg RNA from each sample. 32P-dCTP-labeled Bcl-3 cDNA
was used as a probe. 18s rRNA is shown as a control for equivalent
loading.
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After growth factor deprivation of TF-1 cells for 24 hours, in 3 experiments, 55% to 60% of cells were determined to be in G0/G1 of
the cell cycle, and the rest of the cells were in S or G2/M phase. The
percentage of cells in S phase increased significantly to a mean peak
of 52% ± 2% at 4 to 10 hours after growth factor stimulation
(P  .02). The percentage of cells in G2 and M phase also
increased significantly, and the increase peaked at 16 to 20 hours
after growth factor stimulation (P < .002). The doubling time
of TF-1 cells was 24 hours. No significant differences were noted in
the cell cycle status of cells after GM-CSF compared with Epo
stimulation. It is noteworthy that the increase in Bcl-3 protein and
mRNA preceded the entrance of the majority of cells into S phase, but
in TF-1 cells, Bcl-3 expression did not correlate with a specific phase
of the cell cycle.
Nuclear expression of Bcl-3 is enhanced by hematopoietic growth
factors.
Previously, transiently transfected Bcl-3 protein has been
predominantly localized in the nucleus.2,3,9 The N-terminal part of Bcl-3 resembles a nuclear localization signal.2
Although this sequence is not perfectly conserved compared with other
nuclear localization signals, deletion of this sequence abolished the nuclear localization of Bcl-3.2 The subcellular
localization of endogenous Bcl-3 in a physiological setting, such as in
hematopoietic growth factor-stimulated proliferation, remains unclear.
To determine the subcellular localization of Bcl-3 in TF-1 cells,
nuclear and cytoplasmic extracts were isolated from GM-CSF-induced or
Epo-induced cells and Western blotting analysis was performed. Bcl-3
protein was weakly detectable in both cytoplasm and nucleus before
growth factor stimulation (Fig 3). The
effect of growth factor stimulation on the subcellular localization of
Bcl-3 was determined. Growth factor stimulation greatly enhanced the
level of cytoplasmic Bcl-3 (Fig 3). However, in addition, the nuclear
level of Bcl-3 also was greatly increased, suggesting that growth
factors enhance Bcl-3 nuclear translocation. To control for the quality
of subcellular fractionation, nuclear and cytoplasmic extracts were
examined for IB and E47 localization. IB, a cytoplasmic
protein, was not detected in the nuclear fraction (Fig 3). E47, a basic
helix-loop-helix transcription factor,25 was primarily
detected in the nucleus (Fig 3).
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| Fig 3.
GM-CSF and Epo enhance Bcl-3 nuclear translocation.
Growth factor-induced TF-1 cells (1 × 107) were used for
nuclear or cytoplasm separation. Twenty-five micrograms of nuclear or
20 µg of cytosolic protein extracts was loaded on each lane of a 12%
polyacrylamide gel and subjected to Western blotting with anti-Bcl-3
antibody. As a control, 15 µg of nuclear or cytoplasmic extract was
loaded on a 10% polyacrylamide gel to detect IB. For
GM-CSF-stimulated cells, 30 µg of nuclear or cytoplasmic extract was
loaded on each lane of 12% gel and detection was with anti-E47
antibody as another control for quality of subcellular fractionation.
Representative results are shown from 3 independent experiments.
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Bcl-3 expression in normal human erythroblasts is correlated with
proliferation.
The expression of Bcl-3 protein was determined in normal erythroid
proliferation and differentiation. Human BFU-E-derived erythroid
precursors were removed from culture on days 7, 10, and 14 of
maturation.14 Day-7 cells have a large proliferative capacity, day-10 cells are only partially hemoglobinized with decreased
proliferative capacity, and day-14 cells are largely terminally
differentiating normoblasts. Western blotting assay was performed using
whole cell lysates from day-7, -10, and -14 cells
(Fig 4A). In day-7 cells, which are rapidly
proliferating, Bcl-3 had the highest expression level and decreased as
erythroid precursors terminally differentiated. This dynamic pattern
suggests that Bcl-3 expression is associated with normal erythroid
proliferation rather than differentiation. Although Bcl-3 was minimally
detectable in day-14 cells, IB showed little decline during
differentiation and substantial quantities were still present at day
14. In day-10 BFU-E-derived cells, subcellular localization studies
with nuclear and cytoplasmic extracts also showed that Bcl-3 was
present in both nucleus and cytoplasm, whereas IB was primarily
cytoplasmic (Fig 4B).
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| Fig 4.
Bcl-3 expression in day-7, -10, and -14 BFU-E-derived
erythroblasts. (A) Normal human BFU-E-derived erythroblasts were
harvested and the whole cell lysates from 4 × 105 (Bcl-3)
or 2 × 105 (IB) cells were separated on a 10%
polyacrylamide gel. Western analysis was performed with anti-Bcl-3 or
anti-IB antibodies and ECL. (B) Nuclear and cytoplasmic extracts
were separated from day-10 cells. Fifty micrograms of nuclear (N) or
cytoplasmic (C) extract was loaded onto each lane of a 10% gel and
subjected to Western blotting. Two experiments were performed with
anti-Bcl-3 or anti-IB antibodies with identical results.
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Bcl-3 is hyperphosphorylated in TF-1 and BFU-E-derived
erythroblasts.
Bcl-3 mRNA has previously been shown to be expressed in several
mammalian tissues.3 Expression of Bcl-3 protein was
examined here in 7 human tissues. Bcl-3 was highly expressed in heart, skeletal muscle, and erythroid precursors; weakly detectable in liver,
kidney, and spleen; and barely detectable in the brain (Fig 5). Parallel Northern blotting
(Clontech) showed that the pattern of Bcl-3 protein correlated with
mRNA expression (data not shown) and a single mRNA band was observed.
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| Fig 5.
Bcl-3 is expressed in many human tissues. Protein
extracts from normal human tissues were prepared as described in the
Materials and Methods. Thirty micrograms of protein was loaded on each
lane of a 10% polyacrylamide gel and detection was with anti-Bcl-3 antibody.
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The molecular weight of Bcl-3 deduced from the protein sequence is 47 kD.1 Bcl-3 protein detected from normal human tissues such
as heart, skeletal muscle, spleen, and liver showed a slightly higher
molecular weight (Fig 5). Western blotting analysis showed multiple
isoforms of Bcl-3 in GM-CSF- or Epo-stimulated TF-1 cells, and the
same result was obtained with BFU-E-derived erythroblasts (Figs 1, 3,
and 4). In BFU-E-derived erythroblasts (Fig 5) or TF-1 cells
(Fig 6), Bcl-3 was larger than that in
other human tissues. It is known that Bcl-3 is a proline- and
serine-rich protein with high phosphorylation potential.3,4
To test if the observed heterogeneity in size was due to protein
phosphorylation, TF-1 and BFU-E-derived erythroblast cells and normal
heart were treated with CIP. CIP treatment resulted in molecular weight
reduction of Bcl-3 in both TF-1 (Fig 6, top) and BFU-E-derived (Fig 6,
bottom) cells. However, CIP had no effect on the molecular weight of
Bcl-3 from human heart tissue. In Fig 6, the highest molecular weight band was thought to represent a nonspecific protein, because it was
inconsistently observed. These data demonstrate that the different size
of Bcl-3 protein in tissue compared with TF-1 or BFU-E-derived cells
is at least partially due to differential Bcl-3 phosphorylation. GM-CSF
and Epo stimulated the appearance of higher and lower molecular weight
Bcl-3 isoforms (Fig 1) and both are found in TF-1 and BFU-E nucleus and
cytoplasm (Figs 4 and 6). The functional significance of Bcl-3
hyperphosphorylation in these hematopoietic-derived cells remains to be
determined, but in other systems Bcl-3 dephosphorylation resulted in
decreased activity.3,4
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| Fig 6.
Bcl-3 is hyperphosphorylated in TF-1 cells and
BFU-E-derived erythroblasts. (Top) Whole cell lysates (W) and nuclear
(N) and cytoplasmic (C) extracts were prepared from Epo-induced or
GM-CSF-induced TF-1 cells. Thirty micrograms of each extract was
incubated with or without 26 U of CIP at 37°C for 40 minutes and
then subjected to Western blotting assay with anti-Bcl-3 antibody and
ECL. (Bottom) Thirty micrograms of whole cell lysate (W) from normal
human heart tissue or 50 µg from day-10 BFU-E-derived erythroblasts
was also incubated with or without 26 U CIP and subjected to Western
blotting with anti-Bcl-3 as described in the Materials and Methods.
Three experiments were performed with similar results. (+) with CIP; () without CIP.
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Overexpression of Bcl-3 in TF-1 cells activates an HIV-1 B
enhancer.
To determine whether Bcl-3 has a role in gene activation in TF-1 cells,
functional reporter gene assays were performed with an HIV-1
B-TATA-luciferase reporter plasmid.19,22 This plasmid was cotransfected into proliferating TF-1 cells (cultured in the presence of GM-CSF) along with cDNA for Bcl-3, p50, or p52. In these
experiments, indicated amounts of plasmids expressing p50, 52, or Bcl-3
were transfected either separately or in combination along with 0.5 µg of HIV-1 B-TATA-luciferase reporter plasmid into TF-1 cells.
The total amount of transfected DNA (2.0 µg) was kept constant by
adding appropriate amounts of expression vector without insert. The
pGL2 vector was transfected as the negative control. As shown in
Fig 7, a high level of B-TATA-luc was
expressed when this reporter plasmid was transfected alone. This result
is consistent with our finding that endogenous Bcl-3 and other NF-B
factors are induced in TF-1 cells by the growth factor
GM-CSF.26 Significant further induction of luciferase was
observed in TF-1 cells cotransfected with the B-TATA reporter plasmid and Bcl-3 together with p50 or p52 (P < .05). These
results demonstrate that both endogenously expressed and transfected
Bcl-3 and NF-B factors p50 or p52 are capable of positively
stimulating gene expression from the HIV-1 B enhancer in TF-1 cells.
View larger version (16K):
[in this window]
[in a new window]
| Fig 7.
Activation of an HIV-1 B-TATA-luciferase reporter
plasmid after overexpression of Bcl-3 in TF-1 cells. A total of 0.5 µg of plasmids expressing Bcl-3, p50, or p52 was cotransfected with 0.5 µg of the HIV-1 B-TATA-luciferase reporter plasmid into TF-1 cells separately or in combination. The pGL2 basic vector (0.5 µg)
was used as negative control. Where noted, 1.0 µg of Bcl-3 was
cotransfected. The total amount of transfected DNA was kept constant by
adding appropriate amounts of expression vector without insert. At 48 hours after transfection, cells were collected for luciferase assay.
Results are expressed as the mean ± SEM (×103 cpm).
Five experiments were performed. *A significant increase above the
B-TATA reporter plasmid (P .05).
|
|
In control experiments (not shown), 1.5 µg of plasmid expressing
NF-B factors p50 or p52 was cotransfected with 0.5 µg of HIV-1
B-TATA-luciferase reporter plasmid in TF-1 cells. Luciferase activity was not significantly different from cotransfection with the
reporter plasmid alone.
Bcl-3 binds to and transactivates a B site in the
c-myb promoter.
Previous studies have shown that transiently transfected Bcl-3 or
purified Bcl-3 proteins interact with both p50 and p52 subunits of NF-B/Rel proteins.2,3,4,9,10 Depending on
experimental conditions, different functional results have been
reported. Bcl-3 protein has been observed to either inhibit the DNA
binding activity of NF-B p50 and p52 in vitro3 or
facilitate p52 or p50 activity by forming a complex with these NF-B
proteins on DNA.4,9,10 To examine the role of Bcl-3 in B
binding under physiological conditions, the interaction of endogeneous
Bcl-3 with NF-B transcription factors in growth factor-stimulated
TF-1 cells was examined. EMSA was performed using an NF-B binding
site that recently was identified from the promoter region of the human
c-myb gene and nuclear extracts from TF-1 cells stimulated with
GM-CSF for 24 hours. This site was chosen because induction of
c-myb mRNA in response to GM-CSF stimulation was observed in
these TF-1 (data not shown) and c-myb has previously been shown
to have an important role in erythropoiesis.27-30 Three
major protein/DNA complexes were detected
(Fig 8, C1, C2, and C3). Antibody
supershift assays showed that all 3 complexes, C1, C2, and C3,
immunoreacted with the anti-p52 antibody. The anti-p50 antibody
supershifted both C2 and C3 complexes, whereas anti-p65 (RelA)
supershifted the C2 complex. None of these complexes was immunoreactive
with antibodies for RelB or c-Rel. These results suggest that p50 and
p52 bind to the NF-B site as either homodimers or heterodimers with
each other or with p65. Formation of the C1 complex was reproducibly
inhibited by anti-Bcl-3 antibody, suggesting that Bcl-3 is a part of
this complex, which is also composed of p52. In nonstimulated TF-1
cells, the C1 complex containing Bcl-3 and p52 was much weaker than
that detected in induced cells (Fig 8B). These results are
consistent with the hypothesis that induction of Bcl-3 expression by
GM-CSF contributes to enhancement of Bcl-3 DNA binding and increased
formation of C1.
View larger version (50K):
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| Fig 8.
Bcl-3 is associated with NF-B p52 in TF-1 cells
(EMSA). (A) Six micrograms of nuclear extracts prepared from TF-1 cells
stimulated with GM-CSF for 24 hours was incubated with different
NF-B antibodies for 10 minutes before adding a
32P-labeled c-myb B binding oligonuclear probe.
Three DNA-protein complexes were generated. Complexes 2 and 3 (C2 and
C3) were supershifted by anti-p50; C1, C2, and C3 were shifted by
anti-p52. C1 was reproducibly inhibited by anti-Bcl-3 antibody
(with a long exposure, a supershifted band was also visible). However,
anti-RelB and anti-c-Rel had no effect on any of these complexes.
Similar results were observed in 3 experiments. (B) EMSA was performed
with growth factor-deprived TF-1 cells or TF-1 cells induced with
GM-CSF for 24 hours. The C1 complex was greatly increased by GM-CSF
stimulation, whereas other complexes had no significant change. This
experiment was repeated 3 times with similar results.
|
|
To further examine whether Bcl-3 has a role in regulating c-myb
expression, the c-myb B-TATA-luciferase plasmid was
cotransfected into proliferating TF-1 cells (cultured with GM-CSF)
along with cDNA for Bcl-3, p50, or p52. Methods are as described for
the HIV-1 B-TATA-luciferase reporter plasmid. As shown in
Fig 9, significant levels of c-myb
B-TATA-luciferase were expressed when this reporter plasmid was
transfected alone. This is consistent with our finding that Bcl-3 and
other NF-B factors are induced by GM-CSF26 and that
significant amounts of c-myb mRNA are present in these cells
(data not shown). A significant induction of luciferase was observed
when the TF-1 cells were transfected with the c-myb reporter
plasmid along with Bcl-3 together with p52 or 50 (P .05).
These results demonstrate that endogenously induced and transfected
Bcl-3/NF-B factors are capable of inducing gene expression from the
c-myb NF-B site in vivo in TF-1 cells.
View larger version (15K):
[in this window]
[in a new window]
| Fig 9.
Overexpression of Bcl-3 and p50 or p52 activates a
c-myb B-TATA-luciferase reporter plasmid. A total of 0.5 µg of plasmids expressing Bcl-3, p50, or p52 was cotransfected with
1.0 µg of c-myb B-TATA-luciferase reporter plasmid into
TF-1 cells separately or in combination. The pGL2 basic vector was used
as negative control. The total amount of transfected DNA (2 µg) was
kept constant by adding appropriate amounts of expression vector
without insert. At 48 hours after transfection, cells were collected
for luciferase assay. Results are expressed as the mean ± SEM
(×103 cpm). Three experiments were performed. *A
significant increase above the c-myb B-TATA reporter plasmid
(P < .05).
|
|
| |
DISCUSSION |
GM-CSF and Epo induce erythroid proliferation by triggering a cascade
of signal transduction events. Upon binding to their specific
receptors, these growth factors induce activation of second messengers,
including the protein tyrosine kinase JAK2, Stat5, ras, Raf-1, and MAP
kinase.31-36 Induction of these signaling proteins results
in activation of specific transcription factors, including GATA-1, SCL
and other basic helix-loop-helix (bHLH) transcription factors, NF-E2,
and RBTN2, which in turn control erythroid proliferation and
differentiation.25,37-42 In this study, involvement of the
proto-oncogene Bcl-3 in the signaling mechanisms of GM-CSF and
Epo is demonstrated. The expression of Bcl-3 is greatly
enhanced by hematopoietic growth factor stimulation at the level of
both transcription and translation. A dynamic expression pattern of
Bcl-3 in normal erythroid cells is demonstrated, suggesting that Bcl-3 is also involved in normal erythropoiesis.
The induction of Bcl-3 by both GM-CSF and Epo involves enhanced
expression of Bcl-3 mRNA, suggesting that these growth factors induce transcription of the Bcl-3 gene. A number of potential regulatory sequences have been found in the 5-flanking region of
the Bcl-3 gene.1 These sequences include binding
sites for Sp1, AP-1, AP-2, and NF-B, indicating possible involvement
of these transcriptional factors in regulation of Bcl-3 gene
expression.1,3 In addition to these elements, we observed
two other potential regulatory sequences, CAGCTG and
CAACTG.1 These two DNA sequences are highly similar to the
E-protein binding site CANNTG, termed E-box.43
E-proteins belonging to the bHLH family of transcription factors form heterodimers with tissue-specific transcription factors such as SCL, which then bind to E-box motifs to participate in the
transcriptional regulation of genes involved in cell
growth.39,43,44 We and others have previously shown that
SCL, E47, and HEB are involved in BFU-E-derived human erythroid
proliferation and differentiation.39,40,44 GM-CSF
stimulation of TF-1 cells induces E47 expresssion (Fig 3), and this was
also observed for HEB and E2-2 (data not shown). Bcl-3
transcription is likely controlled through recognition of binding sites
in the promoter region of Bcl-3 by growth factor-regulated transcription factors involved in erythroid proliferation and differentiation, which may include SCL heterodimers.
The studies reported here demonstrate that endogenous Bcl-3 is located
in both the nuclear and cytoplasmic compartments. Similar results have
been obtained by immunofluorescent staining of TF-1 cells (not shown).
These results are not in full agreement with previous reports in which
the transiently transfected Bcl-3 is primarily expressed in the
nucleus.2,3,9 Such discrepancy suggests that the nuclear
expression of Bcl-3 may be regulated under physiological conditions.
The cytoplasmic retention of endogenous Bcl-3 in TF-1 and
BFU-E-derived cells may be due to physical association with other
proteins or to posttranslational modifications. Bcl-3 has been shown to
form nuclear complexes with NF-B p50 and p52,9,10,11 but
it is not known what factors form cytoplasmic complexes with Bcl-3. Our
studies also demonstrate that Bcl-3 is hyperphosphorylated in both TF-1
cells and normal erythroblasts. High and low molecular weight isoforms
are present in nucleus and cytoplasm; therefore, the phosphorylation
state does not appear to determine subcellular localization. Higher and
lower molecular weight isoforms are also induced by growth factor
stimulation. The function of different phosphorylation states of Bcl-3
is not clear at this time. Phosphorylation of Bcl-3 has previously been
shown to be important for full activity in other cell
types.3,4 For example, dephosphorylation of Bcl-3 in
thymocytes greatly decreased the ability of Bcl-3 to augment the DNA
binding activity of endogenous p50 homodimers.4
GM-CSF and Epo not only induce the expression of Bcl-3 but also
enhance nuclear translocation upon stimulation of TF-1 cells with these
hematopoietic growth factors. Interaction of Bcl-3 with NF-B/Rel
transcription factors has been demonstrated in a number of previous
studies, although the functional consequences of these interactions
remains controversial.3,5,9-12 Although some studies
demonstrated that Bcl-3 inhibits the DNA binding activity of NF-B
proteins, other studies showed that Bcl-3 binds to a B enhancer
together with p52 or p50 and serves as a transcriptional activator.
Consistent with the latter finding, we demonstrated here that
overexpression of Bcl-3 along with p50 or p52 in TF-1 cells results in
induction of both HIV-1 and c-myb B-TATA-luciferase reporter
genes and that Bcl-3 has a function in gene activation in vivo in TF-1
cells. These results provide evidence that Bcl-3 may play a positive
role in transactivation of B-containing genes in erythroid cells. In
TF-1 cells, GM-CSF stimulates increased c-myb mRNA expression.
We also present data that suggest that Bcl-3 forms a complex with
NF-B p52 on a B element present in the promoter of the
c-myb gene and that formation of this complex is enhanced by
stimulation with GM-CSF. Bcl-3 interaction with nuclear NF-B
proteins, including p52, may contribute to induction of expression of
specific genes involved in erythroid proliferation, including
c-myb. The importance of c-myb in erythropoiesis has previously been demonstrated.27-30 The reported increase in
Bcl-3 in response to mitogenic signaling, associated with
induction of immediate genes c-fos and c-myc, is
consistent with our observations.1
| |
FOOTNOTES |
Submitted September 29, 1997;
accepted April 17, 1998.
Supported by National Institutes of Health Grants No. DK46778 (B.A.M.),
CA 68471 (S.-C.S.), and MO1 RR10732 (GCRC grant) and by a grant from
The Pennsylvania State University Cancer Center. S.-C.S. is a scholar
of the American Society of Hematology. B.A.M. is the recipient of an
American Cancer Society Faculty Award.
Address reprint requests to Barbara A. Miller, MD, Department of
Pediatrics, The Milton S. Hershey Medical Center, PO Box 850, Hershey,
PA 17033-0850; e-mail: bamll{at}psu.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Timothy W. McKeithan for providing the
Bcl-3 cDNA and Dr Warner C. Greene for the antipeptide specific antisera for NF-B/Rel proteins. We are grateful to Dr Toshio Kitamura for providing TF-1 cells. The authors thank Maxine Gerberich for careful preparation of the manuscript. We appreciate the technical assistance of Carol Stine.
| |
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Abstract
Introduction
Abstract