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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3369-3378
Regulated Expression and Functional Role of the Transcription Factor
CHOP (GADD153) in Erythroid Growth and Differentiation
By
Margaret Coutts,
Kunyuan Cui,
Kerry L. Davis,
Joan Cleves Keutzer, and
Arthur J. Sytkowski
From the Laboratory for Cell and Molecular Biology, Division of
Hematology and Oncology, Department of Medicine, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, MA.
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ABSTRACT |
The hematopoietic growth factor erythropoietin (Epo) triggers
changes in the expression of genes that encode important regulators of
erythroid cell growth and differentiation. We now report that Epo
markedly upregulates chop (gadd153) expression and that
this transcription factor plays a role in erythropoiesis. Using a
differential hybridization assay, we isolated a full-length cDNA of
chop as an Epo upregulated gene in Rauscher murine
erythroleukemia cells. RNase protection assays demonstrated that Epo or
dimethyl sulfoxide induction increased steady-state mRNA levels 10- to
20-fold after 24 to 48 hours. Western blot analysis confirmed a marked
increase in CHOP protein. Among the other c/ebp family members,
only c/ebp  was also upregulated during erythroid
differentiation. Among normal hematopoietic cells examined,
steady-state mRNA levels were highest in erythroid cells, with levels
peaking during terminal differentiation. Transient overexpression of
chop in Rauscher cells resulted in a significant increase in
Epo- or dimethyl sulfoxide (DMSO)-induced hemoglobinization, further
linking chop upregulation to erythroid differentiation.
Artificial downregulation of chop in normal murine bone marrow
cells with antisense oligodeoxynucleotides inhibited colony-forming
unit-erythroid (CFU-E)-derived colony growth in a
concentration-dependent manner. Burst-forming unit-erythroid (BFU-E)-derived colony growth was not affected. Using a Far Western type of analysis, we detected several potential CHOP binding partners among the nuclear proteins of Rauscher cells. Importantly, the number
and relative abundance of these proteins changed with differentiation. The results strongly suggest that CHOP plays a role in erythropoiesis, possibly through interactions with both C/EBP and non-C/EBP family members.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE INTRACELLULAR signaling network
triggered by the hematopoietic growth factor erythropoietin (Epo)
modulates the expression of genes encoding important regulatory
proteins. For example, the association of Epo with its receptor
triggers activation of phospholipase-C- and protein kinase
C .1 This signaling pathway is required for Epo's
upregulation of the transcription factor c-myc2-4
and is associated with Epo's mitogenic effect. Docking of Epo with its
receptor also results in the activation of the JAK/STAT pathway. JAK2
kinase associates with the receptor's cytoplasmic domains and
phosphorylates STAT5.5 The activated STAT5 dimerizes and
translocates to the cell's nucleus. Erythroid differentiation also
requires the downregulation of c-myb.6,7 In some
experimental systems, artificial downregulation of Myb protein in the
absence of erythropoietin is sufficient to induce hemoglobinization.8 Other regulatory proteins important to erythroid differentiation include GATA-19,10 and
NF-E2.11 These transcription factors regulate globin gene
expression in a coordinate fashion; however, it is not known which
signal transducers activate them. One approach to understanding how
Epo-induced signaling leads to the establishment of erythroid-specific
patterns of gene expression is to compare the species of mRNAs present
before and after Epo treatment.
In the present study, we used a differential hybridization technique to
identify Epo-regulated genes. We report that, in Rauscher murine
erythroleukemia cells, both Epo and dimethyl sulfoxide (DMSO) markedly
upregulate chop (gadd153), a member of the c/ebp family
of transcription factor genes.12,13 We further
show that an increase in chop transcript levels is associated
with terminal differentiation of normal erythroid cells in vivo and that antisense chop oligodeoxynucleotide treatment of bone
marrow cells inhibits colony-forming unit-erythroid (CFU-E) but not
burst-forming unit-erythroid (BFU-E) colony growth. In addition to the
possibility of CHOP-C/EBP interactions, our data also suggest that
there may be other, non-C/EBP proteins in erythroid cells with which
CHOP interacts. Moreover, these potential alternative binding partners change during erythroid differentiation.
CHOP was first isolated as growth arrest and
DNA-damage inducible gene 153 (gadd153).12,14 CHOP was also isolated on the basis
of dimerization with C/EBP.13 CHOP and the
other gadd genes are induced by a wide variety of treatments
that cause metabolic stress or cessation of mitosis. Some of these
inducers are DNA alkylation, nutritional deprivation, oxidative stress,
and treatments that perturb endoplasmic reticulum
function.15-18 These treatments also tend to induce
cessation of mitosis. Cessation of mitosis also occurs when cells
undergo terminal differentiation: in fact, direct expression of CHOP
can lead to cell cycle arrest.19 CHOP mRNA increases
dramatically when dividing 3T3-L1 fibroblasts are induced to
differentiate into amitotic, lipid-rich adipocytic cells13,15,16 and during keratinocyte
differentiation.20 However, because CHOP is ubiquitously
expressed (see Results), it is likely that it plays a role in the
differentiation of many tissues.
Other members of the C/EBP family have been studied more extensively in
terms of the role they play in differentiation. In adipocytic
differentiation, the  , , and  C/EBP isoforms are expressed
with complex kinetics.21 These C/EBP protein isoforms may
interact in specific hierarchical patterns that change as differentiation proceeds.22 Differentiating monomyelocytic
cells have their own distinct pattern of C/EBP isoform
expression.23 Similarly, C/EBP levels fluctuate during
differentiation of hepatoma cells,24 ovarian
follicles,25 and gut epithelium,26 as do levels
of C/EBP levels during B-cell27 and eosinophilic differentiation.28 Expression of the recently described
C/EBP may be particularly important in the development of
granulocytic cells.29 CHOP is an unusual member of this
family, in that it does not appear to form homodimers and it has a
noncanonical DNA-contact domain.30 Hence, its function and
mechanism may be substantially different than other C/EBPs.
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MATERIALS AND METHODS |
Cell culture.
Rauscher murine erythroleukemia cells31,32 were cultured in
Dulbecco's modified Eagle medium (DMEM), 10% fetal bovine serum (FBS;
heat-inactivated), 36°C, 95% air/5% CO2. Cells were
induced with recombinant human Epo (50 to 100 U/mL) or with DMSO (0.7% to 1.0%, vol/vol).
Construction and screening of the cDNA library.
A Rauscher cell cDNA library was prepared by Clontech (Palo Alto, CA)
using the  DR2 vector. RNA was isolated from cells treated with Epo
(50 U/mL) and cycloheximide (10 µg/mL) for 1 hour using the acid
guanidinium thiocyanate method of Chomczynski and Sacchi,33
except that lithium precipitation was not performed. The cDNA library
was screened using differential plaque hybridization. Approximately
40,000 clones were plated in 10-cm dishes at 8 pfu/cm2.
Nitrocellulose filters (Millipore, Bedford, MA) were
lifted off each plate in duplicate. One set of nitrocellulose lifts was hybridized to 32P-labeled cDNA prepared from untreated
Rauscher cells. The duplicate set was hybridized to
32P-labeled cDNA prepared from cells treated with Epo (50 U/mL) and cycloheximide (10 µg/mL) for 1 hour. The cDNA was prepared using Superscript reverse transcriptase (GIBCO-BRL, Gaithersburg, MD) according to the manufacturer's instructions.
Hybridizations were performed in 50% formamide, 6× SSC, 5×
Denhardt's, 0.5% sodium dodecyl sulfate (SDS), 100 µg/mL salmon
sperm ssDNA at 42°C. Filters were washed three times in 1×
SSC, 0.5% SDS at 60°C for 1 hour. DNA sequencing was performed
using a USB DNA sequencing kit (USB, Cleveland, OH).
RNA analysis.
RNase protection assays were performed essentially as directed by the
Ambion RNase Protection Assay kit (Ambion, Inc, Austin, TX) except that
20 U RNase T1 and 0.5 U RNase A were used in a sample volume of 100 µL. The antisense probes protected the chop cDNA sequence
from nucleotide 470 to 782; the glyceraldehyde 3-phosphate dehydrogenase (gapdh) probe protected nucleotide 1120 to 1228. Northern blots were performed on poly A+ RNA isolated from
1 mg of total RNA using standard methods. Electrophoresis was through
formaldehyde-agarose gels. Washes were 20 minutes each of 2× SSC,
1× SSC, and 0.1× SSC, all with 0.1% SDS at 60°C.
Expression of recombinant CHOP.
For expression of CHOP protein in Escherichia coli,
BamHI and EcoRI restriction sites were introduced into
the chop cDNA 5' and 3' ends, respectively, by
polymerase chain reaction (PCR) using the following two primers,
5'-TTAAGGGATCCCAGCTGAGTCCCTG-3' and
5'-TTCGGAATTCCTATGTGCAAGCCGA-3'. The PCR product was
cloned into the BamHI and EcoRI sites of the pTrc-His
expression vector (InVitrogen, Carlsbad, CA) in
the reading frame, resulting in a histidine tagged CHOP protein
when expressed in E coli. The histidine-tagged
protein was purified using Ni-NTA resin (Qiagen, Valencia,
CA). Briefly, cells from 3 L of culture were collected by
centrifugation and resuspended in sonication buffer (50 mmol/L sodium phosphate, 300 mmol/L NaCl, pH 8.0). The cells were
disrupted by sonication, and the sonicated mixture was centrifuged at
10,000g for 20 minutes. The supernatant was collected, and 6 mL
of 50% slurry of Ni-NTA resin, previously equilibrated in sonication buffer, was added and stirred at 4°C for 60 minutes. The resin was
poured into a 1.5-cm diameter column and washed with sonication buffer
followed by 50 mmol/L sodium phosphate, 300 mmol/L NaCl, pH 6.0, until
the A280 of the effluent was less than 0.01. The proteins
were eluted with a pH gradient from 6 to 3.5. The fractions containing
CHOP protein were collected, and the pH was adjusted to 7.4 with
phosphate buffer.
For expression of the CHOP-glutathione-S-transferase (GST) fusion
protein, the chop PCR product was cloned into the pGEX-3x vector (Amersham Pharmacia Biotech, Inc, Piscataway, NJ).
CHOP-GST protein was purified following the
manufacturer's procedure (Pharmacia). Briefly, cells were centrifuged,
resuspended in phosphate-buffered saline (PBS) buffer (140 mmol/L NaCl,
2.7 mmol/L KCl, 10 mmol/L Na2HPO4, 1.8 mmol/L
KH2PO4, pH 7.3), disrupted by sonication, and
centrifuged. Glutathione Sepharose 4B equilibrated with PBS buffer was
added to the supernatant of the sonicated mixture, and the mixture was
incubated with gentle agitation at room temperature for 1 hour. The
mixture was then packed into a small column and washed with PBS buffer
until the A280 of flow-through was less than 0.01. The
CHOP-GST fusion protein was eluted with 10 mmol/L reduced glutathione
in 50 mmol/L Tris-HCl, pH 8.0.
Western blot analysis.
Cells were harvested by centrifugation, washed once with ice-cold
Dulbecco's PBS, and lysed directly in SDS sample buffer. A 10:1 ratio
of sample buffer to cell pellet was used. After boiling the samples for
5 minutes, they were passed repeatedly through a 23-gauge needle and
syringe to fragment the DNA. Cell lysate protein (100 µg/lane) was
electrophoresed on a 13% SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) gel and was transferred electrophoretically (100 mA for 2 hours at 4°C) onto polyvinylidene fluoride (PVDF) filters (Millipore). The filters were allowed to dry after transfer and
then were rewet with methanol. The blots were blocked overnight with
5% nonfat dry milk in PBS, 0.1% Tween 20.
Rabbit polyclonal antiserum against His-CHOP was prepared by Organon
Teknika (Durham, NC) using purified CHOP protein as
antigen. The polyclonal antibody was affinity purified using His-CHOP
as the ligand immobilized on Affi-Gel 15 (Bio-Rad, Hercules,
CA) according to the manufacturer's instructions. For
antibody purification, 150 mL of anti-CHOP rabbit serum diluted in an
equal volume of buffer A (10 mmol/L Tris-HCl, 0.1 mol/L NaCl, pH 7.5)
was applied to the affinity column by circulation overnight. The column
was washed sequentially with buffer A and buffer B (10 mmol/L Tris-HCl, 4 mol/L NaCl, pH 7.5) sequentially and the eluates were collected. The
antibodies were eluted finally with 4 mol/L MgCl2 followed by 4 mol/L guanidine HCl in buffer A. The elution was monitored by
A280. The collected fractions were dialyzed, concentrated
by ultrafiltration, and stored at  20°C.
Antisense oligodeoxynucleotide experiments.
To demonstrate a role for CHOP in normal erythropoiesis,
loss-of-function experiments were performed. Mouse bone marrow cells were treated with chop antisense (GGACTCAGCGCCATGAC) or
missense (CAGTACCGTCGACTCAGG) oligodeoxynucleotides (oligos; Oligos
Etc, Wilsonville, OR). BFU-E- and CFU-E-derived colony growth was
studied as follows. Bone marrow cells were flushed from the femurs of 7-week-old female C57BL6/J mice (Jackson Laboratories, Bar Harbor, ME)
with Alpha medium (GIBCO-BRL) using a 23-gauge needle and syringe. To
obtain a single-cell suspension, the cells were twice drawn through the
same needle. The cells were washed twice in fresh Alpha medium,
counted, and resuspended in Alpha medium/3% FBS (Hyclone Laboratories,
Logan, UT) at 1 × 106 cells/mL. The cells were
incubated in the absence or presence of specified concentrations of
chop antisense or missense oligos before plating. Further
additions of oligos were made according to the schedules described
below. All experiments were performed in duplicate.
For CFU-E growth, on day 0 the oligos were added to 0.6 mL of the cell
suspension and 2.7 mL MethoCult M3330 medium (Stem Cell Technologies,
Inc, Vancouver, British Columbia, Canada). After 4 hours of incubation
at 37°C in a humidified 5% CO2 incubator, 1.1 mL of
the mixture was plated into each of two 35-mm dishes. On day 1, the
cultures received an addition of oligos equal to 25% of the initial
concentration. On day 2, the cultures were scored. Each 35-mm dish was
placed within a gridded 60-mm dish, and the number of CFU-E-derived
colonies in 2 quadrants of the dish was determined using an inverted
microscope. The counts from duplicate dishes were averaged.
For BFU-E growth, on day 0 the oligos were added to 0.3 mL of the cell
suspension and 3.0 mL MethoCult M3434 medium (Stem Cell Technologies)
and the cells were incubated overnight at 37°C in a humidified 5%
CO2 incubator. Cell suspensions (1.1 mL each) were plated
into replicate 35-mm dishes on day 1. The cultures received an addition
of oligos equal to 25% of the initial concentration on day 1 and on
each successive day. All dishes were scored on day 8 for
BFU-E-derived colonies. The counts for duplicate dishes were averaged.
Isolation of hematopoietic cells.
Populations of T cells, B cells, and erythroid cells were isolated
using antibody-coated plates using standard methods.34,35 Antibody-coated plates were prepared using polystyrene bacteriological petri plates. Antibodies were diluted to 100 µg/mL in 50 mmol/L Tris,
pH 9.5. Ten milliliters was poured into 100 × 15 mm plates and
allowed to incubate overnight at 4°C. Plates were then washed 4× in PBS. B cells were recovered using antimouse Ig. Nonadherent cells were then panned on plates coated with an antithymocyte antibody.
Cells negative to both selections were harvested as erythroid cells.
Ten murine spleens were prepared as a single-cell suspension in RPMI
1640 with 5% FBS at 2 × 107 cells/dish. Cells were
allowed to incubate for 30 minutes. Nonadherent cells were removed by
decanting, and adherent cells were then washed 5 times in PBS with 1%
FBS. Cells were removed by repeated pipetting with a pasteur pipette
using the wash buffer. Erythroid precursors were isolated and
fractionated as described.36 Spleen cells were isolated
from Epo-treated mice and washed in Hank's Balanced Salt Solution
without magnesium or calcium (GIBCO). Spleens were crushed and filtered
through a mesh filter to obtain a single-cell suspension. The cell
suspension was sedimented through Ficoll-Hypaque (Pharmacia) to remove
lymphocytes. The pellet from the Ficoll-Hypaque was then layered onto a
discontinuous Percoll gradient that consisted of 6-mL layers of 45%,
65%, 70%, 77%, and 90% Percoll in magnesium- and calcium-free
Hank's Balanced Salt Solution. Approximately 1 × 108
cells were loaded onto the 30-mL gradient and were subjected to
centrifugation at 5,000g for 20 minutes at 4°C. After
centrifugation, fractions of cells were collected using a peristaltic
pump and then washed in DMEM. Cells were counted and characterized by
microscopic examination and benzidine staining.37
Differential counts were performed before and after the separation.
Cell numbers were quantified using a hemacytometer.
Cell transfection.
One day before transfection, Rauscher cells were plated at a density of
1 × 105 cells/mL in 10-cm tissue culture dishes in
DMEM/10% FBS. Cells were harvested by centrifugation, suspended in
DMEM without FBS at a density of 3 × 106 cells/mL,
and transfected with pSVK3-chop or
pSVK3 alone (20 µg DNA/transfection) by electroporation
using a GenePulser II (Bio-Rad) according to the manufacturer's
instructions. Cells were grown for 48 hours after transfection and
analyzed. The transfection efficiency ranged from 10% to 20% in
several experiments, as monitored by transfection of
pCMV-gal control plasmid.
Detection of CHOP binding partners.
A variation on the nuclear extraction procedure of Dignam et
al38 was used. Extract preparation was performed at
4°C. Cells were harvested by centrifugation, washed in ice-cold
PBS, resuspended in a small volume of PBS, and recentrifuged in
microfuge tubes. Five packed-cell volumes of Dignam's buffer A were
added to the pellet, which was gently resuspended by low-speed
vortexing. The cells were then incubated at 0°C for 10 minutes. The
cells were pelleted at low speed in a microfuge, and the supernatant
was discarded. Five volumes of buffer A were added, and the cells were
broken by vigorous vortexing. The nuclei were pelleted, and four
original cell volumes of Dignam's buffer C were added. The nuclei were
lysed with 20 strokes of a Dounce homogenizer (type B pestle). The
lysate was stirred for 30 minutes at 0°C. The nuclear extract was
obtained by centrifugation of the broken nuclei (10 minutes in a
microfuge at 4°C). Five hundred micrograms of nuclear protein
extract was loaded onto a 8-mm wide lane of a 13%/0.35% acrylamide/bis gel and electrophoresed. Molecular weights were assigned
by comparison to prestained molecular weight markers (GIBCO-BRL). The
proteins were transferred electrophoretically to a PVDF membrane
(Millipore Immobilon P; 0.45 µm) for 90 minutes at 100 mA in 39 mmol/L glycine, 48 mmol/L Tris-HCl, 0.037% SDS, 20% methanol. After
blotting, each lane was cut into two strips, with half being used to
test for either control (GST) or experimental (CHOP-GST fusion) protein
binding (modified from the procedure of Ferrell and
Martin39). The PVDF strips were incubated in 7 mol/L
guanidine HCl, 50 mmol/L Tris-HCl, 50 mmol/L dithiothreitol (DTT), 2 mmol/L EDTA, pH 8.0, at room temperature with
gentle rocking for 1 hour. Blots were rinsed with and then incubated with 1% bovine serum albumin, 250 mmol/L KCl, 20 mmol/L potassium phosphate, 2 mmol/L DTT, 0.2 mmol/L EDTA, pH 7.4 (4°C overnight with gentle rocking). Blots were then blocked for 3 hours with 5%
bovine serum albumin, 50 mmol/L KCl, 20 mmol/L potassium phosphate, 2 mmol/L DTT, 0.2 mmol/L EDTA, pH 7.9. To each blocked strip was added
either GST protein or CHOP-GST fusion protein (15 µg/mL in blocking
buffer), and the proteins were allowed to bind overnight. The blots were washed twice for 10 minutes in blocking buffer. Goat
anti-GST antibody (Pharmacia) diluted 1/100 in blocking buffer was
added and allowed to bind for 1 hour at room temperature with gentle
rocking. The blots were washed twice for 10 minutes in blocking buffer.
The second antibody (peroxidase-conjugated rabbit antigoat IgG,
1/1,000; Cappell-Organon Teknika 55358) was added. The blots were again
allowed to incubate at room temperature for 1 hour and were washed as
described before. ECL (Amersham Pharmacia Biotech, Inc)
chemiluminecent detection of the bound complexes was performed
according to the manufacturer's instructions.
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RESULTS |
Identification of Epo-regulated genes.
To identify genes regulated by Epo, we performed a differential
screening process. We constructed a phage cDNA library from Epo-induced Rauscher murine erythroleukemia cells and prepared duplicate nitrocellulose lifts. One lift was hybridized to
[32P]cDNA prepared from uninduced Rauscher cell mRNA. The
other was hybridized to [32P]cDNA prepared from cells
induced with Epo (50 U/mL) in the presence of cycloheximide (10 µg/mL) for 1 hour. After washing and autoradiography, careful visual
comparison of the duplicates showed members of the library that
corresponded to candidate Epo-regulated genes. Approximately 40,000 clones were screened initially.
To verify the authenticity of the Epo-upregulation, the cDNA inserts of
the tentative positives were isolated and used as probes in Northern
blot assays of RNA prepared from Rauscher cells induced with Epo but
without cycloheximide. Three of the initial tentative positives were
authentically Epo-upregulated. DNA sequencing showed that one of the
Epo-upregulated cDNAs bore exact identity to the transcription factor
chop.12,13 It contained the entire coding region
and complete 3' untranslated region. The 815-bp chop cDNA
hybridized to an mRNA of approximately 1.1 kb (not shown). The other
two genes were novel and will not be discussed further.
chop is upregulated during erythroid differentiation of
Rauscher cells.
We determined the steady-state levels of chop mRNA in Rauscher
cells after induction with Epo or DMSO using nuclease protection assays. Rauscher cells were induced with either Epo (100 U/mL) or DMSO
(0.7%) for 0 to 72 hours. During this interval, the cells differentiated as evidenced by hemoglobin synthesis. Both Epo and DMSO
induction resulted in a marked increase in steady-state levels of
chop mRNA (Fig 1A and B). In some
experiments, chop mRNA increased modestly (50% to 100%) after
1 to 3 hours of induction. However, in all experiments, chop
mRNA increased 10- to 20-fold after 24 to 48 hours and remained at
these high levels throughout the 72-hour induction. Similar results
were obtained in several replicate experiments.
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| Fig 1.
chop is upregulated during erythroid
differentiation of Rauscher cells. (A) Epo induction. (B) DMSO
induction. RNase protection assays for chop and gapdh
(glyceraldehyde-3 phosphate dehydrogenase) were performed as described
in Materials and Methods and Results.
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The Epo- or DMSO-induced upregulation of chop mRNA was
unrelated to cell density or to nutrient depletion. The same basal level of chop was detected in uninduced cells (0 time) and in cells maintained in standard culture conditions for 72 hours without Epo or DMSO. Moreover, when cells were induced with Epo or DMSO but
maintained at subconfluent densities (by dilution with Epo-or DMSO-supplemented media to 0.5 × 106 cells/mL),
chop upregulation was equally striking. In contrast to results
reported with adipocytic cell lines,16 reduced glucose levels did not effect chop induction. Rauscher cells cultured in glucose 2 mmol/L showed the same pattern of chop induction as cells cultured in 10 mmol/L glucose (data not shown).
CHOP protein expression.
We confirmed that CHOP protein levels increase markedly during Rauscher
cell differentiation (Fig 2). Purified,
bacterially expressed recombinant CHOP (rCHOP; 2.5 or 0.5 ng) or
SDS-solubilized protein extracts from DMSO uninduced (0 hours) or
induced (48 or 72 hours; 100 µg/lane) were subjected to SDS-PAGE and
were analyzed by Western blot using affinity-purified rabbit anti-CHOP antibodies (left panel) or preimmune rabbit IgG (right panel). The
anti-CHOP antibodies specifically recognized 2.5 ng but not 0.5 ng of
rCHOP. Preimmune IgG did not recognize rCHOP. Both anti-CHOP and
preimmune IgG cross-reacted nonspecifically with several minor protein
species from Rauscher cells (lanes 0, 48, and 72). However, despite
this background, only the anti-CHOP antibodies identified a prominent
band in differentiating cells (left panel, 72 hours) that migrated to
the same position as rCHOP. This increase in CHOP protein correlates
well with the increase in CHOP mRNA (Fig 2).
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| Fig 2.
CHOP protein increases during erythroid differentiation
of Rauscher cells. Purified recombinant CHOP protein (rCHOP) or lysates
from uninduced (0 hours) or DMSO-induced (48 and 72 hours) cells were
subjected to SDS-PAGE and electrophoretic transfer. They were probed
with either affinity-purified anti-CHOP antibodies (left panel) or
purified preimmune IgG (right panel). Note the prominent increase in
CHOP protein detected after 72 hours of induction (left panel, 72 hours).
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Expression of c/ebp isoforms during Rauscher cell
differentiation.
In some cell types, the C/EBP isoforms are known to
heterodimerize and to have changing interactions during
differentiation.23-25 Therefore, it was important to
determine which c/ebp isoforms are expressed in erythroid cells
and whether their expression changes during erythropoiesis. Northern
blots were prepared with polyA+ RNA from uninduced and
DMSO-induced Rauscher cells and were probed with c/ebp , c/ebp
, c/ebp , or chop (Fig 3A
and B). The blots were probed subsequently with gapdh cDNA.
c/ebp and c/ebp mRNA levels were extremely low
and did not change during differentiation. In contrast, c/ebp
and chop mRNA increased 8.5- and 9.5-fold, respectively, during induction, as determined by laser densitometry.
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| Fig 3.
Expression of c/ebp isoforms during erythroid
differentiation. (A) Autoradiograms of Northern blot analyses. (B)
Densitometric analysis of data in (A) normalized to 0 hours. Note the
increase in both chop and c/ebp. In contrast,
c/ebp and c/ebp were unchanged.
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These blots were also used to approximate the levels of the different
c/ebp isoforms relative to each other. In uninduced Rauscher
cells, chop mRNA was fivefold to 10-fold more abundant than
c/ebp mRNA (based on the use of equal amounts of probe with
equal specific activity, assaying equal amounts of polyA+
mRNA). The levels of c/ebp and c/ebp were
conspicuously lower than chop and c/ebp . Thus, at
all times during induced differentiation, chop steady-state
mRNA levels were in large excess over the other c/ebps.
Tissue distribution of chop mRNA.
To determine whether fully differentiated cells in other tissues
express chop, RNase protection assays were performed from a
variety of adult mouse tissues (Fig 4).
Although chop mRNA was detectable in all tissues, its abundance
varied greatly. Uninduced Rauscher cells had chop mRNA levels
10- to 30-fold higher than most of the other tissues. Among the 14 tissues tested, only testis had conspicuously high chop mRNA
levels, approaching that found in Rauscher cells.
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| Fig 4.
chop expression in adult mouse tissues. RNase
protection assay for chop or gapdh was performed as
described in the text. Ten micrograms of RNA was used for each
tissue.
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CHOP expression in normal hematopoietic cells.
Next, we compared chop expression in Rauscher cells with that
seen in normal murine hematopoietic cells
(Fig 5). Among the normal cells studied,
erythroid cells isolated from the spleens of Epo-treated mice had the
highest level of chop mRNA. This level was not as high as that
seen in Rauscher cells. However, the splenic erythroid population is
heterogeneous, consisting of cells at various stages of
differentiation. This analysis was complicated by the rather large
differences in gapdh mRNA seen among these various cell types
despite even loading of the gels demonstrated by ethidium bromide
staining. Interestingly, the chop/gapdh ratio seen in normal
erythroid cells was as high as that seen in Rauscher cells.
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| Fig 5.
chop mRNA expression in Rauscher cells and a
variety of normal murine hematopoietic cells. Splenic T, B, and
erythroid cells were isolated using antibody-coated plates, as
described.
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Because the pronounced upregulation of chop in Rauscher cells
correlated with induction of hemoglobinization, we measured chop mRNA levels in differentiating normal murine erythroid
precursors. As erythroid precursors mature, they decrease in cell
volume and undergo chromatin condensation and enucleation. These
changes result in increasing cell density, a characteristic that can be used as the basis of a separation technique. Normal splenic erythroid precursor cells were separated on a 5-step discontinuous Percoll gradient. The cells from each were evaluated for chop
expression by Northern blot, for their degree of hemoglobinization by
benzidine staining, and for whether they had differentiated to the
point that they had condensed and extruded their nucleus by microscopic examination. The least dense fraction contained the least mature population of precursors. There were only 18% hemoglobinized
(Hb+) cells and virtually all (99%) retained their nuclei.
chop mRNA levels were relatively low in this least dense
fraction. The third of the five fractions contained 90%
Hb+ cells, of which 56% retained their nuclei.
Importantly, this stage of terminal differentiation was associated with
a significant increase in chop mRNA, which is very similar to
the association of chop upregulation and hemoglobinization of
Rauscher cells. The increase was fourfold to fivefold over the least
dense fraction. The two most dense fractions contained 99%
Hb+ cells and 4% and 1% nucleated cells,
respectively. chop mRNA began to decrease in these more
mature erythroid cells (Fig 6).
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| Fig 6.
chop mRNA levels in normal murine erythroid
precursors. Cells were separated by discontinuous Percoll density
fractionation. Cells in each fraction were characterized for the
percentage of hemoglobinized cells and the percentage of nucleated
cells.
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Ectopic expression of chop in Rauscher cells.
We obtained evidence of a functional role for chop in
erythropoiesis by increasing its expression through transient
transfection (Table 1). chop
cDNA was subcloned into a pSVK3 expression vector and was
transfected into Rauscher cells. After 24 hours, cells were incubated
in the absence or presence of Epo or DMSO for 48 hours, and
differentiating cells were identified by staining for hemoglobin.
Nontransfected cells induced with Epo or DMSO for 48 hours were 34% ± 4% and 49% ± 5% hemoglobinized (Hb+),
respectively. In contrast, cells transfected with and overexpressing chop were increased significantly to 43% ± 5% and 62% ± 5% Hb+, respectively, strongly suggesting that
increased chop enhanced erythroid differentiation (P < .02). Overexpression of CHOP protein was confirmed by Western blot
analysis (not shown). Cells transfected with vector alone had responses
to Epo or DMSO virtually identical to those of nontransfected cells. It
is important to note that Rauscher cells are relatively resistant to
transfection. Only 10% to 20% of cells were transfected on average as
monitored by Gal-4 transfection. The absolute 9% to 13% increase in
hemoglobinization seen in chop-transfected cells correlates
well with the transfection efficiencies achieved.
Artificial downregulation of chop with antisense
oligodeoxynucleotides inhibits CFU-E colony growth.
Increased chop expression correlates with erythroid
differentiation, and ectopic expression of chop increased Epo-
and DMSO-induced differentiation of Rauscher cells. Therefore, we
reasoned that downregulating chop levels should result in
reduced differentiation. In the experiment shown in
Fig 7, normal murine bone marrow cells were
grown in methyl cellulose culture in the absence or presence of
specified concentrations of antisense or missense chop oligos using conditions required for growth of CFU-E- or BFU-E-derived colonies. Further additions of oligos were made daily. CFU-E colonies were scored after 2 days, and BFU-E colonies were scored after 8 days.
Antisense chop oligos inhibited CFU-E colony growth in a
concentration-dependent manner. The maximum antisense oligo concentration used (200 µg/mL) reduced CFU-E colony numbers
to 42% of control values. Importantly, missense chop oligo
treatment had no effect on CFU-E growth, providing evidence for the
specificity of the antisense effect. In other, less detailed
experiments, sense chop oligo treatment also had no effect on
CFU-E growth. In contrast to the inhibitory effect seen on CFU-E colony
growth, neither antisense nor missense (nor in pilot experiments,
sense) chop oligos inhibited BFU-E colony growth. Similar
results were obtained in other experiments.
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| Fig 7.
Effect of antisense chop oligodeoxynucleotides on
normal murine erythropoiesis in vitro. (A) CFU-E-derived colony
growth. (B) BFU-E-derived colony growth. ( ) Antisense oligos; ( )
missense oligos. Note the specific concentration-dependent inhibition
of CFU-E colony formation by antisense chop oligos. Each point
is the mean of duplicate determinations.
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Because of technical difficulties in quantifying chop
transcript levels in erythroid colonies grown in methyl cellulose, we used Rauscher cells grown in suspension culture to verify the action of
antisense chop oligos (Fig 8).
Rauscher cells were incubated in the absence or presence of 100 µg
antisense chop oligo/mL for 24 hours. RNA was extracted and a
Northern blot analysis was performed. Antisense chop oligo
treatment caused a marked reduction in chop steady-state
transcript levels. No change was observed using missense oligos (not
shown).
CHOP binding partners during erythroid differentiation.
Because of reports that C/EBP isoforms can interact with non-C/EBP
proteins,40-42 we used a protein-renaturation method to identify potential alternative CHOP binding partners and to determine whether they changed during erythropoiesis. Nuclear extracts from uninduced and induced cells (0.7% DMSO, 48 hours) were prepared, subjected to denaturing electrophoresis, transferred to blotting membranes, and then treated with a denaturing guanidine buffer. The
denaturing buffer was washed off, and the membrane-bound proteins were
allowed to renature overnight. The membranes were then probed with
CHOP-GST fusion protein or GST alone (Fig
9). The CHOP-GST fusion protein interacted strongly with a number of
the nuclear proteins, whereas the GST protein binding was negligible.
The relatively large number of potential alternative binding partners was surprising. Nonetheless, similar observations were made in repeat
experiments. Interestingly, the nuclear extract from induced cells had
a number of new electrophoretically distinct species that bound
CHOP-GST. In six experiments, CHOP-GST bound to proteins of 14.3, 17.5, 38.5, and 54 kD that were unique to the nuclear extracts from induced
(differentiating) cells. There was also a number of CHOP binding
proteins in uninduced cells, the abundance of which increased markedly
during differentiation. They had molecular weights of 16, 27, 32, and
34 kD. Two proteins of approximately 66 and 95 kD, respectively,
appeared to decrease in abundance during differentiation.
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| Fig 9.
Far Western analysis shows numerous potential CHOP
binding partners in erythroid cells and changes in them during
erythropoiesis. See Results.
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In addition to nuclear extracts, we also examined cytosolic fractions
of both uninduced and induced cells for CHOP binding partners. There
were a few proteins of very low abundance that appeared to bind
CHOP-GST or GST proteins weakly. There were no differences between
induced and uninduced cells (data not shown).
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DISCUSSION |
In this study, we have identified the transcription factor chop
as an Epo-regulated gene and have obtained evidence that it plays a
role in erythroid differentiation. c/ebp was also
upregulated during erythropoiesis. We found that chop message
levels in Rauscher cells increased 10- to 20-fold after treatment with
natural or chemical inducers of erythroid differentiation. Distinct
signal transduction pathways are activated in response to EPO or
DMSO,2 but treatment with either inducer results in
upregulation of chop. This suggests that inducers of erythroid
differentiation converge on chop upregulation. We also found
that chop message is particularly abundant in erythroleukemia
cells, relative to terminally differentiated adult tissues, and that
its abundance in normal erythroid cells is modulated during terminal
differentiation. Furthermore, manipulation of CHOP levels perturbs
normal erythropoiesis. This regulated expression is suggestive of a
significant functional role for chop in red blood cell development.
In normal hematopoietic cells, the highest levels of CHOP were found in
erythroid cells, with levels peaking during terminal differentiation.
Rauscher cells have attributes of late BFU-E and early
CFU-E.43 Upon induction with Epo or DMSO, they resemble differentiating CFU-E. This later point in Rauscher cell
differentiation is when chop levels were determined to be the
highest. We demonstrated that CFU-E colony growth was inhibited by
antisense chop treatment, whereas BFU-E colony growth was
unaffected. The inhibition of CFU-E colony development by antisense
chop is consistent with the absolute Epo requirement for CFU-E
differentiation and strongly supports a role for CHOP in the terminal
differentiation of erythroid cells. This finding compliments those
described with Epo and EpoR-null mice, ie, there is an
essential role for Epo in regulating the survival and terminal
differentiation of CFU-E.44 Because we have identified
chop as an Epo-upregulated gene, it would be expected that its
downregulation would block differentiation at the CFU-E stage, as we
have shown. However, the apparent lack of antisense chop oligo
effect on BFU-E colony numbers and morphology is less easily
understood. Epo addition to BFU-E cultures can be delayed somewhat
without affecting BFU-E colony development significantly. However,
ultimately, Epo addition is essential for the final appearance of
mature BFU-E-derived colonies. If BFU-E differentiation in vitro
passes through a CFU-E stage identical to that characterized by CFU-E
progenitors harvested from the bone marrow, then it would be expected
that antisense chop oligos should inhibit terminal differentiation of this CFU-E-like cell, resulting in small and less
robust BFU-E-derived colonies (even if actual colony numbers were not
affected). Our results are consistent with the hypothesis that BFU-E
differentiation in culture may exhibit characteristics unique to the in
vitro environment. This may also include relative resistance to oligo
uptake by in vitro CFU-E-like cells. Taken together, our studies of
Rauscher and normal erythroid cells show that chop expression
is most robust in terminally differentiating CFU-E progeny and that
CHOP plays an important role in the regulation of erythroid differentiation.
CHOP's action is not the same in all cell types. Increased
chop facilitates erythroid differentiation. This effect is the opposite of that seen in adipogenic differentiation, in which increased
chop expression attenuates differentiation and interferes with
the induction of c/ebp and .45 In adipocyte
systems, chop is induced by nutrient depletion in the culture
medium and may slow down differentiation in response to metabolic
stress (particularly the condition of low glucose). In contrast,
chop expression in Rauscher cells was unaffected by altered glucose conditions; also, its induction was not related to cell
density. CHOP expression can also be induced by
treatments that adversely affect function of the endoplamic
reticulum.18,46
At present, the precise mechanism of CHOP's action in erythroid
differentiation is unknown, as is the mechanism by which Epo upregulates chop's expression. Of note is the existence of a
possible GATA-1 binding site at nucleotide 415 in the hamster
chop gene14 and of nucleotide 435 in the
human chop gene.47 The human site is
a perfect, prototypical GATA site, and the hamster site conserves the
GATA-1 core. Indeed, the Epo receptor itself,48 known to be
transactivated by GATA-1,9,49 contains a rather
nonconforming TTATCT sequence. Also of obvious interest (in view of
Epo's chop upregulation concurrent with c/ebp
upregulation) are the numerous NF-I16 (C/EBP) sites in both
chop genes.
The coordinate upregulation of both chop and c/ebp
during erythroid differentiation is suggestive of a mechanistic
relationship. In this regard, it has recently been shown that C/EBP
participates in regulating the pro-B cell-specific enhancer
(PBE).50 In this system, CHOP and C/EBP interact in a
developmental stage-specific pattern. These factors associate and form
an inactive complex in mature B-cell lines, whereas pro-B-cell lines
have active complexes of C/EBP proteins, but no detectable CHOP
protein. This is reminiscent of our results in erythroid cells. CHOP
levels increase as the cells mature. It may well be that the
CHOP-C/EBP has a negative regulatory function in some contexts.
Earlier experiments using cell lines suggested that C/EBP activates
transcription of the IL-6 promoter.51 However, results from
the c/ebp knock-out mouse show lymphoproliferative and
myeloproliferative histologies similar to mice overexpressing
IL-6.52 This in vivo result suggests that C/EBP might
negatively regulate the IL-6 gene. Differentiation requires restriction
in developmental potential, and downregulation of the canonical
CCAAT/enhancer sites may be part of this process. Global transcription
that is characteristic of the undifferentiated state can be suppressed
by increasing the amount of repressors such as CHOP, LIP, and,
possibly, C/EBP. Once physiologically relevant targets of CHOP
regulation are defined, the exact role of CHOP in erythroid
differentiation can be addressed, ie, whether it acts as a
dominant-negative regulator of C/EBP binding, directing CHOP-C/EBP
heterodimers away from canonical C/EBP binding sites, or whether CHOP
activates transcription from other classes of target genes.
Interactions between the different C/EBP proteins (including CHOP) may
be critical to their mechanism and function during differentiation. All of the family members have a
well-conserved C-terminus that includes five heptad repeats that form
an amphipathic helix. Dimerization of these amphipathic
helices allows formation of a leucine zipper. All of the C/EBP family
members contain this motif, and most can form homodimers and
heterodimers. CHOP is an unusual member of this family in that it does
not form homodimers and it has a noncanonical DNA-contact domain. In
some instances, CHOP acts as a dominant negative regulator of other
C/EBPs.13 Through heterodimerization, it inhibits the
binding of its partner to the canonical C/EBP target sequence. In
other instances, the CHOP/C/EBP heterodimer can recognize and
activate noncanonical DNA binding sites.30 Although a
prefered DNA binding sequence has been defined, regulated genes have
not been characterized.
The present studies point to other proteins that interact with CHOP.
The Far Western assay (Fig 9) suggested that CHOP's binding partners
change and become more numerous during differentiation. Other systems
have shown complex and often cell-specific interactions between
different C/EBP family members. There is potential for homodimerization
and heterodimerization of some of the C/EBP isoforms and the
possibility of regulatory interactions in trans (eg, the promoter of
the c/ebp gene has a C/EBP binding site). In addition, interactions between C/EBP and non-C/EBP proteins have been
documented.40-42 In this regard, additional data obtained
by us using the yeast two hybrid system confirms that erythroid cells
(and, presumably, other cells) express non-C/EBP proteins that interact
specifically with CHOP in vitro and in vivo53,54
(manuscripts in preparation). Further studies in this
area will help to elucidate the role of CHOP in erythropoiesis and may
identify novel regulatory pathways.
Because of the association of chop upregulation and ER stress,
Zinszner et al55 performed targeted disruption of
chop in murine ES cells and produced animals with a chop
/ phenotype.55 Somewhat surprisingly,
these mice were described as phenotypically normal and exhibited normal
fertility. Unfortunately, no data regarding the hematopoietic system
were reported. However, these animals, as well as embryonic fibroblasts
derived from them, exhibited a defect in the development of apoptosis
after treatment with agents that cause ER stress. This observation that
a lack of CHOP prevents apoptosis would seem to contradict the
demonstration that Epo, which increases chop expression,
prevents apoptosis of erythroid cells. This apparent paradox mirrors
the dissimilar effects of chop upregulation in adipogenic
differentiation and in erythropoiesis and further supports the
hypothesis that CHOP's role in cell growth and differentiation is
dependent on the context of cell lineage and stage as well as the other
binding partners available to it.
| |
ACKNOWLEDGMENT |
The authors thank Alan Friedman for the generous gift of the
c/ebp , , and cDNAs and Cindy Miller for her tutorial
on methyl cellulose culture. We also acknowledge the expert editorial work of Rosemary Panza.
| |
FOOTNOTES |
Submitted November 2, 1998; accepted January 13, 1999.
Supported by National Institutes of Health (NIH) NRSA F32 DK08986
(M.C.), NRSA F32 DK09201 (K.C.), NRSA F32 HL08563 (J.C.K.), and NIH R01
DK38841 and by US Navy Grant No. N00014-93-1-0776 (A.J.S.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Arthur J. Sytkowski, MD, Laboratory for
Cell and Molecular Biology, Division of Hematology and Oncology,
Department of Medicine, Beth Israel Deaconess Medical Center, 1 Deaconess Rd, 21-27 Burlington Bldg, Room 548, Harvard Medical School,
Boston, MA 02215.
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REFERENCES |
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ABSTRACT
INTRODUCTION