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Prepublished online as a Blood First Edition Paper on December 19, 2002; DOI 10.1182/blood-2002-04-1039.
 Previous Article | Table of Contents | Next Article 
Blood, 15 April 2003, Vol. 101, No. 8, pp. 3265-3273
PHAGOCYTES
Regulation of neutrophil and eosinophil secondary granule gene
expression by transcription factors C/EBP and
PU.1
Adrian F. Gombart,
Scott H. Kwok,
Karen L. Anderson,
Yuji Yamaguchi,
Bruce E. Torbett, and
H. Phillip Koeffler
From the Division of Hematology/Oncology, Cedars-Sinai
Medical Center, Los Angeles, CA; the Burns and Allen Research
Institute, University of California at Los Angeles School of Medicine;
the Departments of Molecular and Experimental Medicine and Immunology,
The Scripps Research Institute, La Jolla, CA; and the Center for Sleep
Respiratory Disorders, Fukoka, Japan.
 |
Abstract |
In the bone marrow of C/EBP  / mice, expression of
neutrophil secondary and tertiary granule mRNAs is absent for
lactoferrin (LF), neutrophil gelatinase (NG), murine cathelinlike
protein (MCLP), and the cathelin B9; it is severely reduced for
neutrophil collagenase (NC) and neutrophil gelatinase-associated
lipocalin (NGAL). In addition, the expression of eosinophil granule
genes, major basic protein (MBP), and eosinophil peroxidase (EPX) is absent. These mice express C/EBP , C/EBP , and C/EBP in the bone marrow at levels similar to those of their wild-type counterparts, suggesting a lack of functional redundancy among the family in vivo.
Stable inducible expression of C/EBP and C/EBP in the murine
fibroblast cell line NIH 3T3 activated expression of mRNAs for B9,
MCLP, NC, and NGAL but not for LF. In transient transfections of
C/EBP and C/EBP, B9 was strongly induced with weaker
induction of the other genes. C/EBP and C/EBP proteins weakly
induced B9 expression, but C/EBP induced NC expression more
efficiently than the other C/EBPs. The expression of MBP was
inefficiently induced by C/EBP alone and weakly induced with
C/EBP and GATA-1, but the addition of PU.1 resulted in a striking
cooperative induction of MBP in NIH 3T3 cells. Mutation of a predicted
PU.1 site in the human MBP promoter-luciferase reporter construct
abrogated the response to PU.1. Gel-shift analysis demonstrated binding of PU.1 to this site. MBP and EPX mRNAs were absent in a PU.1-null myeloid cell line established from the embryonic liver of
PU.1/ mice. Restitution of PU.1 protein expression
restored MBP and EPX protein expression. This study demonstrates that
C/EBP is essential and sufficient for the expression of a particular
subset of neutrophil secondary granule genes. Furthermore, it indicates the importance of PU.1 in the cooperative activation of eosinophil granule genes.
(Blood. 2003;101:3265-3273)
© 2003 by The American Society of Hematology.
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Introduction |
Transcription factors play a major role in the
development of specific lineages in the hematopoietic
system.1,2 Mature cells of the myeloid lineage, including
peripheral blood monocytes, tissue macrophages, and neutrophilic
and eosinophilic granulocytes, derive from a common myeloid
precursor. Of the numerous transcription factors involved in
myelopoiesis, C/EBP, C/EBP, and PU.1 are critical for normal
granulocytic differentiation. Targeted inactivation of these genes in
mice demonstrates their importance in the development, function, and
maturation of neutrophils and eosinophils.2-6
In the 2 murine models for PU.1 deficiency, mice either die at late
gestation or survive for approximately 48 hours after birth and succumb
to septicemia.5,6 Both models have defects in the myeloid
and lymphoid lineages. A lack of mature macrophages, neutrophils,
dendritic cells, osteoclasts, B cells, and T cells is
observed.5-7 Antibiotic treatment allows mice that survive birth to live up to 2 weeks, during which time some cells with the
characteristics of neutrophils develop by day 3.6 These neutrophils appear normal by morphology and express neutrophil markers
such as Gr-1 and chloroacetate esterase, but they fail to mature
completely as indicated by the lack of secondary granule gene
expression.8 In addition, neutrophils from PU.1 knockout mice are functionally impaired with defects in superoxide production, bacterial uptake, and killing.8 Restoration of
PU.1 gene expression in a myeloid cell line derived from the
embryonic livers of these mice reinstated expression of the secondary
granule genes, and functions of normal terminal neutrophil maturation
were acquired.9
Mice lacking the C/EBP protein displayed abnormalities in liver,
adipose tissue, and lungs and died within the first few hours of birth
because of impaired glucose metabolism.10,11 Analysis of
C/EBP-deficient mice revealed a loss of mature neutrophils and
eosinophils in peripheral blood and in fetal and newborn
livers.12 Most white blood cells resembled immature
myeloid cells with a block at the myeloblast stage.12 This
was associated with a dramatically reduced expression of the
granulocyte-colony-stimulating factor (G-CSF) and interleukin-6
(IL-6) receptor mRNAs, whereas mRNA levels of the macrophage-CSF
(M-CSF) and granulocyte macrophage-CSF (GM-CSF) receptors were
unaffected.12,13
Like C/EBP-deficient mice, C/EBP knockout mice displayed defects
in granulopoiesis.3 An increase in granulocyte progenitors occurred in the C/EBP-deficient bone marrow. In addition, the peripheral blood from C/EBP-deficient mice displayed increased numbers of hyposegmented morphologically atypical
neutrophils.3 This indicated a block at a later stage of
granulocytic differentiation not observed in the
C/EBP-deficient mice. In contrast to C/EBP-deficient mice,
C/EBP-deficient mice developed normally and were fertile but
eventually died of opportunistic infections likely resulting from
neutrophil cell dysfunction.3 Neutrophils from
C/EBP-deficient mice are defective in chemotaxis, disaggregation,
receptor up-regulation, superoxide production, and bactericidal
activity.3,14 C/EBP-deficient mice lacked or displayed
severely reduced bone marrow expression of mRNAs encoding secondary and
tertiary granule proteins. These included lactoferrin (LF), neutrophil
collagenase (NC), neutrophil gelatinase (NG), neutrophil
gelatinase-associated lipocalin (NGAL), cathelicidin B9/NGP (neutrophil
granule protein), and murine cathelinlike peptide
(MCLP/CNLP).14,15 C/EBP-deficient mice also exhibited abnormalities in their eosinophils.3
Phenotypic and functional defects of neutrophils and eosinophils
observed in C/EBP-deficient mice closely paralleled those of
neutrophil-specific granule deficiency (SGD), a rare congenital disorder in humans.3,14,15 Determination of the mutational status of the human C/EBPE locus in 2 patients with SGD
revealed that it results from an autosomal recessive inheritance of
frame-shift mutations in the C/EBPE gene.16-18
Because of these numerous deficiencies and functional defects, SGD
patients and C/EBP-deficient mice are immunocompromised and acquire
frequent bacterial infections, including infection from
Pseudomonas aeruginosa and Staphylococcus aureus.
Together with in vitro studies of gene expression, these murine models
aid in the identification of target genes for these transcription
factors. A number of myeloid-specific genes contain functional C/EBP-
and PU.1 binding sites in their promoters, making them potential
targets for C/EBP or C/EBP. These include the primary granule
proteins neutrophil elastase (NE), proteinase 3, and myeloperoxidase
(MPO), the receptors for G-CSF, M-CSF, and GM-CSF, and the secondary
granule protein LF.19-24 A number of these genes show
reduced or absent expression in the knockout mice.25,26
The absence of expression of a particular gene indicates that it may be
a target gene directly activated by the transcription factor, but it is
necessary to assess whether the gene is directly activated.
Alternatively, the block in granulocytic differentiation observed in
the knockout mouse models may result in an insufficient number of cells
that express the gene in question. On the other hand, ectopic
expression of the transcription factor(s) to induce target gene
expression may promote differentiation in myeloid cell lines that, in
turn, results in the indirect regulation of the "target" gene. In
this report, the hypothesis that C/EBP is essential and sufficient
for the expression of neutrophil and eosinophil secondary granule genes
was tested. Using a combination of studies on cells from the knockout
mice and induction of myeloid-specific gene expression in a
nonhematopoietic murine fibroblast cell line, it was demonstrated that
C/EBP is a key transcriptional regulator of neutrophil secondary
granule genes. Additionally, C/EBP cooperates with PU.1 and GATA-1
to activate eosinophil granule gene expression.
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Materials and methods |
C/EBP/ mice, C/EBP/ mice, and
PU.1/ cell lines
Bone marrow cells were derived from wild-type or
C/EBP/ mice from a 129SV × NIH Black
Swiss background at 6 to 8 weeks of age.3 Bone marrow
cells were derived from C/EBP/ mice at 6 to 8 weeks
of age.4 C/EBP/ mice were kindly
provided by K. Xanthopolous and J. Lekstrom-Himes (Aurora Biosciences,
San Diego, CA; and National Institutes of Health, Bethesda, MD;
respectively), and C/EBP/ mice were kindly provided
by Shizuo Akira (Department of Biochemistry, Hyogo School of Medicine,
Japan). The PU.1/ myeloid cell line and the PU.1 and
M-CSF receptor-transduced derivatives were prepared as described
previously.9
Transfections and generation of stable cell lines
NIH 3T3 cells were maintained in Dulbecco modified Eagle medium
(DMEM; Gibco/BRL, Gaithersburg, MD) containing 10% adult bovine serum
(BS; Gemini BioProducts, Calabasas, CA). For transient transfections, approximately 3 × 105 NIH 3T3 cells were seeded per well
of a 12-well plate. For each DNA combination, 3 µg plasmid DNA was
mixed with 15 µL GenePORTER reagent (Gene Therapy Systems, San Diego,
CA) in 1 mL OptiMEM reduced serum medium (Gibco/BRL) as directed by the
manufacturer. One third of the DNA mixture was added per well of a
12-well plate (3 wells per DNA combination). After a 5-hour incubation,
2 mL culture medium was added. For stable transformation, approximately 0.5 × 106 NIH 3T3 cells were plated in a 35-mm well and
were transfected with 3 µg plasmid (pMTCB6+, pMT-32,
or pMT-) using 10 µL GenePORTER reagent. At 48 hours after
transfection, cells were trypsinized and plated into a 100-mm dish in
the presence of 700 µg/mL G418 (Gibco/BRL). A polyclonal population
of G418-resistant cells was obtained from each transfection. These
clones were screened for inducible expression of either
C/EBP32 or C/EBP by Western blot analysis. Expression
vectors were induced by the addition of ZnSO4 (Sigma
Chemical, St Louis, MO) to a final concentration of 0.1 mM. Transient
transfections in 35-mm plates (6-well dishes) were performed as
described for the stable cell lines but were harvested 24 to 48 hours
after transfection.
Expression vectors and promoter-reporter gene constructs
The cDNA encoding human C/EBP30 was
subcloned from pcDNAI-C/EBP30 into pcDNA3 (Invitrogen,
Carlsbad, CA).27 The pcDNA3 expression vector containing
the human C/EBP32 isoform was a generous gift from Dr K. Xanthopoulos (Aurora, San Diego, CA). The pMSV-C/EBP (rat)
expression vector was kindly provided by Dr A. Friedman (Johns Hopkins
University, Baltimore, MD) and the expression vectors
pXM-GATA-128 and pMT2-FOG (friend of gata) (murine)29 were kind gifts from Dr S. Orkin (Harvard
University, Boston, MA). The human major basic protein gene
(hMBP) luciferase reporter construct containing sequence
between positions nucleotide (nt)  117 to +47 of the hMBP
P2 promoter region was described previously.30
Construction of the zinc-inducible C/EBP32 vector pMT-32 was described previously.31 To
construct the zinc-inducible rat C/EBP expression vector, a
1.1-kilobase pair (kbp) NcoI C/EBP cDNA fragment was
blunt-ended with Klenow and subcloned into EcoRV-cut pMT-CB6+ (kindly provided by F. Rauscher).32 The pCMVSPORT
vectors expressing human C/EBP32,
C/EBP30, and C/EBP were generated as described
previously.33
RNA isolation and analysis
Total RNA was isolated by lysis of cells in TRIzol reagent
as described by the manufacturer (Gibco/BRL). For reverse
transcription-polymerase chain reaction (RT-PCR) analysis, total RNA
was treated with RNase-free DNaseI (Promega, Madison, WI) and
synthesized into cDNA with Moloney murine leukemia virus (MMLV) reverse
transcriptase in a 50 µL volume as described by the manufacturer
(Gibco/BRL). For transiently transfected cells, the entire RNA sample
was reverse transcribed; for stably transformed cells, 2 µg RNA was
used. PCR was performed with 1 µL cDNA per reaction using HotStar Taq
polymerase (Qiagen, Chatsworth, CA). After a 15-minute denaturing step
at 94°C, reactions were 94°C for 30 seconds, 55°C, 56°C, or
58°C for 30 seconds, and 72°C for 30 seconds. Cycle numbers for
each primer set are described in the figure legends. Products were
electrophoresed on 2% agarose gels and blotted for Southern analysis
as described previously.17 Primers used for PCR and
Southern blot hybridization were described previously15 or
are summarized in Table 1. Each primer
pair used for RT-PCR was designed to span 1 or more introns; therefore,
amplification of contaminating genomic DNA would result in a product
much larger than expected for amplification of the cDNA. A region 1 probe (N-terminal transcriptional activation domain, more than 90%
similar to murine) of the human C/EBP gene was used to for Northern
hybridization.34
Quantitative real-time-PCR (QRT-PCR) for major basic protein
(MBP) expression was performed using a second primer set that spanned
an intron, as described in Table 1. Triplicate reactions for each cDNA
were set up as described in the previous paragraph, and SYBR
Green I (Molecular Probes, Eugene, OR) was added at a 1:60 000
dilution. After denaturing the template 15 minutes at 95°C, a 4-step
PCR reaction of 95°C for 30 seconds, 60°C for 30 seconds, 72°C
for 30 seconds, and 80°C for 20 seconds was performed. At the last
step, measurement of incorporated SYBR Green I was performed at 2°C
below the empirically determined melting temperature for the PCR
product. This melted all potential nonspecific products while
maintaining the specific product and ensuring that nonspecific products
were not detected. Gel electrophoresis confirmed that nonspecific
amplification was negligible. The balance of the cDNAs was determined
by quantifying the relative levels of 18S RNA in the samples. This was
performed with a TaqMan probe (FAM-agcaggcgcgcaaattaccc-TAMRA) and
amplification using TaqMan Universal PCR Master Mix (PE Biosystems, Foster City, CA). The nucleotide sequence of the PCR primers for 18S
was 5'-aaacggctaccacatccaag-3' (18S-F) and 5'-cctccaatggatcctcgtta-3' (18S-R). The threshold cycle (Ct) for each was determined,
and the expression levels of MBP were normalized to 18S. The fold change (FC) of the expression vector (v)-transfected samples compared with the empty vector (ev)-transfected control was determined by the
following equation: FC = 2 ((Ct
MBPv Ct18Sv) (Ct MBPev Cft18Sev)).
For Northern blot analysis, total RNA was electrophoresed through 1%
agarose/formaldehyde gels and was transferred to Hybond N+ membranes
(Amersham Life Sciences, Arlington Heights, IL). Probes were
synthesized with a Strip-EZ random priming kit (Ambion, Austin, TX)
with the incorporation of 5'-[32P]-dATP (3000 Ci/mmol
[111 000 GBq/mmol]; Dupont/NEN, Boston, MA). Blots were
hybridized overnight in UltraHYB solution (Ambion) and were washed at a
final stringency of 0.1 × SSC (standard saline citrate), 0.1%
sodium dodecyl sulfate (SDS) at 68°C for 20 minutes. Blots were
exposed to Kodak XO-Mat film with an intensifying screen (Eastman-Kodak, Rochester, NY). The probes used for Northern blot analysis are described in the first paragraph of this section or in Table 1.
Protein isolation and analysis
For the stable cell lines, total cell protein was prepared and
Western blot analysis was performed as described
previously.17 For transient transfections, total cell
protein was prepared from the organic phase of the Trizol lysate as
described by the manufacturer (Gibco/BRL). The antiserum against the
amino-terminal half of C/EBP was described
previously.35 Commercially available antibodies against
C/EBP (C-22), C/EBP (14AA), C/EBP (C19), and C/EBP (M17)
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All
antibodies were used at a final concentration of 0.2 µg/mL. The
primary antibody was detected with a donkey-antirabbit horseradish
peroxidase (HRP) conjugate (Amersham Life Sciences) diluted 1:5000.
Antigen-antibody complexes were visualized using the Supersignal
chemiluminescence kit (Pierce, Rockford, IL) and exposure to Kodak
XO-Mat film.
Electrophoresis mobility shift assays
COS-1 cells were transfected with empty or PU.1 expression
vector as described in "Transfections and generation of stable cell
lines." For total cell extract, cells were washed with
phosphate-buffered saline (PBS) and were lysed in NP-40 lysis buffer
(50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40) containing Complete
protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Double-stranded oligonucleotides were end labeled with
32P- adenosine triphosphate (ATP; Dupont/NEN) using T4
polynucleotide kinase (Invitrogen) as described by the manufacturer.
Oligonucleotide sequences (sense-strand) were: (1) wild-type consensus
PU.1 5'-gggctgcttgaggaagtataagaat-3'; (2) mutant consensus
PU.1 5'-gggctgcttgagagagtataagaat-3'; (3) wild-type MBP
PU.1 5'-tctccctgggggaagttcctccaaggcc-3'; and
(4) mutant MBP PU.1
5'-tctccctgggcaaagtttgtccaaggcc-3'. Core
sequences are highlighted as underlined text. Electrophoresis mobility
shift assays (EMSAs) were performed as described
previously.34
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Results |
Absence of secondary granule gene mRNA expression in the
C/EBP-deficient mouse occurred in the presence of other C/EBP family members. The expression of secondary granule gene mRNAs in the bone
marrow RNA is severely reduced (NC) or absent (LF, NG, MCLP, and B9) in
the C/EBP-deficient mouse.15 In addition, NGAL levels were substantially reduced (Figure 1).
Numerous in vitro studies indicate that the different C/EBP family
members have the ability to bind the same DNA sequences and to activate
transcription. This suggests the possibility of a functional redundancy
among family members. To determine whether levels of expression of
other members were altered in the bone marrow of C/EBP-null mice, we performed Northern blot analysis for C/EBP, C/EBP, and C/EBP. In each case, these family members were expressed at levels equal to or
higher than (for C/EBP) those found in the wild-type mouse (Figure
1). These results indicate that the presence of other family members is
not sufficient to compensate for the loss of C/EBP. This supports
the hypothesis that C/EBP, and not the other C/EBP family members,
is required for the expression of those secondary and tertiary granule
genes that are missing in humans and mice lacking functional
C/EBP.
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| Figure 1.
Bone marrow of C/EBP-deficient mice expresses mRNAs
encoding C/EBP, C/EBP, and C/EBP but lacks mRNA expression of
neutrophil secondary granule proteins.
Northern blot of total RNA (5 µg) from one wild-type (WT) and 2 C/EBP-null (KO-1 and KO-2) mice. The blot was hybridized
sequentially with probes for NGAL, C/EBP, C/EBP, C/EBP, and
-actin.
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The bone marrow of C/EBP-deficient mice lacks mRNA expression for
eosinophil granule proteins. The severe reduction of eosinophils in the
bone marrow and peripheral blood of C/EBP-deficient
mice3 and the lack of eosinophil granule protein
expression in SGD patients36 suggested that eosinophil
granule gene expression may be impaired in the C/EBP-deficient mice.
Northern blot analysis of total bone marrow RNA from wild-type and
C/EBP-deficient mice revealed an absence of MBP and eosinophil
peroxidase (EPX) mRNA expression, but normal expression of
myeloperoxidase (MPO) mRNA (Figure 2A). Because the MPO and EPX mRNAs are similar in size and sequence and are
detected by the same probe in Northern hybridizations, RT-PCR analysis
of the samples was performed with primers specific for either MPO or
EPX. As observed by Northern blot analysis, the expression of EPX mRNA
was severely reduced or absent in the C/EBP-deficient mice, whereas
MPO expression was not (Figure 2B). The expression of MBP and EPX was
not reduced in the bone marrow of C/EBP/ mice (data
not shown). These data indicate that C/EBP is required for
eosinophil granule gene expression.
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| Figure 2.
Bone marrow of C/EBP-deficient mice lacks mRNA
expression for the eosinophil granule proteins MBP and EPX.
(A)
Northern blot analysis of total RNA (5 µg) from one wild-type (WT)
and 2 C/EBP-null (KO-1 and KO-2) mice. The blot was hybridized
sequentially with probes MBP, EPX, and -actin. (B) RT-PCR analysis
of MPO and EPX gene expression in the total bone marrow RNA from one
wild-type and 2 C/EBP-null mice. PCR of cDNAs was performed with
primers specific for either MPO or EPX (35 cycles). Products were
electrophoresed on a 2% agarose gel, Southern blotted, and hybridized
with radiolabeled, gene-specific oligonucleotides (Table 1).
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Generation of stable NIH 3T3 cell lines capable of zinc-inducible
expression of C/EBP32 and C/EBP
The overexpression of C/EBP or C/EBP in several leukemia
cell lines promotes granulocytic differentiation.31,37
This, in turn, leads to induction of a number of myeloid-specific genes that may or may not be direct targets of the transcription factors. In
addition, hematopoietic cell lines express transcription factors important in the regulation of myeloid-specific genes, thus making it
difficult to discern the contribution of these other transcription factors in cooperative activation. Myeloid-specific gene expression was
induced in avian fibroblasts by the coexpression of exogenous NFM
(nuclear factor myeloid, also known as avian C/EBP) and
MYB.38,39 This occurs in the absence of differentiation
and eliminates the induction of genes that occurs during this process.
We reasoned that a similar phenomenon would occur in a mammalian
fibroblast cell line such as NIH 3T3. To determine whether secondary
granule genes are direct targets of C/EBP or C/EBP in a mammalian
system, we established cells with stable, inducible expression of each
transcription factor (Figure 3). Levels
of C/EBP and C/EBP protein were undetectable in the empty vector (pMT) cells in the presence or absence of zinc induction (Figure 3A,
lanes 1-2). In contrast, expression of either the
C/EBP32 or C/EBP protein was readily detected after
24 hours of zinc treatment in the pMT-32 and pMT-
stably transformed cell lines (Figure 3A, lanes 3-6). Northern blot
analysis indicated that comparable levels of C/EBP and C/EBP mRNA
were expressed on incubation with ZnSO4 (Figure 3B).
Expression of C/EBP mRNA levels appeared slightly higher than
C/EBP, with some leaky expression detected in the absence of zinc
induction (Figure 3B, lanes 5-6). These results indicate that the
stable NIH 3T3 cell lines express comparable levels of C/EBP or
C/EBP in an inducible fashion.
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| Figure 3.
Induction of neutrophil secondary granule protein mRNAs in stably
transformed NIH 3T3 cell lines expressing inducible human
C/EBP32 and rat C/EBP.
(A) Western blot analysis of NIH 3T3 cell lines stably transformed with
either empty (pMT), C/EBP32, or C/EBP containing
zinc-inducible expression vector. Expression of C/EBP32
and C/EBP was determined for cells incubated in the absence () or
presence (+) of 0.1 mM ZnSO4 for 24 hours (lanes 1-6).
COS-1 cells transfected with the same expression vectors and incubated
with 0.1 mM ZnSO4 for 24 hours were included as negative
and positive controls (lanes 7-9). (B) Northern blot analysis of total
RNA prepared from the stable NIH 3T3 cell lines incubated in the
absence () or presence (+) of 0.1 mM ZnSO4 for 48 hours.
The blot was hybridized sequentially with probes for C/EBP and
C/EBP, B9, and -actin. (C) Stable NIH 3T3 cell lines were
incubated in the absence () or presence (+) of 0.1 mM
ZnSO4 for 48 hours. Total RNA was harvested and subjected
to RT-PCR analysis for the secondary granule genes B9 (33 cycles), MCLP
(35 cycles), NGAL (33 cycles), NC (33 cycles), and the control gene
GAPDH (25 cycles). Total RNA from undifferentiated 32Dcl3 cells was
included as a positive control, but GAPDH expression was not determined
(lane 7).
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Activation of neutrophil secondary granule gene expression in
NIH 3T3
To determine the ability of C/EBP and C/EBP to activate gene
expression of secondary granule genes, the stable cell lines were
incubated in the absence or presence of ZnSO4 for 48 hours. Total RNA was subjected to RT-PCR analysis for genes that are deficient
in the bone marrow of the C/EBP-null mice. Both C/EBP and
C/EBP induced expression of MCLP, NGAL, and NC (Figure 3C). Interestingly, at 30 to 33 cycles, B9 was detected only in the C/EBP-expressing cell line (Figure 3C); however, at 35 cycles, low
levels of B9 were observed in the C/EBP stable line (data not
shown). The induction of B9 by C/EBP was detected by Northern blot
analysis as well (Figure 3B). These results demonstrated that using
both RT-PCR and Northern assays, we could detect the expression of
neutrophil-specific genes in a nonhematopoietic cell system.
Differential induction of neutrophil secondary granule genes
mediated by C/EBP family members
The C/EBP family members are all capable of binding the same DNA
site and activating the same promoters in promoter-reporter assays. To
compare the ability of the different C/EBP family members to activate
secondary granule gene expression in NIH 3T3, cells were transiently
transfected with expression vectors either for C/EBP32,
C/EBP30, C/EBP, C/EBP, or C/EBP. RT-PCR
analysis for the genes MCLP, NC, or B9 was performed (Figure 4, top
panels). Western blot analysis of the
proteins extracted from the organic phase of the TRIzol lysate prepared
from the transfected cells was performed to demonstrate that the
respective proteins were expressed (Figure 4, lower panels). A cDNA
prepared from 32Dcl3 cells served as a positive control (+) and a cDNA
from empty vector transfected cells was used as a negative control
().
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| Figure 4.
Differential
induction of neutrophil secondary granule genes mediated by C/EBP
family members.
(A) NIH 3T3 cells were transiently transfected with 3.0 µg empty
expression vector (), human C/EBP32, or human C/EBP. RT-PCR
analysis for the genes MCLP, NC, or B9 was performed. A cDNA prepared
from 32Dcl3 cells served as a positive control (+). Products were
analyzed on 2% agarose gels, Southern blotted, and hybridized with
radiolabeled, gene-specific probes (Table 1). The lower panel
represents a Western blot of the protein extracted from the organic
phase of the TRIzol lysate prepared from the transfected cells. (B) NIH
3T3 cells were transfected with expression vectors for human
C/EBP32, C/EBP30, C/EBP, and C/EBP
and were analyzed for expression as described above. The lower panel
represents a Western blot simultaneously probed for each family member
of the protein extracted from the organic phase of the TRIzol lysate
prepared from the transfected cells.
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Both C/EBP and C/EBP efficiently induced the expression of B9
(Figure 4A) in a dose-response fashion (data not shown) and weakly
induced MCLP and NC expression (Figure 4A and data not shown). The
expression of MCLP and NC was not as easily detected in the transient
transfections as in the stable transformants (Figure 3), probably
because of the lower efficiency of transient transfections. The p30
isoform of C/EBP, which activates reporter gene expression
several-fold less efficiently than the p32 isoform,40 induced B9 expression less efficiently (Figure 4B). In contrast, C/EBP was unable to induce significantly the expression of any of
the secondary granule genes (Figure 4B). Interestingly, C/EBP was
unable to induce either B9 or MCLP, but it induced NC gene expression
significantly better than the other C/EBP family members (Figure 4B).
All family members induced NGAL expression (data not shown). These
results demonstrate differences in the ability of the C/EBP family
members to activate particular target genes, suggesting a lack of
functional redundancy.
Cooperative transcriptional activation of MBP gene expression in
NIH 3T3 cells by C/EBP32, GATA-1, and PU.1
Overexpression of C/EBP in NIH 3T3 cells alone was unable to
induce MBP expression efficiently (Figure
5). Previous studies demonstrated that
GATA-1 and C/EBP synergistically activate the human MBP-P2
promoter.41 Because MBP expression is deficient in the
bone marrow cells of the C/EBP/ mouse (Figure 2) but
not in the C/EBP/ mouse (data not shown), we
cotransfected C/EBP with GATA-1. A slight increase in MBP levels
over either C/EBP or GATA-1 alone was observed (Figure 5A). We
hypothesized that an additional factor was required for the induction
of high levels of MBP expression. Alignment of the nucleotide sequence
of the murine and human MBP promoter regions revealed
conservation of the C/EBP and GATA-1 sites (Figure
6). In addition, 2 GGAA core sequence
motifs (1 and 2) in a purine-rich region of the promoters indicated a
potential PU.1 binding site (Figure 6). The PU.1 core element 2 was
perfectly conserved between the murine and human promoters, but core
element 1 was not. This suggested the possibility that PU.1 may be
involved in the regulation of MBP gene expression.
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| Figure 5.
Cooperative transcriptional activation of MBP gene
expression in NIH 3T3 cells by C/EBP32, GATA-1, and
PU.1.
(A) NIH 3T3 cells were seeded in 6-well plates at approximately 70%
confluence and were transfected with the combinations of expression
vectors indicated below the graph. The total amount of expression
vector was kept equal in each transfection with the addition of empty
vector. The combinations of expression vectors and the ratio of FOG
expression vector to GATA-1 expression vector are indicated on the
x-axis. At 24 hours after transfection, the cells were lysed in TRIzol
reagent, total RNA was isolated and treated with DNaseI, and cDNA was
synthesized. Quantitative RT-PCR using SYBR Green was performed in
triplicate for each sample. The levels of MBP expression were
normalized to 18S. Results are presented as fold changes (± SD)
compared with cells transfected with empty vector. (B) Southern blot
analysis of the amplification products from single (, , G, and P)
or 3 expression vector (+G+P)-transfected NIH 3T3 cells. PCR was
performed for MBP (33 cycles) and GAPDH (25 cycles). indicates
empty; (), C/EBP; (P), PU.1; (G), GATA-1; (F), FOG.
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| Figure 6.
Conservation of the C/EBP, GATA-1, and PU.1 binding sites in the human
and murine MBP-P2 promoter regions.
Genomic DNA sequences for the human and murine MBP promoter
regions (GenBank accession numbers M34462 and L46768, respectively)
were aligned using the Align X feature of the Vector NTI 6.0 software program (Informax, Bethesda, MD). Nucleotide numbers are
indicated in parentheses. Previously characterized binding sites for
C/EBP and GATA-1 are shaded.30,41 The putative TATA box
and transcription start site (arrow) are indicated. Conserved core
consensus sequences 1 and 2 for PU.1 (GGAA) are shaded. Gaps in the
alignment are denoted by . Conserved nucleotides are indicated
by the consensus sequence.
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To determine whether PU.1 cooperates with C/EBP and GATA-1 in the
activation of MBP expression, combinations of the 3 expression vectors
were transfected into NIH 3T3 cells and the induction of MBP expression
was monitored by RT-PCR. PU.1 alone was unable to induce gene
expression efficiently (Figure 5A-B). However, a striking induction was
observed when C/EBP, GATA-1, and PU.1 were cotransfected (Figure
5A-B). FOG, an inhibitor of MBP gene expression,41 repressed this induction in a dose-response
fashion (Figure 5A). To determine whether this occurred with the human promoter, a MBP promoter-luciferase construct
[pMBP(-117)-LUC]30 was cotransfected with
the same combinations of vectors. The induction of luciferase activity
reflected the results seen with the induction of the endogenous murine
gene (Figure 7). Taken together, these data indicated that the MBP promoter required a combination
of C/EBP, GATA-1, and PU.1 for efficient activation.
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| Figure 7.
Cooperative transcriptional activation of the human
MBP promoter by C/EBP32, GATA-1, and PU.1.
NIH 3T3 cells were cotransfected with combinations of expression
vectors indicated on the x-axis. In addition, a human MBP-P2
promoter-luciferase construct [pMBP(-117)-LUC] was
included in each transfection.30 At 24 hours after
transfection, lysates were prepared, and luciferase activity (RLU) was
measured and normalized to Renilla luciferase to control for
transfection efficiency. The graph represents the average (± SD) of 2 experiments performed in triplicate.
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Mutation of the conserved PU.1 site in the human MBP-P2 promoter
abolishes cooperative transcriptional activation with PU.1
To determine which core element in the conserved PU.1 site was
required for the cooperative activation of the human MBP
promoter, either core 1 (mutant [mt] 1), core 2 (mt 2), or both (mt
1-2) core sequences were mutated in the reporter construct
pMBP(-117)-LUC. Wild-type or mutant reporter constructs
were cotransfected with expression vectors for C/EBP, GATA-1, or
PU.1 alone or in various combinations. The mt 2 and mt 1-2 reporter
constructs lost response to PU.1, but not C/EBP or GATA-1, compared
with the wild-type or mt 1 reporter constructs (Figure
8). In addition, the synergistic activation by all 3 factors was abrogated in the mt 2 and mt 1-2 reporter constructs (Figure 8). These results indicate that the highly
conserved core 2 sequence is required for activation of the
MBP promoter by PU.1. In transfections with expression
vectors for C/EBP or GATA-1, the mt 2 and mt 1-2 reporter constructs activated less efficiently. This may be attributed to the loss of
binding by ETS-like factors found in NIH 3T3 cells to the mutated promoters whereas their binding would occur in the wild-type and mt 1 promoter constructs.
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| Figure 8.
Mutation of the conserved PU.1 site in the human MBP-P2
promoter abolishes cooperation with PU.1.
Mutations were introduced to the PU.1 core elements 1 and 2 of the
pMBP(117)-luciferase construct, as indicated in the figure. These
were cotransfected with various combinations of the expression vectors
for PU.1, GATA-1, and C/EBP32. At 24 hours after
transfection, lysates were prepared and luciferase activity (RLU) was
measured and normalized to renilla luciferase to control for
transfection efficiency. The graph represents the average (± SD) of 2 experiments performed in triplicate.
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The loss of response to PU.1 was predicted to result from the loss of
PU.1 binding to the promoter. Electrophoretic mobility shift assay
(EMSA) revealed that PU.1 expressed in COS-1 cells was able to bind to
its consensus site and that this binding was blocked by the addition of
anti-PU.1 antibody (Figure 9, left panel). The binding of PU.1 to the
consensus site was competed by the unlabeled wild-type consensus and
the wild-type MBP PU.1 site, but not when the PU.1 sites were mutated
in either of these oligonucleotides (Figure 9, middle panel). Finally,
PU.1 binding to the wild-type MBP PU.1 site was detected in COS-1
extracts expressing PU.1, and this binding was blocked by the addition of anti-PU.1 antibody (Figure 9, right panel). These data demonstrate the binding of PU.1 to the predicted site in the MBP promoter and,
together with the reporter construct data, indicate that the loss of
promoter activation by PU.1 results from a lack of PU.1 binding to the
mutated core 2 site.
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| Figure 9.
PU.1 binds the predicted site in the MBP-P2 promoter.
COS-1 cells were transfected with empty () or PU.1 (+) expression
vector, and total cell extracts were prepared 24 hours after
transfection. Wild-type consensus PU.1 (left and middle panels;
5'-gggctgcttgaggaagtataagaat-3') or wild-type MBP PU.1
(right panel;
5'-tctccctgggggaagttcctccaaggcc-3')
double-stranded oligonucleotides were end-labeled with
[32P]- adenosine triphosphate (ATP). Labeled
oligonucleotides (0.02 pmol) were incubated with 5 µg total cellular
extract in the presence or absence of anti-PU.1 (1 µg) antibody (left
and right panels) or unlabeled (0.2 pmol) wild-type or mutant
oligonucleotides (consensus,
5'-gggctgcttgagagagtataagaat-3'; MBP PU.1,
5'-tctccctgggcaaagtttgtccaaggcc-3'). Arrows at
the right of each panel indicate the positions of the complexes
containing PU.1. Wt indicates wild-type; Mt, mutant; CON, consensus
PU.1 binding site; MBP, major basic protein promoter PU.1 binding site;
Ab, anti-PU.1 antibody (sc-352; Santa Cruz Biotechnology).
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Eosinophil granule gene expression is absent in
PU.1-deficient cells
To determine the role of PU.1 in the expression of eosinophil
granule genes, levels of MBP and EPX mRNA were determined in a myeloid
cell line derived from the embryonic liver cells of PU.1-deficient
mice.8,9 Like the bone marrow of C/EBP-deficient mice,
these cells lacked expression of neutrophil secondary granule mRNAs
such as LF, NG,8 and B9 (Figure 10, lanes
1-3), but they expressed primary
granule mRNAs including MPO and NE8 (Figure 10, lanes
1-3). Expression of the eosinophil granule mRNAs MBP and EPX was
deficient in the PU.1-null cell line (Figure 10, lane 3). When PU.1
expression was reinstated by retroviral transduction, neutrophil9 and eosinophil granule gene expression was
restored (Figure 10, lanes 4-5). This was not observed on the
restoration of M-CSF receptor expression by retroviral transduction
(Figure 10, lane 6). These results indicate that both C/EBP and PU.1
are important for the expression of eosinophil granule proteins in the mouse.
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| Figure 10.
Lack of eosinophil granule gene expression in a
PU.1-null myeloid cell line derived from the embryonic liver of the
PU.1/ mouse.
The cDNAs were prepared from total RNA from the bone marrow of
wild-type and C/EBP/ mice, a PU.1-null cell line, 2 PU.1-null cell lines with restored PU.1 expression, and one with
restored M-CSFR expression. Primers specific for the primary granule
gene NE, the secondary granule gene B9, and the eosinophil granule
genes MBP and EPX were used (35 cycles for all). Products were analyzed
by electrophoresis through a 2% agarose gel and ethidium bromide
staining. The 18S rRNA was amplified (15 cycles) as a control for the
balance and integrity of the cDNAs.
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Discussion |
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Abstract
Introduction