From the Department of Cell Biology & Biochemistry,
Texas Tech University Health Sciences Center, Lubbock, Texas 79430, the ¶ Department of Microbiology, Michigan State University,
East Lansing, Michigan 48824-1101, the
Laboratory of
Leukocyte Biology-Division of Basic Science, ** Intramural Research
Support Program, Scientific Applications International Corporation,
Frederick, Maryland 21702-1201, and the
Advanced Biosciences Laboratories-Basic
Research Program, National Cancer Institute-Frederick Cancer Research
and Development Center, Frederick, Maryland 21702-1201
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ABSTRACT |
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C/EBP is a member of the CCAAT/enhancer
binding protein family of basic region/leucine zipper transcriptional
activators. The C/EBP
protein is highly conserved between rodents
and humans, and its domain structure is very similar to C/EBP
. In
mice C/EBP
mRNA is only detected in hematopoietic tissues,
including embryonic liver and adult bone marrow and spleen. Within the
hematopoietic system, C/EBP
is expressed primarily in myeloid cells,
including promyelocytes, myelomonocytes, and their differentiated
progeny. To identify potential functions of C/EBP
, cell lines
over-expressing the C/EBP
protein were generated in the P388
lymphoblastic cell line. In contrast to the parental cell line,
C/EBP
-expressing cell lines displayed lipopolysaccharide-inducible
expression of the interleukin-6 and monocyte chemoattractant protein 1 (MCP-1) genes as well as elevated basal expression of the MIP-1
and
MIP-1
chemokine genes. In the EML-C1 hematopoietic stem cell line,
C/EBP
mRNA levels increased as the cells progressed along the
myeloid lineage, just preceding activation of the gene encoding the
receptor for macrophage-colony-stimulating factor (M-CSFR). M-CSFR
expression was stimulated in C/EBP
-expressing P388 cell lines, when
compared with either the parental P388 cells or P388 cell lines
expressing either C/EBP
or C/EBP
. These results suggest that
C/EBP
may be an important regulator of differentiation of a subset
of myeloid cell types and may also participate in the regulation of
cytokine gene expression in mature cells.
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INTRODUCTION |
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The CCAAT/enhancer binding protein
(C/EBP)1 family of
transcription factors consists of four highly related members, named C/EBP, C/EBP
, C/EBP
, and C/EBP
, along with the less closely related Ig/EBP (C/EBP
) and CHOP (gadd153) proteins (reviewed in Ref.
1). The C/EBP proteins are basic region/leucine zipper transcription
factors that bind to the consensus sequence 5' ATTGCGCAAT 3' and
variants thereof (2). Individual members of this family have been
implicated as regulators of differentiation in multiple cell types,
including hepatocytes, adipocytes, and certain blood cell types. In the
hematopoietic system, C/EBPs are expressed primarily in myeloid cells,
including monocytes, macrophages, granulocytes, and their precursors
(3, 4). C/EBP
mRNA has been detected in primitive
(CD34+ and CD38
) progenitor cells, and its
expression is specifically up-regulated when these cells are induced to
differentiate along the granulocytic lineage but not the monocytic
lineage (5). Analysis of granulocytic differentiation of the 32DCl.3
hematopoietic progenitor cell line suggested that C/EBP
acts prior
to the terminal stages of differentiation in this cell lineage (4). In
support of this hypothesis, C/EBP
nullizygous mice lack mature
granulocytes and contain large numbers of immature myeloid blast cells
in their blood (6).
Expression of both C/EBP and C/EBP
is up-regulated during
terminal differentiation of cells in both the granulocytic and monocytic lineages, and C/EBP
appears to be the predominant C/EBP protein in mature granulocytes and macrophages (3, 4).
Differentiation-specific functions for C/EBP
and C/EBP
have not
been clearly defined as yet, although both are capable of collaborating
with wild type and oncogenic forms of the Myb transcription factor to
activate macrophage-specific genes in heterologous cell types (7). The primary functions of C/EBP
and C/EBP
within the hematopoietic system appear to be in the regulation of a number of cytokine and other
genes during inflammatory responses (3, 8-11). Mice carrying a null
mutation in the C/EBP
gene display essentially normal myeloid
development but exhibit marked defects in bacterial killing and
cytostatic functions (12, 13). Despite the fact that C/EBP
has been
proposed to be a critical regulator of cytokine gene expression in
activated macrophages, LPS-dependent induction of cytokine
gene expression was essentially identical in macrophages isolated from
wild type and mutant mice. The relatively normal pattern of cytokine
gene expression in C/EBP
null mice is likely to be due to functional
redundancy among family members (14).
A detailed molecular analysis of the roles of individual C/EBP proteins
over the entire spectrum of myeloid differentiation has been hampered
by technical difficulties in isolating and culturing progenitor cells
from bone marrow and by the transformed phenotype of most immortalized
cell lines. An alternative approach is now possible with the recent
establishment of an immortalized hematopoietic progenitor cell line,
named EML-C1 (15). The EML-C1 cell line was derived by infecting mouse
bone marrow cells with a retrovirus expressing a dominant negative
retinoic acid receptor molecule and proliferates in culture in the
presence of stem cell factor (SCF). The dominant negative retinoic acid
receptor molecule specifically blocks differentiation along the myeloid
lineage; however, the addition of relatively high levels of retinoic
acid (10
5 M) overcomes this block and induces
granulocytic and monocytic differentiation. Consequently, using the
EML-C1 cell line, it is possible to determine the temporal pattern of
expression of any gene over the entire myeloid differentiation process
and to predict functions for genes of interest based on this
information.
We previously reported the cloning and partial characterization of a
gene encoding rat C/EBP, then named C/EBP-related protein 1 (CRP1)
(16). We have now completed the characterization of the C/EBP
gene
and report that it encodes a 281-amino acid transcriptional activator
that binds to the same DNA target as other C/EBPs. C/EBP
is
expressed almost exclusively in hematopoietic tissues in mouse, specifically in myeloid cells, and its expression is up-regulated during myeloid differentiation of the EML-C1 cell line. Ectopic expression of C/EBP
confers novel transcriptional properties on a
heterologous, lymphoid cell line, including the ability to support
LPS-stimulated expression of multiple cytokine genes and activation of
the gene encoding the receptor for macrophage-colony-stimulating factor.
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EXPERIMENTAL PROCEDURES |
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Cloning and Sequencing--
Isolation of the rat C/EBP
genomic clone was described previously (16). A mouse C/EBP
cDNA
was isolated by PCR amplification of cDNA generated from total RNA
isolated from the P388D1(IL-1) macrophage cell line using the amplimers
CRP1 ATG-4 (GACGGCCCATGGCCCACGGGACCTAC) and S1PCRp1
(GACGGCAAGCTTGGCTCAGCTGCAGCCCCC). This PCR product was subsequently
used to screen a mouse 129SV genomic library (Stratagene, La Jolla,
CA), and nine positive clones were isolated which together spanned 18 kilobase pairs of the C/EBP
locus, including the complete coding
sequence. DNA sequencing was carried out using the Sequenase kit
(Amersham Pharmacia Biotech) under standard conditions, except that in
double-stranded sequencing reactions, nonspecifically terminated
fragments were extended by incubation in the presence of terminal
deoxynucleotidyltransferase and all four nucleotides prior to addition
of the stop solution. DNA sequences were analyzed using the SEQED,
BESTFIT, PILEUP, and GELASSEMBLE programs of the University of
Wisconsin Genetics Computer Group package.
Total RNA Isolation and Northern Analysis--
Total RNA was
prepared from tissues and cells using a modified guanidine
isothiocyanate/phenol extraction procedure and analyzed by Northern
blotting and hybridization as described previously (17). The C/EBP
probe was an 850-bp NcoI/HindIII fragment
containing the complete murine cDNA, and the MCP-1 probe was a
580-bp murine cDNA (18).
Reverse Transcription-PCR-- RT-PCR was carried out essentially as described (19). Briefly, 2 µg of total RNA was reverse-transcribed using 0.2 µg of oligo(dT)18 and 400 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) in a total volume of 50 µl for 1 h at 37 °C. 3 µl of the RT reaction was then mixed with 1 µl of 10× Buffer (500 mM Tris-HCl (pH 8.3), 2.5 mg/ml crystalline bovine serum albumin, and MgCl2 at 10, 20, or 30 mM (Idaho Technology Inc., Idaho Falls, ID)), 0.2 mM each dNTP, 150 ng of each primer, and 1.25 units of Taq DNA polymerase (Fisher) in a total volume of 10 µl. Reactions were loaded into a capillary tube, and PCR cycles were carried out using the Rapidcycler Thermal cycler (Idaho Technology). Annealing temperatures and Mg2+ concentrations were initially optimized for each primer pair. In most cases, PCR was carried out at 2 mM MgCl2 with the following cycle parameters: 94 °C, 15 s, 1 cycle; 94 °C, 0 s, 60 °C, 0 s, 72 °C, 25 s, 30 cycles; 72 °C, 30 s, 1 cycle. Reaction products were visualized in ethidium bromide-stained agarose gels, and images were captured as PICT files using UVP ImageStore 7500 Gel Documentation software, cropped in Adobe Photoshop version 3.0.4, and annotated using Macromedia Freehand version 7.0. Accuracy and linearity of RT-PCR results was confirmed by varying input cDNA concentration, cycle number, and by comparing results with those from quantitative Northern blots (see Fig. 2 for example). The primers used in this study are shown in Table I.
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Cell Culture--
P388, P388D1(IL-1), M1, WEHI-3, and IC-21 cell
lines were maintained in RPMI 1640 (Mediatech, Herndon, VI)
supplemented with 5% fetal bovine serum (Hyclone, Logan, UT),
penicillin, and streptomycin. 32DCl.3, FDCP-1, NFS-58, and DA-3 cell
lines were cultured in the same medium supplemented with 20% WEHI-3
conditioned medium as a source of IL-3. M-NFS-60 cells were maintained
in RPMI containing 10% fetal bovine serum supplemented with 10% L
cell conditioned medium as a source of M-CSF. GG2EE and WEHI-274.1 were
maintained in RPMI containing 10% fetal bovine serum. MPRO cells were
cultured in Dulbecco's modified Eagle's medium containing 20 ng/ml
granulocyte/macrophage-colony-stimulating factor (PeproTech, Rocky
Hill, NJ). EML and EPRO cells were maintained in Iscove's modified
Dulbecco's medium containing 20% horse serum and 200 ng/ml SCF
(PeproTech) or 20 ng/ml granulocyte/macrophage-colony-stimulating factor, respectively. Myeloid differentiation of EML-C1 cells was
initiated by washing and replating EML cells at 1 × 105 cells per ml in Iscove's modified Dulbecco's medium
containing 20% horse serum, IL-3, SCF and 1 × 105
M retinoic acid (Sigma) for 6 days. Cultures were harvested
at various time points for RNA preparation. For LPS inductions, cells were washed twice with phosphate-buffered saline and fed with fresh
medium containing 20 µg/ml LPS (Escherichia coli serotype 026:B6, Sigma).
Purification of Primary Blood Cells-- Normal murine bone marrow cells were aspirated from BALB/c mouse femurs with Iscove's modified Dulbecco's medium. Animal care was provided in accordance with the procedures outlined (43). Granulocytes and erythroid cells were purified by fluorescence-activated cell sorting from normal BM cells stained with GR-1-FITC (20-30% of BM) or TER-119-FITC (20-30% of BM) antibodies, respectively, with their isotype matched controls to gate for brightly stained cells. B cells were purified by fluorescence-activated cell sorting from a cell suspension made from spleens stained with B220-FITC antibodies and an isotype matched control to gate for positively stained cells. T cells were purified by fluorescence-activated cell sorting from a cell suspension made from thymus stained with CD-3-FITC antibodies and an isotype matched control. Bone marrow derived macrophages were purified as described (14).
Western Blotting and Electrophoretic Mobility Shift Assays
(EMSA)--
Total cellular protein was prepared from selected cell
lines, and Western blotting was performed as described previously (17). Western blots were probed with a C/EBP-specific antiserum (C-22, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Preparation of nuclear
extracts and EMSA procedures have been described previously (20), and
the oligonucleotide probe used here contains a consensus C/EBP
DNA-binding site. Supershift assays were carried out by preincubating
the nuclear extract with a rabbit polyclonal C/EBP
-specific antiserum directed against amino acids 266-279 of the rat
polypeptide.
Plasmid Construction--
The complete C/EBP coding sequence,
including the intron, was amplified from the rat genomic clone by PCR
using two oligonucleotide primers (CRP1 ATG-4 and S1PCRp1, see above)
that overlap the initiation codon and termination codon, respectively.
The resultant PCR product was digested with NcoI and
HindIII (incorporated as part of the amplimers) inserted
into the pMEX eukaryotic expression vector (16) to generate
pMEXC/EBP
p34. The amplified region was sequenced to check for errors
and was shown, after transient transfection into a number of cell
types, to direct the synthesis of a 34,000 molecular weight protein
that was recognized by a C/EBP
-specific antiserum (data not shown).
pMEXC/EBP
p34 was digested with HindIII, and the 5'
overhangs were made blunt using Klenow, and a BamHI linker
was added. This construct was then digested with BamHI, and
the full coding sequence was inserted into the pSV(x) expression vector
(20) to generate pSV(x)C/EBP
p34. Inserted genes in pSV(x) are
expressed from the Moloney murine leukemia virus long terminal repeat
with a subgenomic splicing product expressing the neomycin resistance
gene for selection of stably transfected cell lines.
Generation of C/EBP Expressing P388 Cell
Lines--
106 P388 cells were transfected with 5 µg of
pSV(x)C/EBP
p34 expression vector using 40 µg of Lipofectin (Life
Technologies, Inc.) as described previously for the generation of
C/EBP
expressing P388 cell lines (20). Selection was carried out at
three concentrations of G418 (150, 250, and 350 µg/ml), and four
independent pools of resistant cells (named P388-C
-1 to C
-4) were
established. Expression of the C/EBP
transgene was confirmed by
RT-PCR, Western blotting, and EMSA.
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RESULTS |
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Characterization of the C/EBP Gene--
The sequence of 2391 bp
of a rat C/EBP
genomic clone is presented in Fig.
1A along with the predicted
peptide sequence of the C/EBP
protein. The C/EBP
gene contains a
single intron with two potential splice acceptor sites (indicated as
underlined AG dinucleotides). Sequencing of RT-PCR products generated
using C/EBP
-specific primers confirmed that the more 5' splice
acceptor sequence is apparently exclusively utilized, at least in
hematopoietic cells (see below). Examination of the 5' end of the
C/EBP
coding sequence revealed an in frame translation initiation
codon located 96 bp upstream of the AUG codon described previously
(16). Consequently, conceptual translation of the longest open reading
frame predicts that the full-length C/EBP
protein is 281, rather
than 249, amino acids in length. The 5' end of C/EBP
mRNA was
determined by sequencing of 5'-RACE products generated from adult mouse
spleen cDNA. Several potential start sites were identified;
however, only three of these appeared in multiple subclones and are
included in Fig. 1A. These sites are likely to be bona fide
start sites as they map close to start sites identified for the P
promoter of the human C/EBP
gene (21). The sequences around the cap
sites are purine-rich and lack common promoter elements such as TATA
and CCAAT boxes, a feature shared with many genes expressed in myeloid cells.
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C/EBP Is Expressed in Hematopoietic Tissues--
Previously, we
were unable to detect C/EBP
mRNA in a number of adult mouse
tissues, suggesting that C/EBP
is expressed in a limited temporal or
spatial pattern or in tissues omitted from our original survey. In an
expanded Northern blot survey, we detected a strong signal at
approximately 1.8 kilobase pairs in RNA prepared from adult bone marrow
(Fig. 2A, lane 5), with weak
signals in embryonic liver and adult spleen (lanes 1 and
3). Because C/EBP
appears to be expressed at low levels,
even in positive tissues, we used reverse transcription-PCR (RT-PCR) to
carry out a more extensive survey. Primers were chosen to flank the
single intron within the C/EBP
gene to ensure that PCR products
generated from cDNA could be distinguished from products
synthesized from a genomic template. In agreement with the Northern
analysis, C/EBP
mRNA was detected in embryonic liver and adult
bone marrow and spleen, each of which are hematopoietic tissues.
C/EBP
mRNA was essentially absent from all other tissues
analyzed although low levels were detected in adult lung, which may be
due to infiltration of this tissue with blood cells. In this and all
subsequent RT-PCR assays, a single amplified product was observed which
corresponds to a spliced mRNA utilizing the 5' splice acceptor site
described above.
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C/EBP Is Primarily Expressed in Myeloid Cells--
By having
shown that C/EBP
is expressed in hematopoietic tissues, and at
highest levels in bone marrow, RNA from a panel of rodent cell lines
was analyzed by RT-PCR to determine which cell types within the
hematopoietic system express C/EBP
(Fig. 3). C/EBP
mRNA was detected in 12 out of 13 cell lines of myeloid origin, the lone exception being the
immature myeloid FDCP-1 cell line (Fig. 3A). The highest
level of C/EBP
mRNA was detected in cell lines arrested at the
promyelocyte stage of myeloid development (EPRO and MPRO) and two
IL-3-dependent myeloid cell lines (NFS-58 and DA-3),
whereas lower levels were detected in two myelomonocytic leukemic cell
lines (M1 and WEHI-3). C/EBP
mRNA was also detected in five
monocytic/macrophage cell lines including M-CSF-dependent M-NFS-60 cells and the factor-independent cell lines WEHI-274.1, GG2EE,
P388D1(IL-1), and IC-21. C/EBP
mRNA was not detected in cell
lines representative of erythroid, mast cell, basophil, or T cell
lineages and was present in only one cell line (P388) derived from the
B cell lineage (Fig. 3B). As further confirmation of the
restricted pattern of expression of C/EBP
, RNA was prepared from
purified populations of primary murine blood cells (Fig. 3C). High levels of C/EBP
mRNA were detected in
granulocytes and macrophages but not in B cells, T cells, or
erythrocytes.
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Establishment of C/EBP-expressing P388 Cell Lines--
P388 is
a murine B lymphoblastic cell line that does not express detectable
levels of any C/EBP proteins, despite the presence of C/EBP
mRNA
(see Fig. 4 and below). This cell line was previously used to analyze
the functions of C/EBP
, C/EBP
, and C/EBP
(14, 20). We
established four G418-resistant lines using a C/EBP
-expression vector. C/EBP
mRNA levels in three of the four cell lines
(P388-C
-1, C
-3, and C
-4) were elevated compared with those in
the control P388-Neo cell line as assessed by RT-PCR (Fig.
5A) and Northern blotting
(data not shown). In addition, C/EBP
mRNA levels were unchanged
in C/EBP
-expressing P388-C
cells (Fig. 5A, compare lanes 1 and 2) or in P388 cell lines expressing
either C/EBP
or C/EBP
(data not shown), consistent with our
previous findings that overexpression of one C/EBP protein in P388
cells does not activate expression of other family members (14). To
test whether the elevated mRNA levels resulted in expression of
C/EBP
protein in these cell lines, an EMSA was performed using a
radiolabeled oligonucleotide containing a consensus C/EBP-binding site,
and a representative result showing P388-C
-3 is shown in Fig.
5B. A number of protein-DNA complexes were observed in
nuclear extracts from control P388-Neo cells; however, none were
affected by addition of a C/EBP
-specific antiserum (compare
lanes 1 and 2) or antisera recognizing other
C/EBP family members (14). Two new complexes were observed using
nuclear extracts from P388-C
-3 cells (open arrowheads, lane
3). The upper complex was completely supershifted by addition of
C/EBP
antiserum (closed arrowhead, lane 4) and, based on
its co-migration with bacterially produced, recombinant C/EBP
(data
not shown), appears to correspond to a C/EBP
homodimer. The mobility
of the lower complex, which presumably represents a heterodimer with an
unidentified C/EBP partner (20, 22), was only slightly affected by the
C/EBP
antiserum. Similar binding patterns were observed in both
P388-C
-1 and P388-C
-4 (data not shown).
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C/EBP Participates in LPS-regulated Expression of IL-6 and MCP-1
Genes--
The expression of a number of inflammatory cytokine genes
is up-regulated following lipopolysaccharide treatment of macrophage cell lines such as P388D1(IL-1) while these genes are not normally induced in P388 lymphoblasts. To test whether C/EBP
might
participate in the regulation of these genes, cultures of each of the
cell lines described above were exposed to LPS for 18 h, and RNA
was prepared and analyzed by RT-PCR using oligonucleotides specific for
MCP-1 and IL-6. IL-6 and MCP-1 mRNAs were barely detectable in
control P388-Neo cells and were essentially unresponsive to LPS
stimulation (Fig. 6A, lanes 1 and 2). Basal expression levels of IL-6 or MCP-1 were
elevated in each C/EBP
-expressing cell line with the relative level
of target gene mRNA being approximately proportional to the level
of C/EBP
mRNA and protein (i.e. higher in lanes
5, 9, and 11 than in lane 7).
C/EBP
-expressing lines also displayed LPS-dependent
increases in both cytokine mRNAs, a feature that is most evident in
the P388-C
-2 cell line for IL-6 (lane 8) and P388-C
-2,
C
-3, and C
-4 for MCP-1 (lanes 8, 10, and
12). Finally, in support of our previous results (20), both
genes were also induced in the P388-C
cell line (lanes 3 and 4).
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Ectopic C/EBP Expression Increases Basal Level Expression of
MIP-1
and MIP-1
--
Having shown that C/EBP
was capable of
regulating genes previously identified as C/EBP
target genes, we
attempted to identify unique target genes for C/EBP
using the P388
cell lines. From a survey of several potential targets, we determined
that both C/EBP
and C/EBP
were capable of activating expression
of the genes encoding the
-chemokines, MIP-1
and MIP-1
(Fig.
7). MIP-1
mRNA was detected at low
levels in control P388-Neo cells but was significantly increased after
exposure to LPS for 2 h (lanes 1 and 2). The
basal level of MIP-1
mRNA was significantly increased in both
P388-C
-4 and P388-C
in comparison to P388-Neo cells but was
relatively unaffected by LPS treatment, a pattern similar to that seen
in P388D1(IL-1) cells (lanes 3-8). MIP-1
mRNA was undetectable in P388-Neo cells and slightly increased by LPS treatment. Again basal rather than LPS-dependent mRNA levels were
primarily affected by ectopic C/EBP
and C/EBP
, although levels
did not approach those observed in P388D1(IL-1) cells (compare
lanes 3-6 with 7 and 8). Similar
results were obtained in four independent experiments.
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Regulation of C/EBP Expression during Myeloid
Differentiation--
Having shown that C/EBP
is capable of
activating the expression of genes expressed primarily in mature cells,
we next examined whether it might also play a role in regulating gene
expression during myeloid differentiation. As a first step, we
determined the temporal pattern of C/EBP
expression during myeloid
differentiation using the EML-C1 hematopoietic progenitor cell line.
EML-C1 cells were incubated in the presence of SCF, IL-3, and retinoic
acid, which induces their differentiation along both the macrophage and
neutrophil lineages. Cells were harvested for RNA preparation at
various time points over a 6-day period, and C/EBP
mRNA levels were determined by RT-PCR (Fig. 8). Low
levels of C/EBP
mRNA were detected in the uninduced parental
cells and remained low during the first 8-16 h after retinoic acid
addition. C/EBP
mRNA levels began to increase after
approximately 16 h and reached maximal levels at the 120-h time
point, which were similar to those seen in MPRO promyelocyte cells. A
similar pattern of C/EBP
expression has been observed in multiple
independent induction experiments and was found to be unaffected by the
use of either pure recombinant SCF or conditioned medium from an
SCF-overexpressing cell line (data not shown). The low level of
C/EBP
mRNA at early time points may be explained either by weak
basal expression of C/EBP
in hematopoietic progenitor cells or by
the presence of a small number of myeloid precursors that escape the
dominant negative retinoic acid receptor block. However, because the
initial rapid proliferation of EML-C1 cells slows and appearance of
myeloid progenitors occurs around 24 h after retinoic acid
addition, the increase in C/EBP
at this time suggests that it may
activate the expression of genes required for establishment of certain myeloid cell lineages.
|
M-CSFR Expression Is Specifically Activated by
C/EBP--
Based on their similar expression patterns, and the
fact that the promoter of the M-CSFR gene is known to contain a
functional C/EBP-binding site (23), we tested whether M-CSFR expression was affected by ectopic C/EBP
expression in P388 cells. For
comparison, we analyzed the P388-C
cell line described above and
four independent cell lines expressing C/EBP
(P388-C
-1 to
C
-4). M-CSFR mRNA levels were assessed by RT-PCR using RNA
prepared from unstimulated cells (Fig.
9). M-CSFR mRNA was undetectable in
control P388-Neo cells but, in two independent experiments, was present
in each of the four C/EBP
-expressing cell lines at levels
proportional to the level of C/EBP
mRNA (see Fig.
6A), albeit at lower levels than in P388D1(IL-1) cells. This
effect was essentially specific to C/EBP
as M-CSFR mRNA levels
were only slightly elevated over background levels in the P388-C
or
P388-C
cell lines, despite the fact that both cell lines attain the
ability to activate other C/EBP-dependent genes (see Figs.
7 and 8 and Ref. 14).
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DISCUSSION |
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Our understanding of the molecular mechanisms controlling the
development of mature blood cells from uncommitted progenitor cells has
greatly advanced over the past few years with the identification of
transcription factors that display temporally and spatially restricted
patterns of expression and which are, in many cases, absolutely
required for the development of specific cell types or lineages (24).
Three members of the C/EBP family, C/EBP, C/EBP
, and C/EBP
,
are expressed primarily, but not exclusively, within the myeloid
lineage in the hematopoietic system (3, 4, 25) and function both in the
maturation of specific cell types and in the activation of cytokine
gene expression in macrophages. In this report, we show that a fourth
member of the C/EBP family, named CRP1 or C/EBP
, is also expressed
in the hematopoietic system. Apart from sharing with C/EBP
the
ability to confer lipopolysaccharide-responsive expression of certain
cytokine genes onto a lymphoid cell line, C/EBP
is also capable of
activating the expression of the M-CSFR gene. This finding indicates
that C/EBP
may be required for both the function and development of
macrophages.
Structural Analysis of the C/EBP Gene in Rodents--
Unlike in
humans, where multiple C/EBP
mRNA species have been detected
(21, 26, 27), Northern analysis has revealed the presence of a single
C/EBP
mRNA species of approximately 1.8 kilobase pairs in murine
hematopoietic tissues and cell lines. The simpler pattern in mice
appears to be due to lack of alternate splicing, combined with the use
of a single promoter, albeit one that uses multiple transcriptional
start sites. In addition, despite the existence of internal
translational initiation codons in the C/EBP
coding sequence, we
have only detected a single C/EBP
protein in extracts from murine
cells. This protein is 32 amino acids longer than previously reported
due to the presence of an in frame methionine codon located 96 nucleotides upstream of the site predicted in our earlier study (16).
Although a detailed examination of the structure of the C/EBP
protein has not been reported, our preliminary data indicate that it is
an efficient transcriptional activator, and its domain structure is
very similar to C/EBP
.3
The differential expression of C/EBP
mRNA and protein in P388 cells suggests that expression of the C/EBP
gene may be controlled at the post-transcriptional levels in certain cell types. The mechanism
underlying this mode of regulation is unclear but does not appear to
involve short open reading frames such as those proposed to modulate
translation of both the C/EBP
and C/EBP
mRNAs (28). However,
the fact that a C/EBP
transgene lacking the 5'-untranslated region
is efficiently expressed in stably transfected P388 cells suggests that
other sequences in this region may inhibit C/EBP
translation.
Myeloid Specific Expression and Functions of C/EBP--
The
highly restricted pattern of expression of C/EBP
in both mouse and
human strongly supports the idea that its primary function is within
the myeloid lineage of hematopoietic cells. However, before detailing
the putative myeloid-specific functions of C/EBP
, it may be
worthwhile to consider the possibility that it may also be expressed,
and function, outside the myeloid lineage. For example, C/EBP
mRNA has been detected at low levels in tissues such as mouse lung
or human ovary (27), although these signals could theoretically be due
to the presence of blood cells. To date, there are two exceptions to
the myeloid-specific expression of C/EBP
in hematopoietic cell
lines, namely the human Jurkat T cell line (27) and murine P388
lymphoblasts. We have confirmed the presence of C/EBP
mRNA in
Jurkat cells; however, we have not detected C/EBP
mRNA in any
other human lymphoid cell lines (data not shown), and it is not
expressed at significant levels in the thymus. The C/EBP
gene has
been mapped in both human and mouse and is located on human and mouse
chromosome 14 close to a compound locus containing the T cell receptor
/
genes and other genes expressed in T cells (27, 29). Multiple
rearrangements within this region have been associated with T cell
leukemias or lymphomas in humans, and it is possible that the presence
of C/EBP
mRNA in Jurkat cells is related to the origin or
activation state of this T cell line (30). The significance of C/EBP
mRNA in P388 cells is unclear, particularly as Western analysis,
gel shift assays, and functional tests indicate that C/EBP
protein is not expressed in these cells. However, the strong expression of
C/EBP
in mouse spleen, a major source of B cells, and the reported
existence of a common precursor for both macrophages and B cells (31),
may indicate a role for C/EBP
in B cell development and/or
function.
Redundant Functions of C/EBP Proteins: the Involvement of C/EBP
in Cytokine and Chemokine Gene Expression--
The promoters of many
cytokine genes contain composite DNA elements consisting of binding
sites for C/EBP proteins and NF
B transcription factors that are
required for activation of these promoters during inflammatory
responses (32). Functional cooperativity between C/EBP proteins and
NF
B requires physical interaction between their bZip and Rel
homology domains, respectively (33, 34). A number of lines of evidence,
including the demonstration that ectopic expression of C/EBP
in the
P388 lymphoblastic cell line conferred LPS responsiveness onto the IL-6
and MCP-1 genes (20), suggested that C/EBP
was the primary family
member involved in cytokine gene regulation. However, it now appears
that other family members must be capable of participating in cytokine
gene regulation in vivo because LPS-inducible expression of
most cytokine genes, including IL-6, is not greatly affected in
C/EBP
null mice (13). C/EBP
is a good candidate for performing
these functions in mice because, like C/EBP
, it is capable of
conferring LPS inducibility on both IL-6 and MCP-1 genes in P388 cells.
The generation of mice carrying targeted mutations in multiple C/EBP
genes should address the relative importance of each protein in
cytokine gene regulation.
C/EBP as a Critical Regulator of Macrophage Development:
Activation of M-CSFR Expression--
The specific functions of each
C/EBP protein within the hematopoietic system are rapidly being
determined through the combinatorial use of experimentally
manipulatable cell culture systems and the disruption of C/EBP genes in
mice. From these studies, it has been shown that the C/EBP proteins are
expressed in temporally distinct patterns during myeloid
differentiation and have implicated C/EBP
as a critical regulator of
neutrophil development and C/EBP
as an important factor for
macrophage function (3, 4, 6, 21). The activation of C/EBP
expression in EML-C1 cells at a critical time point during myeloid
differentiation when colony-forming units (granulocyte/macrophage)
first become evident in the culture, along with the coordinate
activation of M-CSFR expression, has led us to predict that C/EBP
may be a critical regulator of macrophage development. This hypothesis
is further supported by the specific activation of M-CSFR gene
expression by C/EBP
in P388 cells, when compared with either
C/EBP
and C/EBP
. The promoter of the M-CSFR gene also contains
binding sites for PU.1, AML-1, and probably other unidentified factors,
and the activation of M-CSFR expression may be dependent upon selective
interactions between C/EBP
and other promoter-bound transcription
factors. Elucidation of the specific functions of C/EBP
during
myelopoiesis should be achieved through analysis of cellular systems
such as EML-C1 in combination with the disruption of the C/EBP
gene
in mice.
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ACKNOWLEDGEMENTS |
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We thank Carrie Cantwell, Nicholas Angerer, and Mauricio Dominguez for excellent technical assistance; Steven Collins and Schickwann Tsai of the Fred Hutchinson Cancer Research Center for the EML-C1 cell line; and Jeannine Lincoln, Curt Pfarr, and Dan Hardy for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants from the Texas Affiliate of the American Heart Association and Texas Tech University Health Sciences Center (to S. C. W.), from the American Cancer Society Grant DB-110, and the Michigan State University Biotechnology Research Center (to R. C. S.), and by the NCI, DHHS, National Institutes of Health, under contract with ABL and SAIC.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF034716.
§ To whom reprint requests should be addressed: Dept. of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 3601 4th St., Lubbock, TX 79430. Tel.: 806-743-2524; Fax 806-743-2990; E-mail: cbbscw{at}ttuhsc.edu.
1 The abbreviations used are: C/EBP, CCAAT/enhancer binding protein; LPS, lipopolysaccharide; CRP1, C/EBP-related protein 1; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; M-CSF, macrophage-colony-stimulating factor; M-CSFR, M-CSF receptor; IL, interleukin; SCF, stem cell factor; EMSA, electrophoretic mobility shift assays; RACE, rapid amplification of cDNA ends; bp, base pair; FITC, fluorescein isothiocyanate.
2 Y. Du and S. C. Williams, unpublished observations.
3 N. Angerer and S. C. Williams, unpublished observations.
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