From the Institute of Anatomy II, Medical School, Friedrich Schiller University, D-07740 Jena, Germany, the § Department of Virology, Medical School Charité, Humboldt University, D-10098 Berlin, Germany, and the ¶ Department of Genome Analysis, Institute of Molecular Biotechnology, D-07749 Jena, Germany
Received for publication, July 24, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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The molecular mechanisms underlying the
regulation of interleukin (IL)-10 transcription in monocytic cells by
various stimuli during inflammation and the stress reaction are not
fully understood. Recently, we provided evidence that stress-induced
IL-10 promoter activation in monocytic cells is mediated by
catecholamines via a cAMP-dependent signaling pathway
including CREB/ATF (cAMP-responsive element binding protein/activating
transcription factor) binding to two CRE motifs. However, the mutation
of these sites diminished cAMP responsiveness by only 50%, suggesting
a role for additional transcription factors and elements in the
cAMP-dependent regulation of the human IL-10 promoter. Here, we
analyze the functional role of one such factor, C/EBP, in two cell
lines of myelomonocytic origin, THP-1 and HL-60, which are known
to differ in their differentiation status and C/EBP protein content. We
show that the level of basal as well as cAMP-stimulated IL-10
transcription depends on the expression of C/EBP An appropriate balance between pro- and anti-inflammatory
cytokines during the immune response is critical in the resolution of
many pathological conditions. In this context, interleukin (IL)1-10 is of special
interest because of its anti-inflammatory and immunosuppressive
properties. It is known that IL-10 synthesis in mononuclear cells can
be induced or enhanced by various inflammatory stimuli and by
sympathetic activation during the stress reaction or injury (1, 2), but
the molecular mechanisms underlying the regulation of these processes
are not fully understood. Recent reports demonstrate that
lipopolysaccharide-induced IL-10 transcription may involve Sp1
activation by p38 mitogen-activated protein kinase (3) or the binding
of Stat3 to the 5'-flanking promoter region of IL-10 (4). We showed
previously that stress-induced IL-10 expression in monocytic cells is
mediated by catecholamines via Using THP-1 and HL-60 cells, we demonstrate that C/EBP Electrophoretic Mobility Shift and Assay (EMSA) and Supershift
Assay--
Preparation of nuclear extracts and electrophoretic
mobility shift assays were performed essentially as described elsewhere (16, 17). 10 µg of nuclear proteins extracted from THP-1 cells were
incubated with 1 ng of radiolabeled oligonucleotides in a 20-µl
reaction for 20 min at room temperature. The location of C/EBP binding
motifs and the sequences of the used oligonucleotides are listed in
Table I with the exception of CRE1 and CRE4, which we have described
previously (6). For competition or supershifting, 100 ng of unlabeled
oligonucleotide or 1.5 µl of the specific antiserum were added,
respectively. The C/EBP Plasmids--
Firefly luciferase reporter gene constructs
containing 1308 bp (pGL2-1308) and 376 bp (pGL2-376) from the region
upstream of the IL-10 translation start codon as well as the double CRE mutant of the 1308-bp fragment (pGL2-mutCRE1,4) have been described elsewhere (6). To obtain each single mutant pGL2-mutC/EBP1, pGL2-mutC/EBP3, and pGL2-mutC/EBP5, the plasmid pGL2-1308 was subjected to in vitro mutagenesis using the
GeneEditor (Promega) and oligonucleotides as indicated in Table I.
Successful mutagenesis was monitored by SpeI digestion,
because the nucleotide exchange introduced an additional restriction
site. The triple mutant pGL2-mutC/EBP1,3,5 was generated by cloning of
a SacI fragment from pGL2-mutC/EBP3 into pGL2-mutC/EBP5
followed by digestion with Bsu36I and ligation of the
resulting fragment into pGL2-mutC/EBP1. pGL2-AvrIImutC/EBP5 was made from pGL2-mutC/EBP5 by digestion with AvrII and
SmaI followed by blunt-ended ligation after refilling of the
3' overhang and comprises 397 bp of DNA from upstream of the start
codon. The sequences of triple mutated and AvrIImutC/EBP5
fragments were confirmed by sequence analysis (BigDye Terminator cycle
sequencing kit, version 2.0, Applied Biosystems). The pMSV/EBP Cell Culture, Transfection, and Luciferase Assay--
THP-1 and
HL-60 cells were purchased from Deutsche Sammlung von Mikroorganismen
und Zellkulturen (Braunschweig, Germany) and maintained in RPMI 1640 medium with 10% fetal calf serum certified for low endotoxin and free
of mycoplasma (Biochrom, Berlin, Germany). Transfections were performed
by electroporation using 10 µg of reporter gene plasmid and 5 × 106 cells/0.25 ml in 0.4-cm electroporation cuvettes with a
gene pulser (Gene zapper 450/2500, IBI, Cambridge, United Kingdom) at
1200 microfarads, 100 ohm, and 300 V. Transfection efficiencies were found to be similar in THP-1 and HL-60 cells as determined by the
expression of the green fluorescent protein (pEGFP-C1, Clontech). Dot blot analysis also showed comparable
results for different reporter gene constructs. In co-transfection
experiments, 5 µg of expression plasmid were used unless stated
otherwise. HL-60 cells transiently transfected with 5 µg of
pMSV/EBP Computational Analysis of the IL-10 Promoter Region--
Human
and murine genomic sequences were retrieved from the University of
California, Santa Cruz Genome Browser (genome.ucsc.edu) datasets
from April 2002 and February 2002, respectively. GenBankTM
accession numbers for the other sequences are AF120030 (Marmota monax genomic IL-10 sequence), BG538741 (human EST), NM_000572 (human mRNA), Z30175 (human IL-10 promoter), NM_010548 (murine mRNA), and AF012909 (M. monax mRNA). Pairwise
comparative analyses were performed with the program DNA Block Aligner
(DBA) (18). The DBA input files were masked with Repeatmasker
(ftp.genome.washington.edu/RM/RepeatMasker.html). The MatInspector
Professional program (www.genomatix.de) was used to predict
transcription factor binding sites (TFBS). All of the positions in the
promoter/enhancer refer to the translation start site of IL-10.
C/EBP cAMP-stimulated IL-10 Promoter Activation in THP-1 Cells Involves
C/EBP cAMP-stimulated IL-10 Promoter Activation in THP-1 Cells Depends on
the Integrity of C/EBP Binding Sites--
To understand the
functional role of each individual C/EBP motif, we assessed the impact
of C/EBP site mutations on cAMP-mediated IL-10 promoter activation in
THP-1 cells. The oligonucleotides chosen for in vitro
mutagenesis (Table I) were tested for loss of their C/EBP protein
binding activity by EMSA (data not shown). In Fig.
4, A and B, the
deleted or mutated promoter fragments are shown together with their
residual promoter activity following cAMP-stimulation in THP-1 cells
relative to the native fragment representing 100% activity. The
mutation of C/EBP5, which lies closest to the translation start point,
decreased cAMP-responsiveness to 22%, whereas the mutation of C/EBP3
and C/EBP1 reduced promoter activity to 42 and 57%, respectively.
Subsequent experiments with the triple mutant pGL2-mutC/EBP1,3,5
revealed no further reduction compared with pGL2-mutC/EBP5. The
truncated promoter/enhancer fragment comprising 376 bp upstream from
the translation start point and lacking CRE and C/EBP motifs with the
exception of C/EBP5 still showed 48% residual activity. Abolition of
C/EBP5 in a deletion mutant of similar length
(pGL2-AvrIImutC/EBP5 comprising 397 bp) reduced cAMP
responsiveness further to 17%. These results emphasize a major
functional role for the C/EBP5 motif in the trans-activation of the
IL-10 gene by C/EBP C/EBP C/EBP Efficient IL-10 Promoter Activation by cAMP Depends on
C/EBP Comparative Analysis of the IL-10 Promoter/Enhancer
Region--
The transcription start site of the human IL-10 gene has
not yet been identified experimentally, but a human EST and 5' ends from mRNA of other species (Sus scrofa L20001,
Ovis aries Z29362, and Trichosurus vulpecula
AF026277) suggest that the transcription start site lies at position
With this study, we add new results to our previous findings
concerning the important role of cAMP and cAMP-inducing agents in the
trans-activation of the human IL-10 gene in monocytic cells. Here, we
show that the binding of the transcription factors C/EBP To study the role of C/EBP In contrast to pro-inflammatory cytokines, which are mainly regulated
by transcription factors such as NFkB and activating protein-1, the
transcription of the prototypic anti-inflammatory cytokine IL-10 in
monocytic cells, as shown here, is activated by cAMP or cAMP-inducing
agents, e.g. catecholamines during sympathetic activation
through transcription factors C/EBP and CREB. The mechanism of their
interaction remains to be investigated.
and
and their
binding to three motifs in the promoter/enhancer region. The C/EBP5
motif, which is located between the TATA-box and the translation start
point, is essential for the C/EBP-mediated constitutive and most of the
cAMP-stimulated expression as its mutation nearly abolished IL-10
promoter activity. Our results suggest a dominant role of C/EBP
transcription factors relative to CREB/ATF in tissue-specific and
differentiation-dependent IL-10 transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenoreceptors linked
to a cAMP-dependent signaling pathway (5), which activates
CREB/ATF by phosphorylation. These transcription factors act through
two cAMP-responsive elements (CRE), CRE1 and CRE4, located within 1308 bp upstream from the translation start codon of the IL-10 gene (6).
Yet, site-directed mutagenesis of these two CRE sites resulting in loss
of protein binding was not sufficient to abolish cAMP responsiveness
with 50% activity remaining. However, we previously described an
additional putative CRE site (CRE3) that binds proteins that do not
belong to the CREB/ATF or the activating protein-1 families of
transcription factors. Although mutation of this CRE3 motif had no
effect on protein binding or on promoter stimulation by cAMP, deletion
of CRE3 by 5'-exonuclease digestion resulted in a significant reduction in activity (6). Therefore, we concluded that other cAMP-responsive transcription factors bind to CRE3, even if mutated, or to hitherto unknown regulatory elements in this region during
stress/cAMP-dependent activation of the IL-10 promoter. At
first, we examined the involvement of three putative activating
protein-2 sites exhibiting protein binding, but we were not able to
confirm their specificity in supershift assays. We next investigated
CCAAT/enhancer-binding proteins (C/EBP), because these transcription
factors are shown to be involved in cAMP-dependent gene
expression, e.g. of IL-6 and IL-1, as well as of enzymes of
catabolic metabolism in the liver (7-9). It is well established that
C/EBP proteins mediate cAMP responsiveness by indirect mechanisms,
which include their increased expression and trans-location into the
nucleus in response to elevated cAMP levels. Moreover, they possess
domains that contain cAMP-inducible activities that are independent of
direct phosphorylation by protein kinase A (PKA) (10, 11). Similar to
CREB/ATF, C/EBP proteins belong to the group of basic region/leucine
zipper transcription factors that have the potential to form
heterodimers with each other and bind to motifs of either specificity
(10, 12). C/EBP proteins are known to regulate adipocytic and monocytic
differentiation, and their expression patterns vary depending on the
state of cellular differentiation (13-15). Recently, we found that
promonocytic THP-1 cells harbor significantly more C/EBP
than the
myelomonocytic progenitor cell line HL-60 (16) and that THP-1 cells,
unlike HL-60 cells, produce high levels of IL-10 protein after cAMP
treatment (data not shown). To gain insight into the components
involved in cAMP/stress-induced IL-10 expression, we asked whether
C/EBP transcription factors contribute to promoter stimulation through the CRE3 site and four putative C/EBP binding sites, which were predicted by computer analysis.
and
are
critical in both basal and cAMP/stress-dependent regulation of IL-10 expression during monocytic differentiation. As targets, we
identified three previously unknown C/EBP motifs in the
promoter/enhancer of the IL-10 gene, one of which corresponds to the
recently described CRE3 site.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
antisera and
oligonucleotides corresponding to C/EBP and CRE consensus binding sites
were obtained from Santa Cruz Biotechnology.
and
pMSV/EBP
expression plasmids that contain the respective cDNAs
from rat under the control of the mouse sarcoma virus (MSV) long
terminal repeat were kindly provided by S. McKnight (University of
Texas Southwestern Medical Center, Dallas, TX). To obtain the
MSV promoter control plasmid pMSV, the pMSV/EBP
plasmid was
digested with NcoI followed by religation. The expression
plasmids for rat p30, pcDNA3/p30, and CREB133 (pCMV/CREB) were
supplied by A. Leutz (Max-Delbrück-Centrum, Berlin, Germany) and
Clontech (BD Biosciences), respectively. The mock
plasmid pcDNA3 was from Invitrogen and pCMV was obtained by
digestion with XhoI and subsequent religation.
were designated as HL-60/EBP
. To account for possible
promoter interference of reporter gene and expression plasmid and to
equalize DNA concentrations, the reaction mixture in the transfection
cuvette was topped up with mock plasmid. After electroporation, 1 × 106 cells/ml/well were cultured in the absence or
presence of 500 µM dibutyryl-cAMP (dbcAMP, Sigma) unless
indicated otherwise. Cells were harvested 24 h after transfection
and assayed for luciferase expression using the Promega luciferase
assay kit and the Autolumat LB 953 (Berthold, Bad Wildbad, Germany).
The expression level of C/EBP
in THP-1 and HL-60 was verified by
Western blotting. Specific luciferase expression or promoter activity
was expressed in arbitrary units (AU) and assessed by normalizing
relative light units (raw data) for the protein content of the lysate
measured by the Bradford protein assay (Sigma). The luciferase
stimulation factor was calculated from the ratio of luciferase activity
in dbcAMP-treated or co-transfected cells versus basal
activity without dbcAMP treatment or transfected with the respective
mock plasmid obtained in the same experimental setting. Conversely, the
inhibition factor was evaluated as the ratio between the higher basal
activity (AU with mock plasmid) and the reduced activity, resulting
from expression of p30 or CREB133. An example for the calculation of the experimental results is given in Table II. Significance was estimated by the Man-Whitney-U test.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
Bind to Three Motifs in the IL-10
Promoter/Enhancer Region--
In this study, we analyzed
five putative C/EBP sites named C/EBP1 to C/EBP5 according to their
position in the enhancer region upstream of the human IL-10 promoter
(Table I). Four of these motifs
(C/EBP2-5) were newly predicted by the MatInspector Professional program with core similarity of 0.7 and optimized matrix similarity. One motif, C/EBP1, is identical to the previously analyzed CRE3 site,
which was shown not to be involved in CREB/ATF-induced promoter activation (6). Oligonucleotides harboring presumptive C/EBP motifs
(Table I) were initially examined for their ability to compete with the
consensus sequence using THP-1 nuclear extracts that are known to
contain considerable amounts of C/EBP proteins. Unlabeled
oligonucleotides consisting of C/EBP1, 3, 4, and 5 competed efficiently
with the C/EBP consensus oligonucleotide for C/EBP protein binding
(Fig. 1A, lanes 2, 4, 5, and 6), whereas C/EBP2 failed to compete effectively
(lane 3). Supershift assays with labeled C/EBP1, 3, and 5 oligonucleotides revealed that DNA-protein complexes contained C/EBP
(Fig. 1B, lane 2, 4, and 9)
and C/EBP
but not C/EBP
. Data obtained using C/EBP5
oligonucleotide are shown as a representative example for the reaction
with antisera against C/EBP
and
(lanes 10 and
11). The proteins shifting the C/EBP4 oligonucleotide failed
to react with C/EBP
or
antiserum (Fig. 1B,
lanes 6 and 7). Since we found that the C/EBP1
motif, which is identical to the previously analyzed CRE3 site binds C/EBP
and
, we wanted to exclude this possibility for the other CRE motifs. Competition experiments revealed that protein binding to
labeled C/EBP3 does not interfere with unlabeled CRE consensus, CRE1 or
CRE4 oligonucleotides (Fig. 1D, lanes 3,
4, and 6). Supershift analysis confirmed that
protein complexes bound to CRE1 and CRE4 oligonucleotides do not
contain C/EBP
or
(data not shown). The location of C/EBP binding
sites in the 1308-bp IL-10 promoter/enhancer fragment is shown with
respect to CRE motifs and the TATA box in Fig.
2.
C/EBP sites, their positions in the 5'-untranslated region
of the IL-10 gene and oligonucleotides used in EMSA and site-directed
mutagenesis
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Fig. 1.
Protein binding to putative C/EBP motifs of
the IL-10 promoter/enhancer. Radiolabeled oligonucleotides as
indicated at the bottom were incubated with nuclear extracts
from THP-1 cells and subjected to competition with a 100-fold molar
excess of unlabeled oligonucleotide (panels A and
C) or supershifted with specific antiserum (panel
B). Competitor oligonucleotides and antibodies are shown at the
top. The shifted DNA·C/EBP protein complex is indicated by
one asterisk, and the supershifted complex is
indicated by two asterisks. The same binding patterns with
C/EBP1, C/EBP3, and C/EBP5 oligonucleotides were distinguishable.
Therefore, the reaction of C/EBP - and
-specific antisera with
C/EBP5 oligonucleotide is given as a representative example.
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Fig. 2.
1308-bp fragment of the IL-10
promoter/enhancer region as cloned in pGL2-basic. The C/EBP and
CRE motifs within the IL-10 promoter/enhancer region are delineated in
relation to the TATA box (black box) and the IL-10
translation start (arrow) point. Gray circles and
squares represent CRE and C/EBP motifs, respectively.
--
To investigate the role of C/EBP
in
cAMP-induced IL-10 promoter activation in THP-1 cells, we
co-transfected increasing amounts of an expression plasmid for the
30-kDa truncated isoform of C/EBP
(pcDNA3/p30) along with the
reporter plasmid pGL2-1308. This isoform acts as a partial C/EBP
antagonist by retaining its binding capacity but with significantly
reduced trans-activation ability (19-21). Overexpression of p30 in
THP-1 cells significantly reduced both the cAMP stimulation of the
IL-10 promoter and its basal activity. The cAMP stimulation rates
adjusted from the p30 inhibition of the basal promoter activity
are shown in Fig. 3 (see also Table II). These results indicate that the
binding of endogenous C/EBP
to the enhancer region and its
trans-activation play a yet unknown role in constitutive and
cAMP-stimulated IL-10 expression.
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Fig. 3.
Influence of p30 overexpression on basal and
cAMP-stimulated IL-10 promoter activity in THP-1 cells. Cells were
co-transfected with pGL2 1308 along with increasing amounts of
pcDNA3/p30 and incubated in the absence or presence of dbcAMP.
Positive values indicate fold stimulation of the basal promoter
activity obtained with the mock plasmid or the respective concentration
of pcDNA/p30 by cAMP. Negative values represent the ratio
(inhibition factor) between the basal and the reduced activity obtained
with the mock plasmid and pcDNA/p30 in the absence of cAMP. Mean
values ± S.D. of three independent experiments are shown.
Inhibition of basal and cAMP-stimulated promoter activity in THP-1
cells by p30
and
. As outlined in panel C, the mutation of C/EBP motifs also reduced the basal promoter activity in
THP-1 cells, suggesting that endogenous C/EBP proteins are involved
in stimulated as well as in basal activity. Finally, as shown in
Fig. 5, cAMP-induced promoter activity in
THP-1 cells was almost completely abolished (residual activity 12%)
when the binding of both C/EBP
and
as well as CREB/ATF was
prevented by co-transfection of pGL2-mutCRE1,4 and pcDNA3/p30.
Overexpression of p30 inhibited the cAMP effect on promoter activity of
pGL2-1308 to the same degree as the destruction of all three C/EBP
motifs (25 and 23% residual activity), but as expected, it had no
effect on the activity of pGL2-mutC/EBP1,3,5 (Fig. 5).
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Fig. 4.
Involvement of C/EBP binding sites in basal
and cAMP-stimulated IL-10 promoter activity in THP-1 cells and in
C/EBP -transfected HL-60 cells
(HL-60/EBP
). Luciferase reporter
gene plasmids containing mutated or deleted promoter fragments
(A) were transfected into THP-1 and into HL-60/EBP
cells.
THP-1 cells were incubated in the presence or absence of dbcAMP.
Promoter activity of the mutants is expressed as the percentage of
cAMP-stimulated (B) and of basal (C) luciferase
expression in THP-1 cells or as a fraction of C/EBP
-mediated
luciferase expression obtained in HL-60/EBP
cells (D)
with pGL2-1308 (representing 100%). Mean values ± S.D. of ten
experiments are presented along. Gray circles and
squares represent CRE and C/EBP motifs, respectively;
blank symbols indicate mutated ones.
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Fig. 5.
cAMP-induced IL-10 promoter activation is
prevented by double mutation of CRE1 and CRE4 motifs and simultaneous
overexpression of p30 in THP-1 cells. Cells transfected with
pGL2-1308, pGL2-mutCRE1,4, or pGL2-mutC/EBP1,3,5 were either
co-transfected along with pcDNA3/p30 or pcDNA3 and incubated in
the presence of dbcAMP. Promoter activity is expressed as a percentage
of cAMP-stimulated luciferase expression obtained with pGL2 1308
and pcDNA3. Results (mean values ± S.D.) of at least five
independent experiments are shown.
Influences Constitutive IL-10 Promoter Activity
in HL-60 and THP-1 Cells--
We recently showed that the
pro-myelomonocytic cell line HL-60 contains significantly less
endogenous C/EBP
compared with THP-1 cells (16). To study the
influence of C/EBP on basal IL-10 promoter activity, we co-transfected
pGL2-1308 along with increasing amounts of pMSV/EBP
in HL-60 cells.
The data revealed that the expression of C/EBP
enhanced IL-10
promoter activity in a concentration-dependent manner
up to 17-fold (Fig. 6). In contrast,
C/EBP
expression over and above the high endogenous C/EBP
levels
in THP-1 cells only increased IL-10 transcription approximately 3-fold
(data not shown). Similar results were obtained by overexpression of C/EBP
(data not shown). The respective mock plasmid had no
significant effect, indicating that the observed promoter activation
resulted from C/EBP expression and was not the result of promoter
interference of co-transfected plasmids. To examine the role of
constitutively phosphorylated endogenous CREB in the C/EBP-mediated
promoter activation, we co-transfected the expression plasmids for
C/EBP
and CREB133 in HL-60 cells. This dominant negative CREB
comprises a serine-to-alanine mutation, is therefore unable to be
phosphorylated, and thus known to almost completely inhibit
CREB-dependent trans-activation (22, 23). As shown in Fig.
7, the C/EBP-mediated IL-10 promoter activation was significantly impaired but not completely prevented by
CREB133. Considering the reduced basal activity in the presence of
CREB133, the genuine C/EBP
stimulation rate decreased from 9.6 (994/103) to 5.6 (208/37). The inhibitory effect of CREB133 was
dependent on the molar concentration ratio of both co-transfected expression plasmids (data not shown). These data suggest that the
phosphorylation of CREB is involved in basal as well as in C/EBP
-mediated promoter activation in HL-60 cells. To verify the
role of C/EBP
in constitutive IL-10 promoter activity in HL-60 and
THP-1 cells, we again co-transfected the p30 expression plasmid. As
expected, p30 had a relatively minor influence on the basal promoter
activity in original HL-60 cells because of their low endogenous C/EBP
expression level. On the other hand, p30 significantly reduced
constitutive IL-10 transcription in THP-1 cells in a manner comparable
to HL-60/EBP
cells transiently expressing exogenous C/EBP
(Fig.
8). These results underline the role of
C/EBP
in constitutive IL-10 expression.
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Fig. 6.
Effect of exogenous C/EBP
expression on basal IL-10 promoter activity in HL-60 cells.
HL-60 cells were co-transfected with pGL2-1308 along with
increasing amounts of pMSV/EBP
. Promoter activity obtained by
supplying exogenous C/EBP
is expressed as multiples of the basal
luciferase expression level in the presence of the mock plasmid.
Results of at least three independent experiments are displayed.
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Fig. 7.
Effect of exogenous C/EBP
expression is inhibited by CREB133. Promoter activity (AU)
from HL-60 cells transfected with pGL2
1308 and 5 µg of
pMSV/EBP
or pMSV and additionally co-transfected with 5 µg
of pCMV/CREB133 or pCMV is shown (mean values ± S.D. of seven
independent experiments).
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Fig. 8.
Influence of p30 overexpression on basal
IL-10 promoter activity in THP-1, HL-60, and
HL-60/EBP cells. pGL2
1308 was
co-transfected into THP-1, HL-60, or HL-60/EBP
cells along with
increasing amounts of pcDNA3/p30. Basal promoter activity is
expressed as a percentage of the relative luciferase light units
obtained with pGL2
1308 and pcDNA3 normalized for protein content.
The mean values ± S.D. of at least three experiments are
shown.
-dependent Constitutive IL-10
Promoter Activity Is Mediated Mainly through
C/EBP5--
Our aim was to evaluate the role of each
individual C/EBP motif in C/EBP
-dependent constitutive
IL-10 expression. For this purpose, we used HL-60/EBP
cells because
of the direct correlation between C/EBP expression and promoter
activity. C/EBP5 had the greatest impact on C/EBP-dependent
promoter stimulation, since its mutation nearly abolished promoter
activity (residual activity 7%) while the mutation of C/EBP3 and
C/EBP1 reduced it only to 38 and 56%, respectively (Fig. 4,
panel D). Similar to cAMP-stimulated THP-1 cells, no further
reduction was seen with the triple mutant pGL2-mutC/EBP1,3,5 compared
with pGL2-mutC/EBP5 in HL-60/EBP
cells. The residual activity of the
truncated promoter/enhancer fragment (pGL2-376) in HL-60/EBP
cells
can be attributed to the effect of C/EBP5, because its mutation almost
eliminated promoter activity. This confirms the essential role of
C/EBP5 in the trans-activation of the IL-10 gene.
--
From p30 overexpression experiments and
functional analysis of C/EBP site mutations, we know that C/EBP
has
a significant effect on cAMP stimulation in THP-1 cells. We now wanted
to investigate whether promoter responsiveness is impaired in HL-60
cells because of the low C/EBP protein content in this cell line. In
fact, comparing the cAMP responsiveness of the IL-10 promoter in both
cell lines, we found that the elevation of intracellular cAMP levels
had only a small effect in HL-60 cells, whereas in THP-1 cells,
elevated cAMP levels induced a steep rise in promoter activity (Fig.
9). As already shown in Fig. 6, promoter
activity was substantially increased, supplying HL-60 cells with
exogenous C/EBP
. Starting from a higher level of constitutive
promoter activity, it could be further stimulated by cAMP in
HL-60/EBP
cells up to the same level as that achieved in THP-1 cells
expressing endogenous C/EBP
(Fig. 9). Even though the cAMP
stimulation rate remains nearly unchanged in HL-60 and HL-60/EBP
cells, together, our data indicate that for maximal promoter activity,
both the presence of C/EBP protein and the phosphorylation of CREB are
necessary.
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Fig. 9.
The level of cAMP-stimulated IL-10 promoter
activity depends on the expression of C/EBP .
pGL2
1308 was transfected into THP-1, HL-60, or HL-60/EBP
cells and
incubated with increasing concentrations of dbcAMP. Promoter activity
is expressed in AU assessed by normalizing relative luciferase light
units for protein content. The graph represents results of
ten independent experiments.
59. Three IL-10 sequences were analyzed: human (hs),
murine (mm), and the M. monax (ma)
promoter/enhancer. All of the three sequences contain a TATA-box
located at similar distances from the ATG: hs
89;
mm
87; and ma
86. Pairwise comparison with
DBA revealed two blocks of similarity that appeared in all of the three
pairs: Block A at approximately
500 to
300 bp and Block B from
100 bp to the ATG start codon containing the TATA-box. These
conserved sequences were analyzed by the MatInspector Professional program (core similarity 0.95, optimized matrix). Block A in human, mouse, and M. marmota contains one C/EBP binding site
(binding site matrix CEBP.01 in hs at
439 to
452, in
mm at
450 to
463, and in ma at
422 to
435) and one CREB binding site (binding site matrix ATF.01 in
hs at
401 to
414, CREB.01 in mm at
415 to
422, and CREB.04 in ma at
385 to
396), corresponding
to the C/EBP3 and CRE4 motifs analyzed in the human IL-10 promoter (Fig. 10). In Block B, another C/EPB
site (C/EBP5 in the human sequence) was identified: in hs at
30 to
43 and in ma at
39 to
52 (both matched the
C/EPB.01 matrix). Initially, in the murine sequence, this matrix was
not detected, but after lowering the search parameters, the equivalent
sequence was found at positions
39 to
52 (Fig. 10). The quality
of local alignments (data not shown) and TFBS matrix similarity
suggest a closer relationship of the human and the M. monax IL-10 gene versus human and mouse or mouse and
M. monax.
View larger version (25K):
[in a new window]
Fig. 10.
Localization of DBA blocks and the predicted
TFBS in the IL-10 promoter region. 1.5 kb upstream of the
translation start codon of Homo sapiens (hsIL-10), Mus
musculus (mmIL-10) and M. monax (maIL-10) were compared
with the program DBA. The categories B, C, and
D represent whole DBA block sequence identity (B,
70-80%; C, 80-90%; D, 90-100%).
Boldface letters, nucleotides belonging to the whole TFBS
region (gray boxes) identified by MatInspector;
boldface capitalized letters, nucleotides of the TFBS core
sequence defined by Genomatix. Only the parts of the blocks with the
candidate TFBS are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
to
three motifs in the IL-10 promoter/enhancer region contributes to basal
activity of the IL-10 promoter and is essential for maximal cAMP
stimulation in differentiated monocytic cells. Each of the individual
C/EBP motifs appeared to have a different impact on the
trans-activation of the IL-10 gene. The most important effector site
seems to be C/EBP5. Because of the proximity of C/EBP5 to the TATA box,
an interaction of C/EBP
bound to this motif with the basal
transcription-initiation complex can be envisaged (24, 25). Conversely,
this model explains the comparatively high trans-activating capacity of
the 376-bp fragment lacking CRE1, CRE4, C/EBP1, and C/EBP3 sites but
retaining C/EBP5. The C/EBP3 motif, close to the main CRE4 site, had a
moderate influence, whereas C/EBP1 seems to be less important. C/EBP1
coincides with the CRE3 site that failed to bind CREB/ATF (6) despite
its greater sequence similarity to the CRE rather than to the C/EBP consensus motif. The binding of C/EBP to non-consensus CRE sites and
the functional substitution of CREB by C/EBP family members have been
described previously (7, 26). It is not surprising that the
functionally relevant motifs C/EBP3, CRE4, and C/EBP5 fall into regions
that are evolutionary conserved between human, mouse, and M. monax, while the less important C/EBP1 and CRE1 sites are not
conserved. These findings together with the fact that plasmids with
single site mutation of C/EBP3 and CRE4 (6) retained similar
cAMP-responsiveness as pGL2-376, bearing only the intact C/EBP5 motif
hint at C/EBP-CREB interaction at multiple sites in a hierarchical and
cooperative manner. We assume that the function of C/EBP3 requires
prior occupation of the downstream C/EBP5 motif and the proximal CRE4
site, which implicates potential DNA looping and/or co-activator
protein binding (19, 25, 27).
in basal and cAMP-stimulated promoter
activation, we used the truncated 30-kDa C/EBP
isoform (p30), which
bears a conserved binding domain but lacks N-terminal regions required
for its trans-activation and differentiation potential (19-21).
Overexpressed p30 reduced the cAMP stimulation rate beyond the
inhibition of basal activity and abolished the remaining promoter
responsiveness of the double CRE mutant by competition with endogenous
p42 C/EBP
in THP-1 cells. Data obtained from structure/function
analysis of C/EBP
provide evidence that the mechanisms of
constitutive and cAMP-inducible trans-activation are different and
involve distinct albeit overlapping domains in the N-terminal portion
of the protein (amino acids 55-86 and 52-124, respectively), which
are both deleted in p30 (9, 19, 28, 29). The cAMP-mediated promoter
stimulation was consistently stronger in THP-1 cells than in HL-60
cells, in agreement with their higher differentiation status and
endogenous C/EBP
/
expression levels. When supplementing HL-60
cells with exogenous C/EBP
, we found that basal/constitutive
promoter activity rose in a concentration-dependent manner and the cAMP
response in HL-60/EBP
cells reached similar levels as in THP-1
cells. However, given the substantially higher basal level, the
cAMP stimulation rate seems to be independent of C/EBP
overexpression. The reason for this remains unclear. We suggest that
the increasing stimulation of the basal activity in THP-1 cells in
response to rising cAMP concentrations involves enhanced expression,
trans-location, and the binding of C/EBP
/
as well as
phosphorylation of CREB, C/EBP and cofactors, and their interaction
(11, 30-32). The model of transient overexpression of C/EBP
in
HL-60 cells can only partially reproduce these processes. Co-transfection experiments with the dominant negative inhibitor of
CREB in HL-60/EBP
cells revealed that trans-activation by C/EBP
to some extent depends on phosphorylated CREB being constitutively present in low amounts. Beyond this, C/EBP
overexpression seems to
up-regulate the IL-10 promoter activity by itself. Taken together, our
data from the mutation and co-expression experiments with p30 and
CREB133 indicate a co-operative interaction of CREB and C/EBP
transcription factors both in basal and in cAMP-stimulated promoter
activation. In contrast to CREB, the cAMP-inducible domain of C/EBP
lacks a PKA phosphorylation site; thus, the mechanism of its activation
by cAMP is yet unclear (33). It has been suggested that a co-activator
could interact with the cAMP-inducible domain of C/EBP transcription
factors only after it itself has been phosphorylated by PKA. This model
is similar to that of CREB activation recruiting CREB-binding protein
(CBP)/p300 after PKA phosphorylation with the exception that
phosphorylation of the co-activator rather than the DNA-binding protein
would regulate the interaction (9, 28, 31). In addition, CBP/p300 is a
possible mediator between CREB and C/EBP. Indeed, CBP/p300 comprises a
binding domain for C/EBP
/
(amino acids 1752-1859), which is
distinct from its CREB binding domain (amino acids 552-660) (34). On
the basis of our experimental data, we conclude that CREB/ATF mediates
a relatively low activating potential in the absence of C/EBP
/
proteins, which are necessary for maximal IL-10 promoter activation. In our view, multiple enhancer binding proteins need to be available simultaneously in differentiated monocytes to achieve stable binding site occupancy required for maximally activated IL-10 transcription. This suggestion is in agreement with the observation that C/EBP
expression is tissue-specific and varies during differentiation, while
CREB/ATF is ubiquitously expressed (35, 36). Therefore, C/EBP-mediated
transcription of a gene, in particular IL-10, is regulated in a
tissue-specific manner and depends on the state of differentiation of a
given cell. Experimental data from a mouse model, which revealed
immediate IL-10 production in response to lipopolysaccharide and
cAMP-elevating agents by hepatocytes known to contain high amounts of
C/EBP
, support this hypothesis (37, 38).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank S. L. McKnight (University of
Texas Southwestern Medical Center, Dallas, TX) for providing the
expression plasmids for C/EBP and
, A. Leutz
(Max-Delbrück-Centrum, Berlin, Germany) for pcDNA3/p30, O. Coutelle for critical reading, and M. Platzer for assistance in the
preparation of this paper.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Pl 163/4-3.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.
Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
49-3641-938570; Fax: 49-3641-938552; E-mail:
cplatzer@mti-n.uni-jena.de.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M207448200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IL, interleukin; CREB, cAMP-responsive element binding protein; ATF, activating transcription factor; AU, arbitrary units; CBP, CREB-binding protein; C/EBP, CCAAT/enhancer-binding protein; CRE, cAMP responsive element; dbcAMP, N6,2'-O-butyryl-cAMP; EMSA, electrophoretic mobility shift assay; MSV, mouse sarcoma virus; PKA, protein kinase A; TFBS, transcription factor binding site; DBA, DNA block aligner; hs, H. sapiens; mm, M. musculus; ma, M. monax.
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