(Received for publication, October 10, 1995)
From the
A construct comprising three tandemly repeated copies of the
B element from the interleukin-8 gene linked to chloramphenicol
acetyltransferase (CAT) (3xNF-
BCAT) was transcriptionally
activated in normal human FS-4 fibroblasts by co-transfection with
expression vectors for NF-
B p50, p65, or p52. Unexpectedly, a
significant activation of 3xNF-
BCAT was also seen upon its
co-transfection with the expression vector for CCAAT box enhancer
binding protein
(C/EBP-
) (but not C/EBP-
or
C/EBP-
). Stimulation by C/EBP-
required some other factor(s)
present in FS-4 cells because no transcriptional activation of
3xNF-
BCAT was seen after co-transfection with C/EBP-
in F9
mouse embryonic carcinoma cells, known to be deficient in several
transcription factors. To determine whether transcriptional activation
was the result of interaction with one of the major NF-
B proteins,
we co-transfected C/EBP-
with NF-
B p50, p65, p50 + p65,
or p52 into F9 or FS-4 cells. No cooperative interaction was seen; in
fact, C/EBP-
reduced p65-stimulated transcription, especially in
F9 cells. Electrophoretic mobility shift assay with a
B probe
revealed that the addition of recombinant C/EBP-
protein to
nuclear extracts from untreated FS-4 cells resulted in the appearance
of four bands. Only one of these bands was supershifted by antibody to
p50, whereas antibodies to p65 or other NF-
B proteins had no
effect. Our findings show that C/EBP-
may cause activation of some
B element-containing genes lacking C/EBP binding sites.
NF-B proteins regulate the expression of many different
genes, including genes encoding cytokines and acute phase proteins and
some viral genes(1, 2, 3) . The NF-
B
family includes p50 (NFKB1), p52 (NFKB2), p65 (RelA), RelB, v-Rel,
c-Rel and Drosophila proteins Dorsal and Dif. The N-terminal
portion of NF-
B proteins contains a 300-amino acid domain termed
the Rel homology region. This domain is responsible for DNA binding and
dimerization of NF-
B proteins. Less homology is found within the
C-terminal region of NF-
B proteins that is known to serve as the
activation domain. In their inactive state NF-
B proteins are
usually sequestered in the cytoplasm in a complex with an inhibitory
subunit termed I-
B(1) . NF-
B proteins are activated
by phosphorylation of the I-
B subunit and its subsequent
proteasome-driven degradation leading to the release from the
NF-
B
I-
B complex(4) . NF-
B then
translocates into the nucleus and rapidly activates gene expression.
Another group of transcription factors important in the regulation of
cytokine and acute phase protein genes is the CCAAT box enhancer
binding protein (C/EBP) (
)family(5, 6, 7) . A typical
structural feature of C/EBP proteins is the presence of a domain
comprising a region of basic amino acids and a leucine zipper region
(b-ZIP domain)(8, 9) . The leucine zipper domain is
responsible for dimerization, causing changes in the structural
conformation of the protein that allow the binding of the basic region
to specific DNA sequences. The C/EBP family includes C/EBP-
,
C/EBP-
(also termed IL-6 DBP or LAP; its human analogue is
NF-IL6), C/EBP-
(Ig/EBP-1), C/EBP-
, and
CHOP/GADD/53(7, 10, 11, 12) .
An
important feature of NF-B and C/EBP proteins is their ability to
heterodimerize with each other and with members of other transcription
factor
families(13, 14, 15, 16, 17, 18) .
NF-
B
C/EBP-
heterodimers are formed through the
interaction of the Rel homology region of NF-
B proteins with
leucine zipper domains within b-ZIP regions of C/EBP transcription
factors. Heterodimerization is facilitated by the presence of adjacent
B and C/EBP binding sites in several genes encoding cytokines
(IL-6, IL-8, G-CSF, neutrophil-activating peptide ENA-78,
MGSA/GRO)(16, 19, 20, 21, 22, 23, 24) ,
acute phase proteins (angiotensinogen, serum amyloid A protein,
-acid glycoprotein, TSG-14/PTX-3) (25, 26, 27, 28) or adhesion
receptors (intercellular adhesion molecule-1)(29) . The
presence of C/EBP
NF-
B protein heterocomplexes has been
demonstrated in intact cells(17, 30) .
Heterodimerization between NF-
B and C/EBP proteins can lead to
both cooperative and antagonistic interactions. For example, in the
IL-8 promoter NF-
B and C/EBP proteins were shown to mutually
augment their binding to the adjacent
B and C/EBP binding sites,
resulting in increased gene expression(20) . Similarly,
C/EBP-
and C/EBP-
increased NF-
B p50- or p65-driven
expression of a SAA3-CAT gene construct(31) . On the other
hand, it was found that C/EBP can inhibit promoters with
B binding
sites. Thus, when NF-
B p65 and C/EBP-
were co-transfected
together with an expression vector driven solely by the
B element
from the IL-8 gene, C/EBP-
inhibited p65-stimulated transcription (20) .
In our present study we show that when overexpressed
in human FS-4 fibroblasts, C/EBP- activates transcription from a
CAT reporter construct based on a trimeric repeat of the
B element
from the enhancer region of the IL-8 gene(32) . However,
C/EBP-
did not stimulate transcription of the same construct in
mouse F9 embryonic teratocarcinoma cells, which fail to express
endogenous NF-
B activity(33, 34) . Therefore, an
interaction of C/EBP-
with NF-
B and/or other transcription
factor(s) is likely to be involved in the transcriptional activation of
the
B-driven construct in FS-4 cells. Our studies of
transcriptional activation were confirmed by electrophoretic mobility
shift assays (EMSAs), showing that in the presence of the
B probe
nuclear extracts from unstimulated FS-4 cells formed specific complexes
with recombinant C/EBP-
protein.
Figure 1:
Transactivation
of NF-B-responsive CAT constructs by co-transfection with various
NF-
B or C/EBP proteins. FS-4 cells were transfected with 10 µg
of reporter construct 3xNF-
BCAT comprising three tandemly repeated
copies of the NF-
B site from the IL-8 gene, except for the
experiment shown in panel C in which 10 µg of construct
3xNF-
B(HLA)CAT, comprising three copies of the NF-
B binding
site from the HLA B-7 gene, was used in one half of the cultures, as
indicated. Co-transfected with the reporter construct were expression
vectors encoding various NF-
B or C/EBP family proteins, as
indicated (1 µg of expression vector/culture). Cells were harvested
20 h later, and CAT activity was determined as described under
``Experimental Procedures.'' Fold induction represents the increase in percentage of acetylation over control
cells transfected with the reporter construct alone. Values shown
represent the means of three separate determinations and their standard
deviations. Panel D shows an autoradiogram of a
representative experiment in which FS-4 cells were co-transfected with
the reporter construct 3xNF-
BCAT and expression vectors for p50,
p65, and C/EBP-
, as indicated.
Figure 2:
Co-transfection of C/EBP- with
NF-
B p50 or p52 proteins does not result in a cooperative
stimulation of the 3xNF-
BCAT reporter construct. FS-4 cells were
co-transfected with 10 µg of the 3xNF-
BCAT reporter construct
and the indicated amounts of expression vector pMSV-C/EBP-
(A), or combinations of different amounts of pMSV-C/EBP-
with 1 µg of pCMV-p50 (B) or pCMV-p52 (C). pCMV4T
is the ``empty'' vector.
Figure 3:
Effects of co-transfection with
C/EBP- and NF-
B p65 on 3xNF-
BCAT activity. FS-4 cells
were co-transfected with 10 µg of the 3xNF-
BCAT reporter
construct and increasing amounts of the pCMV-p65 expression vector with
or without 1 µg of pMSV-C/EBP-
expression vector. Numbers
in parentheses indicate concentrations of expression vectors in
µg of plasmid DNA/culture.
Figure 4:
Lack of inducibility of the 3xNF-BCAT
reporter construct by cytokines or TPA in F9 cells. F9 mouse embryonic
carcinoma cells were transfected with 10 µg of the 3xNF-
BCAT
reporter construct. After an overnight incubation the medium was
replaced and 24 h later cells were treated with murine TNF (muTNF) (10 ng/ml), human TNF (huTNF) (10 ng/ml),
human IL-1
(huIL-1
) (2 ng/ml), murine IL-1
(muIL-1
) (2 ng/ml), or TPA (20 ng/ml) for 5 h. Cells were
harvested, and CAT activity was determined.
Figure 5:
Activation of 3xNF-BCAT by
co-transfection of C/EBP-
and/or NF-
B proteins in F9 cells.
F9 cells were co-transfected with 10 µg of the 3xNF-
BCAT
reporter plasmid and various expression plasmids, as indicated. Numbers in parentheses denote µg of plasmid DNA/culture.
Cell lysates were prepared 48 h after transfection and analyzed for CAT
activity. A, co-transfections were performed with expression
plasmids pMSV-C/EBP-
alone, pCMV-p50 alone, or their combinations
as indicated. B, co-transfection with 1 µg of the
C/EBP-
expression plasmid and/or indicated amounts of expression
plasmid for p52. C, co-transfection with expression plasmids
for C/EBP-
and/or p65. D, co-transfection with expression
plasmids for C/EBP-
and p50 or p65, and their
combinations.
Figure 6:
Effect of I-B on NF-
B p65- or
C/EBP-
-induced transcriptional activation. FS-4 cells were
transfected with the 3xNF-
BCAT reporter plasmid (10 µg) and
combinations of pCMV-p65 (1 µg) or pMSV-C/EBP-
(1 µg) with
10 µg of expression plasmid pSVK3-I
B-
. At 24 h after
transfection cell lysates were prepared and analyzed for CAT activity. Control represents CAT activity in cells transfected with the
reporter plasmid alone.
Figure 7:
Formation of protein-DNA complexes with
nuclear extracts from unstimulated FS-4 cells and recombinant
C/EBP- protein. NF-
B probe was used in EMSAs as described
under ``Experimental Procedures.'' Complexes were resolved in
5.5% native polyacrylamide gels. Locations of specific complexes
C1-C4 are indicated by arrows. A, 8 µg of
nuclear protein extract from unstimulated FS-4 cells and/or 200 ng of
recombinant C/EBP-
protein were mixed with 10,000 cpm of the
P-labeled NF-
B oligonucleotide probe. For the
competition assay a 100-fold molar excess of the same unlabeled
oligonucleotide was added to the binding reaction. B, prior to
adding the
P-labeled oligonucleotide probe, the mixture of
FS-4 nuclear extract and recombinant C/EBP-
protein was
preincubated for 30 min at 37 °C with antibody against C/EBP-
or with nonimmune serum. Other conditions were as in panel A. C, polyclonal antibodies against different NF-
B proteins
(as indicated in the legend) were added to the EMSA binding reactions
and preincubated for 30 min at 37 °C before the addition of the
P-labeled oligonucleotide probe. Other conditions were as
in panel A.
In this study we have shown that C/EBP- expression in
human FS-4 fibroblasts stimulates transcriptional activity of the
3xNF-
BCAT reporter construct comprising three tandemly repeated
copies of the NF-
B binding site from the human IL-8 gene. The
stimulatory effect of C/EBP-
was
B sequence-specific, because
C/EBP-
expression did not activate a similar construct,
3xNF-
B(HLA)CAT, comprising three copies of the NF-
B binding
site from the human HLA B-7 gene (Fig. 1). Two lines of evidence
indicate that stimulation of the 3xNF-
BCAT construct was not the
result of a direct binding of C/EBP-
to the
B sequence.
First, C/EBP-
failed to produce transcriptional activation of
3xNF-
BCAT in the murine F9 embryonic carcinoma cell line (Fig. 5) in which the active (nuclear) form of NF-
B is not
inducible (33, 42) and which is deficient in some
other transcription factors(43, 45) . Second, in EMSAs
recombinant C/EBP-
protein failed to bind to a probe comprising
the same
B binding sites from the IL-8 gene as the 3xNF-
BCAT
expression construct (Fig. 7). Our data support the notion that
complex formation of C/EBP-
with other protein factor(s), present
in FS-4 but not in F9 cells, is necessary for the transcriptional
activation of 3xNF-
BCAT. Direct evidence of the formation of
specific complexes between recombinant C/EBP-
protein and proteins
from the nuclei of unstimulated FS-4 cells was obtained in EMSAs (Fig. 7).
The stimulatory effect on the 3xNF-BCAT
construct was specific for C/EBP-
, because no transcriptional
activation was seen on co-transfection with C/EBP-
or -
(Fig. 1B). C/EBP-
is primarily a regulator of
genes involved in energy metabolism (46) and was recently shown
to be critical for energy homeostasis in newborn mice(47) .
C/EBP-
is abundant in the liver and adipose tissue(48) .
In murine 3T3-L1 preadipocytes and various murine fibroblast lines
C/EBP-
is both necessary and sufficient for the induction of
adipogenesis(49, 50, 51) . The role of
C/EBP-
in the acute phase response is a complex one. Although
C/EBP-
was shown to transactivate the serum amyloid protein A3
gene promoter in hepatoma cells, cytokine treatment led to a
down-regulation of C/EBP-
activity with a concomitant increase in
C/EBP-
and C/EBP-
binding activities(27) . An inverse
correlation between C/EBP-
levels and the acute phase response was
also seen with respect to the regulation of the
-acid
glycoprotein gene(25, 52) . Besides the liver and
adipose tissues, high levels of C/EBP-
were also found in
myelomonocytic cells and in granulocytes, but its functional role in
these cells has not been analyzed(53) .
Transactivation of
3xNF-BCAT by C/EBP-
was demonstrated in FS-4 fibroblasts but
not in F9 embryonic carcinoma cells (Fig. 5). Earlier studies
have demonstrated the absence of NF-
B activity in undifferentiated
F9 cells, as demonstrated by a lack of nuclear protein binding to
B DNA probes or failure of
B-mediated transcriptional
activation(33, 42) . F9 cells were also found to be
deficient in some other transcription factor activities, e.g. transcriptional activation through the cAMP-response element (CRE)
was blocked in F9 cells due to the absence of functional protein kinase
A activity(43, 45) . We confirmed the absence of
activable NF-
B in F9 cells by demonstrating that a variety of
stimuli failed to activate the transfected 3xNF-
BCAT construct (Fig. 4). FS-4 and F9 cells also differed in the transactivation
of 3xNF-
BCAT by various exogenously provided NF-
B proteins:
transfection with p50 or p52 alone activated this construct in FS-4
cells ( Fig. 1and Fig. 3), but not in F9 cells (Fig. 5). While p65 was about equally stimulatory in FS-4 and F9
cells, a cooperative effect of p50 with p65 was seen in F9 but not in
FS-4 cells (Fig. 2, Fig. 3, and Fig. 5). In
addition, co-transfection of C/EBP-
with p65 seemed to have a
stronger inhibitory effect in F9 than in FS-4 cells. Hence, FS-4 and F9
cells differ in the makeup of endogenous factors that can affect the
function of exogenously provided NF-
B and C/EBP proteins. Earlier
we showed that EMSAs with extracts from unstimulated FS-4 cells gives
rise to two NF-
B-specific bands, one composed of p50 homodimers
and one comprising p50/p65 heterodimers(32) . The pattern seen
in Fig. 7(especially panel A, lane 1) is in
agreement with these earlier data.
The most likely interpretation of
the observed transcriptional activation of the 3xNF-BCAT construct
by C/EBP-
in FS-4 cells is that C/EBP-
stimulates
NF-
B-regulated expression through the formation of a heteromeric
complex with nuclear protein factors preexisting in FS-4 cells.
Alternatively, transfection with C/EBP-
stimulates de novo synthesis of protein(s) responsible for the transcriptional
activation in FS-4 cells, but this notion is inconsistent with results
of EMSAs, which show that nuclei of unstimulated FS-4 cells contain
proteins capable of forming specific complexes with recombinant
C/EBP-
in the presence of the NF-
B probe. One possible
candidate for complex formation with C/EBP-
was p65 because a
recent report showed that a heterodimer of p65 and C/EBP-
acts as
a potent activator of transcription from both NF-
B and C/EBP
sites(17) . However, we found no evidence for a role of p65.
First, there was only antagonism between p65 and C/EBP-
when the
two factors were co-transfected into cells ( Fig. 3and Fig. 5). Second, no evidence for the presence of p65 protein in
the complexes formed by FS-4 cell nuclear proteins with recombinant
C/EBP-
could be obtained in EMSAs with antibody to p65 (Fig. 7C). On the other hand, results obtained with two
different antibodies specific for the p50 NF-
B protein suggest the
presence of p50 in one of the four complexes formed between recombinant
C/EBP-
and nuclear proteins from FS-4 cells (Fig. 7C). Yet, co-transfection of p50 and C/EBP-
showed no cooperative interaction either in FS-4 (Fig. 2) or in
F9 cells (Fig. 5). Failure of antibodies to p52, RelB, and c-Rel
(data not shown) to interfere with the appearance of any of the four
bands formed between recombinant C/EBP-
and nuclear proteins from
unstimulated FS-4 cells suggests that other common NF-
B proteins
are probably not present in these complexes. In addition, the failure
of transfected I-
B to significantly inhibit the stimulatory effect
of C/EBP-
(Fig. 6) and the failure of p52 to synergize with
C/EBP-
in the activation 3xNF-
BCAT ( Fig. 2and Fig. 5B) also argue against the involvement of other
NF-
B proteins.
The fact that four different complexes were
formed in EMSAs when recombinant C/EBP- was mixed with nuclear
proteins from FS-4 cells (Fig. 7) suggests the presence of
multiple interacting proteins. Besides NF-
B proteins, C/EBP
proteins can heterodimerize with a variety of other transcription
factors, including c-Fos, c-Jun(13) , C/ATF(54) , and
the glucocorticoid receptor(18) . It is conceivable that
complex formation between C/EBP-
and these or some other
transcription factor(s) would alter the binding specificity of the
resulting complex, which then could recognize
``nonclassical'' sites. This notion finds support in the
recent demonstration that the transcription factor CHOP can dimerize
with other C/EBP proteins and that the resulting dimers are directed
away from classical C/EBP sites recognizing instead other specific DNA
binding domains(12) . Also possibly related is the reported
increase in the binding of upstream stimulatory factor (USF) to the USF
element after overexpression of C/EBP-
(but not C/EBP-
),
implicated in the autostimulatory effect of C/EBP-
on its own
promoter, which lacks any C/EBP binding domain (55) . Since
there was no accompanying increase in USF synthesis, C/EBP-
is
likely to increase USF binding by forming a complex with USF. In a
somewhat similar manner, in our system C/EBP-
does not bind to
3xNF-
BCAT, yet it stimulates transcription from this construct,
most likely by complexing with, and thereby altering the specificity
and binding affinity of, some other nuclear factor(s) present in
unstimulated cells. Our findings establish a mechanism whereby
C/EBP-
may cause activation of some
B element-containing
genes that lack C/EBP binding sites.