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INTRODUCTION |
The CCAAT/enhancer-binding proteins are a family of transcription
factors that all possess a nearly identical basic region leucine zipper
DNA-binding motif, but differ significantly in their amino-terminal
transactivation domain (1, 2). The first member of this protein family
to be cloned, C/EBP
,1
displays a tissue-specific pattern of expression, with the highest levels of expression occurring in liver, adipose tissue, and lung (3).
A number of studies indicate that C/EBP
is necessary and sufficient
to induce adipocyte differentiation (4-7), and it transactivates a
number of adipocyte-specific genes, such as 422(aP2) and
SCD1 (8-10). Antisense inhibition of C/EBP
expression in
3T3-L1 preadipocytes was shown to prevent accumulation of
triacylglycerol (7), and adipocytes from C/EBP
knockout mice are
devoid of lipid (11). Besides this important role in adipose
differentiation and lipid metabolism, C/EBP
also appears to be a
critical player in hepatic nutrient metabolism. It transactivates genes
such as phosphoenolpyruvate carboxykinase (PEPCK) (12), serum albumin (13), and alcohol dehydrogenase (14), and C/EBP
knockout mice
display reduced expression of glycogen synthase and low levels of liver
glycogen as well as delayed expression of PEPCK and
glucose-6-phosphatase genes (11). Consequently, the mice die shortly
after birth from hypoglycemia and other complications.
Most of the studies performed to date characterize C/EBP
as a
constitutive transactivator (15-17). Structure/function analyses of
C/EBP
have demonstrated that the amino-terminal region contains motifs that allow for protein-protein interactions with TATA-binding protein and TFIIB, with mutational analysis indicating that these interactions are important for the constitutive activity of C/EBP
(18). Recently, in studies examining the regulatory properties of the
PEPCK promoter, our laboratory uncovered a role for C/EBP
in
mediating hormonal responsiveness via mechanisms that appear to be
distinct from those utilized to confer constitutive transactivation. The PEPCK gene, which is expressed at the highest levels in liver and
kidney, is transcriptionally responsive to cAMP, but shows tissue-specific response patterns; in liver, the gene is robustly activated by cAMP, whereas in kidney, the response is weak (19). The
explanation for this difference appears to reside in the presence of
C/EBP-binding sites in the PEPCK promoter. The cAMP response unit in
this promoter, which is functional only in liver-derived cells,
consists of a typical cAMP response element (CRE), an AP-1-binding site, and three C/EBP-binding sites (20). All five of these cis-elements are required for maximal responsiveness to
cAMP. We have shown that the
isoform, but not the
isoform, of
C/EBP functions in this cAMP response unit (21).
Another critical player in this cAMP response unit is the CRE-binding
protein (CREB), which participates through its binding to the CRE (22).
However, the CRE in the PEPCK promoter is rather unique in that it also
binds C/EBP proteins with high affinity (23-25). In this report, we
show that C/EBP
can substitute for CREB in the cAMP response unit,
i.e. that cAMP responsiveness can occur in the absence of
CREB.
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EXPERIMENTAL PROCEDURES |
Materials--
DNA-modifying enzymes were purchased from Promega
and New England Biolabs Inc. [acetyl-3H]CoA
(10 Ci/mmol) was purchased from NEN Life Science Products. Tissue
culture supplies were from Life Technologies, Inc. HepG2 cells were
acquired from American Type Culture Collection.
Transfection Experiments and Plasmids--
HepG2 cells were
cultured and transfected as described previously (21). An expression
vector for
-galactosidase was cotransfected to monitor transfection
efficiency. Descriptions of the reporter gene plasmids and expression
plasmids have been previously reported (20-22, 24), with the
exceptions provided below. The expression vector for the C/EBP
mutant Y67A,F77A,L78A, driven by the cytomegalovirus promoter, was
described previously (18).
The chloramphenicol acetyltransferase (CAT) reporter genes
68G3 and
68G4 consist of a minimal PEPCK promoter (26) to which were ligated
three or four copies, respectively, of a double-stranded oligonucleotide (27) that contains the recognition site for the yeast
transcription factor Gal4. The CAT reporter gene
68G4A1 was created
by linking the A-site oligonucleotide (20) to the
68G4 vector. The
68C4 reporter gene consists of four copies of the C-site
oligonucleotide (20) linked to a minimal PEPCK promoter. The
68C4A1
reporter gene was created by linking the A-site oligonucleotide to the
68C4 reporter gene.
The Gal4-C/EBP
deletion mutants were based on the parent vector
G
2 (21), which had the C/EBP
transactivation domain (amino acid
residues 6-217) fused in-frame to the Gal4 DNA-binding domain as an
EcoRI/PstI fragment. The general strategy was to
introduce BamHI restriction sites by site-directed
mutagenesis (28) at selected positions within the coding region of the
transactivation domain. In every case, the BamHI site was
introduced such that it encompassed two intact codons, thereby allowing
in-frame fusions between various regions of C/EBP
coding regions to
create the internal deletion mutants. The
EcoRI/BamHI fragments generated were cloned into
the Gal4 vector expression plasmid pMI (29) to generate the
carboxyl-terminal deletion mutants of C/EBP
. The internal deletion
mutants were created by restricting the appropriate carboxyl-terminal
deletion mutant with BamHI and PstI and ligating
to it the appropriate carboxyl-terminal
BamHI/PstI fragment generated by the
site-directed mutagenesis strategy.
The expression plasmid for the Gal4 derivative of Y67A,F77A,L78A was
created by introducing an EcoRI site over codons 5 and 6 of
the mutant transactivation domain (18) in a similar fashion as
described previously (21). The resulting
EcoRI/PstI fragment, which contained codons
6-217 along with the appropriate mutations, was ligated into pM1.
Verification that similar levels of Gal4 fusion proteins of the
appropriate length were expressed in HepG2 cells was determined in
lysates from transiently transfected cells by Western blot analysis
(30) using an antibody specific for the DNA-binding domain of Gal4
(Upstate Biotechnology, Inc.). It should be noted that in the
experiments where transcription factors, either wild type or Gal4
fusions, were overexpressed, the amount of expression plasmid used for
each protein was different and was based upon the amount that provided
the optimal -fold induction. However, we have performed numerous
experiments with a wide concentration range of each plasmid. While the
absolute values were, in some cases, slightly different from those
presented, the general conclusions were not affected.
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RESULTS |
C/EBP
Can Participate in Mediating cAMP Responsiveness in the
Context of the PEPCK Promoter--
Our laboratory previously
demonstrated that five cis-elements make up the cAMP
response unit of the PEPCK promoter and consist of a CRE, a binding
site for AP-1, and three binding sites for C/EBP (see Fig.
2A). Furthermore, using synthetic promoters that reconstitute the cAMP response unit, we observed that no rigid architectural requirements were required for the activity of this hormone response unit (20, 24). Finally, we provided evidence that
supported the hypothesis that the
isoform, but not the
isoform,
of C/EBP participated in this cAMP response unit (21). This latter
conclusion was based on studies using Gal4-C/EBP hybrid proteins that
were tested on an artificial promoter that reconstituted the cAMP
response unit, i.e. contained a binding site for CREB, AP-1,
and three Gal4-binding sites. However, because these observations were
made on an artificial promoter, we decided to test the ability of the
Gal4-C/EBP hybrid proteins to reconstitute cAMP response within the
context of the PEPCK promoter. A PEPCK promoter-CAT reporter plasmid
(
490P3G4) was used for this study, which has the highest affinity
C/EBP-binding site replaced, within the context of the intact promoter,
by a Gal4-binding site (27). In Table I,
it can be observed that the replacement of this C/EBP-binding site by a
Gal4 site resulted in a significant decrease in PKA responsiveness
(compare
490wt with
490P3G4), consistent with previous studies
showing the importance of this cis-element for maximum
hormone response. Expression of the Gal4-C/EBP
hybrid protein G
2
(21) resulted in reconstitution of PKA responsiveness, whereas the
Gal4-C/EBP
fusion protein G
2 (21) had no effect on PKA
responsiveness. These findings, obtained within the context of the
PEPCK promoter, lend further support to our hypothesis that C/EBP
participates in mediating the cAMP response.
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Table I
C/EBP participates in mediating cAMP responsiveness within the
context of the PEPCK promoter
HepG2 cells were transfected with 7 µg of CAT reporter plasmid, 10 ng
of Gal4 expression vector, 2 µg of PKA expression vector, and 2 µg
of -galactosidase plasmid (transfection control) per plate.
490PCK-CAT contains 490 base pairs of 5'-flanking sequences of the
PEPCK promoter; 490P3G4 contains the same sequences, except that a
C/EBP-binding site is replaced by a Gal4-binding site. -Fold activation
was calculated as the ratio of the CAT activity measured in the
presence and absence of PKA catalytic subunit expression vector
cotransfection. The values shown are the means ± S.E. of at least
three experiments.
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To further test this hypothesis, we capitalized on the observation that
C/EBP
levels in HepG2 cells are ~5% of those present in
hepatocytes (13) and therefore provide a suitable cell background in
which to overexpress C/EBP proteins to examine their transcriptional effects. If our hypothesis is correct about the relative roles of C/EBP
isoforms in the cAMP responsiveness of the PEPCK promoter, overexpression of C/EBP
should enhance the transcriptional response of the promoter to PKA. Conversely, overexpression of C/EBP
should either have no effect or, due to its ability to compete with C/EBP
for binding to the promoter (25), even act in a dominant-negative fashion. The results of these experiments are shown in Fig.
1. The PEPCK promoter-CAT plasmid was
induced strongly (25-fold) by overexpression of the catalytic subunit
of PKA. Overexpression of both C/EBP
and C/EBP
had a relatively
small 2-3-fold effect on the basal activity of the PEPCK promoter.
Overexpression of C/EBP
and the catalytic subunit of PKA acted
synergistically to increase PEPCK promoter activity 40-fold, whereas
overexpression of C/EBP
diminished the inducibility of the promoter
in response to PKA.

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Fig. 1.
Effect of overexpression of C/EBP isoforms on
PKA responsiveness of the PEPCK promoter. HepG2 cells were
transfected with 7 µg of 490PCK-CAT in the absence
(solid bars) and presence (hatched
bars) of an expression vector for the catalytic subunit of
PKA (1 µg) as described under "Experimental Procedures." Where
indicated, an expression vector for either C/EBP (100 ng) or
C/EBP (1 µg) was also cotransfected. The values shown are the
means ± S.E. of at least three experiments and are expressed
relative to the CAT activity obtained with transfection of the CAT
reporter plasmid alone, which was set arbitrarily at 1.0.
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C/EBP
Can Mediate cAMP Responsiveness in the Absence of
CREB--
In a previous study (22), we defined a role for CREB in the
cAMP response unit of PEPCK via its ability to bind to the
near-consensus CRE in the promoter (Fig.
2A). In that study, we showed
that when the CRE was replaced by a Gal4-binding site, expression of a
Gal4-CREB fusion protein could reconstitute the cAMP response,
confirming the ability of CREB to participate in this cAMP response
unit. However, the CRE in the PEPCK promoter is rather unique in that C/EBP proteins bind to this sequence with a similar affinity as CREB
(25), allowing the speculation that C/EBP may also be a regulatory
protein working through this site (see "Discussion"). We tested
this hypothesis using our Gal4 system. In Fig. 2B, we show
that G4-PEPCK, a PEPCK promoter derivative that has the CRE replaced by
a Gal4-binding site (Ref. 31; see Fig. 2A), not only had its
PKA responsiveness reconstituted by expression of a Gal4-CREB fusion
protein in HepG2 cells, but a significant restoration of PKA
responsiveness was also obtained by overexpression of G
2 (Fig.
2B). It should be noted that the wild type PEPCK promoter typically shows a 25-60-fold induction by PKA in transfection assays
in HepG2 cells (Table I) (20, 23, 24). Overexpression of G
2 had no
effect on PKA responsiveness (Fig. 2B). Thus, it appears
that cAMP responsiveness can occur in the complete absence of bound
CREB and that C/EBP
can functionally substitute for CREB at the
CRE.

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Fig. 2.
C/EBP can effectively substitute for CREB
in mediating cAMP responsiveness of the PEPCK promoter.
A, shown is a schematic of G4-PEPCK. This modified promoter
is identical to the wild type PEPCK promoter, except that the CRE has
been replaced by a binding site for Gal4. B, HepG2 cells
were cotransfected with 7 µg of G4-PEPCK; a PKA expression plasmid (1 µg); and where indicated, expression plasmids for Gal4-CREB (1 µg),
G 2 (50 ng), or G 2 (1 µg). The -fold activation by PKA
represents the ratio of the CAT activity obtained in the presence and
absence of PKA catalytic subunit expression. The values shown are the
means ± S.E. of at least three experiments.
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To test this hypothesis further without the use of Gal4 fusion
proteins, we constructed a synthetic promoter that contained four
native C/EBP-binding sites and an AP-1 site (linked to a minimal
promoter), rather than the three C/EBP + AP-1 + CRE site combination
making up the cAMP response unit of the PEPCK promoter. This promoter,
called
68C4A1, showed a 23-fold induction in response to coexpression
of the catalytic subunit of PKA in HepG2 cells (Fig.
3). It should be noted that the parent
promoter,
68PCK-CAT, is not responsive to PKA (20). We also examined
the role of the AP-1 site by assessing the responsiveness of a
synthetic promoter containing only the four C/EBP-binding sites. This
promoter, called
68C4, also displayed a significant (9-fold)
induction by PKA, albeit weaker than
68C4A1 (Fig. 3). These results
suggest that C/EBP can independently mediate cAMP responsiveness and
can synergize with AP-1 to mediate a larger response. This latter
observation is consistent with our previous data, which showed that the
AP-1 site in the PEPCK promoter had no intrinsic ability to mediate cAMP responsiveness, but did synergize with other
cis-elements in the promoter to provide a robust response to
cAMP (20).

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Fig. 3.
C/EBP-binding sites can mediate PKA
responsiveness. The reporter gene 68C4 was constructed by
ligating four copies of an oligonucleotide containing a high affinity
C/EBP-binding site to a minimal promoter. Further ligation of a single
AP-1 oligonucleotide to this reporter gene resulted in the formation of
68C4A1. These CAT reporter plasmids (7 µg) were cotransfected into
HepG2 cells along with a PKA expression plasmid (1 µg). The -fold
activation by PKA represents the ratio of the CAT activity obtained in
the presence and absence of PKA catalytic subunit expression. The
values shown are the means ± S.E. of at least three
experiments.
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Because the experiments shown in Fig. 3 could not distinguish which
C/EBP isoform may be binding to the C/EBP sites in the test promoters,
we performed additional experiments using our Gal4 system. Two
promoters similar to those shown in Fig. 3 were constructed that had
the C/EBP sites replaced with the Gal4 sites (Fig.
4A). Using these promoters, we
demonstrated that PKA responsiveness was negligible unless Gal4-CREB or
G
2 was coexpressed (Fig. 4, B and C). However,
there was a notable difference between these two Gal4 fusion proteins.
Gal4-CREB showed no requirement for the AP-1 site in order to mediate
PKA induction, whereas the inducible activity displayed by G
2 was
significantly enhanced by the presence of the AP-1 site (Fig. 4,
compare B and C). Gal4-CREB also possessed a
lower degree of intrinsic constitutive transactivation activity compared with G
2, but had greater PKA-inducible activity. It should
also be noted that the Gal4-C/EBP
fusion protein, G
2, had no
significant ability to mediate PKA responsiveness (Fig. 4, B
and C).

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Fig. 4.
A Gal4-C/EBP fusion protein can
mediate PKA responsiveness and is augmented by AP-1. A schematic
of the promoters used in this experiment is shown (A). These
reporter genes, 68G4A1 (B) and 68G4 (C), were
constructed as described in the legend to Fig. 3, except that an
oligonucleotide containing a Gal4-binding site was used in place of a
C/EBP oligonucleotide. The CAT reporter plasmids (7 µg) were
cotransfected into HepG2 cells along with a PKA expression vector and,
where indicted, an expression vector for Gal4-CREB (1 µg), G 2 (50 ng), or G 2 (1 µg). The CAT activity measured in the presence of
the Gal4 fusion proteins, but in the absence of PKA expression
(solid bars), is expressed relative to the CAT
activity obtained with transfection of the CAT reporter plasmid alone,
which was set arbitrarily at 1.0. The CAT activity achieved by
overexpression by PKA is depicted by hatched
bars. The values shown are the means ± S.E. of at
least three experiments.
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Structure/Function Analysis of the cAMP-inducible Transactivation
Properties of C/EBP
--
Several previous studies have
characterized the domains of C/EBP
that mediate its constitutive
transcriptional activity. We were interested to see whether the domains
in C/EBP
that mediate cAMP-inducible transcription were similar or
distinct from those involved in its constitutive transcriptional
activity, as has been observed for CREB (31). Various carboxyl- and
amino-terminal deletion mutants of the C/EBP
transactivation domain,
as well as those containing internal deletions, were fused to the
DNA-binding domain of Gal4 and tested both for their basal
transcriptional activity and for their cAMP-inducible activity. All of
the Gal4-C/EBP
fusion proteins shown in Fig.
5 were expressed at comparable levels in
HepG2 cells as assessed by Western blot analysis using an antibody specific for Gal4 (data not shown). The PKA-inducible activities of
these fusion proteins were tested using
109G3A1, a synthetic promoter
that reconstitutes the cAMP response unit of the PEPCK promoter used
previously to assess the activity of Gal4-C/EBP fusion proteins (21).
However, because this promoter has a CRE and an AP-1 site, we tested
the basal transcriptional activities of the Gal4-C/EBP
fusion
proteins using
68G3, which has three Gal4-binding sites linked to a
minimal promoter, so the transcriptional activity measured was derived
exclusively from the action of the Gal4-C/EBP
fusion proteins.

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Fig. 5.
Structure/function analysis of the domains in
C/EBP that mediate constitutive and PKA-inducible
transactivation. The construction of the Gal4 fusion vectors and
the transfection assay are described under "Experimental
Procedures." The basal or constitutive activity of the Gal4 fusion
proteins shown was determined by cotransfecting 1 µg of expression
plasmid along with the reporter gene 68G3 (7 µg) into HepG2 cells.
The promoter activity obtained by overexpression of G 2 was
arbitrarily assigned a value of 100. The -fold induction by PKA
conferred by the Gal4 fusion proteins was assessed using the reporter
gene 109G3A1 as described in the legend to Fig. 4. The values shown
are the means ± S.E. of at least three experiments.
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Our analysis first examined the activity of carboxyl-terminal deletions
of C/EBP
, and we identified three domains that contributed to its
constitutive activity: amino acids 176-217, 97-124, and 51-96 (Fig.
5). A putative attenuation domain, residing within amino acids
136-175, was also identified, based upon the observation that the
constitutive activity of N135 was 3-fold greater than that of the
intact transactivation domain. It should be mentioned that examination
of amino-terminal deletions was unsuccessful since all fusion proteins
lacking the amino-terminal 50 amino acids showed no
activity.2 Internal deletion
mutants demonstrated a critical role for amino acid residues 51-95
(Fig. 5). All of these findings are in general agreement with previous
structure/function studies of C/EBP
(15-18).
The PKA-inducible activity of these fusion proteins was next examined.
In general, the carboxyl-terminal deletion mutants that displayed
constitutive activity also demonstrated PKA-inducible activity.
However, it was also evident that the carboxyl-terminal deletions had
differential effects on the constitutive and inducible activities of
C/EBP
. Deletion of amino acids 176-217 resulted in a loss of 65%
of the constitutive activity of C/EBP
without any measurable loss of
its ability to mediate PKA responsiveness (Fig. 5). Further deletion to
amino acid 135 resulted in a large increase in constitutive activity
without affecting its inducible transcriptional activity, and deletion
of an additional 11 amino acids (N124) resulted in a decrease in
constitutive activity without any effect on inducible activity.
Furthermore, comparison of the properties of N175 with those of the
internal deletion mutant
113-134 show that the latter fusion
protein has a higher constitutive activity than N175, but significantly
lower PKA-inducible activity. Finally, comparison of N124 with the
parent compound G
2 demonstrated that the constitutive activity of
C/EBP
can be compromised without necessarily affecting its
PKA-inducible activity. It should be noted that the residual PKA
responsiveness that was detected even when the Gal4 DNA-binding domain
alone was expressed originates from the inherent activity of the test
promoter that, due to the presence of the CRE, does confer some PKA
inducibility (21, 23).
A consistent observation made in the experiments shown in Fig. 5 was
that all C/EBP
fusion proteins that were devoid of constitutive activity (N50,
51-95, and
51-111) also possessed no measurable PKA-inducible transcriptional activity. The common region missing in
all of these mutant proteins was the domain encompassing amino acids
51-95. This region contains the only conserved motif within the
transactivation domain among C/EBP family members, consisting of two
conserved boxes (A and B) separated by a spacer region ranging in
length from 5 to 34 amino acids (18). The B box contains a homology box
2 core, which was previously identified as a transactivation domain
residing within c-Fos, c-Jun, and c-Myc (32, 33). This conserved motif
in C/EBP
, spanning amino acids 55-86, has been shown to mediate
protein-protein interactions with the TATA-binding protein and with
TFIIB, and mutation of conserved amino acids within this motif
significantly reduced the constitutive activity of C/EBP
(18).
Because point mutations represent a less severe approach to protein
structure/function analysis compared with deletion analysis, we decided
to test a triple-point mutant for its PKA-inducible activity. This
C/EBP
mutant, termed Y67A,F77A,L78A, contains alanine
substitutions at tyrosine 67, phenylalanine 77, and leucine 78 and was
previously shown to possess constitutive activity that was ~10% of
the wild type (18). We fused the transactivation domain (amino acids
6-203) containing these mutations to the DNA-binding domain of Gal4
and tested its activity on the reporter plasmid
109G3A1. As
shown in Table II, G
2 displayed
significant constitutive activity when expressed in HepG2 cells, and
its presence also led to an enhancement of the PKA responsiveness of
the reporter gene. When the triple-mutant derivative of G
2
(G
Y67A,F77A,L78A) was expressed, little enhancement of the
basal activity of the reporter gene was detected. More interestingly,
G
Y67A,F77A,L78A retained full capacity to enhance promoter activity
in response to PKA (Table II), providing strong evidence that the
constitutive and PKA-inducible transactivation activities of C/EBP
are mediated by distinct mechanisms.
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Table II
Inactivation of the constitutive activity of C/EBP does not
impair its ability to mediate cAMP responsiveness
HepG2 cells were transfected as described in the legend to Table I,
except that the CAT reporter plasmid was 109G3A1, and 50 ng of the
Gal4 fusion protein expression plasmid was used. Relative basal
activities of the Gal4 fusion proteins were calculated by arbitrarily
assigning the CAT activity of 109G3A1, obtained with transfection of
the reporter plasmid alone, at 1.0. -Fold activation was calculated as
the ratio of the CAT activity measured in the presence and absence of a
PKA catalytic subunit expression vector. The values shown are the
means ± S.E. of at least three experiments.
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DISCUSSION |
While the PEPCK promoter is regulated by a number of hormones
(reviewed in Ref. 34), the induction by cAMP has been intensively investigated due to the strong and rapid nature of the response and its
tissue-specific characteristics (19, 35). The CRE in this promoter was
the first of its kind to be identified (26), and from it arose the
discovery that many promoters contain this cis-element (36).
A transcription factor that bound to the CRE, termed CREB, was
subsequently identified, and it contains a serine residue that is
phosphorylated by PKA, resulting in its activation (37). This initial
simplified model of how cAMP responsiveness occurs has become
significantly more complex with the discovery of a coactivator for CREB
(38) and the finding that CREB is but one member of a large protein
family (reviewed in Ref. 39). However, in the case of the PEPCK
promoter, an additional layer of potential complexity was uncovered
when it was shown that members of the C/EBP protein family can also
bind to this CRE with high affinity (12, 25), although their affinity
for perfect consensus CREs in other promoters is relatively weak (24).
Not only can C/EBP bind to the CRE in the PEPCK promoter, but it also
produces transactivation, thereby creating a controversy as to whether CREB or a C/EBP protein is the physiologically relevant transcription factor at this site.
Several previous studies addressing this issue have produced
conflicting data. For example, in support of a role for CREB at this
site, our laboratory showed that (i) mutation of the CRE to a perfect
consensus sequence, which resulted in a significant decrease in C/EBP
binding affinity but had no effect on CREB binding affinity (24), did
not alter the cAMP responsiveness of the PEPCK promoter; and (ii) a
Gal4-CREB fusion protein, when tethered to the PEPCK promoter by
replacement of the CRE with a Gal4-binding site, was able to
reconstitute the cAMP responsiveness of the promoter (22). Conversely,
in support of a role for a C/EBP protein, it was shown that (i)
mutation of the CRE to a C/EBP-binding site, which reduced CREB binding
affinity without affecting C/EBP binding affinity, did not reduce the
cAMP responsiveness of the promoter (23); and (ii) mice that contain a
targeted inactivation of the CREB gene have no detectable alterations
in PEPCK gene expression or any apparent abnormalities in glucose
homeostasis (40). In this paper, we present data that suggest that the
binding of either CREB or C/EBP
to the CRE allows the promoter to
retain its cAMP responsiveness (Fig. 2B), i.e.
that C/EBP
can functionally substitute for CREB, which provides an
explanation for the apparent contradictory data summarized above. The
fact that C/EBP
can also bind to this CRE, but with a detrimental
effect on cAMP responsiveness (Fig. 2B), suggests that the
prevailing responsiveness of this promoter to cAMP can be exquisitely
regulated depending on the in vivo concentrations of CREB,
C/EBP
, and C/EBP
. An additional level of regulation has been
suggested by the observations that both C/EBP
and C/EBP
can have
their DNA-binding activity modulated by phosphorylation of specific
serine residues lying within the basic region leucine zipper domain
(41, 42).
We also presented experimental data to support the hypothesis that
C/EBP
may function independently as a modest cAMP-inducible regulator. One criticism of these data could be that we demonstrated cAMP-inducible activity for C/EBP
only on promoters containing multiple binding sites for either the intact protein or the Gal4 fusion
counterpart. Indeed, we were able to detect only a residual amount of
PKA responsiveness when a single C/EBP site was linked to a minimal
promoter.2 However, it should be noted that similar
observations have been made with promoters containing a single CREB
molecule tethered to the promoter. Our laboratory has previously shown
that the PEPCK promoter CRE, when tested independently, is essentially devoid of PKA-inducible activity, as is a single molecule of a Gal4-CREB molecule when recruited to a promoter (22-24). In fact, it
takes the multimerization of three CRE cis-elements before detectable activity of the CRE manifests itself (24). As reviewed recently (43), it is in fact a common observation that cAMP responsiveness (and responses to other hormones) is often mediated by
the synergistic interactions of several cis-elements within a promoter, even those that contain typical CREs. Employing several different cis-elements in the hormonal responsiveness
of a promoter may offer unique regulatory opportunities, including an
expansion of the range of responses and the coordination of information from several signaling pathways into an integrated transcriptional response.
If C/EBP
is a cAMP-activated nuclear regulator, what is the
molecular mechanism whereby it mediates this activity? While we do not
have an answer to this question at present, it is known that C/EBP
is not phosphorylated by PKA to any significant extent (41). It is
possible that some as yet unidentified coactivator for C/EBP
, which
itself can be phosphorylated and activated by PKA, allows C/EBP
to
display this hormone-inducible activity. Recently, the CREB-binding
protein (CBP), which is a coactivator for CREB (38), was implicated as
a possible coactivator for C/EBP
(44), although no direct
protein-protein interaction between CBP and C/EBP
has been reported.
Interestingly, the transactivation potential of CBP can apparently be
induced by PKA (38). Thus, CBP may fit the role of this putative
C/EBP
coactivator that mediates the PKA-inducible activity. Arguing
against this hypothesis, however, is that C/EBP
, which does not
function as a cAMP-inducible transcription factor (see Figs. 1,
2B, and 4 (B and C) and Table I), also
appears to utilize CBP as a coactivator (38). How CBP could confer PKA
inducibility to one isoform and not the other is not readily obvious,
although one could speculate that the nature of the protein-protein
interaction could differ, in one case allowing the expression of the
inducible activity and in the other instance masking it. Answers to
these questions should be forthcoming as the coactivator role of CBP
for these two C/EBP isoforms is solidified and the molecular nature of
the protein-protein interactions is characterized.
Any model developed to define the mechanism whereby C/EBP
is able to
mediate cAMP responsiveness must incorporate the observation that the
constitutive and PKA-inducible transcriptional activities of this
factor appear to be exerted via at least partially independent mechanisms. The first evidence for this came from experiments where we
observed that G
2, when expressed in the choriocarcinoma JEG-3 cell
line (i.e. non-hepatoma cells), exhibited PKA-inducible activity, but little if any constitutive activity (21). In the present
study, we have presented additional data in support of this hypothesis
by showing that the C/EBP
mutant Y67A,F77A,L78A remained fully
competent as a cAMP-dependent transactivator even though
its constitutive transcriptional activity was significantly inhibited
(Table II). Deletion analysis (Fig. 5) also provided some indication of
the distinct nature of the constitutive and inducible activities of
this factor. While our analysis cannot identify a precise "domain"
that confers the inducible activity, our data do allow us to conclude
that this activity is fully contained within amino acids 6-124 since
N124 remains fully functional with respect to this activity. Since N96
lost all inducible activity, it was interesting to speculate that
region 96-124 was the PKA-inducible domain. However, our attempts to
analyze this region in isolation were unsuccessful since the presence
of amino acids 6-50 was absolutely required to detect any activity of
the Gal4 fusion proteins. We also recognize that the lack of activity
of N96 does not necessarily indicate the lack of involvement of any of
the remaining amino acids in the PKA-inducible activity since the loss
of activity could be due solely to alterations in tertiary structure as
a result of the deletion. Another conclusion that we can make is that
the sole conserved transactivation motif among C/EBP family members,
which lies between amino acids 55 and 86 (18), is likely not involved
in the PKA-inducible activity of C/EBP
. The triple mutant that we
tested contained mutations in three conserved amino acids found in all
C/EBP isoforms, and the amino acids at positions 77 and 78 are part of
the homology box 2 core (18). Nerlov and Ziff (18) demonstrated that
these residues are involved in the constitutive activity of C/EBP
,
apparently exerting their effects by participating in protein-protein
interactions with the TATA-binding protein and TFIIB. Since C/EBP
also contains these conserved amino acids but does not act as a
cAMP-inducible factor, one would predict that this conserved motif,
which is found in all C/EBP isoforms, does not participate in the
cAMP-inducible activity of C/EBP
. Indeed, our data support this
hypothesis, allowing us to conclude that cAMP-inducible activity is not
mediated via interactions with the TATA-binding protein or TFIIB, but
via interactions with an associated coactivator and/or another target
within the preinitiation complex.
There is ample evidence to indicate that C/EBP
has several important
biological roles, beyond being simply a constitutive transactivator. As
evidenced in this paper, C/EBP
can function as an effector molecule
in the cAMP signaling pathway, leading to the alteration of expression
of target genes. Additionally, we have recently shown that C/EBP
can
participate in mediating triiodothyronine responsiveness (45). Given
its tissue-limited expression pattern (3), it also contributes to the
tissue-specific pattern of gene expression and tissue differentiation,
particularly adipose. Since the expression of C/EBP
is temporally
regulated, with no accumulation occurring until just prior to birth
(3), it also appears to function as a developmental transcription
factor. Its involvement in the activation of the PEPCK gene at
parturition (46) plays a particularly critical role in providing the
newborn with the capacity for glucose synthesis, as this gene codes for the enzyme generally thought to be the rate-limiting enzyme of this
pathway. Many of these important biological roles were highlighted by
examination of mice that contained a targeted deletion of the C/EBP
gene or of adult mice with the gene conditionally disrupted. The
knockout mice died within hours after birth of hypoglycemia; displayed
no ability to store liver glycogen or adipocyte lipid; and had reduced
expression of genes for glycogen synthase, PEPCK, glucose-6-phosphatase, and uncoupling protein of brown adipose tissue
(11). Similar alterations in gene expression were observed in the
conditional knockout mice (47). The findings of the present paper
emphasize an additional role of this transcription factor, that of a
hormone-responsive activator, and provide further evidence of the
important role it plays as a central regulator of energy homeostasis
(48).
We thank Richard Hanson, G. Stanley
McKnight, Steve McKnight, Claus Nerlov, Ivan Sadowski, Shizuo
Akira, and Patrick Quinn for generously providing plasmids. We thank
Gerald Davies for assistance in the Western blot analysis.