(Received for publication, July 31, 1995; and in revised form, November 20, 1995)
From the
The antagonistic effect of cAMP on the insulin-induced expression of fatty acid synthase (FAS) in liver could be mimicked in vitro using H4IIE hepatoma cells, both by measuring the response of the endogenous FAS gene and by assaying expression of transfected reporter genes containing promoter elements of the FAS gene. 5`-Deletion analysis and replacement mutagenesis revealed that an essential element required for cAMP antagonism of the insulin effect is an inverted CCAAT box located between nucleotides -99 and -92. DNase I footprinting and gel shift analysis revealed that this region can bind a protein present in nuclei of liver and spleen, organs that express high and undetectable levels of FAS, respectively. This protein is not a CCAAT/enhancer-binding protein, C/EBP. Thus, the FAS gene appears unusual in that the sequence element required for transcriptional regulation by cAMP is neither a cAMP response element (CRE) nor a binding site for AP-1, AP-2, or C/EBP. These results suggest that essential to the regulation of FAS transcription by cAMP is the interaction of an inverted CCAAT box motif with a constitutively produced trans-acting factor that either itself undergoes modification in response to cAMP or associates with a protein that is produced or modified by cAMP exposure.
It has long been recognized that glycogen metabolism,
gluconeogenesis, glycolysis, fatty acid oxidation, and synthesis are
all coordinately regulated in the liver by short-term regulatory
mechanisms involving primarily allosteric and covalent modulation of
key enzymes(1) . Critical to this short-term regulation are the
opposing actions of insulin and glucagon. Glucagon, via its
intracellular messenger, cAMP, activates protein kinases involved in
the phosphorylation of key enzymes and transforms the liver from a
glycogenic, glycolytic, lipogenic tissue to a glycogenolytic,
gluconeogenic, and fatty acid-oxidizing tissue. Initially, when the
food supply is stopped, serum insulin concentration falls and glucagon
concentration rises, triggering this short-term regulatory mechanism.
On refeeding, insulin concentration rises, glucagon falls, and these
changes are reversed, and the liver reverts to its role as a
glycogenic, glycolytic, lipogenic tissue. It seems likely that
antagonism between insulin and glucagon may also serve in long-term
regulation of these pathways by controlling transcription of key genes (2) . For example, on prolonged fasting, cAMP may eventually
initiate the down-regulation of transcription of lipogenic enzymes.
Indeed, it has been shown that injection of dibutyryl cAMP will prevent
the activation of FAS ()transcription in the liver that
normally accompanies refeeding(3) .
All of the long-term changes in lipogenesis that occur in response to dietary, hormonal, and developmental cues appear to be accompanied by changes in the rate of transcription of the FAS gene in a tissue-specific manner. Elucidation of the sequence of the entire rat FAS gene, including 6.1 kilobases of the 5`-flanking region(4, 5) , has made possible detailed exploration of the mechanisms regulating transcription of this gene, and, recently, sequences required for mediating the effect of insulin have been identified(6, 7) . The objective of this study was to identify sequences in the FAS gene that are essential for mediation of the cAMP effect on transcription. Because cAMP has been reported to antagonize the insulin effect on transcription of the FAS gene in hepatocytes, we identified and utilized a hepatoma cell line that appears to be good model system for this physiological process. Our strategy was to utilize 5`-deletion mutagenesis and replacement mutagenesis of chimeric FAS-reporter genes to narrow down the location of the sequences required for cAMP antagonism of insulin action and to look for trans-acting nuclear factors that might bind to the putative response element. The results of this study are presented in the following report.
For hormonal
treatments, the transfected cells were maintained in the
serum-supplemented media for 16 h, then switched to media containing
0.5% serum for at least 12 h prior to treatment with 20 nM insulin and/or 1 mM cAMP for 48 h. Cells were harvested,
resuspended in 0.2 ml of 0.25 M Tris HCl buffer (pH 7.8),
lysed by freeze/thawing, and soluble extracts were prepared for
-galactosidase assay. The remainder of the supernatant was heated
at 65 °C for 10 min to destroy endogenous esterases. Heat-denatured
proteins were removed by centrifugation, and portions of the soluble
extract were assayed for CAT activity.
Cyclic AMP had no effect on the amount of endogenous FAS present in cells grown in medium containing either 20% or 0.5% serum. Nevertheless, in the presence of cAMP, insulin failed to induce an increase in the endogenous FAS content of H4IIE cells (Table 1). Again, this result is similar to that observed in vivo, since administration of dibutyryl cAMP has been shown to block the insulin-dependent induction of FAS expression in the liver that is normally seen following fasting and refeeding(3) .
The FAS
content of H4IIE cells (0.7% of the cytosolic protein, by weight)
cultured in the presence of insulin (Table 1) is considerably
less than that of normal liver (
6% of the cytosolic protein, by
weight) derived from fasted rats that have been refed a high
carbohydrate diet(19) . Nevertheless, the observation that
H4IIE cells modulate their FAS content in response to insulin and cAMP
in a manner similar to that exhibited by normal hepatocytes encouraged
us to adopt this cell line as a model system for identifying hormone
response elements within the FAS gene.
Figure 1:
Effect of insulin and
cAMP on transcription of the CAT reporter gene driven by the promoter
and various flanking sequences from the FAS gene in H4IIE cells. H4IIE
cells were co-transfected with a -galactosidase expression plasmid
and various chimeric FAS-CAT constructs containing different lengths of
FAS upstream sequences. Cells were incubated for 48 h either with no
hormones, with 20 nM insulin, with 1 mM cAMP, or with
both cAMP and insulin. The fold increase in CAT expression induced by
hormone treatment was normalized to
-galactosidase expression.
Full details are presented under ``Experimental Procedures.'' ND, not determined. The data represent the means ± S.D.
of 4 to 6 experiments. The range of observed CAT activities
corresponded to acetylation of 1 to 6% of the chloramphenicol substrate
per h.
The addition of cAMP alone to H4IIE cells transfected with various chimeric reporter genes and cultured in the presence of 0.5% serum had little effect on CAT expression. However, in the presence of cAMP, the ability of insulin to stimulate CAT expression was compromised. This muting of the insulin effect by cAMP was evident with all deletion constructs that exhibited an insulin effect (Fig. 1), suggesting that the sequence element responsible for sensitivity to cAMP is located between nucleotides -149 and +68 of the FAS gene. Similar results were obtained using two different cAMP analogs, 8-bromoadenosine 3`,5`-cyclic monophosphate and 8-(4-chlorophenylthio)adenosine 3`,5`-cyclic monophosphate.
Figure 2: DNase I footprinting analysis of the FAS promoter region in the presence of nuclear extracts from rat liver. An end-labeled DNA fragment comprising FAS promoter sequences from nucleotides -190 to -23 was digested with DNase I in the presence or absence of a liver nuclear extract from either fasted or fasted-refed rats, and the products were fractionated by electrophoresis on denaturing polyacrylamide gels. The regions of the DNA probe protected from digestion are indicated by a box in the margin. Lane 1, G + A reaction; lanes 2 and 5, bovine serum albumin included, no nuclear extract; lane 3, 50 µg of liver nuclear proteins from fasted rats; lane 4, 50 µg of liver nuclear proteins from fasted-refed rats.
When the -74/-52 footprinted region
was removed from the -249/+68 FAS-CAT chimeric gene (in the
construct -249/+68 FAS-CAT) insulin responsiveness of CAT expression was lost, and,
thus, the response to cAMP could not be evaluated (details not shown).
This finding was consistent with results of an earlier study that
identified nt -68/-52 as essential for mediating insulin
responsiveness of the FAS gene(6) .
The second footprinted region, which contains an inverted CCAAT box motif at nt -99/-92, was evaluated by replacement mutagenesis in the context of the -249/+68 FAS-CAT construct. Although mutation of the inverted CCAAT box had no effect on the ability of the chimeric gene to direct the insulin-sensitive expression of CAT, responsiveness to cAMP was completely abolished (Fig. 3). The results of this experiment indicated that the insulin and cAMP effects on transcription of the FAS gene are mediated by different regions of the promoter and strongly implicated the inverted CCAAT box in the regulation by cAMP. To verify that the inverted CCAAT box motif specifically was responsible for the nuclear protein binding observed in the DNase I protection assay, we employed the electrophoretic mobility shift assay using a radiolabeled probe corresponding to nucleotides -106 to -85 (Fig. 4). Liver nuclear extracts from fasted and fasted/refed rats and spleen nuclear extracts from fasted/refed rats retarded the mobility of the probe to the same extent (slow migrating band, lanes 2, 8, and 5, respectively). Formation of these radiolabeled DNA-protein complexes was abolished in the presence of 100-fold molar excess of an unlabeled oligonucleotide corresponding exactly to nucleotides -106/-85 (lanes 4, 10, and 7), but not when the sequence between -99/-92 of the unlabeled probe was mutated (lanes 3, 9, and 6). When a radiolabeled -106/-85 probe containing the same mutated sequence between -92/-99 was used in the electrophoretic mobility shift assay in the presence of nuclear extracts from either liver or spleen, DNA-protein interactions were not observed (lanes 12-14). Faster migrating radiolabeled DNA-protein complexes were also formed with nuclear extracts from liver and spleen. However, formation of these radiolabeled complexes was reduced in the presence of unlabeled oligonucleotides containing either the intact or the mutated inverted CCAAT box sequence (lanes 2 and 3, 6 and 7, 9 and 10), and these complexes were formed from the radiolabeled mutated oligonucleotide probe (lanes 12-14). It would appear therefore that formation of these fast migrating complexes is not specifically dependent on the presence of the inverted CCAAT box sequence. However, from the properties of the slow migrating DNA-protein complex, one could conclude unequivocally that rat liver and spleen contain nuclear protein(s) that specifically interact with the inverted CCAAT box located at nt -99/-92 of FAS promoter.
Figure 3: Effect of mutation of the inverted CCAAT box on mediation of cAMP responsiveness. H4IIE cells were transfected with either the wild type (WT) or mutated (Mutant) -249/+68 FAS-CAT constructs and subjected to the hormone treatment for 48 h. 0, no hormones; +Ins, plus insulin; +Ins/cAMP, plus insulin and cAMP. In the mutated FAS-CAT constructs, nucleotides -99 to -92, 5`-CATTGGCC-3`, were changed to 5`-GTCCAAGG-3`. Values represent means with standard deviation for 4 independent experiments.
Figure 4: Demonstration that nuclear proteins bind to the the inverted CCAAT box region of the FAS gene. Electrophoretic mobility shift assays were performed using nuclear proteins from livers (L) or spleens (S) of fasted (F) or fasted-refed (R) rats. The end-labeled DNA probes comprising FAS promoter sequences from nucleotides -106 to -85 were either identical with the wild type sequence (Wt) or contained mutations (mu) in the region -92 to -99 (see Fig. 3). Some incubations also included 100-fold molar excess of unlabeled competitive oligonucleotides, representing either the wild type (Wt) sequence -106 to -85 or the mutated (mu) sequence.
The possibility
that the trans-acting factor binding to the inverted CCAAT box
might be a member of the well characterized family of C/EBP
transcription factors was evaluated and discounted because of the
following evidence. Firstly, neither the electrophoretic mobility nor
the amount of the DNA-protein complex formed between liver nuclear
extract and a radiolabeled oligonucleotide probe representing the
inverted CCAAT box was affected in the presence of either authentic
anti-C/EBP antibodies or an unlabeled oligonucleotide representing
the consensus binding site for C/EBP
(Fig. 5, lanes
1-4). Secondly, formation of the DNA-protein complex between
liver nuclear extract and a radiolabeled oligonucleotide probe
representing the inverted CCAAT box was prevented when the extract was
preheated for 10 min at 65 °C, conditions under which the C/EBP
proteins are stable. (Fig. 5, lanes 2 and 7).
Thirdly, authentic C/EBP
, expressed as a recombinant protein in E. coli, did not bind to a probe containing the FAS-inverted
CCAAT box (Fig. 5, lane 8), although it did bind to an
oligonucleotide containing the consensus binding site for C/EBP
;
the electrophoretic mobility of this complex was supershifted by
anti-C/EBP
antibodies (Fig. 5, lanes 10 and 11), and association of the radiolabeled probe with the
complex was effectively competed out with unlabeled C/EBP consensus
probe (Fig. 5, lanes 10 and 12). Fourthly, the
amount of DNA-protein complex formed between recombinant C/EBP
and
an oligonucleotide containing the consensus binding site for C/EBP was
unaffected by the presence of a large excess of unlabeled probe
containing the FAS-inverted CCAAT box (Fig. 5, compare lanes
10 and 13). The results of these experiments indicate
that it is highly unlikely that the cAMP effect on transcription of the
FAS gene is mediated by a member of the C/EBP family of transcription
factors.
Figure 5:
C/EBP is not the protein involved in
mediating the cAMP effect via the inverted CCAAT box. Two radiolabeled
oligonucleotide probes were employed, one containing the FAS inverted
CCAAT box, FAS nt -106 to -85 (lanes 1-8),
the other containing the consensus binding site for C/EBP (lanes
9-13). Protein extracts were prepared either from liver
nuclei of fasted or fasted/refed rats or from E. coli cells
that had been transfected with a C/EBP
expression vector. Where
indicated, a polyclonal antiserum raised against C/EBP
was
included for ``supershifting.'' In some incubations,
unlabeled oligonucleotides representing either the consensus C/EBP
binding site, the FAS inverted CCAAT box, or the cAMP response element,
CRE, were included at 100-fold molar excess as potential competitors
for the radiolabeled probe. Lane 1, inverted CCAAT box probe
alone; lane 2, inverted CCAAT box probe plus liver nuclear
extract from fasted/refed rats; lane 3, same as lane
2, plus C/EBP
antibodies; lane 4, same as lane
2, plus unlabeled C/EBP competitive oligonucleotide; lane
5, same as lane 2, plus unlabeled CRE competitive
oligonucleotide; lane 6, inverted CCAAT box probe plus liver
nuclear extract from fasted rats; lane 7, inverted CCAAT box
probe plus heat-treated liver nuclear extract from fasted/refed rats; lane 8, same as lane 1, plus E. coli extract; lane 9, consensus C/EBP binding site probe
alone; lane 10, same as lane 9, plus E. coli extract; lane 11, same as lane 10, plus
anti-C/EBP
antibodies; lane 12, same as lane 10,
plus unlabeled C/EBP competitive oligonucleotide; lane 13,
same as lane 10, plus inverted CCAAT box competitive
oligonucleotide.
The inverted CCAAT box motif is quite dissimilar from the consensus cAMP-responsive element (CRE), TGACCTCA, and an unlabeled oligonucleotide containing the CRE consensus sequence does not compete with the labeled inverted CCAAT box probe for binding to the liver nuclear protein (Fig. 5, lanes 2 and 5); neither does the labeled inverted CCAAT box probe bind to purified CREB protein (details not shown). Based on this evidence, CREB protein does not appear to be involved in mediating the cAMP effect.
In response to insulin and cAMP, the hepatoma cell line H4IIE modulates its endogenous FAS concentration in a manner qualitatively similar to that exhibited by normal liver cells. Thus, the elevation of endogenous FAS protein concentration induced by exposure to insulin can be blocked by simultaneous exposure to cAMP. Using these cells as a model system, we transfected chimeric reporter genes containing various 5`-flanking sequences from the FAS gene and demonstrated that expression of the reporter gene could be enhanced by insulin and that this response could be antagonized by cAMP. In the course of this study, using 5`-deletion analysis of the FAS-reporter gene, we initially localized the insulin response element between nt -124 and -30 (details not shown) and subsequently identified within this region a sequence element between -74/-52 that was protected from DNase I action in the presence of H4IIE nuclear extract. Deletion of the footprinted region from the chimeric FAS-reporter gene resulted in the loss of insulin responsiveness indicating that this region of the promoter is required for mediating the insulin response. While these studies were in progress, Moustaïd et al.(6) independently reported that the insulin response element of the FAS gene is located between nucleotides -68 and -52. Thus, the results of our studies are in complete agreement with their findings.
By examining the effect of deletion and replacement mutagenesis on the ability of cAMP to antagonize insulin action, we were able to identify a sequence element between nt -99/-92 that is essential for cAMP action. This region was also found to be essential for the binding of a nuclear factor present in the livers and spleens of fasted and fasted-refed rats. The electrophoretic mobilities of the DNA-protein complexes formed with these various nuclear extracts were indistinguishable. Since spleen does not express detectable levels of FAS (4) and expression in the liver is elevated on feeding but drastically lowered on fasting, there is clearly no correlation between the presence in a particular tissue of a nuclear protein binding to nt -99/-92 and the level of FAS expression. More likely then, this protein is a constitutive transcription factor that either itself undergoes covalent modification as a result of exposure of the cells to cAMP or associates with a protein that is either expressed or modified following cAMP treatment. Mutational disruption of the inverted CCAAT box also decreases activity of the FAS promoter approximately 2-fold, in the absence of insulin or cAMP (Fig. 3), indicating that the constitutive transcription factor binding to the inverted CCAAT box plays a role in supporting basal, as well as cAMP-modulated, transcription.
When placed in front of a heterologous promoter, nt -124 to -30 of the FAS gene confer responsiveness to insulin but not to cAMP. This observation indicates that the context of the FAS gene is important in mediating the cAMP effect and raises the possibility that a protein binding to another sequence element within nt -124 and +68 may also be required for mediating the cAMP effect, perhaps through interaction with the protein binding to the inverted CCAAT box.
The FAS appears
to be one of a small group of genes transcriptionally regulated by cAMP
that bear cAMP-responsive sequences distinct from the more common CREB,
AP-1, and AP-2 binding sites. Other genes that fall into this category
include the mouse renin gene(24) , the human myelin basic
protein gene(25) , the bovine steroid hydroxylase
(P-450) gene(26) , the rat c-fos gene(27) , the porcine G-protein
subunit gene(28) , and the human tryptophan hydroxylase
gene(29) . In all of these other examples, cAMP is an inducer
of transcription, whereas in the case of the hepatic FAS it is a
negative regulator. In several of these genes, CCAAT box-binding
proteins have been implicated as mediators of the cAMP-regulated
transcription, notably the c-fos gene which is regulated by
the phosphorylated form of C/EBP
(27) , the G-protein
subunit which is regulated by an unidentified CCAAT
box-binding protein and the human tryptophan hydroxylase gene which,
like the FAS gene, has an inverted CCAAT box that is essential for
mediating the cAMP effect on transcription. In the case of the FAS, it
appears highly unlikely that the protein binding to the inverted CCAAT
box is a member of the C/EBP family of transcriptional factors, based
on its heat lability and immunochemical properties. Clearly, the
purification and characterization of the transcription factor(s)
binding to the inverted CCAAT box motif would provide further insight
into the mechanism by which cAMP regulates transcription of the FAS
gene.