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Molecular Endocrinology, doi:10.1210/me.2004-0105
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Molecular Endocrinology 19 (1): 163-174
Copyright © 2005 by The Endocrine Society

A Distal Region Involving Hepatocyte Nuclear Factor 4{alpha} and CAAT/Enhancer Binding Protein Markedly Potentiates the Protein Kinase A Stimulation of the Glucose-6-Phosphatase Promoter

Amandine Gautier-Stein, Gilles Mithieux and Fabienne Rajas

Institut National de la Santé et de la Recherche Médicale, Unité 449/Institut National de la Recherche Agronomique 1235/Université Claude Bernard Lyon 1, Institut Fédératif de Recherche Laennec, 69372 Lyon cedex 08, France

Address all correspondence and requests for reprints to: Dr. Amandine Gautier-Stein, Institut National de la Santé et de la Recherche Médicale, Unité 449-Faculté de Médecine Laennec, Rue Guillaume Paradin-69372 Lyon cedex 08, France. E-mail: Amandine.Gautier{at}univ-lyon1.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucose-6-phosphatase (Glc6Pase) is the last enzyme of gluconeogenesis and is only expressed in the liver, kidney, and small intestine. In these tissues, the mRNA and its activity are increased when cAMP levels increased (e.g. in fasting or diabetes). We first report that a proximal region (within –200 bp relative to the transcription start site) and a distal region (–694/–500 bp) are both required for a potent cAMP and a protein kinase A (PKA) responsiveness of the Glc6Pase promoter. Using different molecular approaches, we demonstrate that hepatocyte nuclear factor (HNF4{alpha}), CAAT/ enhancer-binding protein-{alpha} (C/EBP{alpha}), C/EBPß, and cAMP response element-binding protein (CREB) are involved in the potentiated PKA responsiveness: in the distal region, via one HNF4{alpha}- and one C/EBP-binding sites, and in the proximal region, via two HNF4{alpha} and two CREB-binding sites. We also show that HNF4{alpha}, C/EBP{alpha}, and C/EBPß are constitutively bound to the endogenous Glc6Pase gene, whereas CREB and CREB-binding protein (CBP) will be bound to the gene upon stimulation by cAMP. These data strongly suggest that the cAMP responsiveness of the Glc6Pase promoter requires a tight cooperation between a proximal and a distal region, which depends on the presence of several HNF4{alpha}-, C/EBP-, and CREB-binding sites, therefore involving an intricate association of hepatic and ubiquitous transcription factors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLUCOSE-6-PHOSPHATASE (Glc6Pase; EC 3.1.3.9) is a major enzyme involved in gluconeogenesis, because it is expressed in three tissues, i.e. the liver, the kidney and the small intestine, and it gives them the capacity to release glucose in blood (1, 2). It must be mentioned that the occurrence of low Glc6Pase expression in other tissues, e.g. islets or brain, is questionable because of the failure to detect Glc6Pase mRNA in these tissues (3). In these three organs, the Glc6Pase mRNA and its activity are increased during physiological situations under the control of glucagon (e.g. in fasting, diabetes and in the very first days after birth); they are then normalized upon refeeding or insulin treatment (4, 5, 6). Furthermore, cAMP increases Glc6Pase mRNA levels in primary hepatocytes without altering the Glc6Pase mRNA stability (6), which suggests that the regulation of this gene by cAMP may occur at a transcriptional level.

The transcriptional regulation by cAMP mainly occurs via phosphorylation of transcription factors belonging to the cAMP-response element (CRE)-binding protein/activating transcription factor (CREB/ATF) family made by the protein kinase A (PKA). This family includes the CREB, the cAMP-response element-binding modulator, and ATF1 (7). The PKA phosphorylation of these transcription factors, bound to their consensus binding site (TGACGTCA), allows the recruitment of the CREB-binding protein (CBP) coactivator, which in turn increases the transcriptional activity. In addition, PKA phosphorylation may increase the binding of CREB to weaker binding sites (8). CREB and CBP factors are ubiquitously expressed and thus are not sufficient to confer tissue-specific responsiveness to cAMP. Moreover, they cooperate with other specific transcription factors to confer the cAMP effect (9). For example, HNF4{alpha} (hepatocyte nuclear factor 4{alpha}) is involved in the cAMP regulation of the tyrosine aminotransferase gene (10) and of the liver carnitine palmitoyl transferase 1 (11). In addition, C/EBP (CAAT/enhancer-binding protein) {alpha} and ß are necessary for the maximal cAMP induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene through three distal binding elements (12).

A cAMP regulation mechanism of Glc6Pase gene transcription has already been characterized in a very proximal region of the gene (–231/+3 bp) (13). This mechanism involves the CREB protein through two CREs (13, 14), and two liver-specific transcription factors: HNF1 (15) and HNF6 (16). CRE1 (–165/–158 bp) binds the CREB protein and is involved in the transactivation of the Glc6Pase gene by cAMP and glucocorticoids in the hepatic H4IIE cells (14). The involvement of CRE2 (–141/–134 bp) in the cAMP regulation is not yet clear: CRE2 binds the CREB protein, and its mutation decreases the induction of the Glc6Pase gene by cAMP in the hepatic HepG2 cells (13). However, in renal LLC-PK cells, this CRE2 site is not able to confer a direct stimulatory effect of PKA on reporter gene expression (15). Regarding tissue- specific factors, the HNF1-binding site (–225/–212), which specifically binds HNF1{alpha} in HepG2 cells or HNF1ß in LLC-PK cells, is crucial for cAMP induction in renal cells, but not in hepatic cells (15). The liver transcription factor HNF6 is involved in the regulation of the Glc6Pase gene by cAMP in HepG2 cells via a binding site located between –113 and –104 bp (16).

Interestingly, Glc6Pase gene expression is delayed at birth in C/EBP{alpha} knockout mice (17), and C/EBPß knockout mice show impaired hepatic glucose production (18), suggesting a role for C/EBP proteins in the transcriptional control of the gene. Moreover, HNF4{alpha} has been involved in the transcriptional stimulation of the Glc6Pase gene by peroxisome-proliferative-activated receptor-{gamma} coactivator-1{alpha} (PGC1{alpha}) (19), suggesting a relationship between HNF4{alpha} and the response to cAMP. In this work, we have thus studied the implication of HNF4{alpha} and C/EBP in the cAMP transcriptional regulation of the Glc6Pase gene promoter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of Two Distinct cAMP/PKA Response Regions in the Glc6Pase Promoter
HepG2 cells were transfected with the Glc6Pase promoter-firefly luciferase (LUC) constructs previously described (20), in the presence of 10 µM forskolin, an activator of adenylate cyclase. In the presence of forskolin, the transcriptional activity of all promoter constructs was induced (Fig. 1AGo). Deletions of the Glc6Pase promoter sequence between –1640 and –694 bp had no effect on the activation by forskolin of the respective Glc6Pase promoter constructs, which were all induced by about 10 times (Fig. 1AGo). In contrast, the deletion of the Glc6Pase promoter sequence from –694 to –500 bp resulted in a marked decrease in the forskolin induction of the Glc6Pase transcriptional activity. The deletion in the Glc6Pase promoter sequence between –500 and –320 bp had no further marked decreasing effect on forskolin-stimulatory action (Fig. 1AGo).



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  		Fig. 1. The Glc6Pase Promoter Region Located between –694 and –500 bp Confers a High Responsiveness to Forskolin and PKA

HepG2 cells were transfected with 1 µg of the 5'-deletion fragments of the Glc6Pase promoter in the presence of 10 µM forskolin in panel A and an expression vector of the catalytic subunit of PKA (100 ng) in panel B, as described in Materials and Methods. Treatment with forskolin for 6 h was performed 24 h after cell transfection in the serum-free medium. Results are expressed as fold induction over basal conditions in the absence of effector. Values shown are the means ± SEM of at least three experiments. *, Significantly different from the –694/+60B construct value; P < 0.05.

 
In comparison, in the presence of an expressing vector of the catalytic subunit of PKA (21), the transcriptional activity of all promoter constructs was markedly induced (Fig. 1BGo). Deletions of the Glc6Pase promoter sequence between –1640 and –694 bp had no effect on the ability of PKA to induce the transcriptional activity of the respective Glc6Pase promoter constructs, which were all stimulated by about 80- to 100-fold. In contrast, the deletion of the Glc6Pase promoter sequence from –694 to –500 bp resulted in a dramatic decrease in the PKA induction of the transcriptional activity. Consistent with the forskolin study, the deletion of the Glc6Pase promoter sequence between –500 and –320 bp had no further decreasing effect on PKA-stimulatory action (Fig. 1BGo). The cAMP responsiveness of the proximal promoter region has already been described (see Introduction); thus we did not consider the effect of further deletion on the forskolin and/or the PKA induction of the promoter activity. Taken together, these data strongly suggested that the region extending from –694 to –500 bp was required for a maximal induction of the Glc6Pase transcriptional activity by cAMP and PKA.

We showed previously that the Glc6Pase promoter shows an HNF4{alpha}-binding site located at nucleotides –668/–647 (20). Furthermore, the sequence analysis of the Glc6Pase promoter region between –694 and –500 bp predicts a potential binding site of C/EBP located at nucleotides –608/–603. Because these two transcription factors were previously shown to be involved in the cAMP regulation of the tyrosine aminotransferase gene (10) and the PEPCK gene (12), we have studied their involvement in the potentiation of the PKA induction of the Glc6Pase gene conferred by the –694/–500 bp region. Because HNF4{alpha} and C/EBP also exhibit binding sites in the proximal region [–79/–67 bp (19), +2/+14 (20) for HNF4{alpha}, and –135/–130 (13) for C/EBP], we have also examined the involvement of these proximal sites in the PKA effect.

HNF4{alpha} Is Involved in the PKA Induction of the Glc6Pase Promoter
To assess the role of HNF4{alpha} in the Glc6Pase transcriptional regulation by PKA, HepG2 cells were transfected with the –694/+60B or –320/+60B promoter constructs in the presence of the PKA expression vector and a dominant negative expression vector of HNF4{alpha} (DN-HNF4{alpha}) (22). The overexpression of DN-HNF4{alpha} had no effect on the basal promoter activity of any of the constructs (Fig. 2AGo). In contrast, the PKA induction of the promoter activity in the presence of DN-HNF4{alpha} was strongly inhibited by more than 90% for the –694/+60B construct and by about 80% for the –320/+60B construct (Fig. 2BGo). These results strongly suggested that HNF4{alpha} is crucial for the induction by PKA in both the proximal and distal regions.



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  		Fig. 2. Involvement of HNF4{alpha} in the PKA Responsiveness of the Glc6Pase Promoter

A, HepG2 cells were transfected with 1 µg of the –694/+60B or –320/+60B constructs of the Glc6Pase promoter in the absence (black bars) or in the presence (gray bars) of DN-HNF4{alpha} plasmid (50 ng). The basal promoter activities are expressed in relation to the control activity in the absence of DN-HNF4{alpha}. B, The same plasmids were cotransfected in the presence of a PKA expression vector (100 ng). Results are expressed as fold induction over basal conditions without PKA. *, Significantly different from the value in the absence of DN-HNF4{alpha}; P < 0.01. C, Cells were transfected with the –694/+60B constructs with mutations of the respective HNF4{alpha}-binding sites and with a PKA expression vector. The location (and the number) of the HNF4{alpha}-binding sites are identified by small gray boxes. Arrows indicate the initiation transcription site. Results are expressed as fold induction by PKA. *, Significantly different from the PKA induction of the wild-type construct; P < 0.01. Values shown are the means ± SEM of at least three experiments.

 
We further characterized the individual implication of the HNF4{alpha}-binding sites in the induction by PKA via site-directed mutagenesis. There was no effect of individual mutations of the HNF4{alpha}-binding sites on the basal promoter activity (data not shown). The mutation of the HNF4{alpha} site 3 (20) on the –694/+60B promoter construct resulted in a 40% decrease in the induction by PKA (Fig. 2CGo). The mutation of the HNF4{alpha} site 1 (20) on the –694/+60B promoter construct also decreased the induction by PKA by 40%. The mutation of the HNF4{alpha}-binding site described by O’Brien and co-workers (–79/–67 bp, named site 2) (19), resulted in a 70% decrease in the induction by PKA (Fig. 2CGo). These results clearly indicated that HNF4{alpha} was involved in the PKA regulation in the proximal and distal regions of the Glc6Pase promoter through its three binding sites.

C/EBP Is Involved in the PKA Induction of the Glc6Pase Promoter
If the C/EBP proteins are involved in the PKA induction of the Glc6Pase promoter, this should be proven: HepG2 cells were transfected with the –694/+60B promoter constructs in the presence of the PKA expression vector and a C/EBP dominant negative expression vector (A-C/EBP) able to prevent the binding of both C/EBP{alpha} and C/EBPß proteins (23). The overexpression of A-C/EBP resulted in a 50% loss of the basal promoter activity of the –694/+60B construct (Fig. 3AGo). In the presence of A-C/EBP, the PKA induction of the promoter activity was inhibited by about 40% (Fig. 3BGo). The mutation of the potential distal C/EBP-binding site (–608/–603 bp, named C/EBPsite2) had no effect on the basal promoter activity (Fig. 3CGo, midpanel), but resulted in a 40% decrease in the PKA induction of the construct (Fig. 3CGo, right panel). In contrast, the mutation of the previously identified proximal C/EBP-binding site (13) (–135/–130, named C/EBPsite1) led to a significant decrease in the basal promoter activity of the construct (Fig. 3CGo, midpanel), but had no significant decreasing effect on its induction by PKA (Fig. 3CGo, right panel). To assess the binding capacity of the C/EBP{alpha} and C/EBPß isoforms to the potential distal binding site of the Glc6Pase promoter, we incubated two radiolabeled oligonucleotide probes, matching, on the one hand, the Glc6Pase-binding site (C/EBPsite2, corresponding to the Glc6Pase promoter sequence –619/–580 bp, Table 1Go) and, on the other hand, a consensus C/EBP-binding site (C/EBPcons, Table 1Go), with protein extracts from both untreated HepG2 cells and HepG2 cells treated with forskolin. Gel shift experiments showed that protein binding to both probes led to the formation of one major complex irrespective of the cell extract used (treated or not with forskolin) (Fig. 4Go, A and B). The unlabeled probe efficiently blocked this binding. In contrast, an unlabeled oligonucleotide matching the specific HNF4{alpha}-binding site of the PEPCK promoter and the unlabeled oligonucleotide C/EBPsite2Mut (Table 1Go) were unable to effectively inhibit the protein binding with the labeled probe (Fig. 4Go, A and B). Furthermore, preincubation of cell proteins with an antibody directed against C/EBP{alpha} or C/EBPß prevented the binding to the probes (Fig. 4Go, A and B). The specificity of the antibodies used was confirmed by Western blot analysis using whole-cell extracts from HeLa cells, HeLa cells transfected with an expression vector of C/EBP{alpha} (pMEX-C/EBP{alpha}), and HeLa cells transfected with an expression vector of C/EBPß (CMV-LAP) (Fig. 4CGo). These results strongly suggested that C/EBP{alpha} and -ß were both involved in the PKA induction of the Glc6Pase transcriptional activity via a distal binding site located at –608/–603 bp and that forskolin treatment did not increase this binding.



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  		Fig. 3. Both C/EBP Isoforms {alpha} and ß Are Involved in the PKA Responsiveness of the Glc6Pase Promoter

A and B, HepG2 cells were transfected with 1 µg of the –694/+60B construct of the Glc6Pase promoter without (black bars) or with the A-C/EBP plasmid (100 ng, gray bars) in the absence (panel A) or in the presence (panel B) of a PKA expression vector (100 ng). Results are expressed as in panels A and B of Fig. 2Go. *, Significantly different from experiments in the absence of A-C/EBP; P < 0.01. C, Cells were transfected with the –694/+60B construct in which the respective binding sites of C/EBP were disrupted (the location and the number of C/EBP sites are identified by a number and gray boxes; arrows indicate the initiation transcription site). Results are expressed in relation to the basal activity of the wild-type construct (midpanel) and as fold induction over basal conditions (right panel). *, Significantly different from the value for the wild-type construct; P < 0.01. Values shown are the means ± SEM of at least three experiments.

 

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  		Table 1. Oligonucleotides Used in Mutagenesis or EMSA Experiments

 


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  		Fig. 4. Both C/EBP Isoforms {alpha} and ß Bind to a Distal Site of the Glc6Pase Promoter

A and B, Gel shift mobility assays were carried out with protein extracts from untreated HepG2 cells (panel A) or HepG2 cells treated with forskolin (panel B) and double-stranded radiolabeled oligonucleotide probes matching the putative distal C/EBP-binding site of the Glc6Pase promoter (C/EBPsite2) or the consensus C/EBP-binding site (C/EBPcons). Competition experiments were performed in the presence of 100 ng of the unlabeled probe, of the oligonucleotide C/EBPsite2Mut, and of an oligonucleotide matching the high-affinity HNF4{alpha}-binding site of the PEPCK promoter. An anti-C/EBP{alpha} antiserum or an anti-C/EBPß antiserum was added in the binding mixture as indicated. Specific DNA complexes are indicated by arrows. Asterisk refers to a putative supershift. NR refers to nonrelevant binding. The data shown are representative of four experiments yielding comparable results. C, The specificity of the antibodies raised against C/EBP{alpha} (left panel) and ß (right panel) was analyzed by Western blot analysis in whole-cell extracts from HeLa cells, HeLa cells transfected with the CMV-LAP expression vector, and HeLa cells transfected with the pMEX-C/EBP{alpha} expression vector. Protein (50 µg) were analyzed in each well. Numbers in the middle of the panel refer to the molecular mass of protein markers, and the specific molecular weight of each protein is indicated by an arrow.

 
Proximal CREB-Binding Sites Are Essential for a High PKA Responsiveness Conferred by the Distal Region
To determine whether the distal promoter region located between –694 and –500 bp was able to confer a cAMP response by itself, we constructed a plasmid containing this promoter region placed upstream of a minimal tyrosine kinase promoter. This construct was not sensitive to the induction by PKA (data not shown). This strongly suggested that this promoter region had to cooperate with another promoter region to confer its PKA-potentiating effect. Because proximal HNF4{alpha}-binding sites participate in the distal response (see above), we have thus considered the involvement of the proximal CREB-binding sites in the potentiated PKA responsiveness conferred by the distal region. Two proximal binding sites (–165/–158 bp, CRE1; and –141/–134 bp, CRE2) were described previously (15). HepG2 cells were transfected with the –694/+60B or the –320/+60B constructs in the presence of the PKA expression vector and a dominant negative expression vector of CREB (A-CREB) (24). A-CREB is able to specifically prevent the binding of CREB or cAMP-response element-binding modulator (25). The overexpression of A-CREB had no effect on the basal promoter activity of any of the constructs (Fig. 5AGo). In the presence of A-CREB, the PKA induction of the transcriptional activity of the –694/+60B construct was virtually abolished (>95% decrease) (Fig. 5BGo), whereas the PKA induction of the –320/+60B construct was reduced by at least 75% (Fig. 5BGo). These data clearly indicated that CREB was essential for the maximal PKA induction of the transcriptional activity of the –694/+60B construct. There was no effect of mutations of the CRE sites on the basal promoter activity (data not shown). In contrast, the mutation of the CRE1 site in the –694/+60B construct resulted in a 90% decrease in the PKA induction of the transcriptional activity, whereas the mutation of the CRE2 site decreased the PKA induction by 40% (Fig. 5CGo). The mutations of both CRE1 and CRE2 resulted in a suppression of the PKA induction comparable to that induced by the mutation of CRE1 alone (Fig. 5CGo). These data strongly suggested that both proximal CRE sites were crucial for the potentiation of PKA induction of the Glc6Pase promoter conferred by the distal region.



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  		Fig. 5. Proximal CREB-Binding Sites Are Crucial for the Potentiated PKA Responsiveness of the Glc6Pase Promoter

A and B, HepG2 cells were transfected with 1 µg of the –694/+60B or –320/+60B constructs of the Glc6Pase gene without (black bars) or with (gray bars) the A-CREB plasmid (100 ng), in the absence (panel A) or in the presence (panel B) of a PKA expression vector (100 ng). Results are expressed as in Fig. 2Go. *, Significantly different from the value in the absence of A-CREB. C, Cells were transfected with the –694/+60B construct in which specific binding sites of CREB (CRE1 and/or CRE2) were disrupted by site-directed mutagenesis (the location and the number of CRE sites are identified by a number and gray boxes; arrows indicate the initiation transcription site). Results are expressed as fold induction by PKA. *, Significantly different from the PKA induction of the wild-type construct; P < 0.01. Values shown are the means ± SEM of at least three experiments.

 
In Situ Association of HNF4{alpha}, C/EBP{alpha}, C/EBPß, and CREB Proteins to the Glc6Pase Promoter In Response to cAMP
The chromatin immunoprecipitation (ChIP) assay was used to determine whether the endogenous rat Glc6Pase gene promoter is occupied by these factors under basal conditions or only after induction of cAMP production. Antibodies directed against HNF4{alpha}, C/EBP{alpha}, C/EBPß, and CREB were used to immunoprecipitate chromatin fragments from rat FAO cells treated or not with forskolin (Fig. 6Go). PCR amplification was conducted using primer pairs specific for the –694/+ 60 bp region of the Glc6Pase gene. The association of tissue-specific factors, such as HNF4{alpha}, C/EBP{alpha}, and C/EBPß, with the Glc6Pase gene promoter was not altered by the forskolin treatment, whereas the association of CREB was observed only upon stimulation with forskolin (Fig. 6Go). Because all these factors are able to interact with CBP, we also performed a ChIP assay with an antibody specific for CBP. As for CREB protein, the association of CBP with the Glc6Pase gene promoter was detected only upon forskolin treatment (Fig. 6Go). None of the mock assays revealed amplification from the primers specific for the Glc6Pase gene promoter (Fig. 6Go). These results strongly suggested that HNF4{alpha}, C/EBP{alpha}, and C/EBPß were constitutively bound to the Glc6Pase gene promoter, whereas CREB and CBP would be bound to the promoter in response to forskolin.



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  		Fig. 6. HNF4{alpha}, C/EBP{alpha}, and C/EBPß Proteins Are Constitutively Bound to the Rat Glc6Pase Gene Promoter, Whereas CREB and CBP Factors Will Be Bound to the Promoter upon Forskolin Treatment

Association of HNF4{alpha}, C/EBP{alpha}, C/EBPß, CREB, and CBP proteins with the endogenous rat Glc6Pase gene promoter was measured by a ChIP assay with untreated rat FAO cells (U) or FAO cells treated with forskolin (F). PCR amplification of immunoprecipitated chromatin fragments was performed using primer pairs specific for the –694/+60 bp region of the rat Glc6Pase gene. The mock lane shows the results of control immunoprecipitations performed in parallel without antibody. The input lane shows the result of samples not subjected to immunoprecipitation. The data shown are representative of three experiments yielding comparable results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The results presented here constitute the first demonstration of the involvement of HNF4{alpha} and of the C/EBP isoforms {alpha} and ß in the cAMP transcriptional regulation of the Glc6Pase gene. We have shown that: 1) a distal promoter region (–694/–500 bp) of the Glc6Pase promoter is required for a maximal transactivation by PKA; 2) an HNF4{alpha}-binding site and a C/EBP-binding site in this region are both involved in this potentiation of the PKA induction; 3) two proximal CREB and two proximal HNF4{alpha}-binding sites cooperate with these distal sites to confer this maximal PKA responsiveness of the Glc6Pase promoter; and 4) HNF4{alpha} and C/EBP{alpha} and -ß proteins are constitutively bound to the rat Glc6Pase gene promoter whereas CREB and CBP factors will be bound upon stimulation by cAMP.

We report here that HNF4{alpha} is crucial for the PKA induction of the Glc6Pase promoter activity, via several sites located within the distal and the proximal region of the promoter. We (20) and others (19) have previously highlighted the importance of HNF4{alpha} in the transcriptional regulation of the Glc6Pase gene. It may appear surprising, therefore, that the overexpression of DN-HNF4{alpha} has no effect on the basal promoter activity. However, one may bear in mind that the Glc6Pase promoter activity is regulated by a number of hepatic transcription factors, which are likely to compensate for the loss of HNF4{alpha} function (13). Moreover, in strong agreement with our result, the basal expression of both the Glc6Pase and the PEPCK genes is not decreased in the liver of HNF4{alpha} knockout mice (26). It is obvious that binding site 2 seems to play a more crucial role than sites 1 and 3 in the PKA regulation (see results of Fig. 2Go), whereas we have previously reported that sites 1 and 3 alone might fully account for the regulation by polyunsaturated fatty acids (20). It has been strongly suggested that binding site 2 is involved in the regulation of the Glc6Pase gene by PGC-1{alpha} in H4IIE cells (19). We should mention that we could not find any evidence of a transcriptional effect of PGC-1{alpha} on the Glc6Pase gene in cotransfection studies in HepG2 cells (data not shown). Moreover, PGC-1{alpha} appears to be an amplifier of transcription rather than a necessary factor for the stimulation by cAMP (27). Taken together, our results strongly suggest that HNF4{alpha} plays a key role in the cAMP induction of the Glc6Pase gene via at least the three sites studied here, with a prominent role played by site 2 in the proximal region.

C/EBP proteins have been involved previously in the transcriptional regulation of numerous liver genes (28). Regarding the gluconeogenic genes, it has been proposed that C/EBP{alpha} –/– mice die soon after birth due to severe hypoglycemia caused by reduced expression of glycogen synthase and decreased levels of the major gluconeogenic enzymes PEPCK and Glc6Pase, which impair the hepatic glucose output (17). The C/EBPß –/– mice show at least two different phenotypes (A and B). Mice of phenotype B die within 24 h after birth because of severe hypoglycemia, resulting from the inability to mobilize hepatic glycogen and express PEPCK (18). Mice of phenotype A survive to adulthood but show fasting hypoglycemia and impaired stimulation of hepatic glucose output in response to glucagon and adrenaline (18, 29). It could be derived from these results that both C/EBP proteins {alpha} and ß might be involved in the transcriptional regulation of PEPCK and Glc6Pase genes in vivo. First, regarding the Glc6Pase gene, we have shown that C/EBP proteins are likely to be involved in the Glc6Pase transcription, because both the basal activity and the PKA induction of the Glc6Pase promoter are markedly decreased in the presence of a dominant negative form of C/EBPs (Fig. 3Go, A and B). Second, we have identified a C/EBP-binding site in a distal region (C/EBPsite2: –608/–603 bp) of the Glc6Pase promoter, and shown that the mutation of this site similarly decreases the PKA induction of the promoter activity. It should be noted that the PKA induction is quantitatively decreased to a very similar extent in the presence of the dominant negative form of C/EBP and in the mutated construct, suggesting that the PKA effect mediated by C/EBP involves this distal binding site only. In agreement with this, the mutation of the proximal C/EBPsite1 previously described by Lin et al. (13) has a decreasing effect on the basal promoter activity, but has no effect on the PKA induction (Fig. 3CGo). C/EBPsite1 has been shown to bind the two C/EBP isoforms {alpha} and ß (13). The fact that both anti-C/EBP{alpha} and anti-C/EBPß antibodies are able to prevent the binding to the DNA probes strongly suggests that C/EBP isoforms {alpha} and ß bind to the Glc6Pase C/EBPsite2 (and to the C/EBPcons probe) as an {alpha}ß-heterodimer. However, the consensus site of C/EBP proteins (C/EBPcons) is known to bind either homo- or heterodimers of both C/EBP isoforms (28). The exclusive binding of the {alpha}ß-heterodimer shown here might thus depend, at least in part, on a specificity of experimental conditions rather than on a definite specificity of the Glc6Pase C/EBPsite2 for the {alpha}ß-heterodimer. Our results are significantly in line with previous reports stating that both isoforms are involved in the cAMP effect on the PEPCK promoter (9, 30). In addition, the expression of C/EBPß in the liver in vivo goes parallel with the Glc6Pase gene variation occurring under nutritional (fasting) and/or hormonal (diabetes) changes (31). The involvement of the C/EBP isoforms in the regulation of the Glc6Pase gene by cAMP demonstrated herein might thus be a mechanism of physiological importance in a more general process of regulation of gluconeogenesis.

Then, we have shown that the potentiated PKA responsiveness of the Glc6Pase promoter requires the participation of proximal CRE sites and cannot be given by the distal part of the promoter alone (see Results). Similarly, the distal PEPCK promoter region(–355/–200 bp) containing the C/EBP-binding sites is not able to confer a maximal cAMP responsiveness to an heterologous promoter but requires the presence of one proximal CRE site (–100/–82 bp) (9, 32). Regarding the Glc6Pase gene, because the mutation of CRE1 in the –694/+60 bp construct almost completely abolishes the potentiated PKA response, CRE1 should have a primary role to play by itself in the whole PKA regulation of the Glc6Pase promoter. Moreover, the mutation of CRE2 in the same construct retains a substantial stimulation by PKA (potentiated with regard to the native –320/+60 bp construct; compare panels B and C of Fig. 5Go). This suggests that CRE2 also plays a role, albeit less important than CRE1, in the potentiation of the PKA response conferred by the distal HNF4{alpha} and C/EBP-regulatory sites.

Finally, we have further improved our study by demonstrating the binding of HNF4{alpha}, C/EBP{alpha}, C/EBPß, and CREB in the context of chromatin in situ. We have shown that HNF4{alpha}, C/EBP{alpha}, and C/EBPß bind constitutively to the rat Glc6Pase gene promoter whereas CREB is recruited after PKA activation. The constitutive binding of HNF4{alpha}, C/EBP{alpha}, and C/EBPß to the promoter is in line with the fact that forskolin does not increase the binding of C/EBP proteins to the promoter and is consistent with previous results performed on the PEPCK gene promoter (33). As a general rule, the CREB protein is often already bound to its target promoters in basal states and is further activated in place by phosphorylation by PKA in the induction situation. However, it has also been reported that PKA can promote the binding of CREB to certain binding sites (8). This might be the case of the CRE sites of the Glc6Pase gene promoter.

In conclusion, we have reported here that an optimal PKA responsiveness of the Glc6Pase promoter requires the cooperation between a proximal region exhibiting two CRE- and two HNF4{alpha}-binding sites, and a distal region exhibiting one C/EBP- and one HNF4{alpha}-binding site. It has also been shown that two other hepatic factors, HNF6 and HNF1 (the latter only in a kidney cell line), are involved in the cAMP regulation of the Glc6Pase gene (15, 16). The whole Glc6Pase cAMP response unit might thus be tightly associated to tissue-specific factors, which will all induce a potentiated responsiveness to cAMP. It has been shown that the ubiquitous CBP transcriptional activator coordinates the response to cAMP of the PEPCK gene through its interaction with CREB and C/EBP (34). HNF4{alpha} can also interact with CBP and enhance the transcription of the apolipoprotein CIII gene in HeLa cells (35). It is likely that the presence of several binding sites for CREB, HNF4{alpha}, C/EBP{alpha}, and C/EBPß in the Glc6Pase promoter, by providing multiple anchoring possibilities for CBP, will further stabilize the assembling of the preinitiation complex of transcription. Here, we have documented the latter hypothesis by demonstrating that CBP is recruited to the Glc6Pase gene promoter upon stimulation by cAMP (results in Fig. 6Go). This may account for the dramatic potentiation of the stimulation by PKA given by the distal sites. These results point out, for the first time, the role of C/EBP{alpha} and C/EBPß, HNF4{alpha}, and CBP in the cAMP regulation of Glc6Pase, a crucial liver target gene. They also provide a sound molecular basis for the marked induction of Glc6Pase observed under nutritional and hormonal states in which the cAMP level is increased (i.e., in fasting or diabetes).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reporter Plasmids, Expression Vectors, and Antibodies
The rat Glc6Pase promoter constructs containing regions up to nucleotide –1640, relative to the transcription start site, and cloned into the pGL2 basic vector (names by B in construct names; Promega, Madison, WI) upstream of a luciferase reporter gene, were previously described (20). Mutant forms of the Glc6Pase promoters were generated by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), as recommended by the supplier and with specific mutagenic oligonucleotides. Mutagenic oligonucleotides are detailed in Table 1Go. All mutant constructs were confirmed by sequencing. Expression vectors A-CREB and A-C/EBP encode the dominant negative proteins of CREB and C/EBP, respectively, under the control of the cytomegalovirus promoter (generous gift from C. Vinson) (23, 24). These dominant negative inhibitors were constructed by fusing a designed amino acid extension onto the N terminus of the CREB or the C/EBP leucine zipper domain. The acidic extension of the dominant negative inhibitors interacts with the basic region of the nature factors forming a coiled-coil extension of the leucine zipper and thus prevents the basic region of wild-type CREB or C/EBP from binding to DNA (23, 24, 25). The expression vector pCR3-HNF4{alpha} encodes the HNF4{alpha} protein, and the expression vector PKA encodes the catalytic subunit of the protein kinase A (generous gifts from M. Raymondjean and B. Viollet) (21). The recombinant expression vector pDGT.23–1 encodes a dominant negative form of the HNF4{alpha} protein (generous gift from T. Leff) (22). The recombinant expression vector pMEX-C/EBP{alpha} encodes the C/EBP{alpha} protein (generous gift from J. Auwerx), and the recombinant expression vector CMV-LAP encodes the rat C/EBPß protein (generous gift from P. Gos) (36). Plasmids used for transfection were purified using the Plasmid maxi kit (Jet Star, Genomed GmBH, Löhne, Germany).

The following commercially available antibodies were used for EMSA assays, ChIP assays, and Western blots: CBP sc-369, HNF4{alpha} sc-8987, C/EBP{alpha} sc-9315 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), C/EBPß (Cell Signaling Technology, Inc., Beverly, MA), and CREB (New England Biolabs, Inc., Beverly, MA).

Cell Culture and Transfection
HepG2 human hepatoma cells were grown in DMEM supplemented with 6% fetal bovine serum, 5 mM glutamine, streptomycin (1 µg/ml), and penicillin (1 U/ml) at 37 C in a humidified 5% CO2/95% air atmosphere. For transient transfection, 1 d before the transfection, 200,000 cells were plated out in 35-mm wells in six-well cell culture plates. The complete medium was refreshed 1 h before transfection. HepG2 cells were transfected by the calcium-phosphate transfection method as previously described (20), with 1 µg of the Glc6Pase-LUC plasmid, 1 ng of pCMV-RL (Renilla luciferase, Promega) to correct for transfection efficiency, and expression vectors, as indicated (50 or 500 ng). Matching empty vectors were transfected as a control. The total amount of DNA (2.2 µg) was kept constant by addition of pBluescript SK+ plasmid. For forskolin treatment, HepG2 cells were treated 24 h after transfection for 6 h with 10 µM forskolin in serum-free medium as indicated. Cells were then washed three times with PBS and lysed with passive lysis buffer (Promega). After 15 min incubation, cells were scraped and centrifuged at 10,000 x g for 5 min at 4 C to eliminate cell debris. RL and LUC activities were determined with a BCLBook luminometer (Promega) using the Dual-Luciferase kit Assay Reagent (Promega). The levels of LUC activities were normalized by means of the RL activities. Statistical analyses were performed by an ANOVA followed by a Student’s t test for unpaired data.

EMSAs
EMSAs were performed with whole-cell protein extracts (37) from HepG2 or HepG2 treated with 100 µM forskolin. Binding reactions (20 µl) contained end-labeled double-stranded oligonucleotide probe (0.1 ng, 100,000 cpm; see sequence in Table 1Go), 1 µg of polydI.dC in the binding buffer (10 mM Tris-HCl, pH 7.9; 2.5 mM MgCl2; 0.25 mM EDTA; 0.25 mM dithiothreitol; 0.25 µg/µl BSA; 4% glycerol) and 15 µg of whole-cell extract (0.1 M HEPES). Reaction mixtures were incubated for 20 min at room temperature. Free DNA and DNA-protein complexes were separated in a 5% nondenaturing polyacrylamide gel (acrylamide-bisacrylamide, 19:1), in 0.5x TBE buffer (45 mM Tris-borate; 1 mM EDTA, pH 8.3). In competition experiments, 100 ng of the competitor DNA (see sequence in Table 1Go) was incubated in the mixture before the addition of the cellular extracts. For gel supershift assays, 1 µl of anti-C/EBP{alpha} antibody or 1 µl of anti-C/EBPß antibody was preincubated with cell extracts 20 min before the addition of the free probe. These antibodies were directed against epitopes present only in one of the isoforms and not in the other counterpart, thus making the possibility of cross-reaction between the two forms unlikely. After electrophoresis, gels were dried and analyzed on a PhosphorImager SI (Molecular Dynamics, Inc., Sunnyvale, CA) of the CECIL (Centre Commun d’Imagerie de Laennec).

ChIP Assay
Rat hepatoma FAO cells (4 x 3.106 cells/condition) had been treated for 6 h in the presence (or the absence) of 10 µM forskolin in a serum-free medium. Cross-linking was performed with 1% formaldehyde in phosphate buffer saline at room temperature for 5 min. To arrest cross-linking, glycine was added directly to the medium at a final concentration of 125 mM for 10 min, and the cells were rinsed twice with ice-cold PBS. Cells were harvested with cell lysis buffer (0.5 ml/plate of 10 mM Tris-HCl; 1 mM EDTA, pH 8; 0.5% Nonidet P-40; 1 mM phenylmethylsulfyl fluoride; 1 µg/ml aprotinin; 1 µg/ml leupeptin) and chilled in liquid nitrogen. Lysates were then transferred to 15-ml tubes containing 25 µm diameter glass beads. To shear chromatin, the lysate/bead mixture was sonicated (Branson Sonifier 250 with a 3-mm microtip probe; Branson Ultrasonics Corp., Danbury, CT) in ice (eight pulses of 1 sec at a setting of 30%), yielding chromatin fragments of 500-1000 bp in size. Samples were centrifuged at 14,000 x g for 4 min at 4 C to remove detritus, and the supernatant was collected. To provide a positive control (input), 200 µl of the supernatant was retained. Each immunoprecipitation was performed with {approx}25µg of chromatin in RIPA buffer (140 mM NaCl; 1 mM EDTA; 10 mM Tris-HCl; 1 mM phenylmethylsulfonylfluoride; 1% Triton X-100; 0.1% sodium dodecyl sulfate; 0.1% Na-Deoxycholate, pH 8). To reduce nonspecific background, each chromatin sample was precleared with 60 µl of a solution of protein A-sepharose (100 mg/ml RIPA buffer), supplemented with 300 µg/ml sonicated salmon sperm DNA (Fermentas, Inc., Hanover, MA) and 1 mg/ml BSA, for 1 h at 4 C on a rotating wheel. Chromatin complexes were immunoprecipitated for 16–18 h at 4 C while rotating with 10 µg of primary antibody or without antibody (mock) to provide negative controls. Immune complexes were collected with 40 µl of a solution of protein A-sepharose (100 mg/ml), supplemented with 300 µg/ml sonicated salmon sperm DNA (Fermentas) and 1 mg/ml BSA for 3 h at 4 C on a rotating wheel, followed by centrifugation at 14,000 x g for 30 sec at 4 C. The beads were washed for 10 min at 4 C five times with RIPA buffer, once with LiCl wash buffer (10 mM Tris-HCl, pH 8; 250 mM LiCl; 1 mM EDTA, pH 8; 0.5% Nonidet P-40; 0.5% Na-Deoxycholate), and twice with TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 8). To digest RNA and proteins present in the samples, ribonuclease (50 mg/ml), sodium dodecyl sulfate (1%), and proteinase K (400 µg/ml) were added, and the samples were incubated overnight at 37 C. To reverse cross-linking, the samples were further incubated for 6 h at 45 C. DNA was purified by phenol/chloroform extraction and precipitated in the presence of 100 µg of glycogen. PCR amplification was performed using primers specific for the –694/+60 bp region of the Glc6Pase gene (S: 5'-TTATCAGTTGCCAGGTGGG-3'; AS: 5'-CCAAAGTCGTGGAGCACGTTC-3').

Western Blot
Whole-cell protein extracts (50 µg) from HeLa cells, HeLa cells transfected with pCMV-LAP, or HeLa cells transfected with pMEX-C/EBP{alpha} for 48 h were separated by SDS-10% PAGE and transferred to Immobilon membrane (Millipore Corp., Bedford, MA). Immunoblotting was performed using an anti-C/EBPß antibody (Cell Signaling Technology) or an anti-C/EBP{alpha} antibody (Santa Cruz Biotechnology) at a 1:500 dilution and revealed by electrogenerated chemiluminescence.


   ACKNOWLEDGMENTS
 
We thank C. Vinson for providing the A-CREB and A-C/EBP expression vectors; M. Raymondjean and B. Viollet for providing the HNF4{alpha} and the PKA catalytic subunit expression vector; T. Leff for providing the DN-HNF4{alpha} expression vector; J. Auwerx for providing the pMEX-C/EBP{alpha} expression vector; and P. Gos for providing the pCMV-LAP expression vector. We are also grateful to E. Lalli for helpful discussion during this work and Y. Gosmain for his help in establishing the ChIP assay protocol. We thank C. Zitoun for her technical help in some experiments and C. Limoge and C. Beaufrere for their kind help in editing the manuscript.


   FOOTNOTES
 
This work was supported in part by a grant from the Institut Appert (to A.G.-S.).

First Published Online September 23, 2004

Abbreviations: A-C/EBP, Dominant negative form of C/EBP; A-CREB, dominant negative form of CREB; ATF, activating transcription factor; CBP, CREB binding protein; C/EBP, CAAT/enhancer-binding protein; ChIP, chromatin immunoprecipitation assay; CMV-LAP, expression vector of C/EBPß; CRE, cAMP response element; CREB, CRE binding protein; DN-HNF4{alpha}, dominant negative form of HNF4{alpha}; Glc6Pase, glucose-6-phosphatase; HNF, hepatocyte nuclear factor; LUC, firefly luciferase; PEPCK, phosphoenolpyruvate carboxykinase; PGC1{alpha}, peroxisome-proliferative-activated receptor {gamma} coactivator-1 {alpha}; PKA, protein kinase A; RL, renilla luciferase.

Received for publication March 12, 2004. Accepted for publication September 16, 2004.


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 DISCUSSION
 MATERIALS AND METHODS
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