3',5'-Cyclic Adenosine Monophosphate Response Element-Binding Protein and CCAAT Enhancer-Binding Protein Are Dispensable for Insulin Inhibition of Phosphoenolpyruvate Carboxykinase Transcription and for Its Synergistic Induction by Protein Kinase A and Glucocorticoids
David Yeagley and
Patrick G. Quinn
Pennsylvania State University College of Medicine, Department of Cellular and Molecular Physiology, Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: Patrick G. Quinn, The Pennsylvania State University, College of Medicine, Department of Cellular and Molecular Physiology, C4718, 500 University Drive, Hershey, Pennsylvania 17033. E-mail: pquinn{at}psu.edu.
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ABSTRACT
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Phosphoenolpyruvate carboxykinase (PEPCK) transcription is induced by cAMP/protein kinase A (PKA) and glucocorticoids [dexamethasone (Dex)] and is inhibited by insulin to regulate blood glucose. Recent reports suggested that CCAAT enhancer binding protein (C/EBP) binding to the PEPCK cAMP response element (CRE) plays a role in Dex induction and that insulin-induces inhibitory forms of C/EBPß to inhibit transcription. Here, we assessed the roles of CRE-binding protein (CREB) and C/EBP factors in mediating hormone-regulated transcription. Neither cAMP nor insulin regulated the phosphorylation of C/EBP. Cycloheximide did not block insulin inhibition, indicating that alternate translation of C/EBPß is not required. Dominant-negative CREB or C/EBP blocked induction by PKA, but neither affected regulation by Dex or insulin. Tethering the activation domains of CREB or C/EBP to a CRE
Gal4 (G4) site mediated varying extents of basal and PKA-inducible activity, but neither activation domain affected induction by Dex or inhibition by insulin. Surprisingly, synergistic induction by PKA and Dex did not require the CRE and was unaffected by dominant-negative CREB or C/EBP. PKA and Dex also synergistically induced a minimal 3xglucocorticoid response element promoter, but inhibited Dex induction of the mouse mammary tumor virus and IGF-binding protein 1 promoters, even though PKA alone did not regulate these promoters. These results suggest that PKA modifies the activity of other factors involved in Dex induction to mediate synergistic induction or inhibition in a promoter-specific manner. Our data indicate that the roles of CREB and C/EBP are restricted to mediating PEPCK induction by PKA, and that other factors mediate PEPCK induction by Dex, synergism between PKA and Dex, and inhibition by insulin.
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INTRODUCTION
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THE ENZYME PHOSPHOENOLPYRUVATE carboxykinase (PEPCK) catalyzes a rate-limiting and irreversible step in hepatic gluconeogenesis (1, 2, 3, 4). PEPCK transcription is tightly regulated by hormones to maintain glucose homeostasis, because PEPCK enzyme activity is not regulated by allosteric binding of metabolites or covalent modification (1, 2, 3, 4). In vivo, PEPCK transcription is induced during a fast by glucagon (via cAMP) and by glucocorticoids, and induction is inhibited by insulin secreted in response to feeding (1, 3, 4, 5). Identical patterns of hormonal regulation are observed in animals and in H4IIe hepatoma cells treated with cAMP [or the catalytic subunit of protein kinase A (PKA)], glucocorticoids [dexamethasone (Dex)], and insulin (6, 7). In contrast, expression of exogenous PEPCK in HepG2 cells is induced by cAMP/PKA (8, 9, 10, 11, 12, 13, 14, 15) but is not inhibited by insulin (10, 14). The prevailing model is that induction by glucagon or cAMP is mediated by phosphorylation of cAMP response element (CRE)-binding protein (CREB), which must interact with CCAAT enhancer binding protein (C/EBP) and other factors bound to an upstream accessory enhancer (AC) (8, 9, 10, 11, 14, 16, 17). Together, the CRE and AC enhancer form a cAMP and insulin response unit that mediates both induction by cAMP and inhibition by insulin (Fig. 1A
) (8, 9, 11, 14, 16, 17). PKA-mediated phosphorylation of CREB is retained in cells treated with both cAMP and insulin, in which PEPCK transcription is inhibited (17), suggesting that insulin modifies the activity of an AC-associated factor or coactivator involved in induction to inhibit transcription (14). Maximal induction by glucocorticoids requires the interaction of several factors bound to two glucocorticoid response elements (GREs) and several accessory factor sites that comprise a glucocorticoid response unit (GRU) (18, 19, 20, 21).

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Fig. 1. Effects of cAMP and/or Insulin Treatment of H4IIe Cells on CRG-Mediated Expression of G4-PEPCK and Phosphorylation of C/EBP and c-jun
A, Diagrammatic representation of the PEPCK promoter showing known regulatory elements and known (above) and putative (below) factors binding them. B, H4IIe cells were cotransfected with the CRG expression vector and the G4-PEPCK luciferase reporter in the absence and presence of a PKAc expression vector, as described in Materials and Methods. Each precipitate was split into two dishes, and half of them were treated with 10 nM insulin for the final 20 h of the experiment. C, H4IIe cells were incubated with 32Pi for 3 h to label the intracellular ATP pool and treated with cAMP and/or insulin during the final 30 min, a time at which hormonal effects upon transcription are maximal (6 ). Nuclear extracts were prepared and proteins were immunoprecipitated as described in Materials and Methods with antibodies against C/EBP , C/EBPß, c-jun, or ATF-2, as indicated. The gels containing C/EBP and c-jun were exposed to film for 60 h, whereas that containing ATF-2 was exposed to film for 4 h. Results of a typical experiment are shown. Similar results were obtained with independent preparations of nuclear extracts.
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It has been suggested that the PEPCK CRE is among these GRU accessory factor-binding sites (22) and that it is bound by C/EBP during glucocorticoid induction and by CREB during induction by cAMP-mediated activation of PKA (20, 23, 24). In addition, alternate translation of an inhibitory form of C/EBPß (LIP), which lacks a transactivation domain, has been proposed to mediate insulin inhibition by competing for the binding of the active form of C/EBPß (LAP) (24). This seems somewhat at odds with an earlier demonstration that regulation of run-on transcription of the PEPCK gene by cAMP/PKA and insulin was shown to be unaffected by cycloheximide treatment, and thus independent of de novo protein synthesis (6).
CREB and C/EBP are members of the b-ZIP [basic zipper DBD (DNA binding domain)] family of transcription factors, which interact in many ways to regulate transcription of target genes (25). Several types of interactions among b-ZIP factors, which can be classified into subfamilies containing CREB/adenovirus transcription factor (ATF), activator protein 1 (AP-1), and C/EBP factors, have been characterized (26, 27). Dimerization occurs between the leucine zipper regions of subfamily members and, in some cases, between subfamilies to generate diversity in gene regulation (26, 27). Added complexity arises from the fact that several of these factors are uniquely regulated by protein kinases in response to different extracellular signals (28, 29, 30, 31, 32). In addition, b-ZIP factors, each bound to their cognate recognition sites, cooperate in the recruitment of transcription-regulatory complexes. For example, phosphorylated-CREB and C/EBP cooperate in recruiting CBP to the PEPCK promoter (33).
The DNA recognition sites of b-ZIP factors comprise an inverted repeat, providing half-sites for each dimerization partner. The consensus sites of different subfamilies are sufficiently related that the sites for one subfamily may be recognized by the factors of another, at least in vitro (25, 27). Both CREB and C/EBP bind the PEPCK CRE in vitro, leading to the suggestion that they may bind interchangeably (8, 9, 11, 23, 34, 35). In addition, cotransfection of fusion proteins containing the activation domains of either CREB or C/EBP isoforms fused to the Gal4 (G4) DBD were able to support, albeit to varying extents, cAMP or glucocorticoid activation of a G4-PEPCK reporter, in which the CRE was changed to a G4 site, (10, 11, 12, 14, 15, 17, 23, 24). A significant problem with the hypothesis that C/EBP mediates PKA induction by binding the CRE is that direct modification of C/EBP by PKA has not been demonstrated.
The acidic zipper (synthetic) DBD (A-ZIP) proteins, which target specific subclasses of b-ZIP factors (CREB, C/EBP, and AP-1), have been particularly useful in discriminating between their contributions to transcription regulation in cells (14, 36, 37, 38). In the A-ZIP factors, the leucine zipper region mediating dimerization specificity is fused to an acidic extension with charge complementary to the basic residues that mediate site-specific DNA recognition (25, 36, 37, 38, 39). The A-ZIP:b-ZIP heterodimers formed are far more stable than the native dimers and prevent binding of the native factor (25, 36, 37, 38, 39). This effectively neutralizes the endogenous factors in transfected cells and interferes with the genes regulated by them (14, 36, 37, 38). We previously reported that neutralization of C/EBP with A-C/EBP diminished, but did not eliminate, induction by PKA, consistent with the proposed role of C/EBP as an accessory factor (14). In contrast, neutralization of CREB with A-CREB abolished PKA induction, indicating that CREB binding to the CRE is required to regulate cAMP/PKA-induced PEPCK transcription.
In vivo, the PEPCK gene maintains a low level of basal transcription that is rapidly increased by PKA or glucocorticoids to increase glucose synthesis and availability, all of which can be as rapidly inhibited by insulin (3, 5). The critical importance of modulating regulation of PEPCK transcription between low and high levels, depending upon nutritional status, raises the question of whether the binding of heterotypic factors, such as C/EBP, to the CRE is functionally important.
The current study was undertaken to examine the interactions between the PKA and glucocorticoid signaling pathways and to establish the respective roles of CREB and C/EBP in mediating hormonal regulation of the PEPCK gene by cAMP, Dex, and insulin, acting alone or together. We previously showed that cAMP stimulates phosphorylation of CREB, concomitant with induction, and that insulin does not promote dephosphorylation of CREB (17), suggesting that another factor is modified by insulin-stimulated kinases. Here, we examine the potential for cAMP/PKA or insulin to regulate the phosphorylation state of C/EBP and AP-1 factors, in light of suggestions that C/EBP or AP-1 factors regulate activity of the cAMP/insulin response unit (16). The role of insulin-regulated alternate translation of C/EBPß was evaluated by examining the dependence of hormone regulation on protein synthesis in cycloheximide-treated cells. For gain of function experiments, the activation domains of CREB, C/EBP
, and C/EBPß were tested for their ability to mediate basal and inducible activity of a PEPCK reporter gene by fusing them to the G4 DBD. For loss of function experiments, the role of various b-ZIP factors in hormonal regulation was examined using dominant negative A-ZIPs.
Our results indicate that the roles of CREB and C/EBP factors are restricted to cAMP/PKA induction of transcription. Induction by Dex was independent of the factor bound to the CRE, and ablation of CREB or C/EBP activity had no effect on regulation by Dex or insulin. Surprisingly, synergistic activation by PKA and Dex was independent of CRE binding and was also unaffected by ablation of CREB or C/EBP, indicating that PKA promotes synergism through modification of the binding or activity of other factors. Analysis of other Dex-responsive promoters demonstrated that PKA can either synergize with or oppose induction by Dex, depending upon the promoter context. Taken together, our results suggest that CREB and C/EBP are critical for induction of PEPCK gene transcription by cAMP/PKA, but that regulation by insulin, glucocorticoids, and synergism between PKA and glucocorticoids is regulated by modification of other factors and/or cofactors.
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RESULTS
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Effects of cAMP and Insulin on PEPCK Expression and Phosphorylation of C/EBP and AP-1 Factors
As in our previous studies, we used H4IIe cells, in which endogenous and exogenous PEPCK promoters are regulated as in vivo by cAMP, Dex, and insulin, rather than HepG2 cells, in which PEPCK promoter activity is not inhibited by insulin (10, 14). We (14, 17) and others (8, 9, 11) showed previously that the CRE is necessary, but not sufficient, to mediate induction. As shown in Fig. 1B
, cotransfection with a CREB-Gal4 (CRG) fusion protein restores induction to a G4-PEPCK promoter, in which the CRE is changed to a Gal4 site. This induction is lost upon mutation of the PKA phosphorylation site in S133A, indicating that phosphorylation of CREB Ser 133 is necessary for induction by PKA. Given that studies of the PEPCK promoter in HepG2 cells indicated that C/EBP and AP-1 factors cooperate with CREB in promoting induction by PKA (11, 16) and that there is evidence that insulin can phosphorylate these factors (32, 40), we asked whether C/EBP or AP-1 phosphorylation was regulated in H4IIe cells. Immunoprecipitation of ATF-2, a b-ZIP factor that is phosphorylated by stress-related kinases (41, 42) to regulate PEPCK gene transcription (43), was used as a control. As indicated in Fig. 1C
, treatment of H4IIe cells with cAMP or insulin for 30 min, a time at which their effects on run-on transcription are maximal (6), had no effect on the overall phosphorylation of C/EBP
or C/EBPß. It is formally possible that offsetting changes in phosphorylation result in no net change, but the experiments described below also contraindicate a role for C/EBP in insulin regulation. An important internal control is provided by the reversible control of phosphorylation of c-jun by cAMP and insulin. As indicated in the figure, the upper two bands, which were decreased by cAMP and restored by concomitant treatment with insulin, represent the transcriptionally active forms of c-jun (44). Thus, the immunoprecipitation assay is capable of monitoring hormone-induced changes in phosphorylation. However, the observed pattern is opposite of that expected if c-jun participated in cAMP/PKA-mediated induction. Phosphorylation of ATF-2 was not regulated by cAMP or insulin, consistent with a report that pharmacological inhibition of p38MAPK did not prevent stress- or insulin-mediated inhibition of PEPCK transcription (45). Our experiments provide no evidence for hormonal regulation of phosphorylation of C/EBP and AP-1 factors that would account for induction of PEPCK transcription by cAMP or its inhibition by insulin.
Effects of Inhibition of Protein Synthesis on Regulation of PEPCK Expression by Glucocorticoids and Insulin
A recent study suggested that an increase in the abundance of an inhibitory isoform of C/EBPß (LIP, liver inhibitor protein) mediates insulin inhibition of PEPCK transcription by competing for binding of the active isoform (LAP, liver activator protein) (24). Because LIP and LAP are most likely produced by alternate translation of a common mRNA, the above hypothesis would require insulin to stimulate preferential translation of LIP to inhibit transcription. Direct studies of PEPCK transcription by run-on assay in H4IIe cells showed that induction by cAMP and inhibition of induction by insulin were independent of protein synthesis (6). Here, we evaluated the contribution of de novo protein synthesis to induction of PEPCK mRNA synthesis by Dex and its inhibition by insulin. Cells were pretreated with cycloheximide to inhibit protein synthesis before treatment with hormones for 3 h, a time at which hormone-regulated PEPCK mRNA changes are maximal, after which PEPCK mRNA was isolated and quantitated (Fig. 2
). Dex treatment of the cells induced PEPCK mRNA accumulation, and concomitant treatment with cAMP produced synergism. Pretreatment of the cells with cycloheximide enhanced hormone-induced increases in PEPCK mRNA amount, which may result from stabilization of mRNA. However, insulin inhibited basal and hormone-induced PEPCK mRNA accumulation in the presence and absence of cycloheximide. This result demonstrates that de novo protein synthesis is not required for regulation of PEPCK mRNA accumulation by Dex or insulin, in the presence or absence of cAMP. Together with the earlier demonstration that cycloheximide had no effect upon regulation of run-on transcription of PEPCK by cAMP and insulin in these cells (6), the data show that hormonal regulation of PEPCK transcription is independent of de novo protein synthesis. Therefore, insulin-induced alternate translation of C/EBPß to produce inhibitory forms is not required for insulin inhibition of PEPCK transcription.

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Fig. 2. Effects of Cycloheximide on Hormone-Regulated PEPCK Gene Expression
H4IIe cells were pretreated with 10 µM cycloheximide for 30 min before the initiation of treatment with 0.1 mM 8-(4-chlorophenylthio)-cAMP, 0.5 µM dexamethasone, and/or 10 nM insulin. After 3 h of hormone treatment, a time at which maximal changes in PEPCK mRNA are observed (6 ), total RNA was isolated from the cells with Trizol reagent. Primer extension and phosphor imager analysis was used to quantify hormone-induced changes in PEPCK transcripts. The mean for two experiments is shown.
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Effects of Dominant-Negative CREB and C/EBP on Hormonal Regulation of the PEPCK Promoter
C/EBP has been postulated to contribute to Dex induction of PEPCK by binding the CRE and interacting with factors associated with the glucocorticoid response element (GRE) and its accessory factor sites (20, 23, 24). This conclusion was derived from experiments in which C/EBP was overexpressed. However, determining whether regulation still occurs when the factor is neutralized provides a more compelling test of its role in regulation. The specificity of the A-ZIP factors for differing subfamily members (CREB, C/EBP, or AP-1) has been established (25, 36, 37, 38). We previously evaluated plasmids expressing dominant-negative A-ZIP factors to show that CREB and C/EBP were required for induction by PKA (14). Here, we extend that analysis to evaluate the roles of b-ZIP factors in regulation of PEPCK transcription by Dex and insulin (Fig. 3
). Cotransfection of cells with A-Fos, which dimerizes with c-jun and prevents the formation of productive AP-1 complexes, modestly stimulated induction of PEPCK-Luc by PKA, Dex, or PKA + Dex. This is consistent with a report that overexpression of fos inhibited PEPCK expression without affecting the pattern of hormonal regulation (46). In contrast, cotransfection of A-CREB abolished induction by PKA, without affecting regulation by Dex (/+ PKA) or insulin. Cotransfection of A-C/EBP, which will neutralize the activity of both C/EBP
and C/EBPß, inhibited induction by PKA, as in our previous study (14), but had no effect on the extent of induction by Dex (/+ PKA). A-C/EBP would have been expected to abolish insulin inhibition of Dex-induced transcription if alternate translation of C/EBPß (LIP) played an obligatory role in mediating insulin inhibition of PEPCK transcription. However, neutralization of C/EBP activity with A-C/EBP did not impair insulin inhibition of basal or hormone-induced transcription. These results demonstrate that CREB and C/EBP contribute to induction by PKA but that neither is required for inhibition by insulin or induction by Dex.

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Fig. 3. Effects of Cotransfection of Dominant-Negative CREB, C/EBP, and AP-1 Factors upon Hormonal Regulation through the PEPCK Promoter
H4IIe cells were cotransfected with the indicated A-ZIP expression vector and a luciferase reporter gene under control of the complete PEPCK promoter (PEPCK-Luc), in the absence and presence of a PKAc expression vector, as described in Materials and Methods. Each precipitate was split into two dishes, and half of them were treated with 10 nM insulin for the final 20 h of the experiment. The results shown represent the mean ± SEM of six independent experiments. In some cases, the SEM was too small to be represented in the figure.
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It has been tacitly assumed that synergistic induction by PKA and Dex would involve the PKA-regulated factor CREB bound at the CRE, or in alternate schemes, occupation of the CRE by C/EBP. An unexpected, yet important, feature of our data is that A-C/EBP or A-CREB, which inhibit PKA induction, had no effect whatsoever on synergistic induction of PEPCK promoter activity by PKA and glucocorticoids. This result indicates that PKA promotes synergism through regulation of the binding and/or activity of another factor or cofactor involved in regulation by Dex.
Effects of Chimeric Transcription Factors Tethered to a CRE
G4 Site on Hormonal Regulation of the PEPCK Promoter
The capacity of the activation domains of CREB, C/EBP
, or C/EBPß to regulate a G4-PEPCK reporter in response to PKA, Dex, and/or insulin was evaluated by fusing them to the Gal4 DBD. Cells were cotransfected with a G4-PEPCK-Luc reporter, in which the CRE is replaced with a Gal4 site and vectors expressing G4-AD fusion proteins, in the presence and absence of PKAc, Dex, and insulin, to assess the contributions of the activation domains to regulation (Fig. 4
and Tables 1
and 2
). CRG contains the G4-DBD fused in place of the C-terminal CREB-DBD. In the remaining fusion proteins, the respective activation domains are fused to the carboxy terminus of the G4-DBD and lack their native DBDs. C/EBPß-108 contains a functional activation domain, truncation of which abolishes activity in C/EBPß-25 (13). As shown in Fig. 4A
, the parent PEPCK-Luc is induced by either PKA or Dex, which exhibit synergistic effects when present together. Mutation of the CRE to a Gal4 site in G4-PEPCK (Fig. 4
, B and C, and Tables 1
and 2
) abolished induction by PKA and reduced both basal and Dex-induced activity by half, as previously reported (22, 23, 24, 47). Consistent with the results obtained with dominant negatives, PKA augmented Dex induction, even when cells were cotransfected with G4-DBD or G4-C/EBPß-25, both of which lack activation domains. Cotransfection of cells with chimeric factors containing the CREB activation domain fused to the G4-DBD at its carboxy terminus (CRG), as in the native protein, or at its amino terminus (G4-CREB), provided induction by PKA but did not increase induction by Dex beyond that seen with G4-DBD (Fig. 4B
). In contrast, cotransfection of G4-C/EBP
stimulated an increase in basal transcription, but did not mediate substantial induction by PKA or increase induction by Dex (Fig. 4C
). Cotransfection of G4-C/EBPß-108, which contains the C/EBPß activation domain, also increased basal expression and mediated some induction by PKA, although to a lesser extent than either of the CREB chimeras (Fig. 4C
). In all cases, insulin inhibited hormone-induced transcription, regardless of the factor bound to the Gal4 site, indicating that it regulates other factors to disrupt the inducing complex. Comparison of the extent of induction indicates that synergism between PKA and Dex occurred regardless of what, if any, activation domain was tethered to the Gal4 site replacing the CRE. Furthermore, the observation that synergy between PKA and Dex persists in the presence of G4-DBD at the CRE
G4 site is consistent with the lack of effect of ablation of CREB or C/EBP described above and also suggests that PKA modifies the activity of other factors and/or cofactors to promote synergism in activation.

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Fig. 4. Effects of Chimeric Transcription Factors upon Regulation of a PEPCK Promoter with the CRE Replaced by a G4 Site, G4-PEPCK
In all cases, H4IIe cells were cotransfected with the indicated reporter and expression plasmids and treated as described in Materials and Methods. The results shown are the mean ± SEM of six replicate experiments. In some cases, the SEM was too small to be represented in the figure. The results are separated into groups to aid comparisons. A, Regulation of PEPCK-Luc by endogenous transcription factors. B, Regulation of G4-PEPCK-Luc by CREB-G4 chimeras in either orientation. C, Regulation of G4-PEPCK-Luc by G4-C/EBP chimeras.
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Effects of PKA on Glucocorticoid-Responsive Promoters
The unexpected finding of CRE- and CREB-independent synergy between PKA and Dex led us to examine their combined effects on regulation of other promoters induced by glucocorticoids, but not PKA. A minimal promoter under control of three copies of the TAT GRE (3xGRE, Fig. 5B
) mediated strong induction by Dex. This 3xGRE promoter was unresponsive to PKA alone, but PKA augmented induction by Dex, as in the case of PEPCK (Fig. 5A
). In contrast, the mouse mammary tumor virus (MMTV) and IGF-binding protein 1 (IGFBP1) promoters (Fig. 5
, C and D) mediated dramatic induction by Dex, but PKA, which had no effect alone, inhibited Dex induction to different extents. From this small sample, it is clear that the effects of PKA upon Dex induction are highly variable and promoter specific, suggesting modulation by PKA of the interaction of the glucocorticoid receptor (GR) with other factors and/or cofactors.

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Fig. 5. Effects of Dex and PKA on Glucocorticoid-Responsive Promoters
H4IIe cells were cotransfected with the indicated luciferase reporter gene (3xGRE, MMTV, or IGFBP1) in the absence and presence of a PKAc expression vector, as described in Materials and Methods. The results shown represent the mean ± SEM of three to six independent experiments. In some cases, the SEM was too small to be represented in the figure.
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DISCUSSION
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In the present report, we evaluated the interactions of the PKA and glucocorticoid signaling pathways in mediating synergism and the respective roles of CREB, AP-1, and C/EBP factors. Various experimental strategies have indicated that each of these factors plays one or more roles in mediating regulation of PEPCK transcription by PKA, Dex, and insulin. We found that neither cAMP nor insulin regulate the phosphorylation state of C/EBP or AP-1 factors in a way that could account for cAMP/PKA induction or insulin inhibition of PEPCK gene transcription. Previous experiments, employing G4-AD fusion factors and G4-PEPCK, were interpreted as evidence that C/EBP plays a role in induction by cAMP/PKA or Dex when bound to the CRE (12, 15, 23, 24). Here, we found that the CREB activation domain most effectively mediated induction by PKA, whereas induction by Dex did not require CREB, C/EBP, or any activation domain to be bound at the CRE. A more surprising finding is that synergistic activation by Dex and PKA is independent of any activation domain bound to the CRE and is also independent of either CREB or C/EBP, regardless of their binding sites. Also, dominant-negative A-CREB and A-C/EBP, which block or reduce PKA induction, respectively, had no effect upon regulation by Dex, in the absence or presence of PKA, or upon inhibition by insulin. Thus, factors other than CREB and C/EBP must mediate both synergistic induction by PKA + Dex and inhibition by insulin.
The mechanism of activation of CREB by PKA, phosphorylation of Ser133, has been substantiated by a number of groups (28, 29, 30), and mutation of Ser133 to an Ala abolishes PKA responsiveness (14, 17, 28, 48). In contrast, no specific PKA phosphorylation site has been mapped in C/EBP, and there is no major change in overall phosphorylation, as demonstrated here. In addition, there is no evidence that mutation of any specific phosphorylation site in the C/EBP activation domain affects regulation by PKA, with or without compensatory changes in phosphorylation of another site. A recent report showing that CBP is phosphorylated by PKA when bound to the C/EBP activation domain (49) may provide an explanation for the modest induction that is seen with C/EBPß (Fig. 4C
). Here, as in our previous experiments (14), neutralization of C/EBP only partially inhibited induction by PKA, whereas neutralization of CREB abolished induction, which is consistent with C/EBP acting as an accessory factor through AC. Our data for regulation of G4-PEPCK (G4-CREB
G4-C/EBP for maximal response) are in agreement with other reports (12, 15), but we interpret them differently. It is important to emphasize that the degree of response to inducers, as opposed to the absolute magnitude of expression, is most important for regulation of a gene such as PEPCK, which changes its relative levels in response to changes in blood glucose. Our data show that tethering of the CREB activation domain to the CRE most closely reproduces the response to PKA seen in the endogenous PEPCK gene. Duong et al. (24) recently suggested that insulin-induced changes in the ratio of LIP and LAP, inhibitory and stimulatory isoforms of C/EBP arising from alternate translation start sites in the C/EBPß mRNA, were responsible for inhibition of PEPCK gene expression. However, this mechanism for an increase in the LIP/LAP ratio would require de novo protein synthesis. Here, we show that cycloheximide did not block the ability of insulin to inhibit hormone-induced PEPCK gene expression. Furthermore, A-C/EBP, which would neutralize both LAP and LIP, had no effect upon insulin inhibition of PKA-, Dex- or Dex + PKA-induced transcription. Thus, we suggest that the effects of either overexpressed LIP or A-C/EBP arise from disruption of an inducing complex required to mediate induction by cAMP/PKA. Fass et al. (33) have demonstrated that effective recruitment of CBP to the PEPCK promoter requires its interaction with both phosphorylated CREB bound at the CRE and C/EBP bound at the AC enhancer. Our data and those of Duong et al. (24) are consistent with a model in which CREB and C/EBP facilitate recruitment of CBP, and possibly other coregulators, during induction by cAMP/PKA. This model is also consistent with studies in mice, where knockout of C/EBP
expression resulted in neonatal death of hypoglycemia (50). Knockout of C/EBPß expression produced the same phenotype in half the mice, but the half that survived the neonatal period, presumably as a result of compensation by C/EBP
, were able to regulate gluconeogenesis under normal conditions (51). It was noted that they became hypoglycemic upon fasting, due to defective cAMP-PKA signaling. However, there was no evidence of any defect in insulin regulation of gluconeogenesis in those mice, as would be expected if insulin signaling had been disrupted. Therefore, C/EBP bound to the AC enhancer is necessary for maximal PKA-induced activity, but C/EBP is not involved in insulin inhibition of PEPCK transcription.
Perhaps the most interesting aspect of the present study is that ablation of CREB or C/EBP activity, which markedly reduced induction by PKA, had no effect upon induction by Dex, either alone or in combination with PKA. Induction of PEPCK expression by glucocorticoids has been extensively characterized, and the factors binding the GRU have been identified by Granner and colleagues over the last several years (18, 19, 20, 21). Some of these reports suggested that C/EBP binding to the CRE also plays a role in induction by glucocorticoids (20, 23, 24). Two complementary experimental designs, loss of function, using A-ZIP, and gain of function, using G4-PEPCK + G4-AD fusion factors, both indicate that neither CREB nor C/EBP needs to be bound at the CRE or any other site to mediate induction by Dex or synergism by Dex and PKA. The GR has been reported to interact with the DBD of CREB (22), and the lack of this interaction in CREB-G4 may account for the reduced level of activation by PKA and Dex in G4-PEPCK, relative to the PEPCK promoter. However, even in G4-PEPCK, synergism is independent of the activation domain tethered at the CRE. In particular, the observation that synergism between Dex and PKA occurs with G4-DBD provides additional evidence that the fundamental synergism between PKA and Dex in stimulating PEPCK expression is due to effects of PKA on other factors or cofactors associated with the promoter. PKA has also been demonstrated to synergize with glucocorticoids in regulating other genes in a manner dependent upon GRU binding proteins, but the mechanism is not known for any of these systems (52, 53). GR has been shown to be phosphorylated by PKA in vitro (54), and this may play a role in negative regulation by GR and nuclear factor-
B (55). However, it has also been reported that PKA can influence induction by glucocorticoids without affecting GR phosphorylation directly (56). Here, we show that PKA can regulate induction by Dex in three additional promoters that are not directly regulated by PKA. Depending upon the promoter context, PKA augmented or inhibited induction by Dex. Thus, our data suggest that PKA regulates the activity of other transcription factors, or the coregulators interacting with them, in a gene-specific manner to provide synergistic activation or inhibition by cAMP and glucocorticoids.
In summary, the experiments reported here provide evidence that both CREB and C/EBP play essential but restricted roles in mediating hormonal responsiveness of the PEPCK promoter. Phosphorylation of CREB bound to the CRE appears to initiate induction by cAMP/PKA, in concert with C/EBP bound to the AC enhancer, most likely by facilitating the recruitment of CBP and other factors. C/EBP is required for maximal PKA induction, but the phosphorylation state of C/EBP is not regulated by cAMP or insulin, and C/EBP is not required for induction by glucocorticoids or for inhibition by insulin. The factor(s) mediating insulin inhibition of induction by PKA and Dex may be the same or different, but still remain to be identified. Our study also provides evidence that synergism between PKA and Dex is not due to simple combinatorial regulation between the two pathways, because ablation of CREB activity or the CRE has no effect. It will be interesting and important to determine what factors and/or cofactors are modified by PKA to provide synergism between PKA and glucocorticoids in regulation of PEPCK gene expression.
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MATERIALS AND METHODS
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H4IIe Cell Culture and Transfection Analyses
H4IIe cells were grown and transfected as previously described (10, 57). In brief, the cells were transfected in solution with 10 µg luciferase (firefly) reporter and 1 µg of each expression plasmid plus 1 µg pRL-SV (Renilla luciferase, Promega Corp., Madison, WI) reporter to correct for differences in transfection efficiency. Half of the cells were seeded into each of two 60-mm dishes, one of which served as a control whereas the other was treated with 10 nM insulin. Where indicated, cells were cotransfected with an expression vector for the catalytic subunit of PKA, obtained from R. Maurer [Oregon Health Sciences University (58)]. After 4 h, the cells were treated with 20% dimethylsulfoxide for 3 min and washed in PBS, after which medium, with or without 10 nM insulin, was added for the remaining 20 h. Cells were harvested with trypsin/EDTA and lysed, and luciferase activities for the firefly and Renilla luciferase reporters were measured with the Dual Luciferase Kit (Promega Corp.), using an ALL Monolight 3010 dual injector luminometer. Variations in PEPCK promoter-firefly luciferase activity due to variations in transfection efficiency were corrected for by measuring Renilla luciferase activity in the same sample. Values were normalized for transfection efficiency, and the mean was computed for several experiments. Data shown in the figures were obtained from independent transfection experiments performed with different preparations of the various plasmids. All figures represent several transfection experiments, each of which was normalized to the untreated control, and the data were combined for analysis. The number of experiments for each figure is indicated in the figure legends.
Vectors
The CRG expression vector contains the activation domain of CREB (amino acids 1277) fused to amino acids 4147 of the Gal4 DBD and has been described previously (48). The G4-C/EBP
, G4-C/EBPß-108, and G4-C/EBPß-25 (Gal4-C/EBP fusion proteins) expression vectors were provided by Roesler et al. (12) and Park et al. (13) and contain the activation domains of C/EBP
and C/EBPß (amino acids 6217 and 1108, respectively) or lack the C/EBPß activation domain (amino acids 125) fused to amino acids 1147 of the Gal4 DBD and have been previously described. The G4-CREB (Gal4-CREB fusion protein) expression vector contains the activation domain of CREB (amino acids 1312) fused to amino acids 1147 of the Gal4 DBD and has been described before (GAL4-CREB
LZ) (59, 60). The PKA expression vector, rous sarcoma virus-C
, contains the cDNA for the catalytic subunit of PKA under control of the rous sarcoma virus promoter (61). The A-ZIP series of expression vectors have been described previously (14, 36, 37, 38). The 3xGRE (pTAT3) was obtained from J. Iniguez-Luhi of the University of Michigan (62). The MMTV promoter was subcloned upstream of luciferase in a modified version of pGL3 (17). The IGFBP-1 promoter has been described previously (63, 64).
Immunoprecipitation of 32P-Labeled Factors
H4IIe cells were preincubated in phosphate-free medium for 30 min before being incubated with 32Pi (6 mCi/T-150 flask) in phosphate-free medium for 3 h to label the intracellular ATP pool. The cells also were treated with 0.1 mM 8-(4-chlorophenylthio)-cAMP, 10 nM insulin, cAMP + insulin, or nothing during the final 30 min, a time at which the effects of cAMP and insulin upon PEPCK transcription are maximal (6). The cells were rapidly harvested, and the remaining procedures were carried out at 4 C. Nuclear extracts were prepared by a modification of the method of Hurst et al. (65), increasing the EDTA to 1 mM and including NaF (20 mM), NaV (1 mM), and okadaic acid (0.25 µM) to inhibit nuclear protein kinase and phosphatase activities. Aliquots of the nuclear extracts were then diluted to 500 µl with RIPA buffer and incubated with 2 µg of the following primary antibodies for 1 h: anti-C/EBP
(Santa Cruz sc-61x; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-C/EBPß (Santa Cruz sc-150x), anti-c-jun (Santa Cruz sc 1694x) or anti-ATF-2 (Santa Cruz sc-187x). An aliquot of 40 µl of a 50% suspension of protein-A/G agarose beads (Santa Cruz sc-2003) was added, and incubation on a rocking platform was continued for an additional hour. The immunoprecipitates were washed as described by Ginty et al. (66) and analyzed by autoradiography after separation of phosphoproteins by 10% SDS-PAGE.
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ACKNOWLEDGMENTS
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We thank Olivia Seymour, Alanna Roff, and Tiffany Adams for excellent technical assistance. We also thank Charles Vinson (National Institutes of Health) for the A-ZIP vectors and J. Iniguez-Luhi (University of Michigan) for pTAT3 (3xGRE).
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FOOTNOTES
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This work was supported by an Individual National Research Service Award (DK09956 to D.Y.) and a grant from the American Diabetes Association (to P.Q.).
First Published Online December 16, 2004
Abbreviations: AC, Accessory; AD, activation domain; AP-1, activator protein-1; ATF, adenovirus transcription factor; A-ZIP, acidic zipper (synthetic) DBD; b-ZIP, basic zipper DBD; CRE, cAMP response element; C/EBP, CCAAT/enhancer binding protein; CREB, cAMP response element binding protein; CRG, CREB-Gal4 fusion of CREB AD to Gal4 DBD; DBD, DNA binding domain; Dex, dexamethasone; G4, yeast Gal 4 factor; GR, glucocorticoid receptor; GRE, glucocorticoid response element; GRU, glucocorticoid response unit; IGFBP-1, IGF-binding protein 1; LAP, liver activator protein; LIP, liver inhibitor protein; MMTV, mouse mammary tumor virus; PKA, protein kinase A.
Received for publication July 9, 2004.
Accepted for publication December 7, 2004.
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