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Molecular Endocrinology, doi:10.1210/me.2006-0085
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Molecular Endocrinology 20 (11): 2831-2847
Copyright © 2006 by The Endocrine Society

Correlation between FOXO1a (FKHR) and FOXO3a (FKHRL1) Binding and the Inhibition of Basal Glucose-6-Phosphatase Catalytic Subunit Gene Transcription by Insulin

Hiroshi Onuma, Beth T. Vander Kooi, Jared N. Boustead, James K. Oeser and Richard M. O’Brien

Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232

Address all correspondence and requests for reprints to: Richard M. O’Brien, Department of Molecular Physiology and Biophysics, 761 Preston Research Building, Vanderbilt University Medical School, Nashville, Tennessee 37232-0615.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin inhibits transcription of the genes encoding the glucose-6-phosphatase catalytic subunit (G6Pase), phosphoenolpyruvate carboxykinase, and IGF binding protein-1 through insulin response sequences (IRSs) that share the same core sequence, T(G/A)TTTT(G/T). The transcription factors FOXO1a and FOXO3a have been shown to bind these elements, but there are conflicting reports as to whether this binding correlates with the action of insulin on gene transcription. Some researchers concluded, from overexpression experiments using FOXO1a, that binding correlated with the insulin response, whereas others concluded, mainly from gel retardation competition experiments using FOXO3a, that it did not. We show here that, although these factors can differentially activate gene transcription in a context-dependent manner, these conflicting data are not explained by a difference in FOXO1a and FOXO3a binding specificity. Instead, we find that gel retardation competition and binding experiments give different results; the latter reveal a correlation between FOXO1a/3a binding and the inhibition of basal G6Pase gene transcription by insulin. In addition, these data show that the binding of FOXO1a/3a to two adjacent IRSs in the G6Pase promoter is cooperative and that promoter context alters the specific IRS base requirements for FOXO1a-stimulated fusion gene expression. Surprisingly, an analysis of insulin action mediated through the G6Pase and IGF binding protein-1 IRSs in the context of a heterologous thymidine kinase promoter reveals that signaling through the latter does not support the accepted model for insulin-stimulated FOXO nuclear exclusion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN LIVER, GLUCOSE-6-PHOSPHATASE catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate, the final step in the gluconeogenic and glycogenolytic pathways (1). Glucose-6-phosphatase is located in the endoplasmic reticulum (ER) membrane and probably exists as a multicomponent enzyme system in which a glucose-6-phosphatase catalytic subunit (G6Pase) has its catalytic site directed toward the lumen of the ER and a G6P transporter serves to deliver G6P from the cytosol to the active site of the catalytic subunit (2, 3).

In vivo studies in liver and in situ studies in liver-derived cell lines or in primary hepatocytes have shown that insulin inhibits basal as well as glucose-, glucocorticoid-, cAMP-, and fatty acid-stimulated G6Pase gene expression (see Ref. 4 for individual citations). In HepG2 cells, the effect of insulin on basal mouse and human G6Pase gene transcription is mediated through a multicomponent insulin response unit that is composed of two regions, designated Regions A and B (5, 6, 7). In the mouse G6Pase promoter, Region A is located between –231 and –199, whereas Region B is located between –198 and –159. Region A acts as an accessory element to enhance the effect of insulin on G6Pase expression mediated through Region B (6). The accessory factor that binds Region A is hepatocyte nuclear factor-1 (HNF-1) (6). Region B contains three insulin response sequences (IRSs), designated IRS 1, 2, and 3 (5, 6). All three IRSs can confer an inhibitory effect of insulin on the expression of a heterologous fusion gene but only IRS 1 and 2 are required for the suppression of basal G6Pase gene expression by insulin (8). The sequence of IRS 1 and 2 resembles that of the IRSs identified in the phosphoenolpyruvate carboxykinase (PEPCK) (9, 10), tyrosine aminotransferase (11), and IGF binding protein-1 (IGFBP-1) (12) promoters.

The identity of the factor that binds these motifs and mediates the action of insulin has been elusive (13), but recently, substantial attention has focused on the potential role of the winged helix/forkhead transcription factor FOXO1a (FKHR) and its orthologs, FOXO3a (FKHRL1) and FOXO4a (AFX). Insulin can inhibit FOXO1a-, FOXO3a-, and FOXO4a-mediated transcriptional activation through the phosphatidylinositol 3-kinase-dependent activation of protein kinase B (PKB), which leads to the phosphorylation and nuclear exclusion of these factors (14, 15, 16, 17, 18, 19, 20, 21). FOXO1a can bind G6Pase IRS 1 and 2 as well as the PEPCK and IGFBP-1 IRSs in vitro (8, 14, 22). In addition, when overexpressed, FOXO1a can stimulate G6Pase, PEPCK, and IGFBP-1 fusion gene expression (8, 22, 23, 24).

Despite these data implicating FOXO1a in the regulation of PEPCK, IGFBP-1, and G6Pase gene transcription, the key question is whether FOXO1a is actually the endogenous factor that mediates the action of insulin on these genes. Thus, other factors have been identified that bind the PEPCK IRS, namely C/EBP (25, 26) and FOXA2 (HNF-3ß) (27, 28), and changing the level of these factors also alters PEPCK gene expression (29, 30). However, detailed mutagenesis studies revealed that the effect of insulin on PEPCK gene transcription does not correlate with the binding of these factors (27). Two groups have performed similar analyses investigating the correlation between FOXO binding and insulin-regulated PEPCK and IGFBP-1 gene expression. Guo et al. (17) generated a series of fusion genes comprising a modified IGFBP-1 promoter containing a single IRS with point mutations in individual IRS bases. They then demonstrated that the ability of overexpressed FOXO1a to stimulate fusion gene expression correlated with the ability of insulin to repress basal fusion gene expression in the absence of overexpressed FOXO1a (17). They also showed that expression of antisense FOXO1a RNA disrupted insulin-regulated gene expression, suggesting that FOXO1a was the endogenous factor mediating insulin action (17). In contrast, Hall et al. (31) generated a series of heterologous thymidine kinase (TK) promoter fusion genes containing either the PEPCK IRS or the two IGFBP-1 IRSs again with point mutations in individual IRS bases. They then performed overexpression and gel retardation competition experiments to assess the ability of FOXO3a to bind to these mutated PEPCK and IGFBP-1 IRS motifs in situ and in vitro, respectively (31). Their studies suggested that FOXO3a binding in vitro only correlated with insulin-regulated fusion gene expression when FOXO3a was overexpressed (31). Hall et al. (31) therefore concluded that, whereas FOXO3a could serve as an insulin response factor when overexpressed, it was not the endogenous factor mediating the action of insulin on these genes. The experiments described in this manuscript were designed to address these conflicting reports by determining whether insulin-regulated G6Pase gene transcription correlates with FOXO1a and/or FOXO3a binding.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FOXO1a and FOXO3a Share an Identical Binding Specificity for G6Pase IRS 1 and IRS 2
A trivial explanation for the conflicting data of Guo et al. (17) and Hall et al. (31) is that FOXO1a and FOXO3a have different binding specificities such that Hall et al. (31) might have seen a correlation between FOXO binding and insulin action had they used FOXO1a rather than FOXO3a. To address this possibility, FOXO binding to G6Pase IRS 1 and 2 was analyzed using the gel retardation assay (Fig. 1Go). When a labeled double-stranded oligonucleotide, designated G6P WT, representing the wild-type G6Pase promoter sequence from –196 to –155 (Table 1Go), was incubated with a crude extract, prepared from bacterial cells expressing a glutathione-S-transferase (GST)-FOXO1a fusion protein, a single isopropylthio-ß-D-galactosidase (IPTG)-induced protein-DNA complex was detected (Fig. 1AGo, arrow). Competition experiments were performed in which a variable molar excess of various unlabeled oligonucleotides representing WT or mutated G6Pase sequences (Table 1Go) were included with the labeled probe. The mutated G6Pase sequences contain point mutations in IRS base numbers 5, 7, and 10 (Table 1AGo). This numbering system originates from the initial definition of the PEPCK IRS as a 10-bp motif (27). The experiments described below focus on the effects of mutating bases 5, 7, and 10 on FOXO1a/FOXO3a binding and insulin action because such analyses formed the basis for the conflicting results of Guo et al. (17) and Hall et al. (31).


Figure 1
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  		Fig. 1. FOXO1a and FOXO3a Share an Identical Binding Specificity for G6Pase IRS 1 and 2

A, A labeled probe, designated G6P IRS WT (Table 1Go), representing the WT G6Pase promoter sequence between –196 and –155, was incubated in the absence (–) or presence of a 250-fold molar excess of various unlabeled competitor DNAs, the sequence of which are shown in Table 1Go. Bacterial extract from either control (–) or IPTG-treated (+) cells transformed with a plasmid encoding a GST-FOXO1a fusion protein was then added and protein binding analyzed using the gel retardation assay as described in Materials and Methods. The specific IPTG-induced protein-DNA complex is indicated by the arrow. In the representative autoradiograph shown only the retarded complexes are visible and not the free probe, which was present in excess. B, Gel retardation experiments were performed as described in panel A except that a variable molar excess of unlabeled competitor DNAs were used as shown, and data were quantitated using a Packard Instant Imager. Results represent the mean ± SEM of at least three experiments. C and E, Gel retardation experiments were performed as described in panel A except that partially purified GST-FOXO3a (C) and His-FOXO1a (E) were used in conjunction with a 50-fold molar excess of the indicated unlabeled competitor DNAs. In the representative autoradiographs shown, only the retarded complexes are visible and not the free probe, which was present in excess. D and F, Gel retardation experiments were performed as described in panel A except that partially purified GST-FOXO3a (pD) and His-FOXO1a (F) were used in conjunction with a variable molar excess unlabeled competitor DNAs as shown and data were quantitated using a Packard Instant Imager. Results represent the mean ± SEM of three experiments. The M5 oligonucleotide in panels A and B, called G6P IRS 1–3 M5 in Table 1Go and G6P TM in a previous publication (8 ), contains point mutations of the M5 bases of IRS 1, 2, and 3, whereas the M5 oligonucleotide used in panels C–F, called G6P IRS 1+2 M5 (Table 1Go), contains point mutations of the M5 bases of IRS 1 and 2 only. We have previously shown that both oligonucleotides behave the same in competition experiments because IRS 3 does not bind FOXO1a (8 ) or FOXO3a (data not shown). The nonspecific (NS) oligonucleotide represents the HNF-1 motif from the ß-fibrinogen gene (Table 1Go).

 

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  		Table 1. Sequences of the Sense Strands of Oligonucleotides Used in Gel Retardation Studies

 
The unlabeled G6P WT oligonucleotide competed effectively for the formation of the GST-FOXO1a-DNA complex (Fig. 1Go, A and B) as did the oligonucleotide containing mutations in base 10 of G6Pase IRS 1 and 2 (Fig. 1AGo). In contrast, the oligonucleotide containing mutations in base 7 of G6Pase IRS 1 and 2 showed an impaired ability to compete, whereas the oligonucleotide containing mutations in base 5 of G6Pase IRS 1 and 2 failed to compete (Fig. 1Go, A and B). This latter result is consistent with our previous studies using crude GST-FOXO1a (8).

When these competition experiments were repeated using partially purified GST-FOXO3a the relative efficiency of competition by the oligonucleotides containing mutations in base 5 and 7 was qualitatively similar, however, competition was achieved using a lower molar excess (Fig. 2Go, C and D). In addition, the oligonucleotide containing mutations in base 5 of G6Pase IRS 1 and 2 now competed for complex formation but less than achieved with the oligonucleotide containing mutations in base 7 of G6Pase IRS 1 and 2; this latter oligonucleotide now competed almost as well as the WT oligonucleotide (Fig. 2Go, C and D). The high (500x) molar excess of unlabeled competitor required to achieve efficient competition when using crude GST-FOXO1a (Fig. 1Go, A and B), even with the labeled probe in excess, is consistent with the kinetics of a low affinity binding protein. We therefore hypothesize that partial purification increases binding affinity by removing factors that would otherwise inhibit GST-FOXO3a binding. A similar phenomenon may explain why we and others have been unable to detect FOXO binding in gel retardation assays using crude nuclear extract. Importantly, when these competition experiments were performed using partially purified His-FOXO1a (Fig. 1Go, E and F) the results were almost identical with those seen with partially purified GST-FOXO3a (Fig. 1Go, C and D). Most notably, the oligonucleotide containing mutations in base 7 of G6Pase IRS 1 and 2 competed almost as well as the WT oligonucleotide (Fig. 1Go, E and F).


Figure 2
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  		Fig. 2. Mutation of G6Pase IRS 1 and IRS 2 at Bases 5 or 7 Abolishes the Effect of Insulin on G6Pase Fusion Gene Expression

HepG2 cells were transiently cotransfected, as described in Materials and Methods, with various G6Pase-luciferase fusion genes (15 µg) and expression vectors encoding Renilla luciferase (0.15 µg) and the insulin receptor (5 µg). The G6Pase-luciferase fusion genes incorporated either the WT promoter sequence, located between –231 and +66, or contained the same promoter fragment with site-directed mutations in the M5, M7 or M10 bases of IRS 1 and 2. After transfection, the cells were incubated for 18–20 h in serum-free medium in the presence or absence of 100 nM insulin. The cells were then harvested and luciferase assays were performed as described in Materials and Methods. In panel A, results are presented as the ratio of firefly luciferase activity, corrected for Renilla luciferase activity in the cell lysate, in insulin-treated vs. control cells, expressed as percent control. In panel B, results are presented as the ratio of firefly luciferase to Renilla luciferase activity in control cells, expressed as a percentage of the value obtained with the WT fusion gene. Results represent the mean ± SEM of three experiments each using an independent preparation of all plasmids with each sample assayed in duplicate. *, P < 0.05 vs. WT.

 
These results suggest that FOXO1a and FOXO3a share an identical binding specificity, a conclusion that is consistent with the results of binding site selection experiments (32, 33). In addition, the results of these competition experiments match those of Hall et al. (31), who found that mutation of the two IGFBP-1 IRSs at base 10 did not affect competition for FOXO3a binding, whereas mutation of these IRSs at base 7 slightly impaired competition.

Mutation of G6Pase IRS 1 and 2 at Base 7 Abolishes the Effect of Insulin on G6Pase Fusion Gene Expression
To investigate the correlation between FOXO binding (Fig. 1Go) and the regulation of G6Pase gene transcription by insulin, a series of G6Pase-luciferase fusion genes were generated that contain the identical IRS mutations as used in the competition experiments described above (Table 1Go). These mutations were all generated in the context of the –231 to +66 G6Pase-luciferase fusion gene that contains both Regions A and B and therefore mediates a maximal repression of basal fusion gene expression by insulin (6). These fusion gene constructs were transiently transfected into liver-derived human HepG2 cells and the effect of mutating G6Pase IRS 1 and 2 at bases 5, 7, and 10 on the repression of basal G6Pase-luciferase fusion gene expression by insulin was investigated. As shown in Fig. 2AGo, insulin repressed basal expression of the WT-G6Pase-luciferase fusion gene and mutation of IRS 1 and 2 at base 10 had no effect on the insulin response. In contrast, mutation of IRS 1 and 2 at base 5 or base 7 abolished the insulin response (Fig. 2AGo). This result suggests a lack of correlation between FOXO binding to the G6Pase promoter and insulin action because the base 7 mutation had little effect on FOXO binding in competition experiments (Fig. 1Go).

The effect of mutating G6Pase IRS 1 and 2 at bases 5, 7, and 10 on basal fusion gene expression in the absence of insulin is shown in Fig. 2BGo. Mutation of IRS 1 and IRS 2 at base 5 or 7 resulted in a decrease in basal G6Pase-luciferase fusion gene expression, whereas mutation of base 10 had no effect (Fig. 2BGo). The combination of the insulin repression and this basal expression data are internally consistent with a model in which insulin inhibits G6Pase gene transcription by disabling the binding of an activator, as previously proposed (8). However, at odds with our previous model (8), the results with the base 7 mutant suggest a lack of correlation between FOXO binding and insulin action, implying that this endogenous activator is a factor distinct from FOXO1a.

Mutation of G6Pase IRS Base 7 Markedly Reduces but Does Not Abolish the Induction of G6Pase Fusion Gene Expression by FOXO1A and FOXO3A
To further explore the correlation, or lack thereof, between FOXO binding and insulin action HepG2 cells were cotransfected with the same G6Pase-luciferase fusion genes described above along with either a control expression vector or the same vector encoding either FOXO1a or FOXO3a (Fig. 3Go). Both FOXO1a and FOXO3a stimulated expression of the WT G6Pase-luciferase fusion gene and mutation of IRS 1 and 2 at base 10 had no effect on this induction (Fig. 3Go). In contrast, mutation of IRS 1 and 2 at base 5 abolished this induction, whereas mutation of IRS 1 and 2 at base 7 markedly reduced, but did not abolish, this stimulation (Fig. 3Go). This result suggested a lack of correlation between the ability of overexpressed FOXO to induce G6Pase fusion gene expression and insulin action. Thus, mutation of IRS 1 and 2 at base 7 impairs FOXO-stimulated fusion gene expression but abolishes insulin action (Figs. 2AGo and 3Go).


Figure 3
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  		Fig. 3. Mutation of G6Pase IRS 1 and IRS 2 at Bases 5 or 7 Markedly Reduces the Induction of G6Pase Fusion Gene Expression by FOXO1a and FOXO3a

HepG2 cells were transiently cotransfected, as described in Materials and Methods, with various G6Pase-luciferase fusion genes (15 µg) and expression vectors encoding Renilla luciferase (0.15 µg) and either pECE (2.5 µg) or pECE-FOXO1a (2.5 µg), or pECE-FOXO3a (2.5 µg). The G6Pase-luciferase fusion genes incorporated either the WT promoter sequence, located between –231 and +66, or contained the same promoter fragment with site-directed mutations in the M5, M7, or M10 bases of IRS 1 and 2. After transfection, the cells were incubated for 18–20 h in serum-free medium. The cells were then harvested and luciferase assays were performed as described in Materials and Methods. Results are presented as the ratio of firefly luciferase activity, corrected for Renilla luciferase activity in the cell lysate, in FOXO1a- or FOXO3a-stimulated vs. control cells, expressed as fold induction. Results represent the mean ± SEM of three experiments each using an independent preparation of all luciferase fusion gene plasmids with each sample assayed in duplicate. *, P < 0.05 vs. WT; **, P < 0.05 M5 vs. M7.

 
Based on similar studies with the IGFBP-1 IRSs, Hall et al. (31) concluded that FOXO3a could act as an insulin response factor (IRF) when overexpressed, but that it was not the endogenous factor mediating the insulin response because gel retardation competition and FOXO3a overexpression experiments demonstrated a lack of correlation between insulin action and both FOXO3a binding and FOXO3a-stimulated expression, respectively. Rather they concluded that their data were consistent with a model in which overexpressed FOXO can displace the endogenous IRF and then mediate insulin signaling. The same conclusion could be reached from these G6Pase IRS analyses because mutation of IRS 1 and 2 at base 7 abolished insulin action (Fig. 2AGo) but had little effect on FOXO binding in gel retardation competition experiments (Fig. 1Go) and only reduced, rather than abolished, FOXO-stimulated fusion gene expression (Fig. 3Go). However, because a number of genetic and molecular experiments support a role for endogenous FOXO1a in mediating the effect of insulin on G6Pase gene transcription (see Discussion), we decided to consider an alternate possibility, namely that the results of these FOXO overexpression and gel retardation competition experiments are misleading. Indeed, the magnitude of the effect of the IRS 1 and 2 base 7 mutation was much greater when assessed in the FOXO overexpression experiments (Fig. 3Go) than in the gel retardation competition experiments using purified FOXO proteins (Fig. 1Go, C–F), indicating that these data are not internally consistent.

Mutation of G6Pase IRS Base 7 Markedly Reduces FOXO1A and FOXO3A Binding to Labeled Oligonucleotide Probes
Based on a consideration of the kinetics of receptor-ligand interactions (34), we considered the possibility that, whereas the IRS 1 and 2 base 7 mutated oligonucleotide was able to compete efficiently for FOXO binding, it may not be able to form a stable complex with FOXO. Such a situation could arise if both the association and dissociation rate of FOXO binding were increased because this would not alter the dissociation constant (34) but would result in the rapid dissociation of the FOXO-DNA complex upon separation of bound and free probe during electrophoresis. If correct, this would imply that the results of the gel retardation competition experiments do not fully reflect FOXO binding ability.

To investigate this possibility, the oligonucleotides described above (Fig. 1Go and Table 1Go) were labeled and incubated with either crude extract prepared from bacterial cells expressing the GST-FOXO1a fusion protein (Fig. 4Go, A and B), with partially purified GST-FOXO3a (Fig. 4Go, C and D), or with partially purified His-FOXO1a (Fig. 4Go, E and F). In each case, the FOXO proteins bound the WT G6Pase probe and mutation of IRS 1 and 2 at base 10 had little or no effect on this binding (Fig. 4Go). In contrast, mutation of IRS 1 and 2 at base 5 abolished binding, whereas mutation of IRS 1 and 2 at base 7 markedly reduced binding (Fig. 4Go). This result confirms the hypothesis that FOXO proteins do not form a stable complex with the IRS 1 and 2 base 7 mutated sequence and that the results of competition analyses are misleading. Such a dissociation between the results of binding and competition experiments has previously been described for signal transducer and activator of transcription 5 binding to the serine protease inhibitor 2.1 promoter (35).


Figure 4
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  		Fig. 4. Mutation of G6Pase IRS 1 and IRS 2 at Bases 5 or 7 Markedly Reduces FOXO1a and FOXO3a Binding to Labeled Oligonucleotide Probes

A and B, Bacterial extract from either control (–) or IPTG-treated (+) cells transformed with a plasmid encoding a GST-FOXO1a fusion protein was added to the indicated labeled probes and protein binding was then analyzed using the gel retardation assay as described in Materials and Methods. The labeled probes represent the WT G6Pase promoter sequence between –196 and –155 or the same sequence but containing mutations in the M5, M7 or M10 bases of IRS 1 and 2 (Table 1Go). A, Representative autoradiograph in which only the retarded complexes are visible and not the free probe, that was present in excess. The specific IPTG-induced protein-DNA complex is indicated by the arrow. B, Mean data ± SEM of three experiments quantitated using a Packard Instant Imager. *, P < 0.05 vs. WT; **, P < 0.05 M5 and M7 vs. M10. C–F, Gel retardation experiments were performed as described in panel A but using partially purified GST-FOXO3a (C and D) and His-FOXO1a (E and F). C and E, Representative autoradiographs; D and F show mean data ± SEM of three to four experiments quantitated using a Packard Instant Imager. *, P < 0.05 vs. WT; **, P < 0.05 M5 and M7 vs. M10; ***, P < 0.05 M5 vs. M7.

 
This result established a strong correlation between FOXO binding ability, as assessed using labeled probes (Fig. 4Go), and the ability of overexpressed FOXO to induce G6Pase fusion gene expression (Fig. 3Go). But although these data correlate, in both assays the magnitude of the effect of the IRS 1 and 2 base 7 mutation is less than that of the base 5 mutation, whereas both mutations abolish the insulin response (Fig. 2AGo). However, an explanation for this apparent lack of correlation between the overexpression data with insulin action can again be provided by a consideration of the kinetics of receptor-ligand interactions and specifically the intracellular abundance of FOXO proteins (34). Because FOXO proteins are expressed at low levels in hepatoma cells (36), we hypothesize that the IRS 1 and 2 base 7 mutation results in the loss of endogenous FOXO binding, and hence insulin action, whereas overexpression of FOXO alters the binding equilibrium such that limited binding of FOXO can now occur to the IRS 1 and 2 base 7 mutated sequence resulting in a small induction of fusion gene expression (Fig. 3Go). Similarly, in gel retardation assays in which the probe is in excess, a low level of binding can be detected to the IRS 1 and 2 base 7 mutated probe (Fig. 4Go), but we hypothesize that this is insufficient to be of functional significance in situ. As such, the observations reported here would be consistent with the conclusion of Guo et al. (17) that endogenous FOXO factors can mediate effects of insulin on gene transcription.

FOXO1a and FOXO3a Bind Cooperatively to G6Pase IRS 1 and 2
Although the data described above support the conclusions of Guo et al. (17), there is a notable difference in the observed effect of the IRS base 10 mutation. Both we (Figs. 2Go and 3Go) and Guo et al. (17) find that mutation of IRS base 5 abolishes the effect of insulin and the ability of overexpressed FOXO1a to induce fusion gene expression. However, Guo et al. (17) found that mutation of IRS base 10 reduced both the effect of insulin and the ability of overexpressed FOXO1a to induce fusion gene expression, whereas we find that this mutation has no effect on either the insulin response (Fig. 2AGo) or FOXO-induced expression (Fig. 3Go). One obvious difference in experimental design that might explain this discrepancy is that the G6Pase fusion genes described above contain two adjacent IRSs that both bind FOXO, whereas Guo et al. (17) used fusion genes comprising a modified IGFBP-1 promoter containing a single IRS. We therefore hypothesized that the difference in results might be explained by cooperative binding of FOXO to the two G6Pase IRS motifs such that mutation of the two IRSs at base 10 was insufficient to destabilize FOXO binding.

To investigate whether such cooperative binding occurs, double-stranded oligonucleotides were synthesized, representing the G6Pase promoter sequence from –196 to –155 encompassing both IRS 1 and 2 (Table 1Go), that contain either a point mutation in G6Pase IRS 2 at base 5 (G6P IRS 2 M5; Table 1Go) or a block mutation in G6Pase IRS 2 (G6P IRS 2 BM; Table 1Go). These oligonucleotides were labeled and incubated with either crude extract prepared from bacterial cells expressing the GST-FOXO1a fusion protein (Fig. 5Go, A and B), with partially purified GST-FOXO3a (Fig. 5Go, C and D), or with partially purified His-FOXO1a (Fig. 5Go, E and F). In each case, the mutation of G6Pase IRS 2 at base 5 markedly reduced the level of FOXO binding relative to that achieved with the WT G6Pase probe despite the presence of the intact IRS 1 motif. The fact that binding is reduced by more than 50% indicates that the complex formed between FOXO and IRS 1 is unstable, which suggests that FOXO binds cooperatively to IRS 1 and 2. In fact, competition experiments show that FOXO1a binds with higher affinity to IRS 1 than IRS 2 (8) so, in the absence of cooperativity, the predicted decrease in binding would have been less than 50%. A further decrease in FOXO binding was detected when the oligonucleotide containing a block mutation in G6Pase IRS 2 was used as the labeled probe, suggesting that the point mutation in IRS 2 does not completely inhibit FOXO binding to this element (Fig. 5Go).


Figure 5
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  		Fig. 5. FOXO1a and FOXO3a Bind Cooperatively to G6Pase IRS 1 and 2

A and B, Bacterial extract from either control (–) or IPTG-treated (+) cells transformed with a plasmid encoding a GST-FOXO1a fusion protein was added to the indicated labeled probes and protein binding was then analyzed using the gel retardation assay as described in Materials and Methods. The labeled probes represent the WT G6Pase promoter sequence between –196 and –155 or the same sequence but containing either a point mutation of IRS 2 at base 5 (M5) or a BM in IRS 2 (Table 1Go). A, Representative autoradiograph in which only the retarded complexes are visible and not the free probe, that was present in excess. The specific IPTG-induced protein-DNA complex is indicated by the arrow. B, Mean data ± SEM of three experiments quantitated using a Packard Instant Imager. *, P < 0.05 vs. WT; **, P < 0.05 M5 vs. BM. C–F, Gel retardation experiments were performed as described in panel A but using partially purified GST-FOXO3a (C and D) and His-FOXO1a (E and F). C and E, Representative autoradiographs; D and F show mean data ± SEM of three experiments quantitated using a Packard Instant Imager. *, P < 0.05 vs. WT; **, P < 0.05 M5 vs. BM.

 
The existence of cooperative binding is also consistent with the observation that only a single complex is detected using crude extract prepared from bacterial cells expressing the GST-FOXO1a fusion protein (Fig. 5AGo), rather than two bands indicative of the binding of one or two FOXO proteins molecules. Multiple bands are detected using partially purified GST-FOXO3a (Fig. 5CGo) and partially purified His-FOXO1a (Fig. 5EGo), but this likely represents the occurrence of proteolysis during purification because the relative abundance of these complexes does not change (Fig. 5Go, C and E).

In contrast with these binding data obtained using the labeled G6P IRS 2 M5 oligonucleotide, gel retardation competition experiments previously indicated that the affinity of FOXO1a binding to this oligonucleotide, that has an intact IRS 1 motif, was only approximately 2-fold lower than binding to the WT sequence (8). As with the labeled G6P IRS 1 and 2 M7 oligonucleotide binding experiments described above (Fig. 4Go), this observation again reveals a lack of correlation between the results of binding and competition experiments. With regards to the G6P IRS 2 M5 oligonucleotide, this lack of correlation between binding (Fig. 5Go) and competition experiments (8) can be explained by a requirement for cooperative binding to IRS 1 and 2 for the formation of a stable FOXO-DNA complex. Similarly, with regards to the G6P IRS 1 and 2 M7 oligonucleotide, the simplest explanation for the lack of correlation between binding (Fig. 4Go) and competition experiments (Fig. 1Go) is that the mutation of base 7 prevents the formation of a stable FOXO-DNA complex by affecting cooperative binding.

Promoter Context Alters the Specific IRS Base Requirements for FOXO1a-Stimulated Fusion Gene Expression
Given the evidence for cooperative binding of FOXO to G6Pase IRS 1 and 2 (Fig. 5Go), the effect of mutating G6Pase IRS 1 at base 10 on FOXO1a-stimulated fusion gene expression in the absence of FOXO binding to IRS 2 was assessed. The rationale here was that this created a situation resembling the single IRS-containing fusion gene used by Guo et al. (17) in their analysis of the correlation between FOXO1a-stimulated fusion gene expression and insulin action. We hypothesized that, in the absence of FOXO1a binding to G6Pase IRS 2 and hence cooperativity, mutation of G6Pase IRS 1 at base 10 might now impair FOXO1a-stimulated fusion gene expression.

G6Pase-luciferase fusion genes were constructed that contained a WT IRS 1 motif or a base 10 mutated IRS 1 motif in conjunction with a point mutation in base 5 of IRS 2 or a block mutation in IRS 2 (Fig. 6AGo). HepG2 cells were cotransfected with these G6Pase-luciferase fusion genes along with either a control expression vector or the same vector encoding FOXO1a. Figure 6AGo shows that FOXO1a induced expression of the WT G6Pase-luciferase fusion gene, whereas mutation of IRS 1 and 2 at base 5 abolished this induction. In contrast, mutation of IRS 2 at base 5 only blunted this induction (Fig. 6AGo), as previously reported (8). When a point mutation was introduced into IRS 1 at base 10 in conjunction with the point mutation in IRS 2 at base 5, there was no further reduction in the magnitude of this induction (Fig. 6AGo). However, the absence of an effect of mutating IRS 1 at base 10 could potentially be explained by residual FOXO binding to the IRS 2 base 5 mutated motif that could still act to stabilize FOXO1a binding to the base 10 mutated IRS 1. The fact that the same result was obtained after the introduction of a block mutation in IRS 2 suggests this is not the case (Fig. 6AGo). Thus, the block mutation of IRS 2 reduced FOXO1a-induced fusion gene expression to the same extent as the IRS 2 base 5 point mutation and when a point mutation was introduced into IRS 1 at base 10 in conjunction with the block mutation in IRS 2, there was no further reduction in the magnitude of FOXO-induced expression (Fig. 6AGo). When the analysis, described above, of the effect of base 10 mutation of G6Pase IRS 1 on FOXO1a-induced fusion gene expression was repeated in rat H4IIE hepatoma cells similar results were obtained (Fig. 6BGo).


Figure 6
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  		Fig. 6. Mutation of G6Pase IRS 1 at Base 10 Does Not Affect the Induction of G6Pase Fusion Gene Expression by FOXO1a Even When IRS 2 Is Mutated

HepG2 cells (A) or H4IIE cells (B) were transiently cotransfected, as described in Materials and Methods, with various G6Pase-luciferase fusion genes (15 µg) and expression vectors encoding Renilla luciferase (0.15 µg) and either pcDNA3 (2.5 µg) or pcDNA3-FOXO1a (2.5 µg). The G6Pase-luciferase fusion genes incorporated either the WT promoter sequence, located between –231 and +66, or contained the same promoter fragment with site-directed point mutations in the indicated bases of IRS 1 and 2 and/or a BM in IRS 2. After transfection, the cells were incubated for 18–20 h in serum-free medium. The cells were then harvested and luciferase assays were performed as described in Materials and Methods. Results are presented as the ratio of firefly luciferase activity, corrected for Renilla luciferase activity in the cell lysate, in FOXO1a-stimulated vs. control cells, expressed as fold induction. Results represent the mean ± SEM of three experiments each using an independent preparation of all luciferase fusion gene plasmids with each sample assayed in duplicate. *, P < 0.05 vs. WT; **, M5/M5 P < 0.05 vs. all other plasmids.

 
These results suggest that the difference between the data reported here and that of Guo et al. (17) with respect to the effect of mutating G6Pase IRS 1 or the IGFBP-1 IRS at base 10 must reflect some context-specific difference inherent to the systems used to study the effect of these mutations. Guo et al. (17) also performed their experiments in HepG2 cells and the mutation introduced, the conversion of a G to a C, was the same in both studies. However, the flanking sequence 5' and 3' of the IRS motifs differ as do the orientation of the elements and the promoters in which these IRSs are located. Promoter context, flanking sequence, and orientation can all significantly influence the behavior of DNA elements (37).

The fact that mutation of these IRS motifs at base 10 can influence the insulin response, at least in some promoter contexts, is perhaps surprising given that binding site selection experiments indicate that FOXO1a binding to DNA can tolerate variation at this base (33). Interestingly, Hall et al. (31) also noted variable results with respect to mutation of IRSs at base 10; mutation of the PEPCK IRS at base 10 had no effect on the insulin response, whereas mutation of the two IGFBP-1 IRSs at base 10 abolished the insulin response (31).

Importantly, the results of these binding experiments (Fig. 5Go) broadly correlate with the analysis of FOXO1a-stimulated G6Pase fusion gene expression (Fig. 6Go). Thus, mutation of IRS 2 reduces both FOXO1a binding (Fig. 5Go) and FOXO1a-stimulated G6Pase fusion gene expression (Fig. 6Go) by much more than 50% indicating that FOXO1a binds cooperatively to IRS 1 and 2 both in vitro and in situ. Interestingly, this correlation is not perfect because the block mutation of IRS 2 reduced FOXO1a-induced fusion gene expression to the same extent as the IRS 2 base 5 point mutation (Fig. 6Go), whereas partially purified FOXO1a binds better to the IRS 2 base 5 point mutation than the IRS 2 block mutant in vitro (Fig. 5Go, C and D). This result emphasizes the influence of promoter context because it implies that one or more other factors can stabilize FOXO1a binding to IRS 1 in situ and overcome the otherwise expected further reduction of FOXO1a-stimulated fusion gene expression as predicted from the cooperative binding seen in vitro (Fig. 5Go).

The Effect of IRS Mutations on Basal Fusion Gene Transcription Is Context Dependent
Given the observation that promoter context can influence the results of IRS mutational analyses, we decided to investigate the effect of mutating G6Pase IRS 1 and 2 in the context of the heterologous TK promoter. Much of the original characterization of the PEPCK IRS was performed in the context of the heterologous TK promoter rather than in the context of the native PEPCK promoter (10, 27). We hypothesized that different results might be obtained in the context of the TK promoter. Double-stranded oligonucleotides representing the WT or mutated G6Pase Region B sequence between –197 and –158 (Table 2Go) were synthesized and ligated into a TK-luciferase vector and the effect of insulin on the expression of the resulting fusion genes was analyzed by transient transfection of the liver-derived rat H4IIE cell line (Fig. 7AGo). We have previously shown that insulin has little effect on reporter gene expression directed by the native TK promoter in H4IIE cells (10, 27) (Fig. 7AGo), whereas it stimulates expression in HepG2 cells (data not shown). Figure 7AGo shows that the WT Region B sequence that contains IRS 1, 2, and 3 (Table 2Go) mediates an inhibitory effect of insulin on fusion gene expression, whereas mutation of IRS 1 and 2 at base 5 abolishes this effect. We have previously shown that the isolated, multimerized IRS 3 motif can also mediate an insulin effect in this context (8), but this element is apparently not insulin responsive in the context of the mutated Region B oligonucleotide. Consistent with the model, whereby insulin can inhibit gene expression by stimulating the nuclear exclusion of FOXO proteins that act as transcriptional activators, basal gene expression decreased upon mutation of IRS 1 and 2 (Fig. 7BGo).


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  		Table 2. Sequences of the Sense Strands of Oligonucleotides Used in TK Vector Studies

 

Figure 7
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  		Fig. 7. The Effect of IRS Mutations on Basal and Insulin-Regulated Fusion Gene Transcription Is Context Dependent

A and B, H4IIE cells were transiently cotransfected, as described in Materials and Methods, with an expression vector encoding Renilla luciferase (0.15 µg) and either the basic TK-pGL3 vector (15 µg) or constructs in which single copies of the oligonucleotides representing the WT (G6P WT) or IRS 1 and 2 M5 mutated (G6P M5) G6Pase promoter sequence from –197 to –158 had been ligated (Table 2Go). C and D, H4IIE cells were transiently cotransfected, as described in Materials and Methods, with an expression vector encoding Renilla luciferase (0.15 µg) and either the basic TK-pGL3 vector (15 µg) or constructs in which single copies of the oligonucleotides representing the WT (BP-1 WT) or M5 (BP-1 M5) mutated IGFBP-1 promoter sequence from –124 to –96 had been ligated (Table 2Go). E and F, H4IIE cells were transiently cotransfected, as described in Materials and Methods, with various IGFBP-1-luciferase fusion genes (15 µg) and an expression vectors encoding Renilla luciferase (0.15 µg). The IGFBP-1-luciferase fusion genes contained distinct lengths of WT promoter sequence, as indicated by the 5' deletion end-points. After transfection, cells were incubated for 18–20 h in serum-free medium in the presence or absence of 10 nM insulin. The cells were then harvested and luciferase assays were performed as described in Materials and Methods. In panels A, C, and E, results are presented as the ratio of firefly luciferase activity, corrected for Renilla luciferase activity in the cell lysate, in insulin-treated vs. control cells, expressed as percent control. In panels B, D, and F, results are presented as the ratio of firefly luciferase to Renilla luciferase activity in control cells, expressed as a percentage of the value obtained with either the TK vector minus insert or the –132 IGFBP-1 fusion gene, as indicated. Results represent the mean ± SEM of three to four experiments using three independent preparations of all plasmids with each sample assayed in duplicate. A–D: *, P < 0.05 vs. TK-pGL3 control vector. E and F: *, P < 0.05.

 
A markedly different result was obtained when this experiment was repeated using the two IGFBP-1 IRSs. Figure 7CGo shows that the WT human IGFBP-1 sequence between –124 and –96, that contains the two IRSs (Table 2Go), mediates an inhibitory effect of insulin on fusion gene expression, whereas mutation of these IRSs at base 5 abolishes this effect. This base 5 mutation has been shown to abolish FOXO1a binding to the IGFBP-1 IRSs (22). In contrast to the data obtained with the G6Pase IRSs (Fig. 7BGo), the accompanying change in basal gene expression associated with mutation of these IRSs is not consistent with the model whereby insulin can inhibit gene expression by stimulating the nuclear exclusion of FOXO proteins. Thus, basal gene expression actually increased upon mutation of the IGFBP-1 IRSs (Fig. 7DGo). This unexpected result with the heterologous IGFBP-1 IRS-TK promoter constructs contrasts with what is seen when the IGFBP-1 IRSs are analyzed in the context of the native IGFBP-1 promoter. Thus, deletion of the IGFBP-1 promoter region between –132 and –103, that encompasses the two IGFBP-1 IRSs, abolished the effect of insulin (Fig. 7EGo) and resulted in a decrease in basal fusion gene expression (Fig. 7FGo) consistent with the loss of FOXO activator binding. These results suggest that the behavior of the IGFBP-1 IRSs differs in the context of the heterologous and native promoters. Potential explanations for this result are discussed below but additional evidence for a difference in IRS/FOXO function in different promoter contexts is apparent from overexpression studies comparing FOXO1a and FOXO3a. Figure 8Go shows that, in H4IIE cells, overexpression of FOXO3a stimulates heterologous G6Pase-TK fusion gene expression more than FOXO1a, whereas in the same cells the effect of FOXO1a on G6Pase-luciferase fusion gene expression is greater than that of FOXO3a. This suggests that the ability of FOXO1a and FOXO3a to induce fusion gene expression is also context dependent.


Figure 8
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  		Fig. 8. The Ability of FOXO1a and FOXO3a to Induce Fusion Gene Transcription Is Also Context Dependent

A, H4IIE cells were transiently cotransfected, as described in Materials and Methods, with expression vectors encoding Renilla luciferase (0.15 µg) and either pECE (2.5 µg) or pECE-FOXO1a (2.5 µg), or pECE-FOXO3a (2.5 µg) and either the basic TK-pGL3 vector (15 µg) or a construct in which a single copy of the oligonucleotide representing the WT G6Pase promoter sequence from –197 to –158 had been ligated (Table 2Go). B, H4IIE cells were transiently cotransfected, as described in Materials and Methods, with a G6Pase-luciferase fusion gene (15 µg) incorporating the WT promoter sequence, located between –231 and +66 and expression vectors encoding Renilla luciferase (0.15 µg) and either pECE (2.5 µg) or pECE-FOXO1a (2.5 µg), or pECE-FOXO3a (2.5 µg). After transfection, the cells were incubated for 18–20 h in serum-free medium. The cells were then harvested and luciferase assays were performed as described in Materials and Methods. Results are presented as the ratio of firefly luciferase activity, corrected for Renilla luciferase activity in the cell lysate, in FOXO1a- or FOXO3a-stimulated vs. control cells, expressed as fold induction. Results represent the mean ± SEM of three experiments each using an independent preparation of all luciferase fusion gene plasmids with each sample assayed in duplicate. Panel A: *, P < 0.05 vs. TK-pGL3 vector. Panel B: *, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
FOXO1a and its orthologs emerged as candidate IRFs indirectly from genetic experiments in Caenorhabditis elegans that identified a homologous transcription factor, Daf-16, as the target of an insulin-like metabolic signaling pathway (38, 39). Subsequently, it was shown that FOXO1a could bind the PEPCK, IGFBP-1, and G6Pase IRSs in vitro (7, 8, 14, 22) and, when overexpressed, stimulate PEPCK, IGFBP-1 and G6Pase fusion gene expression (8, 22, 23, 24). However, a concern with these overexpression experiments is that FOXO is simply displacing an endogenous IRF. In addition, detailed mutagenesis studies produced conflicting data, with Guo et al. (17) concluding that FOXO1a was the endogenous IRF through which insulin regulated IGFBP-1 gene expression and Hall et al. (31) concluding that, whereas FOXO3a could act as an IRF, it was not the endogenous IRF that mediated insulin action through the IGFBP-1 and PEPCK IRSs. The results shown in Figs. 1–5GoGoGoGoGo show that these conflicting data (17, 31) are not explained by a difference in FOXO1a and FOXO3a binding specificity. Instead, we find that gel retardation competition and binding experiments give different results; the latter reveal a correlation between FOXO binding and the inhibition of basal G6Pase gene transcription by insulin.

Several other observations support a role for FOXO1 in the regulation of G6Pase gene transcription. Circumstantial evidence comes from the analyses of mice in which Foxo1 gene expression has been suppressed or in which a constitutively active form of Foxo1 has been overexpressed (40). Both manipulations lead to changes in endogenous G6Pase gene expression (40). However, in these types of experiments one cannot rule out the possibility that the effect of altering Foxo1 expression is indirect. The same caveat applies to the results of Barthel et al. (36) who showed that retroviral overexpression of FOXO1 in H4IIE cells stimulates endogenous G6Pase gene expression. More direct evidence comes from the results of chromatin immunoprecipitation (ChIP) assays. We have previously shown using the ChIP assay that FOXO1a binds the G6Pase promoter within intact HepG2 cells and that insulin treatment reduces this binding (8). The same result is seen in H4IIE cells (8) and mouse immortalized hepatocytes (41).

The studies reported here and the literature cited above strongly support the involvement of FOXO1a in insulin-regulated G6Pase gene transcription. In contrast, whether insulin also acts through FOXO1a to regulate PEPCK gene transcription is controversial. FOXO1a can bind the PEPCK IRS in vitro (22) and, when overexpressed, stimulate PEPCK fusion gene expression in situ (24). In addition, ChIP assays show that Foxo1 binds the PEPCK promoter in hepatocytes (41, 42) and adenoviral overexpression of a Foxo1 carboxy terminal mutant in mice alters PEPCK gene expression (43). However, in 6-month-old mice in which Foxo1 gene expression has been suppressed, or in which a constitutively active form of Foxo1 has been overexpressed, there are changes in G6Pase but not PEPCK gene expression (40). Similarly, Barthel et al. (36) found that retroviral overexpression of FOXO1 in H4IIE cells stimulated G6Pase but not PEPCK gene expression.

These conflicting data regarding the role of FOXO in insulin-regulated PEPCK gene transcription may relate to the observation that insulin can repress the expression of this gene through mechanisms that are independent of the PEPCK IRS motif (9). Thus, alternate PEPCK IRS-independent mechanisms for repression by insulin involving nuclear factor {kappa}B (44), sterol regulatory element binding protein-1c (45), liver-enriched transcriptional-inhibitory protein (46), and Kruppel like factor-15 (47) have been proposed. The existence of alternate mechanisms for transcriptional repression has led to some confusion with several investigators concluding that the PEPCK IRS is not important for the regulation of PEPCK gene expression by insulin (24, 48, 49, 50). A common problem in these studies is that the investigators have studied the ability of insulin to repress the stimulatory effect of glucocorticoids on PEPCK gene transcription. However, the PEPCK IRS is colocalized with an accessory element, designated AF2, that is required for the stimulatory effect of glucocorticoids (51) and mutations that disable the PEPCK IRS but not AF2 have not been identified. Therefore, these studies only show that insulin, working through alternate mechanisms, is able to fully suppress the markedly reduced remaining stimulatory effect of glucocorticoids.

In addition to demonstrating a strong correlation between FOXO binding and insulin-regulated G6Pase gene expression the studies reported here also show 1) that FOXO1a binds cooperatively to G6Pase IRS 1 and 2 in vitro (Fig. 5Go) and in situ (Fig. 6Go), 2) that, based on a comparison of the effect of the IRS base 10 mutation reported here (Fig. 3Go) and by Guo et al. (17), promoter context alters the specific IRS base requirements for FOXO1a-stimulated fusion gene expression, and 3) that promoter context also affects the relative magnitude of FOXO1a- and FOXO3a-stimulated fusion gene expression (Fig. 8Go). Interestingly, the demonstration of cooperative binding of FOXO1a to G6Pase IRS 1 and 2 provides an explanation for paradoxical observation that FOXO1a binds with higher affinity to IRS 1 than IRS 2 but mutation of IRS 1 or 2 is equally deleterious to the insulin response (8). Whether there is a molecular reason for the differential affinity of FOXO1a binding to G6Pase IRS 1 and 2 is unclear, but it may relate to our recent observation that FOXO1a bound to G6Pase IRS 1 but not IRS 2 acts as an accessory factor for glucocorticoid-stimulated G6Pase gene expression (52). The molecular basis for the observed effect of promoter context on FOXO1a- and FOXO3a-stimulated fusion gene expression is also unclear but presumably relates to the differential interaction with, or modification by, other factors at the two promoters examined. Recent studies have shown that FOXO1a/3a can interact with multiple proteins (53) and are subject to complex regulation by acetylation/deacetylation (54) in addition to phosphorylation. Finally, it should be noted that liver cells are responsive to multiple hormones other than insulin such that the constitutive regulation of FOXO factors by endogenous hormones is likely to be much more complex than suggested by experiments in HepG2 cells in which only insulin action is analyzed.

Perhaps the most surprising result reported in this study is that the analysis of insulin action mediated through the G6Pase and IGFBP-1 IRSs in the context of a heterologous TK promoter reveals that signaling through the latter does not support the model for insulin-stimulated FOXO nuclear exclusion (Fig. 7Go). Thus, mutation of the G6Pase IRSs in this context results in a decrease in basal fusion gene expression (Fig. 7BGo), consistent with the loss of FOXO activator binding, whereas mutation of the IGFBP-1 IRSs actually results in an increase in basal expression (Fig. 7DGo). Rechler and colleagues (55) have previously shown that the inhibition of FOXO1-stimulated gene transcription by insulin does not require nuclear exclusion of FOXO1. However, FOXO1 dissociation from a promoter would still result in the inhibition of gene transcription without requiring nuclear exclusion. Two possible explanations for our unexpected results are that 1) the IRF binding the IGFBP-1 IRSs in this promoter context is not a FOXO factor, and 2) a FOXO factor still binds the IGFBP-1 IRSs in this promoter context but acts by a mechanism that involves insulin-stimulated repression rather than promoter dissociation. There is evidence to support both possibilities. For example, Patel et al. (56) have shown that a mammalian target of rapamycin-dependent pathway mediates the action of insulin on IGFBP-1 but not G6Pase gene expression. This would be consistent with a model in which a FOXO factor mediated insulin-regulated G6Pase but not IGFBP-1 gene expression. However, the regulation of FOXO phosphorylation is complex, and it is apparent that insulin can target this factor through kinases other than PKB (57, 58). Whether these unidentified kinases are a mammalian target of rapamycin dependent and whether phosphorylation can result in repression by FOXO factors in the absence of nuclear exclusion remains to be determined. A number of recent papers give circumstantial support for the alternate explanation for our observations, namely that a FOXO factor could function through a mechanism that involves insulin-stimulated repression rather than promoter dissociation in the heterologous promoter context examined. For example, Zhao et al. (59) have shown that FOXO1 can act as a corepressor and several genes have been identified whose expression are directly repressed by FOXO1 (60, 61, 62, 63). Whether insulin acts on these genes to relieve or augment repression mediated by FOXO1 has not been determined.

The promoter-specific characteristics that determine whether a gene is activated or repressed by FOXO1 remain to be determined. Possibilities include the precise complement of coactivators/repressors at a given promoter, but another important parameter may be the acetylation status of FOXO1. cAMP-response element-binding protein-binding protein has been shown to acetylate FOXO factors, a modification that is reversed by the nicotinamide adenine dinucleotide-dependent deacetylase silent information regulator 2 (Sir2) (64, 65, 66). Most reports suggest that acetylation inhibits FOXO action (64, 65, 67). This would be consistent with the recent observation that acetylation directly inhibits FOXO1 DNA binding affinity as well as promoting the phosphorylation of FOXO1 by PKB and hence promoter dissociation (68). However, one report suggests that acetylation stimulates FOXO action (66). Thus, it is apparent that acetylation modulates FOXO1 action, but it is also possible that it may influence whether FOXO1 activates or represses the expression of specific genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Construction
The generation of a mouse G6Pase-luciferase fusion gene, containing promoter sequence located between –231 and +66, relative to the transcription start site, in the pGL3 MOD vector has been previously described (69). This vector is based on the pGL3 Basic firefly luciferase vector (Promega, Madison, WI) but contains a modified polylinker (69). G6Pase-chloramphenicol acetyltransferase (CAT) fusion genes containing site-directed point mutations (SDMs) in the G6Pase IRS 1 and IRS 2 motifs have also been previously described (8). These point mutations were introduced individually or together into the IRSs at base 5 (Table 1Go) to generate the plasmids –231 IRS 1 M5 SDM, –231 IRS 2 M5 SDM and –231 IRS 1 + 2 M5 SDM. These mutations were generated in the context of the –231/+66 G6Pase promoter region (8) and were subcloned from the CAT to the pGL3 MOD vector. A three-step PCR strategy (6, 70) was used to generate G6Pase-luciferase fusion genes designated –231 IRS 1 + 2 M7 SDM and –231 IRS 1 + 2 M10 SDM that contain point mutations introduced into IRSs 1 and 2 at base 7 or 10, respectively (Table 1Go). The same strategy was used to generate the plasmid –231 IRS 2 BM that contains a block mutation of IRS 2 (Table 1Go) and the plasmids –231 IRS 1 M10 + IRS 2 M5 SDM and –231 IRS 1 M10 + IRS 2 BM that contain a point mutation in base 10 of IRS 1 in conjunction with a point or block mutation in IRS 2, respectively. These mutations were generated in the context of the –231/+66 G6Pase promoter region ligated into pGEM7 (Promega) and were then subcloned into the pGL3 MOD vector. The mutations introduced into these plasmids were identical with those used in gel retardation assays. All promoter fragments generated by PCR were completely sequenced to verify the absence of polymerase errors.

The plasmid TKC-VI contains the herpes simplex virus TK promoter ligated to the CAT reporter gene (27, 71). The TK promoter sequence extends from –480 to +51 and contains a BamHI site located between positions –40 and –35. Various double-stranded oligonucleotides representing the WT or mutated mouse G6Pase promoter sequence between –197 and –158 or the WT or mutated human IGFBP-1 promoter sequence between –124 and –96 (Table 2Go) were synthesized with BamHI compatible ends and cloned, as a single copy, in the same orientation as found in the G6Pase and IGFBP-1 genes, into BamHI-cleaved TKC-VI by standard techniques (72). The CAT reporter gene was then replaced with the more sensitive firefly luciferase reporter by reisolating the various TKC-VI promoter constructs, as HindIII-BglII fragments, from the plasmids described above and ligating them into HindIII-BglII digested pGL3 MOD.

The generation of human IGFBP-1-CAT fusion genes, containing promoter sequence located between (–132 and +68) and (–103 and +68), relative to the transcription start site, has been described (73) and were generously provided by Dr. David Powell. The CAT reporter gene was replaced with the more sensitive firefly luciferase reporter by reisolating these IGFBP-1 promoter sequences, as HindIII-XhoI fragments, from the plasmids described above and ligating them into HindIII-XhoI digested pGL3 MOD.

For protein expression in HepG2 and H4IIE cells, a plasmid encoding human FOXO3a (pECE-FOXO3a) and the empty pECE vector were generously provided by Dr. Michael Greenberg (18). A vector encoding human FOXO1a (pcDNA3-FKHR) was generously provided by Dr. Frederic Barr (22). The FOXO1a open reading frame was isolated from this plasmid as a KpnI-XbaI fragment and ligated into KpnI-XbaI digested pECE. A pCDM8 (Invitrogen) expression vector encoding the human insulin receptor was generously provided by Dr. Jonathan Whittaker (74).

For protein expression in bacterial cells, a vector encoding a GST-human FOXO1a fusion protein (pGEX-5X-3-FOXO1a) was generously provided by Dr. David Powell (22). A vector encoding a histidine-tagged variant of full-length human FOXO1a (His-FOXO1a) was constructed by isolating the FOXO1a open reading frame as a KpnI-XbaI fragment from pcDNA3-FOXO1a (22). This fragment was blunt ended using Klenow and ligated into XhoI digested, blunt ended pET-15b (Novagen, San Diego, CA). To generate a plasmid encoding a GST-full-length human FOXO3a fusion protein, the FOXO3a open reading frame was isolated from the pECE-FOXO3a plasmid described above as a HindIII-XbaI fragment and ligated into HindIII-XbaI digested pGEM7 (Promega). The FOXO3a open reading frame was then reisolated as a BamHI fragment and ligated into BamHI digested pGEX 2T (Novagen). All plasmid constructs were purified by centrifugation through cesium chloride gradients (72).

Cell Culture and Transient Transfection
Rat H4IIE hepatoma cells were grown in DMEM containing 2.5% (vol/vol) fetal calf serum and 2.5% (vol/vol) newborn calf serum. Human HepG2 hepatoma cells were grown in the same media supplemented with 5% (vol/vol) Nu serum IV (Becton Dickinson, Inc.). H4IIE and HepG2 cells were transiently transfected in suspension with the plasmids indicated in the figure legends using the calcium phosphate-DNA coprecipitation method as previously described (25, 27, 69). Cells (~3 x 106) were transfected with various firefly luciferase plasmids (15 µg) and simian virus 40-Renilla luciferase (0.15 µg), and where indicated with the pECE (2.5 µg), pECE-FOXO1a (2.5 µg), pECE-FOXO3a (2.5 µg), pcDNA3 (2.5 µg), pcDNA3-FOXO1a (2.5 µg), and insulin receptor (5 µg) expression vectors.

Luciferase Assays
Luciferase assays were performed using the Promega Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions. In these experiments, we saw no effect of insulin or FOXO overexpression on firefly luciferase activity in either HepG2 or H4IIE cells. Therefore, for comparisons of basal, insulin-regulated, and forkhead-stimulated gene expression firefly luciferase activity directed by the various fusion gene constructs was expressed relative to simian virus 40-Renilla luciferase activity in the same sample. Each construct was analyzed in duplicate in multiple transfections, as specified in the figure legends, using three independent plasmid preparations.

Gel Retardation Assay
The plasmid encoding the GST-FOXO1a fusion protein (22) was transformed into Escherichia coli (XL1-Blue). Bacteria were grown to an OD600 of approximately 0.7 in LB supplemented with 200 µg/ml ampicillin and GST-FOXO1A expression was induced by incubation with 1 mM IPTG for 2 h at 37 C. Bacteria were pelleted by centrifugation, resuspended in 50 mM HEPES (pH 7.5), 200 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride and lysed by sonication. His-FOXO1a and GST-FOXO3a fusion proteins were grown and partially purified as previously described (52).

Complementary oligonucleotides representing the WT or mutated G6Pase sequence between –196 and –155 (Table 1Go) were synthesized with BamHI compatible ends, gel purified, annealed, and then labeled with [{alpha}-32P]deoxy-ATP (>3000 Ci mmol–1; NEN Life Sciences, Boston, MA) by using the Klenow fragment of E. coli DNA polymerase I to a specific activity of approximately 2.5 µCi/pmol (72). Labeled oligonucleotide (~7 fmol) was incubated with bacterial lysate containing GST-FOXO1a or partially purified His-FOXO1a and GST-FOXO3a in a reaction volume of 20 µl containing, at final concentrations, 25 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM dithiothreitol, 10% glycerol (vol/vol), 1 mg/ml BSA and 20 ng of poly(dG-dC)·poly(dG-dC). For competition experiments, unlabeled competitor DNA was mixed with the labeled oligomer at the indicated molar excess before the addition of protein. After incubation for 10 min on ice, the reactants were loaded onto a 6% polyacrylamide gel and electrophoresed at 4 C for 120 min at 190 V in 0.5x TBE (72). Gels were pre-run for 30 min at 100 V. After electrophoresis, the gels were dried, exposed to Kodak XAR5 film, and binding was analyzed by autoradiography. Data were quantitated through the use of a Packard Instant Imager (Meriden, CT).

Statistical Analyses
The transfection data were analyzed for differences from the control values, as specified in the figure legends. Statistical comparisons were calculated using an unpaired Student’s t test. The level of significance was P < 0.05 (two-sided test).


   ACKNOWLEDGMENTS
 
We thank Frederic Barr (University of Pennsylvania, Philadelphia, PA), David Powell (Lexicon Genetics Inc., The Woodlands, TX), Michael Greenberg (Harvard Medical School, Boston, MA), and Jonathan Whittaker (Case Western Reserve University, Cleveland, OH) for providing the pcDNA3-FOXO1a, pGEX-5X-3-FOXO1a, pECE-FOXO3a, and insulin receptor expression vectors, respectively. We also thank Craig Vander Kooi (Vanderbilt University, Nashville, TN) and Walter Chazin (Vanderbilt University, Nashville, TN) for providing partially purified the His-FOXO1a and GST-FOXO3a and Lee Limbird (Vanderbilt University, Nashville, TN), Howard Towle (University of Minnesota, Minneapolis, MN), and Roger Colbran (Vanderbilt University, Nashville, TN) for discussions on the kinetics of protein-DNA binding.


   FOOTNOTES
 
Research in the laboratory of R.O’B. was supported by National Institutes of Health (NIH) Grant DK56374 and by NIH Grant P60 DK20593, which supports the Vanderbilt Diabetes Center Core Laboratory. B.V.K. was supported by the Vanderbilt Molecular Endocrinology Training Program (5 T 32 DK07563).

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 13, 2006

Abbreviations: BM, Block mutation; CAT, chloramphenicol acetyltransferase; ChIP, chromatin immunoprecipitation; ER, endoplasmic reticulum; G6Pase, glucose-6-phosphatase catalytic subunit; GST, glutathione-S-transferase; HNF-1, hepatocyte nuclear factor-1; IGFBP, IGF binding protein; IPTG, isopropylthio-ß-D-galactosidase; IRF, insulin response factor; IRS, insulin response sequence; PEPCK, phosphoenolpyruvate carboxykinase; PKB, protein kinase B; SDM, site-directed point mutation; TK, thymidine kinase; WT, wild type.

Received for publication February 16, 2006. Accepted for publication June 29, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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