Inhibition by Insulin of Glucocorticoid-Induced Gene Transcription: Involvement of the Ligand-Binding Domain of the Glucocorticoid Receptor and Independence from the Phosphatidylinositol 3-Kinase and Mitogen-Activated Protein Kinase Pathways
Christophe E. Pierreux,
Birgitte Ursø,
Pierre De Meyts,
Guy G. Rousseau and
Frédéric P. Lemaigre
Hormone and Metabolic Research Unit (C.E.P., G.G.R., F.P.L.)
Louvain University Medical School Christian de Duve Institute of
Cellular Pathology (ICP) B-1200 Brussels, Belgium
Department of Molecular Signaling (B.U., P.D.M.) Hagedorn
Research Institute DK-2820 Gentofte, Denmark
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ABSTRACT
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Insulin can inhibit the stimulatory effect of
glucocorticoid hormones on the transcription of genes coding for
enzymes involved in glucose metabolism. We reported earlier that
insulin inhibits the glucocorticoid-stimulated transcription of the
gene coding for liver 6-phosphofructo-2-kinase (PFK-2). To elucidate
the mechanism of these hormonal effects, we have studied the regulatory
regions of the PFK-2 gene in transfection experiments. We found that
both glucocorticoids and insulin act via the glucocorticoid response
unit (GRU) located in the first intron. Footprinting experiments showed
that the GRU binds not only the glucocorticoid receptor (GR), but also
ubiquitous [nuclear factor I (NF-I)] and liver-enriched [hepatocyte
nuclear factor (HNF)-3, HNF-6, CAAT/enhancer binding protein
(C/EBP)] transcription factors. Site-directed mutational
analysis of the GRU revealed that these factors modulate glucocorticoid
action but that none of them seems to be individually involved in the
inhibitory effect of insulin. We did not find an insulin response
element in the GRU, but we showed that insulin targets the GR.
Insulin-induced inhibition of the glucocorticoid stimulation required
the ligand-binding domain of the GR. Finally, the insulin-signaling
cascade involved was independent of the
phosphatidylinositol-3-kinase and mitogen-activated protein kinase
pathways. Together, these results suggest that insulin acts on the
PFK-2 gene via another pathway and targets either the GR in its
ligand-binding domain or a cofactor interacting with this domain.
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INTRODUCTION
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Insulin and glucocorticoids exert a long term control on glucose
metabolism in liver by regulating the transcription of genes coding for
enzymes involved in glycolysis and gluconeogenesis (reviewed in Refs. 1, 2). Insulin stimulates transcription of several genes coding for
glycolytic enzymes, such as glucokinase and phosphofructo-1-kinase,
while glucocorticoids stimulate transcription of the genes coding for
the gluconeogenic enzymes glucose-6-phosphatase (3),
phosphoenolpyruvate carboxykinase (PEPCK) (4), tyrosine
aminotransferase (TAT) (5), and aspartate aminotransferase (6).
Moreover, insulin can inhibit basal and/or glucocorticoid-induced
transcription of these gluconeogenic genes. The bifunctional enzyme
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) catalyzes
the synthesis and the degradation of fructose 2,6-bisphosphate, a
stimulator of glycolysis and an inhibitor of gluconeogenesis (reviewed
in Refs. 7, 8). Transcription of the gene that codes for liver PFK-2
is controlled by insulin and glucocorticoids in a way similar to the
genes coding for gluconeogenic enzymes. Liver PFK-2 mRNA is barely
detectable in adrenalectomized rats, and it is restored to normal
levels upon treatment of these animals with dexamethasone (9). In rat
hepatoma FTO-2B cells, glucocorticoids stimulate transcription of the
PFK-2 gene and insulin inhibits this stimulation. These effects are
rapid and independent of protein synthesis, which indicates that
glucocorticoids and insulin are acting on preexisting regulatory
proteins (10). The physiological significance of this regulation must
be considered by taking into account the posttranslational
modifications of the product of the PFK-2 gene (reviewed in Refs. 7, 8). Its phosphorylation induced by glucagon inactivates the
6-phosphofructo-2-kinase activity and stimulates the
fructose-2,6-bisphosphatase activity, thereby leading to stimulation of
gluconeogenesis via a decrease in fructose 2,6-bisphosphate. On the
other hand, insulin treatment stimulates fructose 2,6-bisphosphate
synthesis and therefore also glycolysis via stimulation of protein
phosphatase 2A, which dephosphorylates the enzyme.
The mechanism by which glucocorticoid hormones regulate transcription
is fairly well understood. After binding its ligand, the glucocorticoid
receptor (GR) migrates from the cytoplasm to the nucleus and binds to a
glucocorticoid response element (GRE) in regulatory regions of target
genes. The GR then modulates transcription by interacting with
cofactors (11), like glucocorticoid receptor interacting protein-1 or
steroid receptor coactivator-1, which link the GR to the general
transcription factors without binding themselves to DNA. In several
instances, transcription factors bind to DNA in the vicinity of the GRE
and cooperate with the GR, thereby constituting a glucocorticoid
response unit (GRU). The PFK-2 gene contains a GRU in the first intron
(12, 13). Our previous in vivo analysis by genomic
footprinting and our transfection experiments showed that in this GRU,
hepatocyte nuclear factor (HNF)-3 binds to DNA in a
glucocorticoid-dependent way and cooperates with the GR to stimulate
transcription (14).
The way in which insulin regulates transcription is less well
understood. Upon insulin binding, the insulin receptor (IR) becomes
activated. This leads to autophosphorylation of the receptor and to
phosphorylation of substrates, thereby initiating the intracellular
signaling cascades that control various metabolic processes (reviewed
in Refs. 15, 16). In liver, several genes are regulated by insulin
at the transcriptional level via a glucose-dependent pathway. In this
case, insulin stimulates glucose phosphorylation, and a glucose
metabolite indirectly induces a transcriptional response via glucose
response elements that bind transcription factors of the basic
helix-loop-helix (bHLH) family (reviewed in Ref. 17). In addition,
insulin regulates the transcription of several genes via a
glucose-independent pathway that targets an insulin response element
(IRE). Two pathways that transduce the insulin signal from the IR to
the IREs have been identified. The genes coding for PEPCK (18, 19) and
for gene 33 (20) are controlled via a phosphatidylinositol-3-kinase
(PI3K)-dependent pathway. The genes coding for c-fos (21, 22) and for CAAT/enhancer binding protein (C/EBP)
(23) are
controlled via a Ras-Raf-mitogen-activated protein kinase (MAPK)
pathway. The transcriptional stimulation of the c-fos gene
by insulin also depends on protein kinase C isoforms and on a
cross-talk between the Ras and PI3K cascades (24, 25).
Three mechanisms can explain inhibition of transcription by insulin: 1)
displacement of a DNA-bound activator by an insulin-regulated
transcription factor, 2) active insulin-induced repression by a
transcription factor bound to DNA, or 3) inhibition of transcriptional
activity via insulin-induced posttranslational modifications or
protein-protein interactions involving DNA-bound transcriptional
activators, cofactors, or basal transcription factors. The first two,
but not the third, of these mechanisms require the presence of an IRE
in a regulatory region of the target gene. The PEPCK, TAT, and
insulin-like growth factor binding protein-1 (IGFBP-1) genes
contain an IRE that mediates an inhibitory effect of insulin, but the
transcription factor involved has not been identified (4, 5, 26, 27, 28).
In the work reported here we have investigated how glucocorticoids
stimulate transcription of the PFK-2 gene and how insulin inhibits this
effect. We have shown earlier that insulin controls the PFK-2 gene via
a glucose-independent pathway (10). We demonstrate here that both
hormones act via the GRU which, in addition to the GR, binds ubiquitous
and liver-enriched transcription factors. We also show that these
transcription factors modulate the glucocorticoid response, but do not
seem to be individually involved in insulin action. We present evidence
that insulin targets the GR in its ligand- binding domain (LBD) and
that this requires neither the PI3K nor the MAPK pathway.
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RESULTS
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The GRU of the PFK-2 Gene Mediates the Action of Glucocorticoids
and of Insulin
To determine how insulin inhibits the stimulation of the PFK-2
gene by glucocorticoids, we first looked for an IRE in the regulatory
regions of that gene. We stably transfected rat hepatoma FTO-2B cells
with a luciferase reporter construct driven by the PFK-2 gene promoter
alone or linked to the intronic PFK-2 GRU (12). These cells were
treated for 24 h with dexamethasone (1 µM) and/or
insulin (10 nM) and assayed for luciferase activity. No
hormonal response was observed with a construct (pPLLuc138) containing
the PFK-2 promoter alone. In contrast, addition of the
PFK-2 GRU conferred to the construct (pPLLuc138GRU) the stimulation by
dexamethasone and the inhibition of this effect by insulin, without
effect of insulin on basal activity (Fig. 1A
). Thus, the GRU mediated
glucocorticoid and insulin effects similar to those observed on the
endogenous PFK-2 gene. To further explore this phenomenon, we turned to
transient transfection. We chose FTO-2B cells and a nonhepatic cell
line (CHO-IR) that overexpresses the insulin receptor. Since CHO-IR
cells lack endogenous GR, they were cotransfected with a GR expression
vector. Figure 1B
shows that these transiently transfected cells
displayed hormonal responses that were qualitatively similar to those
of stably transfected cells. The effect of insulin was stronger in
transiently transfected CHO-IR than FTO-2B cells, probably because of a
higher IR concentration in CHO-IR cells. The effect of dexamethasone
was greater in transiently than in stably transfected cells, probably
because our nonclonal population of stable transfectants was
heterogenous.

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Figure 1. The GRU of the PFK-2 Gene Mediates the Inhibitory
Effect of Insulin on Induction by Dexamethasone
Relative luciferase activity of stably (A) or transiently (BD)
transfected FTO-2B or CHO-IR cells treated for 24 h with 0.01%
ethanol as a control, 1 µM dexamethasone, 10
nM insulin, or 1 µM dexamethasone in the
presence of 10 nM insulin. E, Structure of the transfected
plasmids. Results are means (± SEM) for at least three
independent experiments for each construct. The inhibitory effect of
insulin on the dexamethasone stimulation was statistically significant
for all the GRU-containing constructs (P <
0.05).
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Having reproduced in two types of transfected cells the hormonal
effects studied here, we tested the specificity of the inhibitory
action of insulin. To do so, we transiently transfected CHO-IR cells
with a luciferase construct driven by a well-known GRE-containing and
glucocorticoid-responsive promoter, namely the mouse mammary tumor
virus (MMTV) promoter (pMMTVLuc). As expected, dexamethasone
treatment stimulated transcription. However, insulin treatment did not
inhibit the glucocorticoid effect (Fig. 1C
). Perhaps the response to
insulin depended on the PFK-2 promoter and/or the distance between the
GRU and the promoter. The PFK-2 GRU was therefore cloned upstream of a
shorter PFK-2 promoter (-36 to +86, pPLLuc36GRU), or the thymidine
kinase (-38 to +51, pTKLuc38GRU), or
-fetoprotein (-80 to +38,
p
FPLuc80GRU) promoters. These constructs were cotransfected with the
GR expression vector in CHO-IR cells, and the cells were treated with
hormones as indicated in Fig. 1D
. The PFK-2 GRU conferred the
glucocorticoid stimulation and the insulin inhibition not only to the
shorter homologous promoter, but also to the heterologous promoters. We
concluded that the glucocorticoid and insulin effects on the PFK-2 gene
are mediated solely by the GRU, irrespective of the promoter and of the
distance.
Transcription Factor Binding to the PFK-2 GRU
To identify the transcription factors that bind to the PFK-2 GRU,
we analyzed this region by in vitro deoxyribonuclease I
(DNase I) footprinting with rat liver nuclear extracts. In addition to
the previously reported binding of the GR, NF-I, and HNF-3 in the
region between coordinates 1 and 70 (Ref. 14 ; Fig. 2
, upper panel) a footprint
[nucleotides (nt) 117151] containing three hyperreactive sites was
detected (Fig. 2
, upper panel, lane 3). These three
hyperreactive sites were localized on sequences that match the HNF-3
consensus (A/GCAAAT/CA)
and were typical of the binding of this factor (29). We therefore
performed the DNase I footprinting experiment on the GRU in the
presence of competing amounts of a HNF-3-binding oligonucleotide. As
shown in Fig. 2
(upper panel, lane 4), this
oligonucleotide reduced the intensity of the hyperreactive
bands to that seen with naked DNA (upper panel, lane 2).
This oligonucleotide also restored the pattern observed without nuclear
extracts in the 5'-half of the footprint, although no HNF-3 consensus
was found in this region. The latter region contains, on the antisense
strand, a AAATCA/CATAA consensus-binding site
for HNF-6 (30 30A ). Moreover, the competing oligonucleotide
used was known to bind not only HNF-3, but also HNF-6 (31). We
therefore used, instead, a competing oligonucleotide known to bind
HNF-6 but not HNF-3 (Fig. 2
, upper panel, lane 5). This
prevented the appearance of the 5'-half of the footprint without
preventing the three HNF-3-induced hyperreactive sites in the 3'-half
of the footprint. Electrophoretic mobility shift assays (EMSAs),
performed on the 117151 footprint region in the presence of
antibodies against HNF-6 or HNF-3 (data not shown), confirmed that
HNF-6 and HNF-3 bind to the GRU as indicated in Fig. 2
(upper
panel). A region partially protected from DNase I by liver nuclear
extracts was detected between nt 90 and 110 (Fig. 2
, upper
panel). When this region was used as a probe in EMSA with liver
nuclear extracts, several complexes were observed (Fig. 2
, lower
panel, lane 1). These were specific as their appearance was
prevented by an excess of cold probe (Fig. 2
, lower panel,
lane 2). Addition of a competing oligonucleotide known to bind C/EBP
(Fig. 2
, lower panel, lane 4) also inhibited complex
formation whereas an excess of unrelated oligonucleotide upstream
stimulatory factor (USF) (Fig. 2
, lower panel, lane
3) did not. We conclude from this experiment that the partial footprint
detected between nt 90 and 110 is due to binding of C/EBP-related
proteins. A schematic representation of the DNA-protein interactions
observed in the GRU is shown in Fig. 2
(upper panel).

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Figure 2. The PFK-2 GRU Binds Several Transcription Factors
in Vitro
Upper panel, DNase I footprinting experiments with liver
nuclear extracts on a GRU-containing radioactive probe. The probe
consisted of an EcoRI-ClaI fragment from
pBS.GRU labeled with [ -32P]dCTP and the Klenow enzyme
at the ClaI site on the sense strand. The competitor
oligonucleotides (oligo) used are
indicated above the lanes. G+A indicates a chemical sequencing of the probe. Bars correspond to protections and arrows to
hyperreactive sites. The dashed bar and the open arrow refer to weak protection and hyperreaction, respectively. The coordinates
correspond to an arbitrary numbering in the GRU (14 ). A schematic representation of the GRU-protein interactions is shown on the
right. The GR binding site indicated could not be seen in DNase I footprinting with crude liver nuclear extracts, but was detected previously with purified
GR (14 ). Lower panel, EMSA with a PFK-2 GRU probe (GRU 90/110) and liver nuclear extracts. Addition of competing unlabeled
oligonucleotides is indicated above the lanes.
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The GRE of the GRU Does Not Function as an IRE
To understand how insulin inhibits the glucocorticoid stimulation
of the PFK-2 gene transcription, we tested the possibility that the GRE
of the gene is a composite GRE/IRE, i.e. a binding site for
the GR and for an insulin-regulated transcription factor that
attenuates the glucocorticoid response. To test this, we replaced the
PFK-2 GRE sequence by a GRE derived from an insulin-insensitive
promoter. If the hypothesis were correct, this swap should abolish the
insulin effect. We chose the MMTV GRE, as we demonstrated above (Fig. 1C
) that it is insulin insensitive. The construct (pPLLuc138GRU M)
containing a GRU modified in this way (mGRU M, Fig. 3A
) was stimulated by dexamethasone, as
expected. However, it was inhibited by insulin like the wild-type GRU
(Fig. 3B
). Since the MMTV GRE became sensitive to insulin when placed
in the context of the PFK-2 GRU, we concluded that the PFK-2 GRE is not
a composite GRE/IRE.

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Figure 3. The GRE of the PFK-2 GRU Does Not Function as an
IRE
A, Schematic representation of the wild-type (GRU wt) and mutant (mGRU)
GRU, and sequences of the GAL4 site (G) and of the GRE in the PFK-2 GRU
(wt) and in the MMTV promoter (M). Arrows indicate the
imperfect palindromic half-sites of the GRE. These GRUs were cloned
upstream of the PFK-2 promoter (-138 to +86) driving the luciferase
reporter gene. B, Relative luciferase activity of transiently
transfected mGRU M and mGRU G in FTO-2B and CHO-IR cells. Results are
means (±SEM) for at least three independent experiments
for each construct. The inhibitory effect of insulin on the
dexamethasone stimulation was statistically significant
(P < 0.05).
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Another way to check whether the GRE is the IRE was to replace it
by an unrelevant sequence that can still bind the GR. We therefore
replaced the PFK-2 GRE by a sequence known to bind the yeast GAL4
activator and cotransfected this construct (pPLLuc138GRU G, Fig. 3A
)
with an expression vector coding for a GAL4/GR fusion protein in which
the DNA-binding domain (DBD) of the GR was replaced by the GAL4 DBD
(32). As expected, this fusion protein activated the pPLLuc138GRU G
reporter construct only in the presence of dexamethasone (Fig. 3B
). The
glucocorticoid induction of this mutated GRU was also inhibited by
insulin. This experiment not only confirmed that the GRE does not
function as an IRE, it also showed that the GR DBD is dispensable for
this action of insulin.
The Binding Sites for NF-I, HNF-3, C/EBP, and HNF-6 in the GRU
Function as Modulators of Glucocorticoid Action but not as IREs
Another mechanism by which insulin could inhibit
glucocorticoid-stimulated gene transcription is by targeting one of the
transcription factors shown above to bind in the vicinity of the GRE.
FTO-2B cells were transiently transfected with constructs containing
mutations (Fig. 4A
) that prevented these
transcription factors from binding to the GRU. The efficiency of the
mutations was controlled by in vitro DNase I footprinting
experiments (data not shown). After hormonal treatment, luciferase
activity was measured in the transfected cells. The results are shown
in Fig. 4B
. These experiments showed that HNF-3 family members have
different functions depending on their location in the GRU. As shown
earlier (14), the absence of HNF-3 binding close to the GRE reduced the
glucocorticoid stimulation (Fig. 4B
, panel e) when compared with the
wild-type GRU (Fig. 4B
, panel a). In contrast, mutation of the three
HNF-3 binding sites at the 3'-end of the GRU (Fig. 4B
, panel b) had no
effect on the hormonal response. By binding to these three sites, HNF-3
could play a structural role (33) and maintain the GRU in the
constitutively open state described previously (14). When the HNF-6
site was mutated, we observed an increase in glucocorticoid stimulation
(Fig. 4B
, panel c). This phenomenon will be discussed elsewhere (C. E.
Pierreux, J. Stafford, D. Demonte, D. K. Scott, J. Vandenhaute, R. M.
OBrien, D. K. Granner, G. G. Rousseau, and F. P. Lemaigre, in
preparation). Mutations of the C/EBP (Fig. 4B
, panel d) and NF-I (Fig. 4B
, panel f) binding sites resulted in a reduced glucocorticoid
response, indicating that these proteins were acting as DNA-bound
cofactors of the GR. Even though the insulin effect was small in the
transiently transfected FTO-2B cells, as discussed above, it was
observed with all the mutated constructs (Fig. 4B
, panels bf). To
confirm this, we transfected the same constructs in CHO-IR cells and
tested their hormonal response. As expected, the effect of insulin in
these cells was more clearly demonstrated than in FTO-2B cells (Fig. 4C
). Insulin inhibited to the same extent the glucocorticoid
stimulation of all the mutated GRUs (Fig. 4C
, panels bf) and of the
wild-type GRU (Fig. 4C
, panel a). These results demonstrated that the
binding sites for NF-I, HNF-3, C/EBP, or HNF-6 do not act as IREs.

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Figure 4. Effects of the Transcription Factors That Bind to
the GRU
A, Schematic representation of the GRU mutants (mGRU) compared with
wild-type GRU (GRU wt). Binding sites are represented by open
rectangles or ovals and mutated sites are indicated by
black filling. These GRUs were cloned upstream of the
PFK-2 promoter (-138 to +86) driving the luciferase reporter gene. B,
Relative luciferase activity in FTO-2B cells transiently transfected
with the constructs described in panel A. The inhibitory effect of
insulin on the dexamethasone stimulation was statistically significant
for all the GRU mutants (P < 0.05, paired
t test). C, Percentage of inhibition by insulin of the
glucocorticoid stimulation of the reporter constructs (described in
panel A) transiently cotransfected in CHO-IR cells with the GR
expression vector. The results are means (±SEM) for at
least three independent experiments for each construct. No statistical
difference was observed when comparing the various GRU mutants (bf)
to the wild-type GRU (a).
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The Ligand-Binding Domain (LBD) of the GR Is Required for the
Insulin Effect
In the absence of a detectable IRE in the GRU, we hypothesized
that the GR or a GR-bound cofactor might be targeted by insulin. We
therefore transfected CHO-IR cells with the pPLLuc138GRU reporter
construct and vectors expressing GR deletion mutants (Fig. 5
). The reporter activity was measured
after treatment with hormones. Consistent with data from Müller
et al. (32), the stimulatory activity of the GR varied among
mutants (see below). The effect of insulin was therefore expressed as
the percentage of inhibition of the GR-induced stimulation. Again
consistent with the data from Müller et al. (32),
GR1-550, GR1-515, and GR1-488,
which are devoid of LBD, were constitutive activators of transcription,
while the activity of GR
77-262 and
GR418-777 was glucocorticoid dependent. In the presence of
wild-type GR (27-fold induction of transcription) and of
amino-terminally deleted GR (GR
77-262 and
GR418-777, which respectively stimulated transcription 9-
and 8-fold in response to dexamethasone), insulin inhibited the
glucocorticoid stimulation by 60%. In contrast, inhibition by insulin
was impaired or absent with GR devoid of LBD (GR1-550,
GR1-515, and GR1-488) despite the fact that
these GR stimulated reporter activity 16-, 13- and 5-fold,
respectively. We concluded from these experiments that the LBD of the
GR is required for insulin to inhibit glucocorticoid stimulation and
that the GR, or a GR-bound cofactor, is the target of insulin
action.

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Figure 5. The GR LBD Is Required for Insulin Inhibition
A, Schematic representation of the wild-type and deletion mutants of
the human glucocorticoid receptor (hGR) shown with its transactivation
domains ( 1 and 2), DBD, and LBD. Deletion start and end points
are indicated. B, Percentage of inhibition by insulin of the
glucocorticoid stimulation of the pPLLuc138GRU reporter construct
transiently cotransfected in CHO-IR cells with the various GR
expression vectors. The results are means (±SEM) for at
least three independent experiments with each construct. The effect of
insulin was significant (P < 0.01) with constructs
a, e, and f, but not with constructs b, c, and d.
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Insulin Signaling to the GRU Is Transduced via a PI3K- and
MAPK-Independent Pathway
As mentioned in the Introduction, regulation of gene
transcription by insulin reportedly involves a PI3K- and/or
MAPK-dependent pathway. To verify whether this applied to the action of
insulin on the PFK-2 GRU, we measured expression of the endogenous
PFK-2 gene in FTO-2B cells incubated with hormones and inhibitors of
the PI3K and MAPK pathways. The cells were incubated for 24 h in
the presence of dexamethasone (1 µM) before addition of
the PI3K inhibitors, wortmannin (100 nM or 1
µM, see Ref. 34) or LY294002 (50 µM, see
Ref. 35). Fifteen minutes later, insulin (10 nM) was added
to the medium containing dexamethasone and the PI3K inhibitor, and the
cells were further incubated for 4 h. Wortmannin becomes less
effective after 3 h (36). We therefore used two different
wortmannin concentrations (100 nM and 1 µM),
as a 4-h incubation is required to monitor changes in PFK-2 mRNA
levels. Cytoplasmic RNA was extracted, and variations of PFK-2 mRNA
concentration were assessed by RT-PCR. As expected (Fig. 6
), dexamethasone increased the PFK-2
mRNA content and insulin inhibited this increase. Neither wortmannin
(Fig. 6A
) nor LY294002 (Fig. 6B
) prevented the insulin effect and
neither drug affected the glucocorticoid response (Fig. 6
, A and B). To
verify that these inhibitors did inhibit the PI3K-dependent cascade in
FTO-2B cells, we measured the activity of pp70S6 kinase, whose
activation requires PI3K (37), rather than the activity of PI3K itself.
Indeed, as LY294002 is not stably bound to PI3K, it may be partially
washed off the enzyme during the immunoprecipitation step, and this may
lead to unduly high PI3K activity (38). FTO-2B cells were incubated
with wortmannin (1 µM) or LY294002 (50 µM)
15 min before addition of insulin (10 nM). Activation of
pp70S6 kinase was measured after 12 min, which corresponds to the time
required for maximal pp70S6 kinase stimulation by insulin under our
conditions. The results (Fig. 6C
) showed that wortmannin and LY294002
were effective in FTO-2B cells as both inhibited the insulin-induced
stimulation of pp70S6 kinase activity.

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Figure 6. Neither the PI3K nor the MAPK Pathway Is Involved
in Insulin Signaling to the PFK-2 Gene
A and B, PI3K is not involved in the insulin-induced inhibition of
PFK-2 gene transcription. FTO-2B cells were incubated for 24 h in
the presence of 1 µM dexamethasone (dex) or with 0.01%
ethanol as a control (ctrl), before addition of 100 nM (d)
or 1 µM (e and f) wortmannin (wt; panel A) or of 50
µM LY294002 (LY; panel B). Insulin (10 nM)
was added 15 min later, and incubation was prolonged for 4 h.
Variations of PFK-2 liver mRNA concentration in these FTO-2B cells were
assessed by RT-PCR and are shown (vertical
columns) as the ratio of coamplified liver mRNA (L) and
internal standard RNA (st). This ratio was measured by the
radioactivity incorporated in the amplified products that were
visualized by ethidium bromide staining, as shown at the top of
the graphs. C, The stimulation of pp70S6 kinase by insulin is
blocked by wortmannin and by LY294002. Immunoprecipitated pp70S6 kinase
activity was assayed from FTO-2B cells treated 10 min with the PI3K
inhibitors (1 µM wt and 50 µM LY) before a
12-min treatment with insulin (10 nM). D, MEK and MAPK are
not involved in the insulin-induced inhibition of PFK-2 gene
transcription. Treatment of FTO-2B cells and assay of PFK-2 liver mRNA
concentration were performed as in panels A and B. The MEK inhibitor
PD098059 was used at 30 µM. E, MAPK activation by insulin
is blocked by PD098059. Immunoprecipitated MAPK activity was assayed
from FTO-2B cells treated 15 min with 30 µM PD098059
before an 8-min challenge with 10 nM insulin. The data are
representative of at least two experiments.
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To determine whether the MAPK cascade is involved in transducing the
insulin signal to the PFK-2 gene, we used PD098059, an inhibitor of
MEK, the kinase that activates MAPK (39, 40). FTO-2B cells were
incubated for 15 min with PD098059 (30 µM) before
addition of insulin (10 nM). Cytoplasmic RNA was extracted
after 4 h, and the relative concentration of PFK-2 mRNA was
determined by RT-PCR. As shown in Fig. 6D
, the MEK inhibitor did not
influence the insulin-induced decrease in PFK-2 mRNA. As a control for
PD098059 efficiency in FTO-2B cells, we measured MAPK activation by
insulin in the presence or absence of PD098059. The data shown in Fig. 6E
demonstrate that PD098059 decreased basal MAPK activity, as assessed
on MBP phosphorylation, and blocked insulin-stimulated MAPK
activity.
These results demonstrated that the insulin regulation of the PFK-2 GRU
is independent from the PI3K and MAPK pathways. These conclusions were
confirmed by testing the effect of wortmannin, LY294002, and PD098059
on luciferase activity in FTO-2B cells transiently transfected with
pPLLuc138GRU and treated with dexamethasone and insulin (data not
shown).
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DISCUSSION
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We have reported here a mode of action of insulin on gene
expression not described so far. Transcription of the gene coding for
liver PFK-2 is stimulated by glucocorticoids, and this stimulation is
inhibited by insulin (10). The present work shows that these hormonal
effects are both mediated by the GRU located in the first intron of the
gene and that this GRU binds not only the GR, but also other
transcription factors. All of them influenced the glucocorticoid
stimulation of the PFK-2 gene. The work also shows that the inhibitory
effect of insulin occurs via a PI3K- and MAPK-independent pathway that
targets the LBD of the GR.
The regulation of gene transcription by insulin has been studied for
several genes in various model systems (reviewed in Ref. 41). The PFK-2
gene regulation by insulin resembles that of the glucose-6-phosphatase,
PEPCK, TAT, aspartate aminotransferase, and IGFBP-1 genes. Indeed,
these genes are all expressed in the liver, and their transcription is
stimulated by glucocorticoids and inhibited by insulin. The
glucocorticoid stimulation of these genes involves a cooperation of the
GR with liver-enriched (C/EBP, HNF-3) and ubiquitous (NF-I)
transcription factors (27, 42). In this respect, the PFK-2 GRU was
regulated by the same transcription factors. The cooperation between
HNF-3 and the GR could involve the same mechanism in the PFK-2 GRU and
in the TAT GRU. Indeed, in both cases HNF-3 binding in vivo
is glucocorticoid-dependent (14, 43).
In contrast to these similarities in transcriptional stimulation by
glucocorticoids, the mechanism by which insulin inhibited this effect
on the PFK-2 gene differed from that described for the PEPCK, TAT, and
IGFBP-1 genes. In the latter, the insulin-induced inhibition of the
glucocorticoid stimulation is mediated by an IRE that corresponds to a
binding site for liver-enriched transcription factors (5, 27). In the
PFK-2 gene, our data show that the target of insulin action is the GR.
Moreover, deletion analysis of the GR demonstrated that the GR LBD is
required for the insulin-induced inhibition of glucocorticoid
stimulation. We therefore postulate that insulin inhibits GR activity
by directly or indirectly targeting the GR LBD. Phosphorylation or
dephosphorylation of the GR LBD could be a mechanism for insulin
action. Several reports indicate that the phosphorylation status of the
GR can influence its function (44, 45). However, the phosphorylation
sites identified so far in the GR are localized in its amino-terminal
part, i.e. not in the LBD. We can also rule out that insulin
treatment prevents binding of the steroid to the LBD or retains the GR
in the cytoplasm since insulin did not prevent dexamethasone from
stimulating the MMTV promoter. Recent data on steroid receptor function
show that these receptors enhance transcription by recruiting an array
of coactivator proteins to the transcription complex (11). Some of
these non-DNA-bound coactivators interact with the GR in a
ligand-dependent way via the LBD (46). Insulin could thus inhibit
glucocorticoid-induced gene transcription by interfering with
GR-coactivator interactions. Our model would be in line with that
proposed by Nakajima et al. (47) to explain how insulin
represses cAMP-responsive genes. These authors demonstrated that
repression by insulin occurs through promotion of an interaction
between pp90RSK and the coactivator CBP. The binding of
pp90RSK to CBP may, in some contexts, interfere with CBP
activity.
Another contribution of the present work was to demonstrate that the
peculiar mode of insulin action defined here hinges on the integrity of
the GRU of the PFK-2 gene. While the glucocorticoid-stimulated MMTV
promoter was insulin-insensitive, the PFK-2 GRU could confer insulin
sensitivity to this promoter and to other heterologous promoters,
irrespective of the distance. We tested the possibility that this
specificity depends on the sequence of the GRE. Indeed, the GR can
adopt different conformations and modes of DNA binding, depending on
the nucleotide sequence of the GRE (48). This suggested that only a
particular GRE-induced conformation of the GR could be
insulin-sensitive. Our results eliminated this possibility, since the
insulin-insensitive MMTV-GRE became insulin sensitive in the context of
the PFK-2 GRU. Moreover, replacing the PFK-2 GRE by a GAL4-binding site
did not prevent insulin from inhibiting glucocorticoid-induced
activity of a GAL4/GR fusion protein. We tested a second model, in
which the specificity of insulin action relies on a GRU context created
by the GR and other factors that bind to the GRU. However, the effect
of insulin was not affected by the individual destruction of the
binding sites for these transcription factors, ruling out that a single
binding site determines the specificity of the insulin effect. The
present results are in line with our in vivo methylation
protection and DNase I footprinting experiments. Addition of insulin
did not modify the in vivo binding pattern seen on the GRU
in dexamethasone-stimulated cells (data not shown). We therefore favor
a mechanism whereby a combination of transcription factors creates an
insulin-responsive unit (IRU) that, for example, recruits specific GR
coactivators on which insulin can act to inhibit the glucocorticoid
stimulation. To confirm this model, we studied (data not shown) the
hormonal response of transfected constructs in which more than one
binding site of the GRU had been destroyed. However, these combinations
of mutations decreased glucocorticoid stimulation too much to allow the
study of insulin inhibition. When insulin inhibits the transcriptional
stimulation of the PEPCK, TAT, and IGFBP-1 genes by glucocorticoids, it
could well do so by targeting the LBD of the GR as described here for
the PFK-2 gene. If so, the sensitivity of these other genes to insulin
could rely on their particular IRE rather than on an IRU, as postulated
for the PFK-2 gene.
To investigate to what extent the insulin regulation of the PFK-2 gene
diverged from that described for other genes, we also studied the
signaling pathway that links the insulin receptor to the PFK-2 GRU. We
ruled out a role of the PI3K pathway or the MAPK pathway. It is
noteworthy that transcription of the PEPCK gene is inhibited by insulin
via PI3K (18, 19). Recent data demonstrated that Stat (signal
transducers and activators of transcription) proteins can mediate
insulin action. Stat 5 interacts with the insulin receptor (49), and it
potentiates glucocorticoid action through binding to the GR (50). Stat
3 mediates insulin signaling to a STAT-response element via a PI3K- and
MAPK-independent pathway (51). We found in the PFK-2 GRU a sequence
(108-TTCTCTGAA-117) that resembles the STAT-binding site consensus.
However, this site was not functional (data not shown). Furthermore,
overexpression of wild-type or dominant negative Stat 5 did not affect
insulin regulation of the PFK-2 gene (data not shown). This ruled out
an involvement of Stat 3 or Stat 5 binding in the insulin control of
PFK-2 gene expression.
In conclusion, our data provide a new mechanism for the hormonal
regulation of gene transcription whereby insulin inhibits
glucocorticoid action by targeting the GR LBD. The identification of
the protein that interacts with the GR LBD should clarify how insulin
exerts its negative effect on the stimulation of gene transcription by
glucocorticoids.
 |
MATERIALS AND METHODS
|
---|
Plasmids
pPLLuc138, which contains the luciferase gene under the control
of the PFK-2 liver promoter (-138 to +86), has been described (52).
pPLLuc138GRU (14) contains a RsaI-RsaI fragment
of the PFK-2 GRU (12) cloned upstream of the PFK-2 liver promoter
(-138 to +86). The RsaI-RsaI fragment of the
PFK-2 GRU was also cloned upstream of a shorter PFK-2 promoter (-36 to
+86, pPLLuc36GRU) or of the thymidine kinase (-38 to +51, pTKLuc38GRU)
or
-fetoprotein (-80 to +38, p
FPLuc80GRU) promoters. All the GRU
mutants were obtained using PCR-directed mutagenesis and were identical
to the wild-type GRU except that in pPLLuc138GRU H the most 5'-HNF-3
binding site of the GRU has been destroyed (14); in pPLLuc138GRU 3H the
three HNF-3 sites (5'-TTGTTTGTTTGTTTGTT-3') were replaced by a
GAL4-binding site (5'-CGGAGTACTGTCCTCCG-3'); in pPLLuc138GRU H6 the
HNF-6-binding site (5'-AAATCCATA-3') was destroyed (5'-AAAGTGCTA-3');
in pPLLuc138GRU C the C/EBP binding site (5'-GTTACAGTTT-3') was
destroyed (5'-TACGTCTAGA-3'); in pPLLuc138GRU N the NF-I binding site
(5'-TGGCAGAACTTTCA-3') was destroyed (5'-TTCTAGAACTTTGC-3'); in
pPLLuc138GRU M and in pPLLuc138GRU G, the PFK-2 GRE was replaced by the
MMTV GRE and by a GAL4 binding site, respectively. pMMTVLuc corresponds
to the pMamNEOLuc reporter vector from CLONTECH (Palo Alto, CA). The
renilla luciferase internal control (pRL138) was constructed by cloning
the PFK-2 promoter (-138 to +86) in the pRLnull vector from Promega
(Madison, WI).
Cell Culture and Transfection
Rat hepatoma FTO-2B cells and chinese hamster ovary cells stably
transfected with the insulin receptor (CHO-IR) were grown as monolayers
in a humidified atmosphere (5% CO2/95% air) in a 1:1
mixture of DMEM and Hams F-12 medium (GIBCO-BRL, Gaithersburg, MD)
supplemented with 10% FCS. Stable FTO-2B transfectants were obtained
by the calcium phosphate coprecipitation method as previously described
(26). For transient transfection, 6 x 105 FTO-2B
cells were plated on a 60-mm dish, and the medium was replaced 24
h later by 3 ml of a solution containing 2.4 ml DMEM devoid of serum
and of antibiotic, 600 µl OPTIMEM containing 10 µg luciferase
reporter plasmid, and 45 µg lipofectin. CHO-IR cells (2 x
105) were plated on a 30-mm dish and transiently
cotransfected 24 h later with a solution containing 0.8 ml DMEM
devoid of serum and of antibiotic, 200 µl OPTIMEM containing 2 µg
luciferase reporter plasmid, 50 ng wild-type GR expression vector
(pRShGR
) or deletion mutants (GR1-550,
GR1-515, GR1-488, GR
77-262,
and GR418-777), 50 ng renilla luciferase internal control
(pRL138), and 10 µg lipofectin. After 16 h, the cells were
washed, incubated in DMEM/Hams F-12 medium plus 0.1% BSA, and
treated with 0.01% ethanol, 1 µM dexamethasone, 10
nM insulin, or 1 µM dexamethasone in the
presence of 10 nM insulin. Twenty four hours after addition
of the hormones, luciferase reporter activities were measured with a
Lumac luminometer and normalized for protein concentration in the
FTO-2B cell extracts or for renilla luciferase (dual-luciferase
reporter assay system, Promega) activity in CHO-IR cell extracts. In
the latter, luciferase activity could not be normalized for protein
concentration because of an increase in protein concentration after
treatment with insulin.
In Vitro DNase I Footprinting and EMSA
DNase I digestions and EMSAs were performed as described (52)
using nuclear extracts from normal rats prepared as described by
Hattori et al. (53). The probe used in DNase I footprinting
was isolated from pBS.GRU as described (14). The double-stranded
oligonucleotide GRU 90/110 used as a probe in EMSA was:
5'-AGCTTAACTGTTACAGTTTCTCTGAAAGA-3'. Double-stranded competing
oligonucleotides were: C/EBP, 5'-TTAAGGACTAACGGGTTAACTTAACTAG-3';
HNF-3, 5'-GTTGACTAAGTCAATAATCAGA-3'; HNF-6,
5'-GATCGCTTTGAAATTGATTTCAAAGC-3'; NF-I,
5'-TCGA-ACCATGGCCTGCGGCCAGAGGGC-3'; and USF,
5'-GTAG-GCCACGTGACCGGG-3'.
Enzymatic Activities
For measurement of pp70S6 kinase activity, FTO-2B cells were
grown in serum-free DMEM/Hams F12 medium containing 0.1% BSA and,
after 24 h, incubated for 10 min without or with 1
µM wortmannin (Sigma Chemical Co., St. Louis, MO) or 50
µM LY294002 (BIOMOL Research Laboratories, Plymouth
Meeting, PA) before a 12-min treatment with 10 nM
insulin. All the subsequent manipulations were conducted at 4 C unless
otherwise stated. Cells were washed twice with PBS and lysed in a
solution containing 50 mM Tris-Cl, pH 7.5, 100
mM KCl, 1 mM EGTA, 1 mM EDTA, 1%
NP-40, 270 mM sucrose, 1 mM
Na3VO4, 10 mM NaF, 10
mM sodium pyrophosphate, 15 mM sodium
ß-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride,
and 1 µg/µl aprotinin and leupeptin. The cell lysates were
centrifuged at 10,000 x g for 10 min, and proteins (1
mg) were immunoprecipitated with anti-pp70S6 kinase antibody (Santa
Cruz Biotechnology, Santa Cruz, CA) and protein A-Sepharose for 2
h. Immunoprecipitates were washed in buffer A (50 mM
Tris-acetate, pH 8, 50 mM NaF, 5 mM sodium
pyrophosphate, 1 mM EGTA, 1 mM EDTA), and the
pp70S6 kinase activity was assayed as described by Krause et
al. (54). For measurement of MAPK activity, the FTO-2B cells were
grown in serum-free DMEM/Hams F12 medium containing 0.1% BSA and,
after 24 h, incubated for 15 min without or with 30
µM PD098059 (BIOMOL). Insulin (10 nM) was
then added for 8 min and cells were washed and lysed as for the pp70S6
kinase assay. Proteins (200 µg) were immunoprecipitated with
anti-ERK-2 antibody (Santa Cruz Biotechnology) and protein A-Sepharose
for 2 h. Immunoprecipitates were washed in buffer A and MAPK
activity was assayed in the same buffer supplemented with 1
µM cAMP-dependent protein kinase inhibitor peptide, 0.5
mg/ml myelin basic protein, and 50 µM
32P-ATP. After 20 min at 30 C, the incubation was loaded
on a 15% SDS-polyacrylamide gel. The dried gel was quantified with an
Instant Imager (Packard Instruments, Meriden, CT).
Relative Concentration of PFK-2 mRNA
This was measured by RT-PCR as described (10). For each
reaction, 500 ng FTO-2B cytoplasmic RNA were added to 0.04 pg of
in vitro synthesized internal standard RNA. The RT was
performed with random hexameric primers. The internal standard RNA and
the PFK-2 liver mRNA were coamplified by PCR using two primers that
recognize both RNAs and yield products of 210 and 274 bp, respectively.
The ratio of the two products reflects the relative concentration of
PFK-2 liver mRNA in FTO-2B cells. [
32P]CTP was added
in the PCR reaction to determine the ratio of the two products by
measuring the radioactivity of gel slices in a scintillation counter or
with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
 |
ACKNOWLEDGMENTS
|
---|
The technical help of N. Aidant and S. Neou is gratefully
acknowledged. We thank G. Verhoeven for pRShGR
, M. Müller for
GR mutants, B. Groner and E. Stöcklin for Stat 5 expression
vectors, C. Szpirer for the
-fetoprotein promoter, and G.
Schütz and E. Clauser for FTO-2B and CHO-IR cells,
respectively.
 |
FOOTNOTES
|
---|
Address requests for reprints to: F. P. Lemaigre, HORM Unit, Box 7529, 75 Avenue Hippocrate, B-1200 Brussels, Belgium. e-mail: lemaigre@horm.ucl.ac.be.
This work was supported by grants from the Belgian State Program on
Interuniversity Poles of Attraction, Prime Ministers Office, Federal
Office for Scientific, Technical and Cultural Affairs; from the
Délégation Générale Higher Education and
Scientific Research, French Community of Belgium; from the Fund for
Scientific Medical Research (Belgium); from the National Fund for
Scientific Research (Belgium); and from the Fonds de
Développement Scientifique (Louvain University). C.E.P. holds a
fellowship from the Fonds pour la Formation à la Recherche dans
lIndustrie et lAgriculture (Belgium), and F.P.L. is Research
Associate of the National Fund for Scientific Research (Belgium).
Received for publication February 12, 1998.
Revision received May 8, 1998.
Accepted for publication June 11, 1998.
 |
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