A Phorbol Ester-Insensitive AP-1 Motif Mediates the Stimulatory Effect of Insulin on Rat Malic Enzyme Gene Transcription
Ryan S. Streeper,
Stacey C. Chapman,
Julio E. Ayala,
Christina A. Svitek,
Joshua K. Goldman,
Alex Cave and
Richard M. OBrien
Department of Molecular Physiology and Biophysics Vanderbilt
University Medical School Nashville, Tennessee 37232
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ABSTRACT
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In liver, insulin stimulates the transcription of
the gene encoding the cytosolic form of malic enzyme (ME) and modulates
protein binding to two putative insulin response sequences (IRSs) in
the ME promoter. One of these IRSs resembles that identified in
the phosphoenolpyruvate carboxykinase (PEPCK) gene, whereas the other
resembles that defined in the glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) gene. To assess the functional significance of these changes in
protein binding, a series of truncated ME-chloramphenicol
acetyltransferase (CAT) fusion genes were transiently transfected into
rat H4IIE hepatoma cells. Deletion of the PEPCK-like IRS motif had no
effect on the stimulation of CAT expression by insulin. Instead, the
stimulatory effect of insulin was mediated through an AP-1 motif and an
Egr-1 binding site that overlaps the GAPDH-like IRS motif. Both the ME
AP-1 motif and the AP-1 motif identified in the collagenase-1 gene
promoter were able to confer a stimulatory effect of insulin on the
expression of a heterologous fusion gene, but surprisingly only the
latter was able to confer a stimulatory effect of phorbol esters.
Instead, the data suggest that AP-1 binds the ME AP-1 motif in an
activated state such that phorbol ester treatment has no additional
effect. The collagenase and ME AP-1 motifs were both shown to bind
mainly Jun D and Fra-2, with similar affinities. However, the results
of a proteolytic clipping bandshift assay suggest that these proteins
bind the collagenase and ME AP-1 motifs in distinct conformations,
which potentially explain the differences in phorbol ester signaling
through these elements.
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INTRODUCTION
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Malic enzyme (ME) catalyzes the oxidative decarboxylation of
malate to pyruvate (1). Three isoforms of ME are found in mammalian
tissues, each of which is encoded by a distinct gene: a cytosolic
NADP+-linked enzyme, a mitochondrial
NADP+-linked enzyme, and a mitochondrial enzyme that can
utilize either NAD+ or NADP+ (2, 3). All three
isoforms are widely expressed, although the relative amounts of each
varies considerably between different tissues (2, 3). The cytosolic
form of ME is most abundantly expressed in liver and adipose tissue
where it plays a central role, in conjunction with the pentose
phosphate pathway, in the generation of NADPH for fatty acid synthesis
(see Ref. 1 for review). The activity of this enzyme is not regulated
either allosterically or by posttranslational modification; however,
the amount of the protein is regulated by a number of factors (1). For
example, in rat liver, insulin stimulates the expression of the
cytosolic form of ME by increasing both the transcription of the gene
(4, 5) and stability of the mRNA (6).
cis-acting elements that mediate the action of insulin on
gene transcription, referred to as insulin response sequences or
elements (IRSs/IREs), have been identified in a number of genes but,
unlike cAMP, which regulates gene transcription predominantly through
one cis-acting element (7, 8), it is already apparent that a
single consensus IRS does not exist (9). Three consensus IRSs have
currently been identified. One of these has the sequence T(G/A)TTT(T/G)
(G/T) and mediates the repression of PEPCK, insulin-like growth factor
binding protein-1, tyrosine aminotransferase, apolipoprotein CIII, and
glucose-6-phosphatase gene transcription by insulin (10, 11, 12, 13, 14). The other
consensus IRSs are the serum response element (SRE; Refs. 9, 15) and
the Ets motif (16), which mediate stimulatory effects of insulin on
several genes. However, whereas the PEPCK-type IRS confers a selective
effect of insulin (9) the serum response element and Ets motif can
mediate the action of other hormones on gene transcription (see Refs.
17, 18 for review). Several additional IRSs have also been
identified, but to date these elements appear to be unique to
individual genes (9). Thus, this situation resembles that for phorbol
esters, which can regulate gene transcription through at least eight
distinct consensus sequences (19).
Data from a recent paper by Garcia-Jiménez et al. (5)
suggested that the gene encoding the cytosolic form of ME may represent
an exception to this complex paradigm. These investigators reported
that sequences similar to the negative IRS identified in the PEPCK gene
(10) and one of the two positive IRSs defined in the GAPDH gene,
designated IRE-A (20), are both present in the ME promoter (5).
Moreover, they demonstrated that nuclear extracts, isolated from the
livers of diabetic rats, exhibited both increased protein binding to
the PEPCK-like IRS motif and decreased binding to the GAPDH-like IRE-A
motif in the ME promoter (5). Insulin treatment of the diabetic rats
stimulated ME gene transcription and reversed these changes in protein
binding back to the level seen in control animals (5). These
observations raised the possibilities that 1) the negative PEPCK-like
IRS motif mediates a positive effect of insulin on ME gene
transcription and therefore represents a consensus IRS for both
positive and negative effects of insulin and 2) that the GAPDH-like
IRE-A motif represents a consensus IRE important for the
insulin-stimulated transcription of genes other than GAPDH. To
determine the functional significance of these observations, we have
analyzed the effect of insulin on the expression of a series of
truncated ME-CAT fusion genes. The results suggest that, in H4IIE
hepatoma cells, an AP-1 motif and an Egr-1-binding site are required
for the stimulatory effect of insulin on ME gene transcription.
However, the ME AP-1 motif is atypical in that, in contrast to the well
characterized AP-1 motif identified in the collagenase-1 promoter, it
fails to confer a stimulatory effect of phorbol esters on the
expression of a heterologous fusion gene. Instead, the data suggest
that AP-1 binds the ME AP-1 motif in an activated state such that
phorbol ester treatment has no additional effect. Based on the results
from a series of experiments, we propose that the selective phorbol
ester signaling through the collagenase and ME AP-1 motifs can be
potentially explained by the observation that these elements bind AP-1
in distinct conformations.
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RESULTS
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Insulin Stimulates ME-CAT Fusion Gene Transcription
To begin to study the regulation of ME gene transcription by
insulin, a ME-CAT fusion gene construct, containing ME promoter
sequence from -882 to +39, relative to the major transcription start
site at +1 (21), was transiently transfected into rat hepatoma H4IIE
cells (Fig. 1
). Insulin stimulated CAT
expression directed by this construct
3-fold under these conditions
(Fig. 1
), which is similar to the magnitude of insulin-stimulated ME
gene transcription in rat liver (4, 5). This result suggests that an
IRS is present in the ME promoter between -882 and +39. The maximal
effect of insulin was seen at 10 nM; increasing the insulin
concentration to 100 nM appeared to be toxic to these
cells, based on their appearance, and significantly reduced CAT
expression (data not shown). Insulin has a similar biphasic action on
the expression of the genes encoding c-fos,
c-myc, and JE (22).

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Figure 1. Insulin Stimulates ME-CAT Fusion Gene Transcription
in H4IIE Cells
H4IIE cells were transiently transfected, as described in
Materials and Methods, with a ME-CAT fusion gene containing
promoter sequence from -882 to +39 (21 ). After transfection, cells were
incubated for 20 h in serum-free medium, in the presence or
absence of various concentrations of insulin. The cells were then
harvested and CAT activity assayed as previously described (14 69 ).
Results are presented as the ratio of CAT activity in insulin-treated
vs. control cells (expressed as fold induction) and
represent the mean of 3 experiments ± SEM.
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To delineate the ME IRS, a series of 5'-deletion mutations of the ME
promoter were constructed in the CAT reporter plasmid pCAT(An) (23),
and the effect of insulin on CAT expression directed by these
constructs was analyzed after transient transfection into H4IIE cells.
Figure 2
shows that insulin-stimulated
ME-CAT gene transcription was reduced when the region of the promoter
between -180 to -152 was deleted and was completely lost upon further
deletion of the promoter sequence between -151 and -124.

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Figure 2. Deletion of the ME Promoter Sequence between -180
and -124 Abolishes the Stimulatory Effect of Insulin on ME-CAT Fusion
Gene Transcription
H4IIE cells were transiently transfected, as described in
Materials and Methods, with a series of ME-CAT fusion genes
with 5' deletion end-points as shown on the abscissa. After
transfection, cells were incubated for 20 h in serum-free medium,
in the presence or absence of 10 nM insulin. The cells were
then harvested and CAT activity assayed as previously described (14 69 ). Results are presented as the ratio of CAT activity in
insulin-treated vs. control cells (expressed as fold
induction) and represent the mean of 410 experiments ±
SEM.
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Insulin Stimulates Protein Binding to an Egr-1 and an AP-1 Motif in
the ME Promoter
To determine whether the stimulation of ME gene transcription by
insulin correlated with changes in protein binding to the ME promoter,
nuclear extracts were prepared from control and insulin-treated H4IIE
cells. Nuclear extract protein binding to three double-stranded
oligonucleotides that span distinct regions of the ME promoter was
assessed using the gel retardation assay (Fig. 3
). When an oligonucleotide representing
the ME promoter sequence between -181 and -145 was used as the
labeled probe, an insulin-stimulated protein-DNA complex was detected
(Fig. 3
; see arrow). Similarly, when an oligonucleotide
representing the ME promoter sequence between -161 and -123 was used
as the labeled probe, two insulin-stimulated protein-DNA complexes were
detected (Fig. 3
; see arrows). In a comparison of nuclear
extracts from control vs. insulin-treated cells, no
consistent difference in protein binding was detected, when an
oligonucleotide representing the ME PEPCK-like motif sequence between
-699 and -664 was used as the labeled probe (Fig. 3
).

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Figure 3. Insulin Stimulates Protein Binding to
Oligonucleotides Representing the -181 to -145 and -161 to -123
Regions of the ME Promoter
Nuclear extracts were prepared from H4IIE cells incubated for 5 h
in serum-free media (C) or serum-free media supplemented with 10
nM insulin (I). Protein binding to the three labeled
oligonucleotide probes shown was analyzed using the gel retardation
assay, as described in Materials and Methods, with three
independent nuclear extract preparations (Preps. 13). In the
representative autoradiograph shown only the retarded complexes are
visible and not the free probe, which was present in excess. The
insulin-stimulated protein-DNA complexes are indicated by the
arrows.
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A series of gel retardation experiments were performed to
investigate the nature of the insulin-stimulated H4IIE cell protein-DNA
complex detected with the -181/-145 oligonucleotide.
Garcia-Jiménez et al. (5) reported that an
insulin-stimulated liver protein-DNA complex could be detected when an
oligonucleotide representing ME sequence from -175 to -156 was used
as the labeled probe in a gel retardation assay. Since this sequence is
included within the -181/-145 oligonucleotide, we hypothesized that
the insulin-stimulated protein-DNA complex detected in H4IIE cells
(Fig. 3
) was the same as that detected by Garcia-Jiménez et
al. in liver (5). Indeed, when an oligonucleotide (ME WT; Fig. 4A
) representing the -175 to -156 ME
sequence was used as the labeled probe in a gel retardation assay, an
insulin-stimulated H4IIE cell protein-DNA complex was detected (Fig. 4B
). In addition, a 100-fold molar excess of the unlabeled -181/-145
oligonucleotide competed effectively for protein binding to the
-175/-156 labeled probe, indicating that the same insulin-stimulated
protein-DNA complex binds both sequences (data not shown).

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Figure 4. The -175 to -156 Region of the ME Promoter Binds
Egr-1
A, Panel A shows the sequence of oligonucleotides used in these
studies. The core IRP-A and IRE-ABP binding motifs (20 24 ) in the
GAPDH IRE-A sequence are boxed as are the IRP-A and Egr-1
binding motifs (25 26 ) in the wild-type (WT) ME -175/-156 sequence.
The altered base pairs in the mutant (MUT) ME -175/-156 sequence are
also boxed. B, The labeled ME WT oligonucleotide probe was
incubated in the absence (-) or presence of a 100-fold molar excess of
the unlabeled oligonucleotide competitors shown before addition of
nuclear extract from control (C) or insulin-treated (I) H4IIE cells
(Prep. 2 from Fig. 3 ). Protein binding was then analyzed using the gel
retardation assay as described in Materials and Methods. In
the representative autoradiograph shown only the retarded complexes are
visible and not the free probe, which was present in excess. The
insulin-stimulated protein-DNA complex, identified in Fig. 3 , is
indicated by the arrow. C, Nuclear extract from control (C)
or insulin-treated (I) H4IIE cells (Prep. 2 from Fig. 3 ) was incubated
with antibody dilution buffer (-) or various amounts of Egr-1
antiserum for 10 min at 4 C, before the addition of the labeled ME WT
oligonucleotide probe and binding buffer and incubation for an
additional 10 min at room temperature. Protein binding was then
analyzed using the gel retardation assay as described in
Materials and Methods. In the representative autoradiograph
shown only the retarded complexes are visible and not the free probe,
which was present in excess. The insulin-stimulated protein-DNA
complex, identified in Fig. 3 , is indicated by the arrow.
100 mM KCl was included in the binding reaction in panel B
but not C which explains the differences in protein-DNA complex
formation.
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Two insulin response elements, designated IRE-A and IRE-B, have been
identified in the GAPDH promoter (20). The IRE-A sequence has been
shown to bind two proteins, designated IRP-A and IRE-ABP (Ref. 24 and
Fig. 4A
). The ME -175 to -156 sequence only contains the core-binding
motif for IRP-A (Fig. 4A
). To determine whether the insulin-stimulated
protein-DNA complex detected with the ME -175/-156 oligonucleotide
represents IRP-A, an oligonucleotide representing the GAPDH IRE-A
sequence was synthesized (Fig. 4A
). A 100-fold molar excess of this
unlabeled IRE-A oligonucleotide did not compete for protein binding
against the -175/-156 labeled probe, indicating that the
insulin-stimulated protein-DNA complex binding this ME sequence is
distinct from IRP-A (Fig. 4B
). An oligonucleotide (ME MUT; Fig. 4A
)
representing the -175 to -156 ME sequence, but containing a mutation
in the core IRP-A motif known to disrupt IRP-A binding to the GAPDH
IRE-A (20), also failed to compete for protein binding against the
wild-type -175/-156 labeled probe (Fig. 4B
). This suggested that the
insulin-stimulated protein-DNA complex contacts this sequence. Further
inspection of the -175 to -156 ME sequence revealed the presence of a
consensus Egr-1 binding motif (see Refs. 25, 26 for review) in
addition to the IRP-A binding motif (Fig. 4A
). The mutation present in
the ME MUT oligonucleotide would be predicted to disrupt both IRP-A and
Egr-1 binding, thus potentially explaining why this oligonucleotide
does not compete for protein binding against the wild-type -175/-156
labeled probe (Fig. 4B
). To confirm that the insulin-stimulated
protein-DNA complex represents Egr-1, a supershift experiment was
performed using a polyclonal antiserum raised against Egr-1 (Fig. 4C
).
The Egr-1 antiserum selectively supershifted the insulin-stimulated
protein-DNA complex without affecting binding of the other protein-DNA
complexes detected with the ME -175/-156 labeled probe (Fig. 4C
).
Additional gel retardation experiments were also performed to
investigate the nature of the two insulin-stimulated H4IIE cell
protein-DNA complexes detected with the -161/-123 oligonucleotide
(Fig. 3
). A 100-fold molar excess of the unlabeled -161/-123
oligonucleotide competed effectively for protein binding against the
-161/-123 labeled probe, indicating that the two insulin-stimulated
protein-DNA complexes detected represent specific interactions (Fig. 5B
; see arrows). By contrast,
when a 100-fold molar excess of unlabeled oligonucleotides representing
the ME Egr-1 motif, either the -181/-145 or -175/-156 sequence
(Fig. 5A
), were incubated with the -161/-123 labeled probe, no
competition was seen (Fig. 5B
). This suggests that neither of the
insulin-stimulated protein-DNA complexes that are binding the -161 to
-123 promoter sequence represents Egr-1.
Inspection of the -161 to -123 sequence revealed the presence of a
consensus AP-1 motif (see Ref. 27 for review) located between -132 and
-126 (Table 1
and Fig. 5A
).
Oligonucleotides representing the ME sequence between -138 and -123,
and also the AP-1 motif sequence between -63 and -78 in the human
collagenase-1 gene (28, 29, 30), were synthesized (Table 1
) and used as
competitors, at a 100-fold molar excess, in a gel retardation assay
with the -161/-123 oligonucleotide as the labeled probe (Fig. 5B
).
Both oligonucleotides competed effectively against the labeled probe
for binding of the two insulin-stimulated protein-DNA complexes (Fig. 5B
; see arrows). Additional oligonucleotides were
synthesized containing a mutated AP-1 motif within the context of the
ME -161/-123, ME -138/-123, and collagenase -63/-78 sequences
(Table 1
). None of these oligonucleotides competed at a 100-fold molar
excess against the wild-type ME -161/-123 labeled probe for binding
of the two insulin-stimulated protein-DNA complexes (Fig. 5B
),
suggesting that both complexes recognize the AP-1 motif. The nature of
the slower migrating insulin-stimulated protein-DNA complex is unknown,
but supershift experiments identical to those described below with HeLa
cell nuclear extracts, using antisera to the various isoforms of fos
and jun, revealed that the major, faster migrating, insulin-stimulated
protein-DNA complex contains members of the AP-1 family of
transcription factors, primarily Fra-2 and Jun D (data not shown).
The ME AP-1 Motif Selectively Mediates Insulin and not Phorbol
Ester Signaling
Figure 2
shows that the effect of insulin on ME-CAT gene
transcription is reduced when the region of the promoter between -180
to -152, which binds Egr-1 (Fig. 4
), is deleted and is completely lost
when the region of the promoter between -151 to -124, which binds
AP-1 (Fig. 5
), is deleted. To determine whether both the Egr-1 and AP-1
binding motifs can act as independent IRSs in H4IIE cells, these
regions were analyzed for their ability to confer an insulin response
in the context of a heterologous promoter. Oligonucleotides
representing these motifs were synthesized and ligated into the
polylinker of a heterologous thymidine kinase (TK) promoter-CAT fusion
gene, containing TK promoter sequence from -105 to +51, and the
resulting constructs were transiently transfected into H4IIE cells.
Insulin had little effect on CAT expression directed by the basic TKCAT
plasmid, but the ME AP-1 motif was able to confer a stimulatory effect
of insulin on CAT expression when ligated to the TK promoter (Fig. 6
). The effect of insulin was not
enhanced by ligating multiple copies of the ME AP-1 motif to the TK
promoter (Fig. 6
). Importantly, when the mutated ME AP-1 motif (Table 1
), which fails to bind either insulin-stimulated protein-DNA complex
(Fig. 5B
), was ligated into the TKCAT polylinker, it also failed to
confer an effect of insulin on the expression of the fusion gene (Fig. 6
). These results were expected since Kim and Kahn (31) had previously
shown, in 3T3-F442A adipocytes, that insulin stimulates the expression
of a heterologous fusion gene containing a multimerized collagenase
AP-1 motif. The collagenase AP-1 motif was also able to confer a
stimulatory effect of insulin on the expression of the TKCAT fusion
gene in H4IIE cells (Fig. 6
). In contrast to the ME AP-1 motif, when
either single or multiple copies of an oligonucleotide representing the
Egr-1 motif (ME WT; Fig. 4A
) were ligated into the polylinker of the
TKCAT fusion gene, they were unable to confer a stimulatory effect of
insulin on CAT expression (data not shown).

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Figure 6. The ME Promoter Sequence between -161 and -123
Can Confer a Stimulatory Effect of Insulin on the Expression of a
Heterologous Fusion Gene
H4IIE cells were transiently transfected, as described in
Materials and Methods, with either the basic TKCAT vector,
designated TK, or constructs, designated ME WT, ME MUT and Coll WT, in
which oligonucleotides representing the wild-type (WT) or mutated (MUT)
ME or collagenase promoter sequence from -161 to -123 and -63 to
-78, respectively, as shown in Table 1 , had been ligated into the
BamHI site of the TK promoter either as a single copy,
in the correct orientation relative to that in the ME gene promoter, or
as multiple (3 4 ) copies. After transfection, cells were incubated for
20 h in serum-free medium in the presence (I) or absence (C) of 10
nM insulin. The cells were then harvested and CAT activity
assayed as previously described (14 69 ). Results are presented as the
ratio of CAT activity in insulin-treated vs. control
cells (expressed as fold induction) and represent the mean of ±
SEM of 311 experiments, in which each construct was
assayed in duplicate.
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While it is known that insulin treatment of various cell types
stimulates the expression and phosphorylation of both c-fos
and c-jun the specific protein kinases involved in these
actions of insulin remain to be defined (see Discussion). By
contrast, the action of phorbol esters on c-jun and
c-fos expression and phosphorylation have been better
characterized (see Ref. 27 for review). Given the long running
controversy concerning the potential role of protein kinase C in
insulin action (see Ref. 32 for review), it was of interest to
determine whether phorbol esters could also stimulate ME gene
transcription. To address this question the full-length -882/+39
ME-CAT fusion gene (21) was transiently transfected into HeLa cells, in
which phorbol ester signaling through AP-1 motifs was initially studied
(29, 30). There was almost no effect of phorbol esters on ME-CAT fusion
gene transcription, whereas phorbol esters stimulated the expression of
a collagenase-CAT fusion gene, containing human collagenase-1
promoter sequence from -518 to +64, more than 100-fold (Fig. 7A
). In HeLa cells, insulin also
stimulated collagenase-CAT gene transcription
16-fold but had almost
no effect on ME-CAT gene transcription (Fig. 7B
). The stimulatory
effects of both phorbol esters and insulin on collagenase gene
transcription require an intact AP-1 motif in the collagenase promoter
(29, 33). Therefore, while these observations were not informative
regarding the role of protein kinase C in insulin action, they raised
the question as to why, in HeLa cells, both insulin and phorbol esters
markedly stimulate collagenase-CAT, but not ME-CAT, gene transcription
even though both gene promoters contain AP-1 motifs.

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Figure 7. Insulin and Phorbol Esters Selectively Stimulate
Collagenase, but not ME, Fusion Gene Transcription in HeLa Cells
A, HeLa cells were transiently transfected, as described in
Materials and Methods, with either a ME-CAT fusion gene
containing promoter sequence from -882 to +39 (21 ) or a
collagenase-CAT fusion gene containing promoter sequence from -518 to
+64. B, HeLa cells were transiently cotransfected with the same
reporter gene constructs as above and an expression vector encoding the
insulin receptor. After transfection, cells were incubated for 20
h in serum-free medium, either (A) in the presence or absence of
various concentrations of PMA or (B) in the presence (I) or absence (C)
of 10 nM insulin. The cells were then harvested and CAT
activity assayed as previously described (14 69 ). Results are
presented as the ratio of CAT activity in PMA or insulin-treated
vs. control cells (expressed as fold induction) and
represent the mean of 37 experiments ± SEM.
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To determine whether these differences in the regulation of ME and
collagenase gene transcription in HeLa cells reflected some inherent
difference between the AP-1 motifs in each promoter, or whether the
context in which the AP-1 motif is located is the relevant factor, the
same heterologous ME-TKCAT and Coll-TKCAT constructs shown in Fig. 6
were analyzed for their ability to mediate phorbol ester and
insulin-stimulated CAT expression in HeLa cells. When these constructs
were transiently transfected into HeLa cells, phorbol esters stimulated
CAT expression directed by the basic TKCAT vector approximately 2-fold,
but this effect was clearly enhanced by the presence of the
collagenase, but not the ME AP-1 motif (Fig. 8A
). In addition, this effect of phorbol
esters was mediated only by the wild-type and not the mutated
collagenase AP-1 motif (Fig. 8A
and Table 1
). This result suggests that
the ME and collagenase AP-1 motifs are inherently functionally distinct
with respect to mediating phorbol ester signaling in HeLa cells (Fig. 8A
), although not with respect to insulin signaling in H4IIE cells
(Fig. 6
).

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Figure 8. Phorbol Esters Selectively Signal through the
Collagenase but not the ME AP-1 Motif. The ME AP-1 Motif Markedly
Enhances Basal TKCAT Gene Transcription
HeLa cells were transiently co-transfected, as described in
Materials and Methods, with a ß-galactosidase expression
vector and either the basic TKCAT vector, designated TK, or constructs,
designated ME WT, ME MUT, Coll WT and Coll MUT, in which
oligonucleotides representing either the wild-type or mutated ME or
collagenase promoter sequence from -161 to -123 and -63 to -78,
respectively, as shown in Table 1 , had been ligated into the
BamHI site of the TK promoter in multiple (3 4 ) copies.
After transfection, cells were incubated for 20 h in serum-free
medium in the presence (P) or absence (C) of 100 nM PMA.
The cells were then harvested and both CAT and ß-galactosidase
activity assayed as previously described (14 69 ). In panel A results
are presented as the ratio of CAT activity, corrected for protein
concentration in the cell lysate, in PMA-treated vs. control
cells and are expressed as fold induction. In panels B and C results
are presented as the ratio of CAT: ß-galactosidase activity in either
control or PMA-treated cells, respectively, and are expressed as
arbitary units. Results represent the mean of ± SEM
of 5 experiments, in which each construct was assayed in duplicate.
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Unfortunately, in HeLa cells, unlike H4IIE cells (Fig. 6
), insulin
markedly stimulates CAT expression directed by the control TKCAT fusion
gene and ligation of multiple copies of the ME or collagenase AP-1
motif into the polylinker of the vector did not enhance the effect of
insulin (data not shown). Therefore, it was not possible to determine
whether insulin can also selectively activate gene transcription
through the collagenase but not the ME AP-1 motif in HeLa cells. [This
variable effect of insulin on TKCAT fusion gene transcription in H4IIE
and HeLa cells is probably due in part to the presence of a cryptic IRS
in the polylinker of this vector that is active in some cell lines but
not others (9, 16).] The fact that there is almost no effect of
insulin on the expression of the full-length ME-CAT fusion gene in HeLa
cells (Fig. 7B
), in contrast to H4IIE cells (Fig. 1
), raises the
possibility that there may be an inherent difference in insulin
signaling through AP-1 motifs in the H4IIE and HeLa cell lines.
Blackshear and colleagues have reported a similar situation with
respect to insulin signaling through the c-fos SRE (15). Thus, in some
cell lines the effect of insulin only required the core serum response
factor binding site in the SRE, whereas in other cells the ternary
complex factor-binding site in the SRE, adjacent to the serum
response factor-binding site, was also required (15).
The ME and Collagenase AP-1 Motifs Bind AP-1 in Distinct
Conformations
A number of parameters were investigated to determine why the ME
AP-1 motif fails to mediate a phorbol ester-dependent increase in gene
transcription when ligated to the heterologous TK promoter. An analysis
of basal ME-TKCAT and Coll-TKCAT fusion gene transcription in HeLa
cells revealed that ligation of the ME, but not the collagenase, AP-1
motif into the TKCAT polylinker markedly enhanced basal fusion gene
transcription (Fig. 8B
). This enhancement of basal fusion gene
transcription was mediated only by the wild-type but not the mutated ME
AP-1 motif (Fig. 8B
and Table 1
). This observation suggests that the
reason why the ME AP-1 motif fails to mediate a phorbol ester-dependent
increase in gene transcription, when ligated to the heterologous TK
promoter, is that AP-1 is already bound in a fully activated state.
Thus, phorbol ester treatment has no additional effect beyond that seen
on the basic TKCAT vector alone (Fig. 8A
). By contrast, ligation of the
collagenase AP-1 motif into the TKCAT polylinker only modestly
increases basal fusion gene transcription (Fig. 8B
), but phorbol ester
treatment enhances expression to a level similar to that obtained with
the ME-TKCAT fusion gene (Fig. 8C
). Based on these observations, the
question as to why the ME AP-1 motif fails to mediate a phorbol
ester-dependent increase in gene transcription when ligated to the
heterologous TK promoter should be restated. The issue is why AP-1
binds the ME, but not the collagenase, AP-1 motif in an activated
state.
Competition experiments revealed that a 100-fold molar excess of the
unlabeled ME -138/-123 oligonucleotide (Table 1
) competed effectively
for specific protein binding when the oligonucleotide representing the
-63/-78 collagenase AP-1 motif (Table 1
) was used as the labeled
probe in a gel retardation assay and vice versa (Fig. 9A
). No difference was seen when a
variable molar excess of the unlabeled ME -138/-123 and collagenase
-63/-78 oligonucleotides were compared for their ability to compete
for protein binding to the labeled ME -138/-123 probe, indicating
that both oligonucleotides bind AP-1 with similar affinity (data not
shown).

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Figure 9. The Collagenase and ME AP-1 Motifs Both Bind the
Same Combination of fos and jun Transcription Factors
A, Protein binding to the two labeled oligonucleotide probes shown was
analyzed using HeLa cell nuclear extracts and the gel retardation
assay, as described in Materials and Methods. Where
indicated, the labeled oligonucleotide probes were incubated in the
absence (-) or presence of a 100-fold molar excess of the unlabeled ME
-138/-123 or Coll -63/-78 oligonucleotides before the addition of
nuclear extract. B, HeLa cell nuclear extract was incubated with
antibody dilution buffer (None) or 1 µg of the indicated antisera for
10 min at 4 C, before the addition of the labeled ME -138/-123 (M) or
Coll -63/-78 (C) oligonucleotide probes and binding buffer followed
by incubation for an additional 10 min at room temperature. Protein
binding was then analyzed using the gel retardation assay as described
in Materials and Methods. In the representative
autoradiographs shown only the retarded complexes are visible and not
the free probe, which was present in excess. The AP-1 complex and a
non-specific (NS) protein-DNA interaction are indicated by the
arrows.
|
|
Since AP-1 is comprised of homodimers and heterodimers of various
members of the fos and jun transcription factor families (34), we
hypothesized that the ME and collagenase AP-1 motifs might selectively
bind specific members of the fos/jun families. To test this hypothesis,
polyclonal antisera to specific fos/jun family members were assayed for
their ability to supershift the specific protein-DNA complex that binds
the ME and collagenase AP-1 motifs (Fig. 9B
). These experiments showed
that the protein-DNA complex detected in the gel retardation assay
contains mainly Fra-2 and Jun D with lesser amounts of c-fos
and Jun B (Fig. 9B
). However, no qualitative or quantitative difference
was detected in AP-1 protein binding to the ME and collagenase probes
(Fig. 9B
). A faint protein-DNA interaction, which migrates slower than
the AP-1 complex, was detected with the collagenase but not the ME AP-1
motif (Fig. 9B
). However, it seems unlikely that this minor interaction
could explain the selective enhancement of basal TKCAT gene
transcription by the ME, but not the collagenase, AP-1 motif. Given the
known action of phorbol esters on AP-1 expression and phosphorylation
(see Discussion), the role of this minor interaction in
phorbol ester signaling through the collagenase AP-1 motif is
unclear.
The proteolytic band shift assay (35) was used to examine the
possibility that the same transcription factors were binding both
probes but in distinct conformations. To study the effect of partial
protease digestion, HeLa nuclear extract was preincubated with either
the labeled collagenase or ME oligonucleotides before the addition of
various concentrations of chymotrypsin (Fig. 10
). Distinct proteolytic products that
selectively bind the collagenase, but not the ME oligonucleotide, probe
were detected (Fig. 10
). Several of these selectively bound products
migrate faster than the nonspecific protein-DNA interaction (Fig. 10
, lower arrow) so it is unclear as to whether these products
are derived from proteolysis of this nonspecific protein-DNA
interaction or the AP-1 complex. However, one of these selectively
bound proteolytic products (Fig. 10
, upper arrow) migrates
slower than the nonspecific DNA-protein interaction and is therefore
likely to be derived through proteolysis of the AP-1 complex. Thus,
this experiment suggests that the proteins bound to the collagenase and
ME AP-1 motifs have different surfaces exposed to proteolytic digestion
indicative of a difference in binding conformation. If so, this
difference in binding conformation could potentially explain the
selective enhancement of basal TKCAT gene transcription by the ME AP-1
motif. It is possible that the faint protein-DNA interaction, which
migrates slower than the AP-1 complex and selectively binds the
collagenase but not the ME AP-1 motif, is the source of the proteolytic
fragments that specifically bind the collagenase AP-1 motif (Fig. 10
).
However, this seems unlikely unless partial proteolysis of this unknown
protein markedly increased its DNA-binding affinity.

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Figure 10. The Collagenase and ME AP-1 Motifs Bind AP-1 in
Distinct Conformations
HeLa cell nuclear extract was incubated with the labeled ME -138/-123
(M) or Coll -63/-78 (C) oligonucleotide probes for 10 min at room
temperature before the addition of various amounts of chymotrypsin and
incubation for an additional 2 min at room temperature. Protein binding
was then analyzed using the gel retardation assay as described in
Materials and Methods. In the representative autoradiograph shown only
the retarded complexes are visible and not the free probe, which was
present in excess. A nonspecific (NS) protein-DNA interaction is
indicated by the arrow as is the AP-1 complex and
proteolytic fragments that specifically bind the Coll -63/-78 labeled
probe.
|
|
 |
DISCUSSION
|
---|
In H4IIE cells the stimulation of ME gene transcription by insulin
is mediated through two cis-acting elements in the promoter,
an AP-1 motif and an Egr-1-binding site (
Figs. 26



). The observation
that insulin stimulates Egr-1 expression has been made in several cell
types (36, 37, 38, 39, 40) but, to the best of our knowledge, ME is the first
insulin-regulated gene identified in which this induction of Egr-1 gene
expression has a specific functional consequence. Unlike the ME AP-1
motif (Fig. 6
), this Egr-1 binding site does not mediate an insulin
response in the context of the heterologous TK promoter (data not
shown). This may indicate that this element only functions in the
context of the native ME promoter. Alternatively, since Egr-1 motifs
can mediate a stimulatory effect of serum when ligated to a minimal
ß-globin promoter (41), it is possible that this element simply
cannot mediate an effect of insulin in the context of the TK promoter.
Such a scenario would be similar to the PEPCK IRS, which can mediate an
insulin response in the context of the heterologous TKC-VI vector (10)
but not TKCAT (42). The inverse is found with the ME AP-1 motif, which
can mediate an insulin response in the context of the TKCAT (Fig. 6
)
but not TKC-VI vector (data not shown).
Various investigators have studied the signaling pathway through which
insulin stimulates Egr-1 gene expression (38, 39, 40, 43). The
phosphorylation of Shc (38) or insulin receptor substrate-1 (IRS-1)
(39) by the receptors for insulin and IGF-I, respectively, both lead to
the induction of Egr-1 gene expression. In fibroblasts, MAP kinase
activation is not sufficient for IGF-I-stimulated Egr-1 gene expression
(39), but it may be required since in 32D hematopoietic progenitor
cells inhibition of MAP kinase blocks insulin-stimulated Egr-1 gene
expression (38). By contrast, in experiments comparing the stimulation
of Egr-1 gene expression by insulin in Rat 1 fibroblasts expressing
either C-terminally truncated or normal insulin receptors, Olefsky and
colleagues (40) found that insulin stimulated Egr-1 gene expression to
the same extent in both cell lines, whereas MAP kinase activation was
almost undetectable in cells expressing the C-terminally truncated
insulin receptors (40). Interestingly, insulin-stimulated
c-fos gene expression was also markedly reduced in the cells
expressing C-terminally truncated insulin receptors, which suggests
that distinct signaling pathways mediate the action of insulin on
c-fos and Egr-1 gene expression (40). Since part of the
effect of insulin on ME gene transcription may be mediated through an
increase in c-fos gene expression (see below), this raises
the possibility that the activation of multiple signal transduction
pathways may be required for the effect of insulin on ME gene
expression. The results of Olefsky and colleagues (40) were surprising
because both the c-fos and Egr-1 promoters contain serum
response elements (SREs) (25), and the effect of insulin on at least
c-fos gene transcription is mediated through the SRE (see
below). Moreover, using the same cell line expressing C- terminally
truncated insulin receptors, Stumpo and Blackshear (43) observed the
predicted result, that insulin-stimulated c-fos and Egr-1
gene expression were both reduced.
In contrast to the results described by Garcia-Jiménez et
al. (5), no difference in protein binding was detected, comparing
nuclear extracts from control and insulin-treated cells, when an
oligonucleotide representing the ME PEPCK-like motif was used as the
labeled probe in a gel retardation assay (Table 1
and Fig. 3
). The same
result was obtained under the assay conditions described in Fig. 3
and
conditions identical to those described by Garcia-Jiménez
et al. (data not shown). This discrepancy could reflect an
inherent difference between extracts isolated from insulin-treated
H4IIE cells (Fig. 3
) and rat liver (5). However, in earlier studies,
using liver nuclear extracts isolated from control, diabetic, and
insulin-treated diabetic rats, we again detected no change in protein
binding to the PEPCK IRS (44). Since these PEPCK IRS-binding proteins
were later shown to represent members of the C/EBP family of
transcription factors (45) and several recent publications report that
insulin regulates expression of the genes encoding C/EBP
, ß, and
(see Ref. 9 for review), the explanation for why we are unable to
reproduce the result obtained by Garcia-Jiménez et al.
(5) is unclear. Clearly though, the analysis of the effect of insulin
on ME-CAT fusion gene transcription (Fig. 2
) indicates that the ME
PEPCK-like IRS motif has no apparent functional role with respect to
insulin action, at least in H4IIE cells.
Insulin markedly induces the expression of several components of the
AP-1 complex including c-jun (22, 37), Jun B (22, 37, 46), and
c-fos (22, 47, 48) but only slightly stimulates Jun D
expression (22, 37, 49). These effects of insulin are seen in multiple
cell types. By contrast, insulin has no effect on Fos B mRNA levels
(22, 37), while the stimulation of Fra-1 expression by insulin is seen
in some (22, 50), although not in all cell types (37). The mechanism
through which insulin induces the expression of these genes is unknown
with the exception of c-fos. Blackshear and colleagues (51)
have shown that the effect of insulin on c-fos gene
transcription is mediated through the SRE in the c-fos
promoter, although the precise trans-acting factor involved
may vary between different cell types (15). Several studies suggest
that the MAP kinase pathway is involved in insulin-stimulated
c-fos gene transcription (52, 53, 54). For example,
overexpression of wild-type p21ras enhances
insulin-stimulated c-fos gene expression (52), whereas
overexpression of dominant negative mutants of p21ras and
Raf-1 block insulin-stimulated c-fos gene expression (53, 54).
The phosphorylation state of c-fos and c-jun is
also stimulated by insulin (31, 55), and several insulin-regulated
protein kinases are known to phosphorylate c-jun, with
either stimulatory or inhibitory effects on c-jun DNA
binding and/or transactivation potential, including MAP kinase, GSK-3,
casein kinase II, and JNK (see Refs. 34, 56, 57, 58 for review).
However, which of these kinases plays the central role in the
regulation of c-jun phosphorylation by insulin in
vivo is unclear. A further potential complication is that the
action of insulin may be cell type specific. For example, while insulin
has been shown to activate MAP kinase in multiple cell types (see Ref.
58 for review), the activation of JNK is seen in some cell types (59)
but not others (33). Surprisingly, despite this wealth of information
and the large number of insulin-regulated genes that have been studied
in detail (see Ref. 9 for review), ME is only the second example, the
first being collagenase (33), of an insulin-stimulated gene whose
transcription is, at least in part, regulated through an AP-1 motif.
Whether the stimulation of collagenase and ME gene transcription by
insulin is principally mediated by an increase in the mass of the AP-1
complex or an alteration in the phosphorylation state of preexisting
fos/jun proteins remains to be determined. In liver, however,
inhibitors of protein synthesis block the induction of ME gene
expression by insulin, suggesting that the former may be the more
likely mechanism (4).
Based on the results of the experiments shown in
Figs. 810

, we
suggest that a difference in AP-1 binding conformation could
potentially explain the selective phorbol ester signaling through the
collagenase AP-1 motif. This difference in AP-1 binding conformation is
proposed to result in AP-1 binding the ME, but not the collagenase,
AP-1 motif in an activated state such that phorbol ester treatment has
no additional effect (Fig. 8B
). Since the ME and collagenase AP-1
motifs share an identical core consensus sequence, TGACTCA, the
difference in AP-1 binding conformation could be explained by the
distinct flanking sequence on either side of the core AP-1 motif (Table 1
). Indeed, Ryseck and Bravo (60) have shown that the flanking sequence
on either side of a consensus AP-1 motif can influence the affinity of
AP-1 binding. Moreover, this effect of the flanking sequence on binding
affinity was not uniform for all fos/jun family members; thus, the
composition of the AP-1 complex would be predicted to change depending
on the precise flanking sequence (60). More recently, various
biophysical approaches have demonstrated that the AP-1 flanking
sequence can affect the conformation of fos/jun binding (61, 62). Our
data suggests that, while the distinct flanking sequences affect the
conformation of AP-1 binding (Fig. 10
), the overall binding affinity of
the AP-1 complex to the collagenase and ME AP-1 motifs are similar
(Fig. 9A
) and that the same fos/jun family members bind both motifs
(Fig. 9B
). Similar observations have been made in studies on the cAMP
response element (CRE) in which the flanking sequence on either side of
a consensus CRE motif can dramatically affect the magnitude of
cAMP-induced gene transcription (63). Interestingly, insulin also
stimulates the transcription of the human glutathione
S-transferase P11 gene (GSTP1) through an undefined
element located in the promoter sequence between -99 and +72 (64). An
AP-1 motif, located between -65 to -59, has identical 5'- and
3'-flanking sequence to the ME AP-1 motif and, as with the ME gene,
this AP-1 motif is unable to mediate a phorbol ester-dependent
stimulation of GSTP1 gene transcription but is critical for high basal
gene transcription (65, 66).
 |
MATERIALS AND METHODS
|
---|
Materials
[
-32P]dATP (>3000 Ci mmol-1) and
[3H] acetic acid, sodium salt (>10 Ci
mmol-1) were obtained from Amersham (Arlington Heights,
IL) and ICN, respectively. Insulin was purchased from Collaborative
Bioproducts. Phorbol 12-myristate 13-acetate (PMA) was obtained from
Sigma Chemical Co. (St. Louis, MO). Specific antisera to
c-fos (sc-52), Fra-1 (sc-183), Fra-2 (sc-604), c-jun
(sc-45), Jun B (sc-46), Jun D (sc-74), and Egr-1 (sc-110) were all
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Plasmid Construction
DNA manipulations were accomplished by standard techniques (67).
An ME-CAT plasmid containing rat ME promoter sequence from -882 to
+39, relative to the major transcription start site, was obtained as a
generous gift from Dr. Vera Nikodem (21). An
XbaI-BamHI fragment of the rat ME promoter,
spanning the promoter sequence from -775 to +38, was isolated from
this plasmid and ligated, in the same orientation as that found in the
endogenous ME gene, into the XbaI-BglII-digested
polylinker of the pCAT(An) expression vector, a generous gift from Dr.
Howard Towle (23). The pCAT(An) vector has polyadenylation signals
located 5' of the polylinker to prevent read-through transcription
(23). Control experiments demonstrated that there was no basal CAT
expression and no effect of insulin when the empty vector, minus the ME
promoter, was transiently transfected into H4IIE cells (data not
shown). A series of truncated ME-CAT fusion genes was generated by
restriction enzyme digestion using XbaI and the one of the
following enzymes: EcoN I, Nae I, SfaN
I and Eco47 III. The Klenow fragment of Escherichia
coli DNA polymerase I was used to fill-in the non-compatible ends;
after blunt-end ligation the resulting plasmids had calculated 5'
end-points of -316, -180, -151, -123 which were confirmed by DNA
sequencing using the USB Sequenase kit.
The plasmid pCol.Luc, containing the human collagenase-1 promoter
ligated to the luciferase reporter gene, was a generous gift from Dr.
Jeremy Tavaré (33). A BglII - HindIII
fragment of the collagenase promoter, spanning the sequence from -518
to +64, was isolated from this plasmid and ligated, in the same
orientation as that of the endogenous gene, into the BglII
digested polylinker of the pCAT(An) expression vector. The
non-compatible 3' HindIII - BglII junction was
filled-in using the Klenow fragment of Escherichia coli DNA
polymerase I before blunt-end ligation.
Plasmid TKCAT contains the herpes simplex virus thymidine kinase (TK)
promoter sequence from -105 to +51 ligated to the CAT reporter gene
and has a unique BamH I site in the polylinker at -105
(68). Various double-stranded complementary oligonucleotides,
representing distinct regions of the ME or collagenase promoters (Table 1
), were synthesized with BamHI compatible ends using a
Perceptive Biosystems Nucleic Acid Synthesis System and were ligated
into BamHI-cleaved TKCAT either as a single copy, in the
correct orientation relative to that in the endogenous gene, or as
multiple copies. The orientation and number of inserts was determined
by restriction enzyme analysis and confirmed by DNA sequencing. All
plasmid constructs were purified by centrifugation twice through cesium
chloride gradients (67).
Cell Culture and Transient Transfection
A) Rat H4IIE hepatoma cells were grown to 40 to 70% confluence
in T150 flasks in Dulbeccos modified Eagles medium (DMEM)
containing 2.5% (vol/vol) fetal calf serum and 2.5% (vol/vol) newborn
calf serum and were then transiently transfected in solution using the
calcium phosphate-DNA co-precipitation method as previously described
(14, 69).
B) Human HeLa cervical carcinoma cells were grown to 90% confluence in
T150 flasks in DMEM containing 10% (vol/vol) calf serum and were
replated the day before use into 75 cm2 culture dishes.
Attached cells were transfected by addition of 0.5 ml of a calcium
phosphate-DNA co-precipitate (69), containing the reporter gene
construct (15 µg) and an expression vector for ß-galactosidase (2.5
µg), to the 10 ml of culture medium. In some experiments (Fig. 7B
) an
expression vector encoding the insulin receptor (5 µg), courtesy of
Dr. Jonathan Whittaker, was co-transfected with the reporter gene
construct (15 µg) and the expression vector for ß-galactosidase
(2.5 µg). After an overnight incubation the media was replaced with
serum-free DMEM supplemented with or without various concentrations of
PMA or 10 nM insulin as indicated in the Figure
Legends.
The cells were then incubated for a further 20 h prior to
harvesting.
CAT and ß-Galactosidase Assays
Cells were harvested by trypsin digestion and sonicated in 300
µl of 250 mM Tris (pH 7.8) containing 2 mM
PMSF. CAT and ß-galactosidase assays were performed exactly as
previously described (14, 69). Since ß-galactosidase is very poorly
expressed in H4IIE cells (14, 69), CAT activity was corrected for the
protein concentration in the cell lysate, as measured by the Pierce BCA
assay and each plasmid construct was analyzed in duplicate in multiple
transfections, as specified in the Figure
Legends. Because phorbol
esters (and insulin) affect RSV-ß galactosidase expression in HeLa
cells, CAT activity was either corrected for the protein concentration
in the cell lysate (Fig. 7
) or for ß-galactosidase activity with the
data obtained from control and PMA-treated cells shown separately (Fig. 8
).
Gel Retardation Assay
A) Labeled probes: double-stranded oligonucleotides representing
various regions of the ME, collagenase, PEPCK or GAPDH promoters were
synthesized with either BamHI (Table 1
) or
HindIII (Fig. 4
) compatible ends, gel purified, annealed and
then labeled with [
-32P]dATP using the Klenow fragment
of Escherichia coli DNA polymerase I to a similar specific
activity of approximately 2.5 µCi/pmol.
B) Nuclear extract preparation: H4IIE and HeLa nuclear extracts were
prepared as previously described (69, 70) except that the nuclear
pellet was extracted with 20 mM HEPES pH 7.8, 0.75
mM spermidine, 0.15 mM spermine, 0.2
mM EDTA, 2 mM EGTA, 2 mM DTT, 25%
glycerol containing 200 mM NaCl, instead of 0.4
M ammonium sulfate, and the supernatant was used directly
in gel retardation assays. The protein concentration of the nuclear
extracts was determined using the Bio-Rad assay and was typically
1
µg µl-1.
C) Standard binding assay: labeled oligonucleotide (7.5 fmol,
30,000
cpm) was incubated with either H4IIE (5 µg) or HeLa (3 µg) nuclear
extract, as indicated in the figure legends, in a final reaction volume
of 20 µl containing 20 mM HEPES pH 7.8, 100
mM NaCl, 0.38 mM spermidine, 0.08
mM spermine, 0.1 mM EDTA, 1 mM
EGTA, 2 mM DTT, 12.5% glycerol (vol/vol) and 1 µg of
poly(dI-dC)·poly(dI-dC). When the ME -181/-145 (Fig. 3
) or ME
-175/-156 (Fig. 4
) oligonucleotides were used as the labeled probes
the HEPES concentration was increased to 40 mM and in some
experiments (Fig. 3
and 4B
) 100 mM KCl was included in the
binding reaction. After incubation for 10 min at room temperature, the
reactants were loaded onto a 6% polyacrylamide gel and electrophoresed
at room temperature for 90 min at 150 V in a buffer containing 25
mM Tris, 190 mM glycine and 1 mM
EDTA. After electrophoresis the gels were dried, exposed to Kodak XAR5
film, and binding was analyzed by autoradiography.
D) Competition experiments: for competition experiments (Figs. 4
, 5
and
9A) or assessment of the relative affinity of binding (data not shown),
unlabeled double-stranded oligonucleotide (1- to 100-fold molar excess)
was mixed with the labeled oligomer before addition of nuclear extract.
Binding was then analyzed by acrylamide gel electrophoresis as
described above.
E) Gel supershift: gel supershift assays were carried out by incubating
H4IIE (5 µg) or HeLa (3 µg) nuclear extract with varying amounts of
antisera, for 10 min at 4 C, before the addition of the labeled
oligonucleotide probe and binding buffer and incubation for an
additional 10 min at room temperature. Where appropriate antisera were
diluted in 10 mM HEPES pH 7.8 containing 1 µg
µl-1 BSA.
F) Proteolytic cleavage: HeLa nuclear extract (3 µg) was
pre-incubated for 10 min at room temperature with either the ME
-138/-123 or Coll -63/-78 labeled probes, as described above,
before addition of various amounts of chymotrypsin for an additional 2
min at room temperature. Proteolytic products were then analyzed by
acrylamide gel electrophoresis as described above.
 |
ACKNOWLEDGMENTS
|
---|
We thank Vera Nikodem and Jeremy Tavaré for the generous
gifts of the rat ME and human collagenase-1 promoters, respectively,
and Howard Towle and Jonathan Whittaker for the pCAT(An) and insulin
receptor expression vector plasmids. We also thank Roland Stein and Rob
Hall for helpful comments on the manuscript and Ron Wisdom, Lynn
Matrisian, Howard Crawford and Linda Sealy for both advice and
providing the AP-1 antibodies. The H4IIE and HeLa cell lines were
kindly provided by Daryl Granner/Cathy Caldwell and Roland Stein/Eva
Henderson, respectively. Data analysis was performed in part through
the use of the VUMC Cell Imaging Resource (CA68485 and DK20593) and the
research was supported by grants from the Mark Collie Foundation and
NIH (RO1 DK52820 to R.OB.).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Richard M. OBrien, Department of Molecular Physiology and Biophysics, 761 MRB II, Vanderbilt University Medical School, Nashville, TN 37232-0615.
Received for publication March 3, 1998.
Revision received July 22, 1998.
Accepted for publication August 13, 1998.
 |
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