From the Departments of Medicine, and Physiology and Biophysics, University of Illinois at Chicago College of Medicine and Veterans Affairs Chicago Health Care System (West Side Division), Chicago, Illinois 60612
Received for publication, September 19, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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Highly related insulin response
sequences (IRSs) mediate effects of insulin on the expression of
multiple genes in the liver, including insulin-like growth factor
binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase
(PEPCK). Gel shift studies reveal that oligonucleotide probes
containing an IRS from the IGFBP-1 or PEPCK gene form a similar
complex with hepatic nuclear proteins. Unlabeled competitors containing
the IGFBP-1 or PEPCK IRS or a binding site for C/EBP proteins inhibit
the formation of this complex. Antibody against C/EBP Highly related insulin response sequences
(IRSs)1 have been found to
mediate effects of insulin on the expression of a number of genes in
the liver, including phosphoenolpyruvate carboxykinase, insulin-like
growth factor binding protein-1 (IGFBP-1), glucose-6-phosphatase, and
apolipoprotein CIII (1-4). This observation has suggested that insulin
may exert effects on the expression of multiple hepatic genes through a
common mechanism (5). Signaling via phosphatidylinositol 3'-kinase is critical for the ability of insulin to suppress
activity of the IGFBP-1, PEPCK, and Glu-6-Pase promoters (6-8) and we have reported that protein kinase B can mediate effects of insulin on
basal IGFBP-1 promoter activity via an IRS downstream from phosphatidylinositol 3'-kinase (6). HNF-3/forkhead proteins can bind to
oligonucleotide probes containing the insulin response sequences from
the IGFBP-1 and PEPCK genes (9, 10). However, these interactions also
involve sequences flanking the IRS, indicating that HNF-3 proteins are
not likely to mediate sequence-specific effects of insulin on promoter
activity via an IRS (10, 11). Recent reports have shown that another
subgroup of forkhead/winged-helix transcription factors,
including FKHR, FKHRL1, and AFX, can stimulate transcription through an
IRS when they are overexpressed in cells, and that phosphorylation by
protein kinase B can disrupt IRS-dependent transactivation
by these proteins (12-16). However, studies to date have not
demonstrated interaction between endogenous FKHR, FKHRL1, or AFX in
nuclear extracts and an IRS in binding assays. Also, Hall et
al. (17) have suggested that these forkhead family members may not
interact with IRSs with the appropriate sequence-specificity to account
for the ability of insulin to regulate promoter activity through an IRS
in cells, indicating that other (as yet unidentified) factors also may
contribute to the ability of insulin to suppress promoter function
through an IRS (17).
CAAT/enhancer-binding proteins (C/EBPs) are enriched in the liver where
they play an important role in the regulation of gene expression (18)
and studies in knockout mouse models reveal that C/EBP proteins play a
critical role in the regulation of hepatic glucose production during
development and in fasting and insulin-deficient mice (19-22). Cell
culture studies have shown that C/EBP proteins also are critically
involved in the differentiation of adipocytes (23-25), indicating that
they play an important role in the integrated regulation of metabolic
processes (26). We have previously reported that a nucleoprotein
complex that interacts with an IRS in the IGFBP-1 promoter may involve
members of the C/EBP family of transcription factors, based on gel
shift studies with an unlabeled competitor containing a known
C/EBP-binding site (11). We now report that a similar complex also
interacts with the IRS in the PEPCK gene and that this complex contains C/EBP Materials and Expression Vectors--
Antibodies against
C/EBP Nuclear Extracts and Gel Shift Assay--
H4IIE and HepG2 cells
were grown to near confluency in polystyrene dishes and refed with
Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 1 g/liter of fatty acid-free bovine serum albumin (Sigma) 24 h
before harvest. Nuclear extracts were prepared from H4IIE hepatoma
cells after the method of Dignam as reported (9), and nuclear
extracts were prepared from HepG2 cells after Nonidet P-40 detergent
lysis (27). Nuclear extracts were prepared from the livers of adult
(200-250 g) male rats after the method of Gorski, as previously
reported (28). Protein content was measured by the Bradford dye-binding
assay (Bio-Rad).
For gel shift and supershift studies, nuclear extracts were
preincubated with unlabeled oligonucleotide competitors or GST-CHOP for
10 min or with antibody for 30 min at 22 °C in 10 µl of binding buffer containing 50 or 5 µg/ml (Figs. 1B, 2B,
and 3A) poly(dI-dC)·poly(dI-dC). Binding reactions were
continued for an additional 30 min following the addition of ~1 × 104 cpm end-labeled double-stranded oligonucleotide
probes, then loaded for 6% native PAGE at 4 °C, as previously
reported (9). Gel shift conditions reported to optimize the binding of
recombinant C/EBP to a probe containing the PEPCK IRS and flanking
sequence (29) were used as noted (Fig. 6A). Gels were dried
prior to autoradiography and/or phosphorimaging.
Double-stranded oligonucleotides containing binding sites for Sp1,
Oct1, and CTF/NF-1 were purchased from Promega, and an oligonucleotide
containing a consensus C/EBP-binding site was from Santa Cruz. An oligo
containing an SRF site was provided by Dr. R. Prywes, and
oligonucleotides containing the high affinity HNF-3 site of the rat
transthyretin gene, the PEPCK IRS and flanking sequence (including
residues 401-422 upstream from the cap site), or residues 85-122
upstream from the RNA cap site of the rat IGFBP-1 promoter were
prepared as previously described (11). Other oligos used in gel shift
studies are described in the text or in figures. Oligonucleotide probes
were end-labeled with T4 polynucleotide kinase.
Partial Purification and Western Blotting of Nuclear
Proteins--
Nuclear proteins from the livers of 3 rats were dialyzed
against 0.1 M KCl, 20 mM Hepes, pH 7.4, 10%
glycerol containing protease inhibitors (0.5 mM
phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.5 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin) and 1 mM dithiothreitol. After dialysis, the salt concentration
was adjusted to 0.1 M KCl based on conductivity, and
proteins were loaded onto a heparin-agarose column (Sigma). The column
was rinsed with low salt buffer, and proteins were eluted in 30 fractions with a linear gradient of 0.1-1 M KCl in 10 mM Hepes, pH 7.4, buffer containing 10% glycerol, protease
inhibitors, and dithiothreitol. Binding activity in every other
fraction was measured by gel shift assay using the
A DNA affinity column was constructed with streptavidin-agarose (Sigma)
and biotinylated oligonucleotides containing three copies of IRSA from
the IGFBP-1 promoter:
5'-biotin-dT-ATACGCAAAACAACATGTCAAAACAAGAGCAAAACAATGC-3' and
3'-TATGCGTTTT GTTGTACAGTTTTGTTCTCGTTTTGTTACG-5'. Proteins were eluted with a 0.1-1 M KCl gradient in 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol
and binding activity was monitored by gel shift assay. Following
dialysis, proteins were re-loaded onto this column with 10 µg/ml
poly(dI-dC)·poly(dI-dC) to reduce nonspecific binding. Proteins were
loaded for a third round of affinity chromatography without
poly(dI-dC)·poly(dI-dC). Gel shift assays were performed with either
the
Partially purified proteins were concentrated with a Centricon-10
concentrator (Amicon) and loaded for 4-20% SDS-PAGE under reducing
conditions, then transferred to nitrocellulose membranes for Western
blotting with antibody against C/EBP Reporter Gene Constructs and Transient Transfection
Studies--
The SauI/HgaI fragment of the rat
IGFBP-1 promoter was cloned into pGL2, and an NheI site was
introduced immediately upstream of the insulin response element by
site-directed mutagenesis, as previously described (6). The
NheI/BamHI fragment containing the insulin
response element was excised and replaced with double-stranded oligonucleotides containing a single IRS ( Initial studies revealed that a 32P-labeled
double-stranded oligonucleotide probe containing the IGFBP-1 insulin
response element (BP1) forms multiple nucleoprotein complexes with
nuclear extracts prepared from rat H4IIE hepatoma cells (Fig.
1A, left panel). The formation
of one complex (solid arrowhead) is inhibited by a 250-fold
molar excess of an unlabeled oligonucleotide competitor containing a
high affinity binding site for HNF-3/forkhead proteins (solid
arrowhead), and we have shown that this complex contains HNF-3 (but not other
C/EBP proteins) supershifts this complex, and Western blotting of
affinity purified proteins confirms that C/EBP
is present in this
complex. Studies with affinity purified and recombinant protein
indicate that C/EBP
does not interact directly with the IRS, but
that other factors are required. Gel shift assays and reporter gene studies with constructs containing point mutations within the IRS
reveal that the ability to interact with factors required for the
formation of this complex correlates well with the ability of insulin
to regulate promoter activity via this IRS (r = 0.849, p < 0.01). Replacing the IRS in reporter gene
constructs with a C/EBP-binding site (but not an HNF-3/forkhead
site or cAMP response element) maintains the effect of insulin on
promoter activity. Together, these findings indicate that a
nucleoprotein complex containing C/EBP
interacts with IRSs from the
IGFBP-1 and PEPCK genes in a sequence-specific fashion and may
contribute to the ability of insulin to regulate gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Gel shift and reporter gene studies with constructs
containing point mutations within the IRS indicate that this complex
interacts with an IRS in a sequence-specific fashion that correlates
well with the ability of insulin to regulate promoter activity. Also, replacing the IRS with a consensus C/EBP-binding site (but not an
HNF-3-binding site or cAMP response element (CRE)) maintains the effect
of insulin on promoter activity. Taken together, these studies indicate
that a nucleoprotein complex containing C/EBP
can interact with IRSs
in both the IGFBP-1 and PEPCK genes and may contribute to the ability
of insulin to suppress promoter function.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(sc-61), C/EBP
(sc-150), CEBP
(sc-151), ATF-1 (sc-270,
which also recognizes CREB 1 p43 and CREM-1), ATF-3 (sc-188),
ATF-4/CREB-2 (sc-200), c-Jun (sc-44, which also recognizes with JunB
and JunD p39), c-Fos (sc-253, which also recognizes FosB, Fra-1, and
Fra-2), Rb p110 (sc-50-G), NF
p50 (sc-7178, which also recognizes
NF
p105), NF
p60 (sc-7151), YY1 (sc-281), FKHR (sc-9809),
FKHRL1 (sc-9813), and the glucocorticoid receptor (sc-1003) were
obtained from Santa Cruz Biotechnology. Antibody against HNF-3
and
HNF-3
were provided by Dr. Robert Costa, and antiserum against
HNF-3
was provided by Dr. Eseng Lai. Rabbit polyclonal antiserum
specific for C/EBP
was provided by Dr. K. Calame, and rabbit
antiserum against the C-terminal and N-terminal regions of C/EBP
were provided by Drs. S. McKnight and P. Johnson, respectively.
Full-length and truncated recombinant C/EBP
was provided by S. McKnight. The bacterial expression vector for GST-CHOP and eukaryotic
expression vectors for CHOP and CHOP-LZ
were provided by
Dr. D. Ron.
IRS.1 oligo as a
probe. Fractions forming the nucleoprotein complex containing C/EBP
were pooled, dialyzed against 0.1 M KCl, 20 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 2.5 mM MgCl2, 0.1% Nonidet P-40, and
conductivity was adjusted to 0.1 M KCl prior to affinity chromatography.
IRS.2 (round 1 and 3) or the
IRS.1 oligo (round 2 of affinity
chromatography) labeled as a probe, thereby ensuring that the purified
proteins would interact both with the affinity matrix and with two
different oligonucleotide probes containing an IRS.
. The membrane was probed with a
second antibody tagged with horseradish peroxidase and the bound
antibody was identified by enhanced chemiluminesence (Amersham
Pharmacia Biotech).
IRS.1 and
IRS.2)
with/without mutations of residues within the IRS (
IRS.1 m1-
IRS.1
m6,
IRS.1M, and
IRS.2M), or with oligonucleotides containing a
consensus binding site for C/EBP (
C/EBP and
C/EBP.2) or HNF-3
proteins (
HNF-3) or the CRE from the PEPCK promoter (
CRE), as
shown in Tables I and II. A functional or mutated IRS or C/EBP-binding site also was cloned into the polylinker region in pGL2 further upstream, as before (6). HepG2 hepatoma cells were transfected with
calcium phosphate precipitates containing 3 µg of reporter gene
constructs, 2 µg of a cytomegalovirus-driven expression vector for
-galactosidase, and 5 µg of empty vector. Transfected cells were refed with serum-free medium with/without 100 nM human
insulin 18 h prior to lysis and analysis of luciferase activity
(6).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
based on supershift studies (9). An unlabeled oligo containing the IRS
and flanking sequences from the PEPCK gene partially inhibits the
formation of this complex, consistent with studies indicating that this
region of the PEPCK gene weakly interacts with HNF-3/forkhead proteins
(8, 9).
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Fig. 1.
Gel shift with oligonucleotide probes.
A, H4IIE nuclear extracts. Nuclear proteins prepared from
H4IIE hepatoma cells (10 µg/lane) were preincubated with/without a
250-fold molar excess of unlabeled competitors prior to incubation with
32P-labeled oligonucleotide probes containing IRSs and
flanking sequences from the IGFBP-1 (BP1) (left) and PEPCK
gene (right). Free probe (first lane in
each panel) or binding reactions were loaded for 6% native
gel electrophoresis. Unlabeled competitors contain the IGFBP-1 insulin
response element or PEPCK IRS, or sequences known to bind
HNF-3/forkhead proteins, DBP, Sp1, or Oct1. (Note: lanes
2-5 of the left panel were included in a previous
publication (11) and are presented with permission of Elsevier
Science.) B, nuclear extracts from H4IIE cells and rat
liver. Nuclear proteins from H4IIE cells (10 µg/lane) (left
panel) or rat liver (1 µg/lane) (right panel) were
preincubated with a 250-fold excess of unlabeled oligonucleotide
competitors prior to incubation with the 32P-labeled probe
containing the IGFBP-1 insulin response element. Nucleoprotein
complexes and free probe were resolved by gel electrophoresis and
identified by autoradiography, as in A.
In contrast, the formation of a complex with lower mobility (Fig. 1, solid arrow) is inhibited effectively by both the BP1 and PEPCK oligos, but not by oligos containing binding sites for HNF-3/forkhead proteins, DBP, Sp1, or Oct1. A complex with similar mobility is formed when the oligo containing the IRS and flanking sequences from the PEPCK promoter is used as a probe (Fig. 1A, right panel, open arrow), and the formation of this complex also is inhibited by competitors containing the IGFBP-1 insulin response element or PEPCK IRS, but not by other competitors. This indicates that this competition is specific and that HNF-3 proteins are not required for the formation of this complex.
A complex with similar mobility also is formed with the BP1 probe and nuclear proteins prepared from rat liver (Fig. 1B). As with extracts from H4IIE hepatoma cells, the formation of this complex is inhibited by an excess of oligo containing the PEPCK IRS, but not by a high affinity HNF-3/forkhead-binding site. The formation of this complex is enhanced with 1 µg of nuclear proteins prepared from rat liver compared with 10 µg of protein from H4IIE hepatoma cells, suggesting some factor(s) required for formation of this complex may be enriched in liver compared with H4IIE cells.
As shown in Fig. 2A, gel shift
studies with the probe containing the IGFBP-1 insulin response element
and nuclear proteins prepared from H4IIE cells (left panel)
or rat liver (right panel) reveal that the formation of this
complex (solid arrow) is inhibited by a 250-fold excess of
the unlabeled BP1 oligo or an oligo containing a C/EBP-binding site,
but not by other competitors, suggesting that C/EBP proteins may
contribute to the formation of this complex. An excess of the C/EBP
oligo (but not other competitors) also inhibits the formation of the
corresponding complex that is formed with the probe containing the
PEPCK IRS (not shown). As shown in Fig. 2B, polyclonal goat
antibody against C/EBP (but not C/EBP
or C/EBP
) supershifts
the complex that is formed by liver nuclear proteins and either the BP1
(left panel) or PEPCK (right panel) probe,
indicating that C/EBP
is present in this complex.
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The sequences flanking IRSs in the BP1 and PEPCK probes are not
conserved (Fig. 1), suggesting that the formation of this complex
involves direct interaction with IRSs in these probes. To explore this
possibility, we created an oligonucleotide probe containing a single
IRS (CAAAACA) which has been shifted upstream from its location in the
BP1 probe to disrupt potential interactions with flanking sequences
(IRS.1), or where the IRS is returned to its initial location, but
flanking sequences are modified (
IRS.2). As shown in Fig.
3A, the
IRS.1 and BP1
probes form a complex with similar mobility. The formation of this
complex is inhibited by an excess of unlabeled oligos containing an IRS
(
IRS.1 and
IRS.2), but not by oligos where the IRS is mutated
(
IRS.1M and
IRS.2M) (Fig. 3A), supporting the concept
that interaction with the IRS is required for the formation of this
complex. Supershift studies performed in the presence of an excess of
the
IRS.1M competitor reveal that the
IRS.1 probe forms a complex
containing C/EBP
with nuclear proteins prepared from H4IIE or
HepG2 hepatoma cells (10 µg) or rat liver (1 µg) (Fig.
3B). Studies with the
IRS.2 probe yielded similar results
(not shown). These results support the concept that the formation of
this complex containing C/EBP
involves interaction with the IRS.
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To characterize this complex further, we partially purified liver
nuclear proteins forming this complex by chromatography with
heparin-Sepharose and three rounds of DNA affinity chromatography using
a matrix constructed with oligonucleotides containing an array of 3 IRSs and monitored binding activity in column eluates by gel shift
assay (Fig. 4A). Goat
polyclonal antibody against the C-terminal region of C/EBP (2 µg/lane) supershifts the major complex formed with partially purified
proteins and the
IRS.1 probe (Fig. 4B, left panel),
similar to results with crude nuclear extracts. Studies with 2 µl/lane rabbit polyclonal antiserum against the C-terminal region of
C/EBP
(C/EBP
CT) also supershifts this complex (Fig.
4B, right panel), although not as completely as 2 µg of
the purified goat antibody. Interestingly, antiserum against the
N-terminal region of C/EBP
(C/EBP
NT) does not disrupt
or supershift this complex, suggesting that this region of C/EBP
may
be masked. Western blotting indicates that 35-, 32-, and 14-kDa forms
of C/EBP
(30) are present in these partially purified proteins (Fig.
4C).
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As shown in Fig. 5A,
competitive binding studies revealed that a 10-fold molar excess of the
unlabeled IRS.1 oligo is sufficient to effectively inhibit the
ability of partially purified proteins to form this complex with the
IRS.1 probe (solid arrow). Higher titers of a competitor
containing a consensus C/EBP-binding site (TTGCGCAA;
C/EBP) are
required to inhibit the ability of the
IRS.1 probe to form this
complex. This result indicates that some factor(s) required for the
formation of this complex interacts preferentially with the IRS
compared with a consensus C/EBP-binding site.
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Also shown in Fig. 5A, partially purified proteins form a
complex with higher mobility when the oligo containing a C/EBP site is
used as a probe (open arrow), and the formation of this
complex is competitively inhibited by a 10-fold excess of the unlabeled C/EBP oligo, but not by the unlabeled
IRS.1 oligo. These findings also support the concept that some other factor(s) in addition to C/EBP
proteins contributes to the formation of the complex that is formed
with the
IRS.1 probe.
C/EBP proteins form hetero- or homodimers via a leucine zipper
dimerization domain and interaction with the DNA-binding domain of both
members of the dimer is required for binding to a canonical C/EBP-binding site (31). As shown in Fig. 5B, a recombinant GST fusion protein containing CHOP, a C/EBP family member which contains a leucine zipper but no functional DNA-binding domain (32),
prevents partially purified proteins from forming a complex with the
probe containing a consensus C/EBP-binding site (C/EBP), but does
not prevent the formation of the complex that is formed with the
IRS.1 probe. This result also supports the concept that proteins
outside the C/EBP family are involved in forming this complex with the
IRS, and indicates that the formation of this complex prevents C/EBP
from interacting with other members of the C/EBP family members via its
leucine zipper dimerization domain.
Gel shift studies performed under conditions previously found to
optimize the binding of C/EBP proteins to the PEPCK probe (10) reveal
that recombinant C/EBP binds directly to probes containing a
consensus C/EBP site or the PEPCK IRS, but does not bind a probe
containing the IGFBP-1 insulin response element (Fig. 6A), consistent with previous
studies (10). Similarly, full-length and truncated C/EBP
(C/EBP
1-276 and C/EBP
132-276,
respectively) also bind the probe containing a consensus C/EBP site
(
C/EBP), but do not bind the
IRS.1 probe under our standard binding conditions (Fig. 6B). Together, these results
indicate that C/EBP proteins do not interact directly with IRSs in the IGFBP-1 promoter, and that another factor present in nuclear extracts is required for C/EBP
to form a complex with either the BP1 or
IRS.1 probe.
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Several transcription factors have been reported to interact with
C/EBP and form nucleoprotein complexes, including ATF4 (also known
as CREB-2) (33), c-Jun and c-Fos (34), YY1 (35), NF
(36, 37), retinoblastoma (Rb) protein p110 (38), and the
glucocorticoid receptor (39). As shown in Fig.
7A, antibodies which recognize
ATF1 (and CREB-1 and CREM-1), ATF3, ATF4/CREB-2, c-Jun (and
JunB and JunD), c-Fos (and FosB, Fra-1, and Fra-2), Rb p110, YY1,
NF
p50 (and p105) or p65 do not disrupt or supershift this
complex. Antibodies against the human glucocorticoid receptor, HNF-3
, -3
, and -3
, or against the N-terminal region of FKHR and FKHRL1, which are expressed in the liver, also do not supershift or
disrupt the formation of this complex or other complexes formed with
this probe (data not shown).
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Previous studies indicate that a complex containing a ~20-kDa protein
and C/EBP interacts with probes containing either the CRE or
the IRS in the PEPCK promoter, and that this complex has ~30-fold
greater affinity for the CRE compared with the PEPCK IRS (29). As shown
in Fig. 7B, unlabeled oligos containing the PEPCK CRE
(
CRE) or the CRE together with flanking sequences from the PEPCK
gene (CRE) do not inhibit the formation of the complex containing
C/EBP
that is formed with partially purified proteins and the
IRS.1 probe. This indicates that formation of this complex does not
require a factor that interacts preferentially with the PEPCK CRE.
To determine whether the complex containing C/EBP might contribute
to the ability of insulin to inhibit promoter activity via an IRS, we
examined the effect of altering individual residues within an IRS
(CAAAACA) on the ability of unlabeled oligonucleotides to interact with
factors required for the formation of this complex in gel shift assays,
and the ability of insulin to suppress promoter activity in reporter
gene studies. Previous studies have shown that altering residue 6 within the IRS (CAAAAgA) disrupts the ability of insulin to suppress
promoter activity via an IRS in both the IGFBP-1 and PEPCK genes (6).
Accordingly, we determined whether mutation of this residue also alters
interactions with factors required for the formation of this complex.
As shown in Fig. 8A, a 5-fold
molar excess of the unlabeled
IRS.1 oligo is sufficient to inhibit
the ability of partially purified proteins to form this complex with
the
IRS.1 probe by 66%, based on PhosphorImaging. Replacing residue
6 of the IRS (
IRS.1m6) impairs the ability of a 5-fold excess of
unlabeled oligo to interact with factors required for the formation of
this complex and inhibit its formation in gel shift assays (Fig.
8A). At the same time, higher titers of the
IRS.1m6
competitor were partially effective at inhibiting the formation of this
complex (Fig. 8A), indicating that differences in binding
activity in oligos containing point mutations within the IRS are most
clear at low titers. Based on this result, we used a 5-fold molar
excess of unlabeled competitors to examine the effects of altering
other residues within the IRS on the ability to interact with factors
required for the formation of this complex.
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As shown in Fig. 8B, altering residues 3, 4, 5, or 6 within
the IRS (IRS.1m3-
IRS.1m6) also reduces the ability of a 5-fold molar excess of unlabeled oligos to interact with factors which are
required for the formation of this complex. In contrast, altering residues 1, 2, or 7 or the IRS does not impair the ability of unlabeled
oligos to inhibit the formation of this complex. As shown in Table
I, altering residues 3, 4, 5, or 6 also
reduces the ability of insulin to inhibit promoter activity via an IRS in reporter gene studies. As shown in Fig.
9, the ability of oligonucleotides containing point mutations within the IRS to interact with factors required for the formation of this complex correlates well with the
ability of reporter gene constructs containing the same mutations to
mediate inhibitory effects of insulin on promoter activity (r = 0.849, p < 0.01). Together, these
results indicate that this complex interacts with the IRS in a
sequence-specific fashion consistent with the concept that this complex
may contribute to the ability of insulin to inhibit promoter activity
via an IRS.
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Since C/EBP is present in this complex, we next asked whether C/EBP
proteins themselves might contribute to the ability of insulin to
inhibit promoter activity. As shown in Table
II, the
IRS.1 reporter gene construct
contains a single IRS located ~100 bp upstream from the transcription
initiation site, and insulin treatment reduces promoter activity in
this construct by 55%. Substituting a single residue (
IRS.1m6) or
several residues (
IRS.1M) within the IRS disrupts the ability of
insulin to suppress promoter activity, indicating that this effect of
insulin is mediated via the IRS, as previously reported (6). Placing a
consensus C/EBP-binding site at the same location (
C/EBP) partially
maintains the ability of insulin to inhibit promoter activity, similar
to an IRS. Placing a consensus HNF-3 site (
HNF-3) or a CRE (
CRE)
at this location does not maintain the ability of insulin to suppress
promoter activity, demonstrating that this effect is specific. Control studies with FKHR expression and antisense vectors (12) confirm that
FKHR forkhead proteins do not interact with this site and that
phosphorylation of FKHR is not required for insulin to regulate promoter activity via this C/EBP-binding site (data not shown), indicating that the ability of insulin to regulate promoter activity via this C/EBP-binding site does not require interaction with FKHR-related forkhead proteins.
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Placing an IRS (but not a mutated sequence) 3 bp further downstream
(IRS.2 versus
IRS.2M) or ~230 bp further upstream
(
IRS.331 versus
IRS.331.m6) to disrupt potential
interactions with flanking sequences also maintains the ability of
insulin to suppress promoter function. In each case, placing a
C/EBP-binding site at the same location also confers an effect of
insulin on promoter activity (
C/EBP.2 and
C/EBP.331), although
not as effectively as an IRS. Together, these results indicate that
C/EBP proteins may contribute to the ability of insulin to suppress
promoter activity.
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DISCUSSION |
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To understand specific mechanisms that may mediate effects of
insulin on gene expression via an IRS, we examined nucleoprotein complexes that interact with oligonucleotide probes containing IRSs
from either the IGFBP-1 or PEPCK gene. Previous studies have shown that
recombinant C/EBP proteins can bind directly to oligonucleotide probes
containing the PEPCK IRS, but not the IGFBP-1 insulin response element
(10), and we confirmed this result. Direct binding of C/EBP proteins to
the PEPCK IRS is thought to require interaction with flanking sequences
and mutation of these sequences does not disrupt the effect of insulin
(10). These observations suggested that C/EBP proteins are not likely
to be involved in mediating the effect of insulin on promoter activity
via an IRS (10). In contrast, we find that C/EBP is present in a
complex of proteins that interacts with residues within an IRS that are
critical for the effect of insulin, and that sequences flanking the IRS
are not essential for the formation of this complex. In addition, replacing the IRS with a consensus C/EBP-binding site maintains an
effect of insulin on promoter activity. To our knowledge, these results
provide the first report indicating that a nucleoprotein complex
containing C/EBP
can interact with an IRS in a sequence-specific fashion and may contribute to the ability of insulin to suppress promoter activity.
C/EBP proteins play an important role in the regulation of gene
expression in the liver (18) and other insulin-responsive tissues (24,
40). Studies in knockout mouse models show that both C/EBP and
C/EBP
contribute to the regulation of hepatic glucose production
(20, 21, 41). C/EBP
contributes to cAMP-responsive expression of
PEPCK (42), a rate-limiting enzyme which plays a critical role in the
regulation of gluconeogenesis, and C/EBP
contributes to the
regulation of the PEPCK promoter by thyroid hormone, glucocorticoids,
and cAMP agonists (43, 44). A recent report by Yeagley et
al. (45) indicates that C/EBP proteins also may play an important
role in mediating effects of insulin on cAMP stimulated activity of the
PEPCK promoter independent of the PEPCK IRS. Together, these findings
indicate that C/EBP proteins play an important role in the
multihormonal regulation of hepatic gene expression.
Competitive binding and functional studies with constructs containing
point mutations within the IRS revealed that 4 residues (AAAC) are
essential for interactions with factors required for the formation of
this complex and for mediating effects of insulin on promoter activity.
Recent studies by Hall et al. (17) indicate that several of
these residues (AAC) also are critical for mediating effects via the
IRS in the PEPCK promoter in cAMP/dexamethasone-stimulated H4IIE
hepatoma cells, supporting the concept that similar factors may
interact with the PEPCK IRS and contribute to the ability of insulin to
regulate that gene. Of note, the consensus HNF-3-binding site
(AAACAAACATT) we introduced into a reporter gene construct (HNF-3)
also contains this core sequence, but does not confer the ability of
insulin to suppress promoter activity. We have reported that the
ability of IRSs in the IGFBP-1 and PEPCK genes to bind HNF-3 proteins
is relatively weak compared with a known high affinity HNF-3-binding
site (9). Presumably, high affinity binding of HNF-3 proteins to this
consensus sequence may prevent interaction with other factors that are
required to mediate the effect of insulin. Based on the present study,
we speculate that interactions with HNF-3 proteins must be relatively
weak to permit IRSs to interact with other factors that are critical
for the ability for insulin to regulate gene expression in
liver-derived cells, and that variations in sequences flanking the AAAC
core are important in determining the relative affinity with which IRSs
interact with insulin-responsive and/or blocking factors.
In this context, it is important to note that this core sequence also
is conserved in a recently identified consensus binding sequence for
FKHR-related forkhead proteins (including FKHR, FKHRL1, AFX, and
DAF-16) (46), which are thought to contribute to effects of insulin on
gene expression (12-16). Taken together with the results of the
present study, these observations suggest the interesting possibility
that multiple factors, including forkhead proteins and the complex
containing C/EBP, may interact with this core AAAC sequence and
contribute to the ability of insulin to regulate promoter activity via
an IRS. This concept also is suggested by previous studies indicating
that IRSs from the apolipoprotein CIII and tyrosine aminotransferase
genes also can interact with factors required for the formation of this
C/EBP
-containing complex (11), but that another IRS from the IGFBP-1
promoter (IRSB) does not (11).
Previous studies have shown that C/EBP may interact with and form
nucleoprotein complexes with a number of factors, including ATF-4
(CREB-2), c-Jun, and c-Fos, Rb p110, and NF
proteins and the glucocorticoid receptor. However, antibodies against
these factors fail to disrupt or supershift the C/EBP
-containing
complex which interacts with the IRS. FKHR-related forkhead
transcription factors are expressed in the liver and can interact
directly with IRSs in the IGFBP-1 and PEPCK genes (12, 14, 15, 47, 48) and it is interesting to speculate that C/EBP
may interact with these forkhead proteins to form a complex with the IRS. Unlabeled competitors containing the high affinity HNF-3-binding site from the
transthyretin gene have been reported to inhibit the binding of
recombinant FKHR to the IGFBP-1 insulin response element (47). However,
a competitor containing this HNF-3-binding site failed to inhibit the
formation of this C/EBP
containing complex (Figs. 1 and 2). Also,
antibodies against HNF-3/forkhead proteins, FKHR and FKHRL1 (which also
are expressed in liver cells), also failed to detect forkhead proteins
in this complex, suggesting that forkhead proteins are not required for
the formation of this complex. However, this negative results does not
entirely exclude this possibility, since epitopes required for
recognition by these antibodies may be masked by the formation of this
complex. At the same time, the observation that a consensus
C/EBP-binding site mediates effects of insulin on promoter activity
similar to an IRS indicates that C/EBP proteins may contribute directly
to the ability of insulin to suppress promoter activity via a mechanism
that is independent of forkhead proteins, and studies reported in an
accompanying paper (48) support this concept.
At the same time, it is important to note that a C/EBP site is only
partially as effective in mediating an effect of insulin on promoter
activity compared with a recognized IRS (Table II). In related studies,
we found that insulin suppresses transactivation by C/EBP, but not
C/EBP
when these proteins are expressed in HepG2 cells (48). Since
both C/EBP
and C/EBP
are expressed in HepG2 cells (49, 50),
C/EBP
may limit the ability of insulin to suppress promoter activity
through a consensus C/EBP-binding site (which can bind hetero- and
homodimers of C/EBP proteins containing C/EBP
and/or C/EBP
)
compared with an IRS (which interacts with a complex containing
C/EBP
, but not C/EBP
). Alternatively, other factors, including
forkhead proteins, may be required to mediate the full effect of
insulin on promoter activity via an IRS in HepG2 cells.
In this context, it is interesting to note that the ability of probes
containing an IRS to form this complex is enhanced in nuclear extracts
prepared from rat liver compared with extracts prepared from H4IIE and
HepG2 hepatoma cells. Since levels of C/EBP proteins are reduced in
hepatoma cells lines compared with normal liver
(49),2 nucleoprotein
complexes containing C/EBP may play a more prominent role in
mediating effects of insulin on hepatic gene expression in
vivo than might be apparent in studies performed in these
transformed cells.
In summary, the results of the present study indicate that a
nucleoprotein complex containing C/EBP can interact with IRSs in the
IGFBP-1 and PEPCK genes. Based on competitive binding data and studies
with recombinant proteins, it appears that C/EBP
does not interact
directly with the IRS in the IGFBP-1 promoter, but that some other
factor(s) is required. The ability of the IRS to interact with factors
required for the formation of this complex correlates well with the
ability of an IRS to mediate inhibitory effects of insulin on promoter
activity, and replacing the IRS with a consensus C/EBP-binding site
maintains the effect of insulin. Additional studies are required to
identify the other factor(s) required for the formation of this
complex, and to examine the relative role that this nucleoprotein
complex and C/EBP proteins play in mediating effects of insulin on
hepatic gene expression.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health, NIDDK, Grant DK41430 and the Department of Veterans Affairs Merit Review Program (to T. G. U.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Section of Rheumatology (M/C 733), Dept. of
Medicine, University of Illinois at Chicago, 900 South Ashland Ave.,
Chicago, IL 60607.
§ Present address: Automated Biotechnology, Merck & Co., Inc., 503 Louise Lane, North Wales, PA 19454.
¶ Present address: Dept. of Pathology, Rm. M158, The University of Chicago, 5841 S. Maryland, Chicago, IL 60637.
Present address: Takeda Pharmaceuticals America, Inc., 475 Half Day Rd., Suite 500, Lincolnshire, IL 60089.
** Present address: Lexicon Genetics, 4000 Research Forest Dr., The Woodlands, TX 77381-4287.
To whom correspondence should be addressed: Rm. 5A122A Research
(MP 151), VA Chicago Health Care System (West Side Division), 820 South Damen Ave., Chicago, IL 60612. Tel.: 312-666-6500 (ext. 57427); Fax: 312-455-5877; E-mail: unterman@uic.edu.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M008541200
2 A. K. Ghosh, T. G. Unterman, and P. F. Johnson, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: IRS, insulin response sequence; C/EBP, CAAT/enhancer-binding protein; CRE, cAMP response element; GST, glutathione S-transferase; HNF-3, hepatocyte nuclear factor-3; IGFBP-1, insulin-like growth factor binding protein-1; PEPCK, phosphoenolpyruvate carboxykinase; Rb, retinoblastoma; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).
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