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INTRODUCTION |
Insulin-like growth factor-I
(IGF-I),1 a conserved
70-residue secreted protein, plays a fundamental role in regulating
somatic growth in mammals and other vertebrate species (1, 2). IGF-I is
synthesized by many cells including osteoblasts (1, 2) and can act as a
growth and differentiation factor within the skeleton as well as in
other tissues (2-4). Production of IGF-I by skeletal cells is
controlled by local and systemic agents, including hormones (5-10).
Both parathyroid hormone and prostaglandin E2
(PGE2) stimulate IGF-I synthesis in cultured osteoblasts by enhancing IGF-I gene expression (11, 12) through mechanisms that are
secondary to hormonal induction of cAMP accumulation (5, 7, 11).
Previous studies have shown that PGE2 stimulates IGF-I gene
transcription in osteoblasts by activating promoter 1, the major IGF-I
promoter in bone (12-14) and in most other tissues (15). We have found
that induction of IGF-I transcription by PGE2 is part of a
primary hormonal response that does not require ongoing protein
synthesis (16), but like other cAMP-activated pathways, does require
the catalytic subunit of cAMP-dependent protein kinase
(13). We recently mapped a functional cAMP response element to the
5'-untranslated region of rat IGF-I exon 1 within a previously
footprinted site termed HS3D (16) and identified CCAAT/enhancer-binding
protein
(C/EBP
) as the principal cAMP-activated transcription
factor in osteoblasts that binds to and transactivates IGF-I promoter 1 through the HS3D site (17).
The C/EBP family comprises a diverse group of transcriptional
regulators with actions on tissue development and regeneration, inflammation, and intermediary metabolism (18). These proteins are
members of the basic leucine zipper family of transcription factors
(18, 19) and share strong amino acid similarity in their COOH-domains,
which contain motifs responsible for protein dimerization and DNA
binding (18). The first C/EBP proteins to be characterized, C/EBP
and C/EBP
(20-22), function as transcriptional activators and play
major roles in adipocyte differentiation and in regulating gene
expression in the liver and other tissues (18, 23-26). C/EBP
also
has been implicated in the control of adipogenesis and in mediating the
acute phase response to inflammatory stimuli (18, 23, 24). Its
potential role in controlling hormone-activated IGF-I synthesis in bone
cells had not been described until our recent report (17).
The current experiments were designed to assess interactions between
C/EBP
and the HS3D DNA element of the major IGF-I promoter from both
physical-chemical and functional perspectives. We find that C/EBP
binds to the HS3D site from the rat IGF-I gene with an affinity
equivalent to that of a known high affinity C/EBP element from the rat
albumin promoter (21, 27) and that, like C/EBP
and C/EBP
(21, 22,
28), it can form protein-protein dimers in the absence of DNA. C/EBP
also binds to HS3D sites from the human and chicken IGF-I genes with
high affinity and functions as a HS3D-dependent
cAMP-inducible transcription factor for the major human IGF-I promoter.
Taken together, our results provide evidence that C/EBP
is a
critical regulator of IGF-I gene transcription in osteoblasts and
potentially in other cell types and species.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Primary osteoblast-enriched cell cultures were
prepared from dissected and collagenase-digested parietal bones of
22-day-old Sprague-Dawley rat fetuses, as described previously (12,
29). Cells from the last 3 digestions were pooled and plated at
4800/cm2 in Dulbecco's modified Eagle's medium containing
20 mM HEPES, pH 7.2, 0.1 mg/ml ascorbic acid, 100 units/ml
penicillin, 100 µg/ml streptomycin (all from Life Technologies,
Inc.), and 10% fetal bovine serum (Sigma).
COS-7 cells (ATCC CRL-1651) were incubated in antibiotic-free
Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were plated at 1 × 105/60-mm
diameter tissue culture dish.
Plasmids--
Rat and human promoter 1-luciferase fusion genes
and rat +4x WT HS3D have been described (13, 15-17). A recombinant
plasmid containing four direct repeats of the 19-bp human HS3D sequence was constructed by ligating the appropriate synthetic double-stranded oligonucleotides into the SacI and MluI sites of RSV-LUC
(30), as outlined previously (17), to generate human +4x WT HS3D.
C/EBP
and C/EBP
expression vectors pcDNA3-C/EBP
, and
pcDNA3-C/EBP
have been described (17). Bacterial expression
plasmids for full-length and internally truncated C/EBP
were
constructed as follows. The 5' end of the C/EBP
coding region was
first modified by polymerase chain reaction by introducing a
BamHI site (underlined) adjacent to the ATG codon (bold)
with oligonucleotides,
5'-GCGGATCCATGAGCGCCGCTCTTTTCAG-3' and
5'-TGTGATTGCTGTTGAAGAGGT-3'. The amplified fragment was purified and
digested by BamHI and NcoI and then was inserted
into BamHI- and NcoI-digested pBS-C/EBP
(17)
to make pBS-C/EBP
/BamATG. After sequencing the amplified portion,
the entire C/EBP
coding region was inserted into BamHI-
and SalI-digested pET29a(+) (Novagen, Madison, WI) to make
pET29a-C/EBP
. In this plasmid, the C/EBP
coding sequence has been
fused in-frame to an NH2-terminal 33-residue S tag. To
generate the internally truncated bacterial C/EBP
expression plasmids, pET29a-C/EBP
-
SacII and
pET29a-C/EBP
-
NcoI, pET29a-C/EBP
was digested with
SacII or NcoI, and the plasmid-containing
fragments were religated. In recombinant protein
C/EBP
-
SacII, amino acids 23 through 152 of C/EBP
have been eliminated, whereas in C/EBP
-
NcoI, residues
2 through 68 have been deleted. A bacterial expression plasmid for
C/EBP
was constructed by directionally cloning rat C/EBP
(22)
into NcoI- and SalI-digested pET29a(+) to
generate pET29a-C/EBP
. In this plasmid, the C/EBP
coding sequence
has been fused in frame to an NH2-terminal 27 amino acid
S-tag.
Gene Transfer Experiments--
Transfection studies using
primary rat osteoblasts were performed as described previously (13,
16). IGF-I promoter 1-luciferase fusion genes were co-transfected with
C/EBP expression plasmids or the empty expression vector and with a
vector carrying the
-galactosidase gene under control of the SV40
promoter (Promega Corp., Madison, WI) to normalize for transfection
efficiency. After transfection, the cells were incubated for 48 h
until reaching confluent density. Cells then were rinsed with
serum-free medium and treated for 6 h with vehicle (ethanol
diluted 1:1000 in serum-free medium) or 1 µM
PGE2 (in serum-free medium). After incubation, the medium
was aspirated and cultures were rinsed with phosphate-buffered saline
and lysed in cell lysis buffer (Promega Corp.), and luciferase activity
was measured as described (13, 16).
Nuclear Protein Extracts--
Confluent osteoblast cultures were
deprived of serum for 20 h. Cells then were rinsed with serum-free
medium and incubated with vehicle (ethanol diluted 1:1000) or 1 µM PGE2 for up to 4 h. Medium was
aspirated, and cultures were rinsed twice with phosphate-buffered saline at 4 °C. Cells were harvested with a cell scraper and gently pelleted, and the pellets were washed with phosphate-buffered saline.
Nuclear extracts were prepared by the method of Lee et al.
(31) with minor modifications (13, 16, 17). Cells were lysed in
hypotonic buffer (10 mM HEPES, pH 7.4, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol) with 1% Triton X-100, phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium fluoride, 0.4 µM microcystin CL), and protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 2 µg/ml leupeptin, 2 µg/ml aprotinin). Nuclei were pelleted and
resuspended in hypertonic buffer containing 0.42 M NaCl,
0.2 mM EDTA, 25% glycerol, and the phosphatase and protease inhibitors indicated above. Soluble proteins released by a
30-min incubation at 4 °C were collected by centrifugation at
12000 × g for 5 min, and the supernatant was dialyzed
for 2 h against 2000 volumes of buffer (20 mM HEPES,
pH 7.4, 100 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 1 mM sodium orthovanadate, 20% glycerol) containing the protease inhibitors listed above. Protein
concentrations were determined using a modified Bradford assay
(Bio-Rad).
C/EBP
and C/EBP
were expressed in transiently transfected COS-7
cells (17). Cells were grown in 150-mm-diameter tissue culture dishes
and were transfected by calcium-phosphate precipitation using 20 µg
of the expression plasmid pcDNA3-C/EBP
. Twenty-four h later, the
medium was changed, and after an additional 24 h, cells were
harvested, and nuclear extracts were prepared as described above.
Antibodies--
Polyclonal antibodies to C/EBP
and C/EBP
were prepared in chickens (Gallina Biotech, Alberta, CA) using purified
S-tagged fusion proteins as antigens. IgY fractions were isolated from eggs by precipitation with polyethylene glycol followed by affinity purification using bacterial-derived antigen immobilized on Sepharose CL-4B (Amersham Pharmacia Biotech).
Western Blotting--
Western immunoblotting was performed after
transfer of electrophoresed proteins to nitrocellulose membranes.
Membranes were incubated in blocking buffer consisting of 5% nonfat
dry milk and 2% fetal bovine serum in TBS-T (20 mM
Tris-Cl, pH 7.6, 137 mM NaCl, 0.05% Tween 20) for 1 h
at 25 °C. Affinity-purified antibody prepared against full-length
bacterially expressed C/EBP
or C/EBP
(diluted 1:500 in blocking
buffer) was then added for 1 h at 25 °C. After washing the
membranes in TBS-T, secondary antibody (rabbit anti-chicken IgY diluted
1:1000 in blocking buffer) was added for 1 h at 25 °C.
Subsequent steps were performed as described previously (17).
Immunoreactive bands were visualized by enhanced chemiluminesence and
exposure to x-ray film.
Preparation of Recombinant C/EBP Proteins--
Recombinant
proteins were generated in bacteria as follows. Plasmids
pET29a-C/EBP
, pET29a-C/EBP
-
SacII,
pET29a-C/EBP
-
NcoI, and pET29a-C/EBP
were
transformed into the BL21(DE3) strain of Escherichia coli.
Bacterial cultures were grown to an A600 of 0.6 in 50 ml of Circlegrow (Bio101, Vista, CA) containing 30 µg/ml kanamycin and 34 µg/ml chloramphenicol and then were induced to express recombinant proteins by addition of
isopropyl-1-thio-
-D-galactopyranoside (Sigma) to a final
concentration of 1 mM for 3 h. Bacterial pellets were
harvested by centrifugation, then were resuspended into 5 ml of
binding/wash buffer (20 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 1% Triton X-100) containing 6 M urea.
Cells were lysed by one cycle of freezing and thawing followed by
sonication. Bacterial debris was removed by centrifugation. The
S-tagged proteins were purified using S-agarose (Novagen), according to
the manufacturer's protocol. Purified proteins were eluted in
binding/wash buffer supplemented with 2 M guanidine
thiocyanate and 2 M urea, followed by dialysis against 20 mM HEPES, pH 7.9, 100 mM KCl, 2 mM
EDTA, 20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride,
0.5 mM dithiothreitol for 2 h at 4 °C. The S tag
was cleaved using biotinylated thrombin as described by the supplier
(Novagen). Recombinant proteins were aliquoted and stored at
80 °C
until use.
Assay for Formation of C/EBP Homodimers--
Two µg of
truncated recombinant C/EBP
protein (C/EBP
-
NcoI)
was incubated with or without 0.01% glutaraldehyde for 10 min at
25 °C. Samples were separated by 8% polyacrylamide gel
electrophoresis followed by Coomassie Blue staining.
DNA-Protein Binding Studies--
Gel mobility shift experiments
followed previously published methods (16, 17). Oligonucleotides and
competitors are listed in Table I.
Radiolabeled double-stranded DNA probes were synthesized by annealing
complementary oligonucleotides followed by fill-in of single-stranded
overhangs with dCTP, dGTP, dTTP, and [
-32P]dATP (800 Ci/mmol, NEN Life Science Products) using the Klenow fragment of DNA
polymerase I. Nuclear protein extracts or recombinant proteins were
preincubated for 30 min on ice with 2 µg of poly(dI-dC) without or
with unlabeled specific or nonspecific DNA competitors in 25 mM HEPES, pH 7.6, 60 mM KCl, 7.5% glycerol,
0.1 mM EDTA, 5 mM dithiothreitol, and 0.05%
bovine serum albumin. After the addition of 5 × 104
cpm of DNA probe for 30 min on ice, samples were applied to 4-12 and
4-20% nondenaturing polyacrylamide gradient gels (Novex, San Diego,
CA) or a 5% nondenaturing polyacrylamide gel. The dried gels were
exposed to x-ray film at
80 °C with intensifying screens.
Quantitative DNA-protein binding studies were performed with a constant
amount of protein (50 ng of bacterial recombinant C/EBP
or 1.3 µg
of COS-7 cell nuclear extract) and increasing concentrations of
radiolabeled probe (0.5 to 100 nM). After electrophoresis, gels were dried, and the radioactivity in bands representing
protein-bound DNA and free probe was measured by phosphoimager
(Molecular Imager System, Bio-Rad). The dissociation constant
(Kd) was calculated from these data as the negative
reciprocal of the slope after results were graphed by Scatchard plot analysis.
Deoxynuclease I (DNase I) footprinting was performed as described (32,
33). End-labeled double-stranded DNA probes flanking the HS3D site in
rat IGF-I promoter 1 were generated by polymerase chain reaction, using
one end-labeled and one unlabeled oligonucleotide primer (the primers
were 5'-CTAAATCCCTCTTCTGCTTG-3' and 5'-AGATAGAGCCTGCGCAAT-3') as
previously described (31, 32). Graded amounts of recombinant bacterial
C/EBP
protein were preincubated for 15 min with poly(dI-dC) in 25 mM HEPES, pH 7.6, 60 mM KCl, 7.5% glycerol,
0.1 mM EDTA, 5 mM dithiothreitol, and 0.05%
bovine serum albumin, followed by the addition of labeled probe
(4.0 × 105 cpm/sample) and incubation for 60 min on
ice. The reaction mixture was then treated with DNase I (final
concentration 0.23 µg/ml, Worthington Biochemical Corp., Freehold,
NJ) in 2.5 mM MgCl2 and 2.5 mM
CaCl2 for 1 min at 25 °C. Nuclease treatment was
terminated by addition of 20 mM EDTA, 200 mM
NaCl, 1% sodium dodecyl sulfate, and 10 µg of yeast transfer RNA,
followed by phenol-chloroform extraction and ethanol precipitation.
Samples were analyzed after electrophoresis on an 8% polyacrylamide, 8 M urea gel and autoradiography for 16 h at
80 °C
with an intensifying screen.
Methylation interference assays were performed by published methods
(34, 35). Double-stranded DNA probes labeled at one end were
synthesized as described above. Labeled probes were methylated by
incubation with 0.2% dimethylsulfate in 50 mM sodium
cacodylate and 1 mM EDTA at 25 °C for 4 min followed by
2 cycles of ethanol precipitation. Recombinant bacterial C/EBP
protein (100 ng) was incubated with methylated labeled DNA for 30 min
on ice, and the DNA-protein complex and free probe were separated by
electrophoresis on a 5% polyacrylamide gel. The wet gels were exposed
to x-ray film, and protein-bound and free probes were isolated and
eluted. Eluted DNA was cleaved by 1 M piperidine for 30 min
at 95 °C followed by 3 cycles of lyophilization and reconstitution.
Samples were analyzed after electrophoresis on an 8% polyacrylamide, 8 M urea gel and autoradiography for 6 h at
80 °C
with an intensifying screen.
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RESULTS |
Our previous studies defined HS3D as an atypical cAMP response
element located in the 5'-untranslated region of rat IGF-I exon 1 that
mediated hormonally activated IGF-I gene transcription in primary rat
osteoblasts (13, 16). We subsequently identified C/EBP
as the
cAMP-regulated transcription factor responsible for hormonally
stimulated gene expression in these cells (17). The current experiments
were designed to investigate the physical-chemical properties of the
interactions between C/EBP
and the HS3D site and to determine
whether C/EBP
was involved as a mediator of cAMP-activated
transcription in IGF-I genes from species other than rats.
HS3D Is a High Affinity Binding Site for
C/EBP
--
Quantitative gel-mobility shift assays were used to
determine the affinity of C/EBP
for the HS3D DNA element following
the methods outlined in "Experimental Procedures." In the first
series of experiments, nuclear extracts from COS-7 cells expressing
C/EBP
(Fig. 1) were used as the source
of recombinant protein, and DNA-protein binding reactions were
performed with a constant quantity of nuclear protein (1.3 µg) and a
200-fold concentration range of 32P-labeled double-stranded
rat HS3D oligonucleotide (0.5-100 nM; Fig.
2A). Binding was saturable,
with an EC50 of ~5 nM DNA. The calculated
Kd of 4.78 nM was very similar to the
value obtained in parallel experiments using the previously described high affinity C/EBP binding site from the rat albumin promoter (Kd of 5.56 nM; Fig. 2B).

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Fig. 1.
Expression of C/EBP
in COS-7 cells. Antibodies to bacterially expressed C/EBP
or C/EBP were raised in chickens and affinity-purified as described
under "Experimental Procedures." Left panel, Western
immunoblots of bacterially expressed C/EBP (lanes 1 and
3) and C/EBP (lanes 2 and 4)
demonstrate that each antiserum recognizes the respective antigen.
Right panel, Western immunoblots of nuclear protein extracts
from COS-7 cells transiently transfected with an expression plasmid for
rat C/EBP (lanes 5 and 7) or for rat C/EBP
(lanes 6 and 8) and probed with antibodies to
each protein.
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Fig. 2.
HS3D is a high affinity binding site for
C/EBP . Quantitative gel-mobility shift
experiments were performed with nuclear extracts from COS-7 cells
transiently transfected with a C/EBP expression plasmid, as
described under "Experimental Procedures." Autoradiography was
performed for 12 h at 80 °C with intensifying screens
(left panels). DNA binding was measured by phosphoimager,
and results of this and two additional experiments are shown in the
center and right panels. Binding
curves are illustrated in the center panels, and
Scatchard plots are illustrated in the right panels
(B/F, protein-bound cpm/unbound cpm). A
32P-labeled rat HS3D double-stranded oligonucleotide probe
was used in panel A, and a 32P-labeled rat
albumin D (Alb D) site probe in panel B.
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Analogous studies were performed using full-length recombinant C/EBP
expressed and purified from E. coli (Fig.
3, lane 1). As seen with COS-7
nuclear protein extracts, binding of 32P-labeled
double-stranded rat HS3D to bacterially derived C/EBP
was saturable,
with an EC50 of ~10 nM and a calculated
Kd of 7.83 nM, approximately half that
obtained using the albumin C/EBP site as the labeled DNA probe
(Kd of 15.67 nM; Fig.
4).

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Fig. 3.
Expression and purification from E. coli of fusion proteins containing full-length and truncated
C/EBP . Photograph of purified fusion
proteins containing an NH2-terminal S-tag derived from
bacteria transformed with expression plasmids pET29a-C/EBP
(lane 1), pET29a-C/EBP - NcoI (lane
2), and pET29a-C/EBP - SacII (lane 3).
Purification on S-agarose was performed as described under
"Experimental Procedures." Approximately 250 ng of protein has been
applied to each lane of this 10% SDS-PAGE gel and
photographed after staining with Coomassie Blue.
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Fig. 4.
HS3D is a high affinity binding site for
bacterially expressed C/EBP . Quantitative
gel-mobility shift experiments were performed with
32P-labeled rat HS3D (A) or rat albumin D
(Alb D) site double-stranded oligonucleotides
(B), and full-length C/EBP was expressed and purified
from E. coli, as described under "Experimental
Procedures." DNA binding was quantified by phosphoimager, and results
of three experiments were plotted as shown. Binding curves are
illustrated in the left panels, and Scatchard plots are
illustrated in the right panels (B/F,
protein-bound CPM/unbound CPM).
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Further evidence that HS3D functioned as a high-affinity binding site
for C/EBP
was obtained from a series of cross-competition gel-mobility shift experiments, using nuclear extracts from COS-7 cells
expressing C/EBP
. As seen in Fig. 5,
unlabeled double-stranded HS3D and albumin D-site oligonucleotides
competed identically with a 32P-labeled HS3D probe for
binding to C/EBP
and competed equivalently with
32P-labeled albumin D-site DNA. Based on these results and
on the information shown in Figs. 2 and 4, we conclude that HS3D is a high affinity binding site for full-length C/EBP
.

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Fig. 5.
Cross-competition between HS3D and an albumin
D (Alb D)-site oligonucleotide. Gel-mobility
shift experiments were performed with 32P-labeled rat HS3D
or albumin D-site double-stranded probes, nuclear protein extracts from
COS-7 cells transiently transfected with a C/EBP expression plasmid,
and the indicated competitor DNAs at 15, 50, 100, and 200-fold molar
excess (HS3D and albumin D-site) or at 200-fold molar excess (Oct-1).
Autoradiography was performed for 6 h at 80 °C with
intensifying screens. DNA binding was quantified by phosphoimager and
was plotted as shown in the lower panels (squares
and solid lines, HS3D competitor; triangles and
dotted lines, albumin D-site competitor). Similar results
were seen in two additional independent experiments.
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C/EBP
Binds to HS3D as a Dimer--
Previous studies had shown
that other members of the C/EBP family, including C/EBP
and
C/EBP
, were able to bind to idealized palindromic recognition sites
as dimers (21, 22, 28). We performed experiments to assess the
stoichiometry of interactions between C/EBP
and the nonpalindromic
HS3D sequence. Bacterial fusion proteins were purified containing
full-length and internally truncated rat C/EBP
fused to an
NH2-terminal S-tag (Fig. 3, lanes 1 and
3). The truncated recombinant protein,
C/EBP
-
SacII, lacked amino acids 23 through 152 of
C/EBP
, which compose part of the transcriptional activation domain
(18). Its absence would not be predicted to alter DNA-protein binding
parameters. Both proteins could bind to the labeled HS3D probe, as
indicated by results of gel-mobility shift experiments pictured in Fig.
6, lanes 2 and 4.
As anticipated, DNA-protein complexes containing truncated C/EBP
(T:T) exhibited faster mobility on native gel electrophoresis than did
complexes with full-length protein (Fig. 6, W:W, compare lanes 4 and 2). When both C/EBP
isoforms were
mixed together before the addition of the labeled oligonucleotide
probe, an additional DNA-protein band of intermediate mobility
(W:T) was detected after gel electrophoresis and
autoradiography (lane 3), indicating formation of a
relatively stable hetero-oligomeric complex containing full-length and
truncated C/EBP
species. Thus, C/EBP
interacted with HS3D as a dimer.

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Fig. 6.
C/EBP binds
to the HS3D site as a dimer. Gel-mobility shift experiments were
performed with 32P-labeled rat HS3D DNA and 100 ng of
full-length (lane 2) or internally truncated (lane
4) S-tagged-C/EBP fusion protein or an equal mixture of both
proteins (lane 3). Autoradiographic exposure was for 4 h at 80 °C with intensifying screens. W, wild type;
T, truncated C/EBP (C/EBP - SacII);
F, free probe. A schematic diagram of full-length and
truncated C/EBP proteins appears below the autoradiogram.
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C/EBP
and C/EBP
have been shown to form protein-protein dimers in
solution even in the absence of DNA (21, 22, 28). To assess the
potential for C/EBP
to self-associate, protein cross-linking studies
were performed with the purified truncated recombinant protein,
C/EBP
-
NcoI (Fig. 3, lane 2), in the absence or presence of low concentrations of glutaraldehyde (0.01%) for 10 min
at 25 °C. As shown in Fig. 7, only the
monomeric protein of ~32 kDa was visualized after gel electrophoresis
in the absence of cross-linker, whereas a larger band of ~65 kDa
additionally was observed after incubation with glutaraldehyde. Similar
results were seen with full-length C/EBP
. Thus, like C/EBP
and
C/EBP
, C/EBP
is able to form protein-protein dimers, which can be
assembled into oligomeric DNA-protein complexes in the presence of a
high affinity element like HS3D.

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Fig. 7.
C/EBP dimerizes in
the absence of DNA. Bacterially expressed
C/EBP - NcoI (3 µg) was incubated with or without
0.01% glutaraldehylde for 10 min at 25 °C. Samples were separated
by 8% polyacrylamide gel electrophoresis followed by Coomassie Blue
staining. Similar results were observed in two additional
experiments.
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Identifying DNA-Protein Contact Sites--
We initially
characterized the HS3D element by in vitro DNaseI
footprinting with rat liver nuclear protein extracts (33) and
subsequently identified a qualitatively similar hormone-inducible DNA-protein interaction using rat osteoblast proteins (13). To
determine whether recombinant C/EBP
could recognize the same segment
of DNA as osteoblast-derived nuclear proteins, in vitro DNaseI footprinting was performed with end-labeled double-stranded DNA
probes derived from rat IGF-I promoter 1 and graded concentrations of
C/EBP
expressed and purified from bacteria. As seen in Fig. 8, recombinant C/EBP
protected the
HS3D site (nucleotides 200-214 on both strands) from nuclease
digestion, effectively recapitulating what was observed previously with
rat osteoblast nuclear proteins (13).

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Fig. 8.
C/EBP binds to the
HS3D site in rat IGF-I promoter 1 as assessed by DNaseI
footprinting. In vitro DNaseI footprinting was
performed as described under "Experimental Procedures" with
end-labeled DNA derived from rat IGF-I promoter 1 and graded
concentrations of bacterially generated C/EBP (5, 15, 50, 100, 200 ng). Autoradiographic exposure was for 16 h at 80 °C with
intensifying screens. The location of the footprint on each DNA strand
is indicated and was calibrated by including a DNA sequencing ladder in
adjacent lanes of the 6% polyacrylamide gel. The
numbers to the left of each panel correspond to
coordinates of rat IGF-I exon 1 as described (15).
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We next performed in vitro dimethylsulfate footprinting to
define some of the nucleotides directly involved in protein-DNA binding. As depicted in Fig. 9, two
guanosine residues on the upper DNA strand and three on the lower
strand that collectively span 8 bp were required for binding by
recombinant full-length C/EBP
. Methylation of these residues
inhibited binding, resulting in the accumulation of modified DNA in the
unbound fraction and its subsequent cleavage by dimethylsulfate and
piperidine. These results are in good agreement with our previous
analysis of the HS3D site by site-directed mutagenesis (16, 17).

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Fig. 9.
Identifying the nucleotide contact points for
C/EBP on HS3D DNA. Methylation
interference assays were performed as described under "Experimental
Procedures." Lanes contain control (C), bound
(B), and free (F) DNA, as indicated.
Autoradiographic exposure was for 6 h at 80 °C with
intensifying screens. Guanine residues important for binding of
C/EBP are in bold type and are marked by
asterisks on the autoradiograph and on the DNA
sequence.
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The HS3D Site Is Structurally and Functionally Conserved in IGF-I
Promoters from Different Species--
Table
II depicts an alignment of the HS3D
region of the rat IGF-I gene with analogous portions of the human,
chicken, and chum salmon genes (36-38). The 25-bp segment shown is
highly conserved, with 1 nucleotide substitution, a G to C
transversion, and 1 deletion in the human and chicken DNAs compared
with rat, and 2 substitutions and 1 deletion in salmon. The human and
chicken sequences are identical, whereas salmon HS3D differs by only a
single nucleotide. Cross-competition gel-mobility shift studies were
performed to determine the relative affinity of the human/chicken HS3D
region for C/EBP
expressed in COS-7 cells. As seen in Fig.
10, both rat and human/chicken
32P-labeled double-stranded probes gave rise to DNA-protein
complexes of identical mobility, and both unlabeled HS3D
oligonucleotides competed equivalently for binding to C/EBP
with
either 32P-labeled DNA sequence. Thus the human and chicken
HS3D sequences behave as high-affinity C/EBP
binding sites.
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Table II
Comparison of IGF-I HS3D sequences
Nucleotide differences from rat IGF-I HS3D are indicated by bold
lettering. Core HS3D sequences are underlined.
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Fig. 10.
The HS3D site of the human IGF-I promoter
binds C/EBP . Gel-mobility shift
experiments were performed with 32P-labeled rat and human
HS3D probes, nuclear extracts from COS-7 cells transiently transfected
with a C/EBP expression plasmid, and unlabeled double-stranded
competitor DNAs at 50, 100, 150, and 200-fold molar excess (except for
Oct-1, which was used at 200-fold molar excess). Autoradiographic
exposure was for 8 h at 80 °C with an intensifying screen.
Representative autoradiographs are shown in the top panel.
The mean of two experiments has been plotted in the lower
panels after quantitation by phosphoimager (squares and
solid lines, rat HS3D competitor; triangles and
dotted lines, human HS3D competitor).
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Functional analyses of the potential role of HS3D in mediating hormonal
regulation of human IGF-I gene transcription were performed with a
chimeric human IGF-I promoter 1-luciferase fusion plasmid. Transient
transfection experiments using rat primary osteoblast cultures showed
that a fragment of human promoter 1 from
1630 to +322 with respect to
the most 5' transcription start site (36) mediated a 6-fold increase in
reporter gene activity after incubation of cells with 1 µM PGE2 for 6 h (Fig.
11A). Co-transfection of the
same plasmid with an expression vector for C/EBP
led to a 5-fold
rise in luciferase activity under basal conditions when compared with
co-transfections with the empty expression plasmid and stimulated a
further 3-fold increase in IGF-I gene activation after treatment with
PGE2 (Fig. 11A).

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Fig. 11.
C/EBP
overexpression stimulates human IGF-I promoter activation in rat
osteoblasts through the HS3D site. A,
osteoblast-enriched cultures were transiently transfected with a
promoterless control plasmid, pOLuc (no promoter), or the
chimeric promoter-reporter gene, hIGF1630-Luc (hIGF-I
promoter) along with either no expression plasmid, the empty
vector, or an expression plasmid for C/EBP . After treatment with
control medium (containing ethanol vehicle) or 1 µM
PGE2 for 6 h, cytoplasmic extracts were prepared, and
luciferase activity was determined and normalized for transfection
efficiency using a co-transfected -galactosidase reporter plasmid.
B, osteoblast-enriched cultures were transiently transfected
with recombinant luciferase reporter genes containing the minimal RSV
promoter or the RSV promoter plus 4 copies of a 19-nucleotide human
HS3D oligonucleotide (RSV promoter + 4X hHS3D). Each
promoter-reporter gene was transiently co-transfected with either no
expression plasmids, the empty vector, or an expression plasmid for
C/EBP . After treatment with control medium (containing ethanol
vehicle) or 1 µM PGE2 for 6 h,
cytoplasmic extracts were prepared, and luciferase activity was
measured and normalized for transfection efficiency using a
co-transfected -galactosidase reporter. Results are shown in
panels A and B from 3 independent experiments
where n = 9. In panels A and B,
asterisks indicate the following: *, significantly different
from control cells (p < 0.05); **, significantly
different from vector-transfected cells (p < 0.05).
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To establish a specific role for the human/chicken HS3D site in
mediating PGE2-activated and C/EBP
-regulated
transcription, a reporter gene containing 4 tandem copies of the 19-bp
natural human/chicken HS3D region cloned 5' to a minimal RSV promoter was transfected into rat osteoblasts. As seen in Fig. 11B,
treatment with 1 µM PGE2 stimulated a 4-fold
increase in luciferase activity but had no significant effect on a
reporter plasmid containing the minimal RSV promoter alone.
Co-transfection with an expression plasmid for C/EBP
led to a
17-fold rise in luciferase activity under basal conditions as compared
with co-transfections with the empty expression vector, and stimulated
an additional 2-fold increase in promoter activity after incubation
with PGE2 (Fig. 11B).
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DISCUSSION |
The studies described in this report define the physical-chemical
properties and functional consequences of interactions between HS3D,
the DNA element in IGF-I promoter 1 that mediates stimulation of IGF-I
gene transcription by cAMP or PGE2 in osteoblasts (13, 16),
and C/EBP
, the key transcription factor responsible for cAMP-activated IGF-I expression in these cells (17). We show that
C/EBP
, expressed in COS-7 cells or purified as a recombinant protein
from E. coli, bound to HS3D with an affinity at least equivalent to that of the albumin D-site, a known high affinity C/EBP
binding sequence (21, 27), and that both DNA elements competed equally
for C/EBP
. C/EBP
bound to HS3D as a dimer, with protein-DNA
contact points located on guanine residues on both DNA strands within
and just adjacent to the core C/EBP half-site, GCAAT. C/EBP
also
formed protein-protein dimers in the absence of interactions with its
DNA binding site, as indicated by results of glutaraldehyde
cross-linking experiments. In conjunction with functional studies,
demonstrating cAMP-inducible transactivation by C/EBP
of human IGF-I
promoter 1 and of a reporter gene with four tandem copies of the
conserved human/chicken HS3D site, our results provide evidence that
C/EBP
is a critical activator of IGF-I gene transcription in rodent
osteoblasts and, potentially, in other cell types and species.
The chemical properties of C/EBP
assessed here resemble those of the
related factors, C/EBP
and C/EBP
. With the addition of our new
studies, it is now clear that all three proteins can rapidly form
dimers in dilute solution without a requirement for the presence of the
specific DNA binding site (Refs. 21, 22, and 28; and this report).
Dimerization is mediated by the COOH-terminal leucine zipper, which
consists a heptad of leucine repeats within a relatively preserved 35 amino acid core (18). This region and the adjacent basic DNA binding
domain are the most conserved portions of C/EBPs, having ~60%
identity among C/EBP
,
, and
(18). Dimerization appears to be
a prerequisite for DNA binding, since in previous studies with
C/EBP
, mutation of any of the leucine residues blocked recognition
of a high affinity C/EBP element (28).
Prior results using the COOH-terminal portion of C/EBP
in
dimethylsulfate footprinting experiments with an idealized
dyad-symmetrical high affinity C/EBP site had identified four
nucleotide contact points and several other sites of enhanced DNA
cleavage (34). Our observations with full-length C/EBP
and HS3D DNA
are very similar. We detected the same four protected nucleotides and
mapped an additional protection to a more 5' guanine on the lower DNA strand (Fig. 9). The slight differences between results may be explained potentially by variation in the DNA binding sites, although the 8-bp central region is identical, or by the different proteins used, a COOH-terminal fragment of C/EBP
previously (34)
versus full-length C/EBP
here.
Our previous results defined HS3D as a functionally important component
in the major IGF-I promoter from the rat (13, 16). The current studies
demonstrate that human IGF-I promoter 1 also is activated by
PGE2 and that a multimerized HS3D element from human or
chicken IGF-I promoter functions as a PGE2-induced and C/EBP
-regulated hormone response element. Based on these
observations and on the similarity of HS3D sites in IGF-I genes from
human, rat, chicken, and salmon (Table II), we tentatively predict that regulation of IGF-I transcription via C/EBP
also is conserved and
postulate that C/EBP
may be a critical intermediate in the hormonal
control of IGF-I synthesis in osteoblasts in several species. Detailed
analysis of recently generated C/EBP
-deficient mice (24) should
provide additional insights into the role of this transcription factor
in regulating production of IGF-I in bone and other tissues.
Other C/EBP isoforms also may play roles in regulating IGF-I gene
expression. We had shown previously that C/EBP
could bind to the
HS3D site and, in co-transfection experiments, could transactivate a
rat IGF-I promoter 1-luciferase reporter gene (17). Because both
C/EBP
and C/EBP
are expressed in liver, fat, and other tissues
(18, 20-22) where IGF-I mRNA also is synthesized (2, 15), it is
reasonable to expect that these proteins also may modulate IGF-I gene
transcription under different physiological conditions.
We previously found that C/EBP
was activated and IGF-I transcription
was stimulated in primary rat osteoblasts by a cyclic AMP-dependent protein kinase-dependent pathway
that did not require ongoing protein synthesis (16). Preliminary
experiments have shown that C/EBP
can be translocated from the
cytoplasm to the nucleus of osteoblasts after PGE2
treatment, even when protein synthesis is
blocked.2 Goals for the
future will be to characterize the pathways responsible for induction
of C/EBP
activity in these cells and to determine the pathways
through which C/EBP
stimulates IGF-I gene transcription.