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Volume 272, Number 50, Issue of December 12, 1997
pp. 31793-31800
(Received for publication, August 8, 1997)
From the Insulin-like growth factor-I (IGF-I) plays a key
role in skeletal growth by stimulating bone cell replication and
differentiation. We previously showed that prostaglandin
E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was
essential for hormonal regulation of IGF-I gene expression. We now
demonstrate that CCAAT/enhancer-binding protein (C/EBP) Insulin-like growth factor-I
(IGF-I),1 a highly conserved
multifunctional peptide with actions on somatic growth, cell survival, tissue differentiation, and intermediary metabolism (1, 2), is produced
by many tissues, including bone (3). IGF-I has been shown to be
synthesized by bone cells, including osteoblasts, and can act as both a
growth and differentiation agent within the skeleton (4-7). Expression
of IGF-I by osteoblasts is regulated by systemic and local factors (8,
9). IGF-I synthesis is enhanced by parathyroid hormone and by locally
produced prostaglandin E2 (PGE2) (8, 9), two
hormones that are involved in coupling the remodeling sequence of bone
resorption and new bone formation (10-15), and is diminished by
glucocorticoids (16), hormones that decrease bone formation. IGF-I thus
has been proposed to play a central role in skeletal growth and in bone
remodeling by acting as a key mediator in hormonal control of these
processes.
Both parathyroid hormone and PGE2 elevate intracellular
levels of cAMP in osteoblasts (9, 17), and it has been shown that
stimulation of cAMP accumulation within osteoblasts enhances production
of IGF-I and increases IGF-I mRNA abundance (8, 9, 17). Previous
studies have demonstrated that PGE2 induces IGF-I gene
transcription in primary cultures of rat osteoblasts (Ob cells) by
activating promoter 1, the major IGF-I promoter in most tissues (18,
19). We have found additionally that stimulation of transiently
transfected IGF-I promoter 1-luciferase reporter genes by
PGE2 can be mimicked by co-transfection with the catalytic
subunit of cyclic AMP-dependent protein kinase and inhibited by a mutant regulatory subunit that is unable to bind cAMP
(20), thus confirming the role of this second messenger in activating
IGF-I gene expression. In recent experiments, we mapped a functional
cAMP response element (CRE) to the 5 We now have identified the principal cAMP-activated transcription
factor in osteoblasts that binds to and transactivates IGF-I promoter 1 via the HS3D site. We show that C/EBP Antibodies to C/EBP Pregnant female Sprague-Dawley rats
were purchased from Charles River Laboratories (Raleigh, NC), and
8-week-old male Sprague-Dawley rats were purchased from
Harlan-Sprague-Dawley (Indianapolis, IN). Rats were housed in
individual cages under a 12-h light-dark cycle with free access to food
and drinking water. All protocols were approved by institutional animal
study committees.
Primary osteoblast-enriched cell cultures (Ob
cells) were prepared from the parietal bones of 22-day-old
Sprague-Dawley rat fetuses, as described previously (23). Cranial
sutures were eliminated during dissection, and the bones were digested
with collagenase for five sequential 20-min intervals. Cells from the last three digestions were pooled and then plated at
4800/cm2 in Dulbecco's modified Eagle's medium containing
20 mM HEPES (pH 7.2), 0.1 mg/ml ascorbic acid, penicillin,
and 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%
heat-inactivated fetal bovine serum. Cells were typically plated at
1 × 105/60-mm diameter tissue culture plate.
Rat IGF-I promoter 1-luciferase fusion genes have
been described previously (20, 22, 24). Other reporter genes were constructed from RSV-LUC, a recombinant vector containing the minimal
Rous sarcoma virus promoter cloned into the promoterless luciferase
plasmid pGL2 basic (Promega Corp., Madison, WI), which was a gift from
Dr. Dwight A. Towler (Washington University, St. Louis, MO) (25). A
pair of 96-bp double-stranded DNA fragments were synthesized containing
either four direct repeats of the 19-bp wild type HS3D sequence
(5 C/EBP C/EBP Table I.
Oligonucleotides for isolating the rat C/EBP Transfection studies using
primary rat osteoblasts were performed as described previously (20,
22). IGF-I promoter 1-luciferase fusion genes were co-transfected with
C/EBP expression plasmids and with a vector carrying the
COS-7 cells were transfected using the calcium phosphate precipitation
method, as described (24). Cells were plated at a density of 1 × 105/60-mm dish in complete medium and incubated overnight.
Four h after the medium was changed, DNA was added. A total of 1 µg
of reporter gene was co-transfected with up to 100 ng of expression plasmid (pcDNA3, pcDNA3-C/EBP Rat osteoblast nuclear extracts were prepared as described previously (20). 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 greater) 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. (28) with minor modifications (20). Cells were lysed in hypotonic buffer (10 mM HEPES, pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol) with phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium fluoride, 0.4 mM microcystin CL), protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 2 µg/ml leupeptin, 2 µg/ml aprotinin), and 1% Triton X-100. Nuclei were pelleted and were 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 12,000 × 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 quantitated using a modified Bradford assay (Bio-Rad). Rat liver nuclear proteins were extracted according to previously published methods (21). Protein concentrations were measured as indicated above. Nuclear extracts were aliquoted and immediately frozen in liquid nitrogen for storage. C/EBP C/EBP proteins were expressed in transiently transfected COS-7 cells.
Cells were grown in 150-mm diameter tissue culture dishes and were
transfected by calcium phosphate precipitation, as outlined above,
using 20 µg of expression vectors (pcDNA3, pcDNA3-C/EBP Gel mobility shift experiments
followed previously published methods (22). Radiolabeled
double-stranded DNA probes were synthesized by annealing complementary
oligonucleotides (Table II), followed by
fill-in of single-stranded overhangs with dCTP, dGTP, TTP, and
[
DNA-nuclear protein cross-linking by UV light was performed according
to published methods (29). Modified radiolabeled double-stranded DNA
probes were synthesized as described above with the substitution of
bromodeoxyuridine triphosphate (Sigma) for TTP. Osteoblast nuclear
protein extracts (30 µg) were preincubated for 30 min on ice as
outlined above for gel mobility shift assays. After the addition of
5 × 104 cpm of bromodeoxyuridine-modified HS3D probe
for 30 min on ice, the reaction mixture was exposed to 300-nm UV light
for 1 h at a distance of 5 cm, as described (29). After
electrophoresis by SDS-PAGE, dried gels were exposed to x-ray film at
Southwestern blotting followed a previously described protocol (30,
31). Osteoblast nuclear proteins (30 µg) were separated by SDS-PAGE
using 15% linear gels, and proteins were transferred to nitrocellulose
membranes. Following a denaturation-renaturation step using
progressively reduced concentrations of guanidine-HCl in
blocking/binding buffer (10 mM HEPES, pH 8.0, 50 mM KCl, 1 mM EDTA, 6.4 mM
MgCl2, 1 mM dithiothreitol), membranes were
incubated in the same buffer for 1 h at 25 °C. Blots then were
hybridized with 1 × 107 cpm of labeled probe in 5 ml
of buffer for 1 h at 25 °C. After washing with the same buffer,
membranes were exposed to x-ray film at Osteoblast nuclear proteins (20 µg) were
separated by SDS-PAGE and transferred to nitrocellulose membranes (32).
After membranes were blocked with 5% nonfat dry milk and 2% goat
serum in 20 mM Tris-Cl, pH 7.6, 137 mM NaCl for
1 h at 25 °C, they were incubated with an antibody to C/EBP In previous studies, we identified HS3D as an atypical cAMP
response element in the 5 Fig. 1. Identification of key nucleotides mediating PGE2-induced nuclear protein binding to the HS3D site. Panel A shows the top DNA strand of wild-type (WT) and mutated HS3D oligonucleotides (mutations in boldface type) used in competition experiments. Panel B illustrates an autoradiograph of a gel mobility shift experiment using a wild-type 32P-labeled HS3D probe, osteoblast nuclear protein extracts, and the oligonucleotides shown in panel A as unlabeled competitors at a 200-fold molar excess. Arrowheads indicate DNA-protein complexes. Autoradiographic exposure was for 18 h at 80 °C with an intensifying screen.
ns, nonspecific band.
[View Larger Version of this Image (75K GIF file)] Fig. 2. Identification of osteoblast nuclear proteins binding to a 32P-labeled HS3D oligonucleotide. Panel A, result of a protein-DNA UV-cross-linking experiment using nuclear protein extracts isolated from osteoblast-enriched cultures treated for 4 h with 1 µM PGE2. Protein-DNA complexes were resolved by electrophoresis through an SDS-polyacrylamide gel (15%), followed by exposure to x-ray film for 3 days at 80 °C with an intensifying screen. Lane 1, no
competitor; lane 2, competition with a 1000-fold molar
excess of unlabeled double-stranded DNA for wild-type HS3D; lane
3, competition with a 1000-fold excess of the HS3D mutant, M6
(22). An arrow points to the protein-DNA complex migrating
at ~40 kDa. Molecular size markers are indicated to the
left. Autoradiographic exposure was for 24 h at
80 °C with an intensifying screen. Panel B,
southwestern blotting experiments were performed as described under
"Experimental Procedures" with nuclear protein extracts isolated
from osteoblast-enriched cultures treated for 4 h with 1 µM PGE2. An arrow points to the protein band migrating at ~35 kDa. Molecular size markers are indicated to the left. Autoradiographic exposure was for
5 h at 80 °C with an intensifying screen.
[View Larger Version of this Image (56K GIF file)]
To begin to characterize the factors interacting with this element, we next performed an additional series of DNA-protein binding experiments. We were able to cross-link osteoblast nuclear proteins by UV light to a double-stranded 32P-labeled HS3D oligonucleotide containing BrdU and show that labeled proteins migrated at ~40 kDa after SDS-PAGE and autoradiography (Fig. 2A). Binding of the probe could be competed by an excess of unlabeled HS3D but was not inhibited by an equivalent excess of a double-stranded oligonucleotide, M6, that contained a mutation in a 4-nucleotide block within the HS3D core that prevented nuclear protein binding (22). A 32P-HS3D oligomer also detected a protein band of ~35 kDa by a filter binding assay (Southwestern blot) of electrophoretically fractionated osteoblast nuclear proteins (Fig. 2B). The difference in mobility between the bands in Fig. 2, A and B, may be accounted for by the mass of the HS3D oligonucleotide covalently coupled to the protein after UV cross-linking. Based on the results in Figs. 1 and 2, we next asked if C/EBP proteins
were expressed in osteoblasts and then examined their potential
presence in DNA-protein complexes. By a ribonuclease protection assay,
both C/EBP Fig. 3. An antibody to C/EBP interferes with the
osteoblast PGE2-induced HS3D gel shift complex. Gel
mobility shift experiments were performed with nuclear protein extracts
isolated from osteoblast-enriched cultures treated with 1 µM PGE2 for 4 h (lanes 1-5)
or from normal rat liver (lanes 6-10) and specific
antibodies as indicated. Lanes 1 and 6 show
results in the absence of antibodies, while other lanes show effects of
the addition of 1 µg of individual antibodies. Arrowheads
indicate DNA-protein complexes. ns, nonspecific band. Autoradiographic exposure was for 12 h at 80 °C with an
intensifying screen.
[View Larger Version of this Image (77K GIF file)]
The D site within the proximal promoter of the rat albumin gene was one
of the first high affinity sequences identified for C/EBP proteins
(36). When used in gel mobility shift experiments with osteoblast
nuclear proteins, the 32P-labeled double-stranded D site
probe shown in Table II gave rise to a DNA-protein complex of identical
mobility as found with the labeled HS3D oligomer (Fig.
4). Cross-competition studies additionally revealed that both sets of protein-DNA complexes could be
inhibited equivalently by a 200-fold molar excess of either homologous
or heterologous competitor, although neither complex was blocked by the
unrelated Oct-1 oligomer. In conjunction with results shown in Fig. 3,
these observations indicate that HS3D contains an authentic C/EBP
binding site and is capable of interacting with both C/EBP Fig. 4. Cross-competition between HS3D and a high affinity C/EBP site. Gel mobility shift experiments were performed with 32P-labeled HS3D or albumin D site probes, nuclear protein extracts from osteoblast-enriched cultures treated with 1 µM PGE2 for 4 h, and unlabeled competitor DNAs at a 200-fold molar excess. Arrowheads indicate protein-DNA complexes. Autoradiographic exposure was for 16 h at 80 °C with an intensifying screen.
[View Larger Version of this Image (79K GIF file)]
As shown previously, treatment of primary rat osteoblasts with
PGE2 induces nuclear protein binding to an HS3D probe (22). As indicated by the Western immunoblot in Fig.
5, PGE2 stimulated the
appearance of a protein antigenically related to C/EBP Fig. 5. PGE2 induces the appearance of C/EBP in nuclear protein extracts. Results are shown of a
Western blot performed with anti-C/EBP antibody (c150) and nuclear
protein extracts from osteoblast-enriched cultures treated with vehicle
(lane 1) or 1 µM PGE2 for 4 h
(lane 2). In vitro translated C/EBP
(lane 3) and C/EBP (lane 4) are included as
negative and positive controls, respectively. An arrow
indicates the detected protein. Molecular size markers are to the
left of the blot.
[View Larger Version of this Image (41K GIF file)]
Next, transient overexpression of C/EBP proteins was tested to see if
further stimulation of IGF-I transcription could be obtained in
osteoblasts. Co-transfection experiments were performed with IGF-I
promoter-luciferase reporter genes and expression plasmids for C/EBP Fig. 6. C/EBP overexpression enhances IGF-I promoter activation in osteoblasts. Osteoblast-enriched cultures were transiently transfected with chimeric promoter-reporter genes, IGF1711b/Luc (containing the HS3D element), IGF1711c/Luc (lacking HS3D through deletion), or promoterless p0Luc, along with expression plasmid pSV7d (parental), pSV7d-C/EBP , or pSV7d-C/EBP , as described under
"Experimental Procedures." After treatment with control medium
(containing ethanol vehicle) or 1 µM PGE2 for
6 h, cytoplasmic extracts were prepared, and luciferase activity
was determined. Results are shown from three independent experiments,
where n = 9. The asterisk indicates values
from C/EBP co-transfections that are statistically different from data
obtained using the parental expression vector (p < 0.05 for a comparison within similar treatments).
[View Larger Version of this Image (25K GIF file)]
Additional experiments were conducted to determine if a neutral
promoter could become responsive to PGE2 by the addition of the HS3D element and to examine transcriptional activation by forced
expression of C/EBP Fig. 7. C/EBP overexpression enhances activation of a neutral promoter containing four tandem copies of the HS3D element. Osteoblast-enriched cultures were transiently transfected with recombinant luciferase reporter genes containing four copies of a 19-nucleotide HS3D oligonucleotide cloned 5 to a minimal RSV promoter.
Construct +4 × HS3D contains wild-type HS3D sequence, while
+4 × mut HS3D contains four tandem copies of a mutant HS3D
oligomer, M6, that was unable to bind osteoblast nuclear proteins (22).
Each promoter-reporter gene was transiently transfected with pSV7d,
pSV7d-C/EBP , or pSV7d-C/EBP , as described under "Experimental
Procedures." After treatment with control medium (containing ethanol
vehicle) or 1 µM PGE2 for 6 h,
cytoplasmic extracts were prepared, and luciferase activity was
measured. Results are shown from three independent experiments, where
n = 9. The asterisk indicates values from
C/EBP co-transfections that are statistically different from results obtained with the parental expression plasmid (p < 0.05 for a comparison within similar treatments).
[View Larger Version of this Image (23K GIF file)]
To confirm the functional studies, osteoblast cultures were transiently
transfected with C/EBP expression plasmids, and nuclear protein
extracts were prepared for gel mobility shift experiments. Consistent
with the results of promoter activation studies, nuclear extracts from
untreated cultures where C/EBP Fig. 8. C/EBP overexpression enhances nuclear protein binding to the HS3D site in osteoblasts. Nuclear protein extracts were isolated from osteoblast-enriched cultures transiently transfected with pcDNA3 or with C/EBP or C/EBP expression plasmids after treatment with vehicle (Control) or 1 µM
PGE2 for 4 h. Gel mobility shift experiments were
performed with a 32P-HS3D oligonucleotide probe.
Autoradiographic exposure was for 24 h at 80 °C with an
intensifying screen.
[View Larger Version of this Image (52K GIF file)]
A nonosteoblast cell line, COS-7, was used to examine C/EBP-stimulated
DNA binding activity toward HS3D in a reconstituted cell culture
system. Transient transfection of a C/EBP Fig. 9. C/EBP proteins expressed in COS-7 cells bind to the HS3D site. Gel mobility shift experiments were performed with nuclear protein extracts isolated from osteoblast-enriched cultures treated with 1 µM PGE2 for 4 h (Ob, lanes 1-3) or with COS-7 cells expressing C/EBP (COS , lanes 4-6) or C/EBP
(COS , lanes 7-9) and specific antibodies, as
indicated. Arrows indicate DNA-protein complexes, and
arrowheads show DNA-protein-antibody complexes.
Autoradiographic exposure was for 14 h (lanes 1-3) or
5 h (lanes 4-9) at 80 °C with an intensifying
screen.
[View Larger Version of this Image (52K GIF file)]
To study functional aspects of C/EBP-regulated gene expression in COS-7
cells, reporter genes containing four copies of either the 19-bp
natural HS3D site or the mutated sequence M6, cloned 5 Fig. 10. C/EBP activates a neutral promoter in
COS-7 cells through the HS3D site. Panel A shows results of
transient co-transfections with a C/EBP expression plasmid or an
empty expression vector (100 ng each) and luciferase reporter genes (1 µg each) containing a minimal promoter (RSV promoter, RSV-LUC), or 4 copies of either a wild type or mutant HS3D site cloned 5 to the
promoter (+4 × WT HS3D and +4 × mut HS3D, respectively).
The asterisk indicates a significant enhancement of reporter
gene expression (p < 0.05) for cells transfected with
+4 × WT HS3D versus RSRV-LUC or +4 × mut HS3D.
Panel B shows effects of co-transfection of the +4 × WT HS3D reporter gene with different quantities of a C/EBP expression plasmid or with 100 ng of either the parental vector, pcDNA3, or a C/EBP expression plasmid. The asterisks
indicate significant increases in reporter gene expression for cells
co-transfected with a C/EBP expression plasmid versus
pcDNA3 (*, p < 0.05; **, p < 0.02; ***, p < 0.01). All experiments in both panels
were performed with the addition of 1 ng of a reporter gene, pRL-CMV, that expresses Renilla luciferase, which was used to correct
for transfection efficiency. Mean ± S.E. is shown of four
experiments performed in duplicate.
[View Larger Version of this Image (17K GIF file)]
The stimulatory effect of C/EBP The studies presented in this report identify C/EBP Members of the CREB/ATF family of nuclear proteins are generally
responsible for activation of gene transcription by cAMP (37). We
previously found that the kinetics of inducible protein-DNA binding at
the HS3D site after PGE2 treatment differed from the constitutive binding seen with an oligonucleotide containing a consensus CRE sequence (22), but we observed that a CRE oligomer could
compete with HS3D for nuclear protein binding. Unlike current results
with an albumin C/EBP probe, where cross-competition with HS3D was
observed, the HS3D oligonucleotide did not displace a labeled CRE probe
from nuclear proteins (22). The HS3D oligomer also did not bind to
bacterially expressed CREB or ATF-1, even after these proteins were
phosphorylated by the catalytic subunit of protein kinase, and
overexpression of CREB or ATF-1 in osteoblast cultures did not
transactivate IGF-I promoter-reporter genes containing an intact HS3D
site.2 These results are now explained with the
identification of C/EBP In some cell types, C/EBP It is surprising that overexpression of C/EBP Hormones that activate cAMP have been shown to stimulate IGF-I gene expression in other tissues, including the liver (43-45). PGE2 and dibutyryl cAMP both enhanced IGF-I synthesis in bone marrow macrophages, although both paradoxically produced a decline in steady-state levels of IGF-I mRNA (46). Similarly, human chorionic gonadotropin, a hormone that increases cAMP production, caused a decrease in abundance of IGF-I mRNA and a slight decline in the rate of IGF-I gene transcription in rat Leydig cells (47). It thus appears that the response of the IGF-I gene to cAMP may depend on as yet uncharacterized cell type-specific factors. In summary, we have demonstrated by multiple criteria that C/EBP * These studies were supported by National Institutes of Health Research Grants 5-RO1-DK37449 and 5-PO1-HD20805 (to P. R.) and 5-RO1-DK47421 (to T. L. M.), and by NASA Grant NAGW 4550 (to T. L. M.).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. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M15647. ¶ To whom correspondence and reprint requests should be addressed: Oregon Health Sciences University, Dept. of Medicine, Molecular Medicine Division, 3181 S.W. Sam Jackson Park Rd., Mail Code: NRC5, Portland, OR 97201-3098. Tel.: 503-494-0536; Fax: 503-494-1933; E-mail: rotweinp{at}ohsu.edu. 1 The abbreviations used are: IGF-I, insulin-like growth factor-I; PGE2, prostaglandin E2; C/EBP, CCAAT/enhancer-binding protein; CRE, cAMP response element; CREB, CRE-binding protein; Ob, osteoblast; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; RSV, Rous sarcoma virus. 2 Y. Umayahara, C. Ji., M. Centrella, P. Rotwein, T. L. McCarthy, unpublished observations. We thank Dr. Steven L. McKnight for the gift
of antisera to C/EBP
Volume 272, Number 50,
Issue of December 12, 1997
pp. 31793-31800
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Y. Umayahara, Y. Kajimoto, Y. Fujitani, S.-i. Gorogawa, T. Yasuda, A. Kuroda, K. Ohtoshi, S. Yoshida, D. Kawamori, Y. Yamasaki, et al. Protein Kinase C-dependent, CCAAT/Enhancer-binding Protein beta -mediated Expression of Insulin-like Growth Factor I Gene J. Biol. Chem., May 3, 2002; 277(18): 15261 - 15270. [Abstract] [Full Text] [PDF] |
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A. M. Delany, D. Durant, and E. Canalis Glucocorticoid Suppression of IGF I Transcription in Osteoblasts Mol. Endocrinol., October 1, 2001; 15(10): 1781 - 1789. [Abstract] [Full Text] [PDF] |
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E. N. Kaytor, J. L. Zhu, C.-I Pao, and L. S. Phillips Physiological Concentrations of Insulin Promote Binding of Nuclear Proteins to the Insulin-Like Growth Factor I Gene Endocrinology, March 1, 2001; 142(3): 1041 - 1049. [Abstract] [Full Text] [PDF] |
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L. Wang and M. L. Adamo Cell Density Influences Insulin-Like Growth Factor I Gene Expression in a Cell Type-Specific Manner Endocrinology, July 1, 2000; 141(7): 2481 - 2489. [Abstract] [Full Text] [PDF] |
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L. Wang, X. Wang, and M. L. Adamo Two Putative GATA Motifs in the Proximal Exon 1 Promoter of the Rat Insulin-Like Growth Factor I Gene Regulate Basal Promoter Activity Endocrinology, March 1, 2000; 141(3): 1118 - 1126. [Abstract] [Full Text] [PDF] |
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T. L. McCarthy, C. Ji, Y. Chen, K. Kim, and M. Centrella Time- and Dose-Related Interactions between Glucocorticoid and Cyclic Adenosine 3',5'-Monophosphate on CCAAT/Enhancer-Binding Protein-Dependent Insulin-Like Growth Factor I Expression by Osteoblasts Endocrinology, January 1, 2000; 141(1): 127 - 137. [Abstract] [Full Text] [PDF] |
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T. L. McCarthy, Changhua Ji, and M. Centrella Links Among Growth Factors, Hormones, and Nuclear Factors With Essential Roles in Bone Formation Critical Reviews in Oral Biology & Medicine, January 1, 2000; 11(4): 409 - 422. [Abstract] [Full Text] [PDF] |
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C. Ji, Y. Chen, T. L. McCarthy, and M. Centrella Cloning the Promoter for Transforming Growth Factor-beta Type III Receptor. BASAL AND CONDITIONAL EXPRESSION IN FETAL RAT OSTEOBLASTS J. Biol. Chem., October 22, 1999; 274(43): 30487 - 30494. [Abstract] [Full Text] [PDF] |
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T. Schinke and G. Karsenty Characterization of Osf1, an Osteoblast-specific Transcription Factor Binding to a Critical cis-acting Element in the Mouse Osteocalcin Promoters J. Biol. Chem., October 15, 1999; 274(42): 30182 - 30189. [Abstract] [Full Text] [PDF] |
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C. Ji, Y. Chen, M. Centrella, and T. L. McCarthy Activation of the Insulin-Like Growth Factor-Binding Protein-5 Promoter in Osteoblasts by Cooperative E Box, CCAAT Enhancer-Binding Protein, and Nuclear Factor-1 Deoxyribonucleic Acid-Binding Sequences Endocrinology, October 1, 1999; 140(10): 4564 - 4572. [Abstract] [Full Text] |
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J.-L. Zhu, C.-I Pao, E. Hunter Jr., K.-w. M. Lin, G.-j. Wu, and L. S. Phillips Identification of Core Sequences Involved in Metabolism-Dependent Nuclear Protein Binding to the Rat Insulin-Like Growth Factor I Gene Endocrinology, October 1, 1999; 140(10): 4761 - 4771. [Abstract] [Full Text] |
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J. P. O'Rourke, G. C. Newbound, J. A. Hutt, and J. DeWille CCAAT/Enhancer-binding Protein delta Regulates Mammary Epithelial Cell G0 Growth Arrest and Apoptosis J. Biol. Chem., June 4, 1999; 274(23): 16582 - 16589. [Abstract] [Full Text] [PDF] |
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Y. Umayahara, J. Billiard, C. Ji, M. Centrella, T. L. McCarthy, and P. Rotwein CCAAT/Enhancer-binding Protein delta Is a Critical Regulator of Insulin-like Growth Factor-I Gene Transcription in Osteoblasts J. Biol. Chem., April 9, 1999; 274(15): 10609 - 10617. [Abstract] [Full Text] [PDF] |
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L. M. Grønning, M. K. Dahle, K. A. Taskén, S. Enerbäck, L. Hedin, K. Taskén, and H. K. Knutsen Isoform-Specific Regulation of the CCAAT/Enhancer-Binding Protein Family of Transcription Factors by 3',5'-Cyclic Adenosine Monophosphate in Sertoli Cells Endocrinology, February 1, 1999; 140(2): 835 - 843. [Abstract] [Full Text] |
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Y. Kim and S. M. Fischer Transcriptional Regulation of Cyclooxygenase-2 in Mouse Skin Carcinoma Cells. REGULATORY ROLE OF CCAAT/ENHANCER-BINDING PROTEINS IN THE DIFFERENTIAL EXPRESSION OF CYCLOOXYGENASE-2 IN NORMAL AND NEOPLASTIC TISSUES J. Biol. Chem., October 16, 1998; 273(42): 27686 - 27694. [Abstract] [Full Text] [PDF] |
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T. L. McCarthy, C. Ji, Y. Chen, K. K. Kim, M. Imagawa, Y. Ito, and M. Centrella Runt Domain Factor (Runx)-dependent Effects on CCAAT/ Enhancer-binding Protein delta Expression and Activity in Osteoblasts J. Biol. Chem., July 7, 2000; 275(28): 21746 - 21753. [Abstract] [Full Text] [PDF] |
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G. Elberg, J. M. Gimble, and S. Y. Tsai Modulation of the Murine Peroxisome Proliferator-activated Receptor gamma 2 Promoter Activity by CCAAT/Enhancer-binding Proteins J. Biol. Chem., September 1, 2000; 275(36): 27815 - 27822. [Abstract] [Full Text] [PDF] |
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J. Billiard, Y. Umayahara, K. Wiren, M. Centrella, T. L. McCarthy, and P. Rotwein Regulated Nuclear-Cytoplasmic Localization of CCAAT/ Enhancer-binding Protein delta in Osteoblasts J. Biol. Chem., April 27, 2001; 276(18): 15354 - 15361. [Abstract] [Full Text] [PDF] |
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J. Billiard, S. S. Grewal, L. Lukaesko, P. J. S. Stork, and P. Rotwein Hormonal Control of Insulin-like Growth Factor I Gene Transcription in Human Osteoblasts. DUAL ACTIONS OF cAMP-DEPENDENT PROTEIN KINASE ON CCAAT/ENHANCER-BINDING PROTEIN delta J. Biol. Chem., August 10, 2001; 276(33): 31238 - 31246. [Abstract] [Full Text] [PDF] |
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S. Gutierrez, A. Javed, D. K. Tennant, M. van Rees, M. Montecino, G. S. Stein, J. L. Stein, and J. B. Lian CCAAT/Enhancer-binding Proteins (C/EBP) beta and delta Activate Osteocalcin Gene Transcription and Synergize with Runx2 at the C/EBP Element to Regulate Bone-specific Expression J. Biol. Chem., January 4, 2002; 277(2): 1316 - 1323. [Abstract] [Full Text] [PDF] |
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