CCAAT/Enhancer-binding Protein delta  Is a Critical Regulator of Insulin-like Growth Factor-I Gene Transcription in Osteoblasts*

Yutaka UmayaharaDagger , Julia BilliardDagger , Changhua Ji§, Michael Centrella§, Thomas L. McCarthy§, and Peter RotweinDagger

From the Dagger  Oregon Health Sciences University, Molecular Medicine Division, Department of Medicine, Portland, Oregon 97201-3098 and § Yale University School of Medicine, Section of Plastic Surgery, New Haven, Connecticut 06520-8041

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin-like growth factor-I (IGF-I) plays a major role in promoting skeletal growth by stimulating bone cell replication and differentiation. Prostaglandin E2 and other agents that induce cAMP production enhance IGF-I gene transcription in cultured rat osteoblasts through a DNA element termed HS3D, located in the proximal part of the major rat IGF-I promoter. We previously determined that CCAAT/enhancer-binding protein delta  (C/EBPdelta ) is the key cAMP-stimulated regulator of IGF-I transcription in these cells and showed that it transactivates the rat IGF-I promoter through the HS3D site. We now have defined the physical-chemical properties and functional consequences of the interactions between C/EBPdelta and HS3D. C/EBPdelta , expressed in COS-7 cells or purified as a recombinant protein from Escherichia 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, and both DNA elements competed equally for C/EBPdelta . C/EBPdelta 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, as determined by methylation interference footprinting. C/EBPdelta also formed protein-protein dimers in the absence of interactions with its DNA binding site, as indicated by results of glutaraldehyde cross-linking studies. As established by competition gel-mobility shift experiments, the conserved HS3D sequence from rat, human, and chicken also bound C/EBPdelta with similar affinity. We also found that prostaglandin E2-induced expression of reporter genes containing human IGF-I promoter 1 or four tandem copies of the human HS3D element fused to a minimal promoter and show that these effects were enhanced by a co-transfected C/EBPdelta expression plasmid. Taken together, our results provide evidence that C/EBPdelta is a critical activator of IGF-I gene transcription in osteoblasts and potentially in other cell types and species.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 delta  (C/EBPdelta ) 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/EBPalpha and C/EBPbeta (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/EBPdelta 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/EBPdelta and the HS3D DNA element of the major IGF-I promoter from both physical-chemical and functional perspectives. We find that C/EBPdelta 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/EBPalpha and C/EBPbeta (21, 22, 28), it can form protein-protein dimers in the absence of DNA. C/EBPdelta 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/EBPdelta is a critical regulator of IGF-I gene transcription in osteoblasts and potentially in other cell types and species.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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/EBPdelta and C/EBPbeta expression vectors pcDNA3-C/EBPdelta , and pcDNA3-C/EBPbeta have been described (17). Bacterial expression plasmids for full-length and internally truncated C/EBPdelta were constructed as follows. The 5' end of the C/EBPdelta 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/EBPdelta (17) to make pBS-C/EBPdelta /BamATG. After sequencing the amplified portion, the entire C/EBPdelta coding region was inserted into BamHI- and SalI-digested pET29a(+) (Novagen, Madison, WI) to make pET29a-C/EBPdelta . In this plasmid, the C/EBPdelta coding sequence has been fused in-frame to an NH2-terminal 33-residue S tag. To generate the internally truncated bacterial C/EBPdelta expression plasmids, pET29a-C/EBPdelta -Delta SacII and pET29a-C/EBPdelta -Delta NcoI, pET29a-C/EBPdelta was digested with SacII or NcoI, and the plasmid-containing fragments were religated. In recombinant protein C/EBPdelta -Delta SacII, amino acids 23 through 152 of C/EBPdelta have been eliminated, whereas in C/EBPdelta -Delta NcoI, residues 2 through 68 have been deleted. A bacterial expression plasmid for C/EBPbeta was constructed by directionally cloning rat C/EBPbeta (22) into NcoI- and SalI-digested pET29a(+) to generate pET29a-C/EBPbeta . In this plasmid, the C/EBPbeta 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 beta -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/EBPdelta and C/EBPbeta 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/EBPdelta . 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/EBPdelta and C/EBPbeta 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/EBPdelta or C/EBPbeta (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/EBPdelta , pET29a-C/EBPdelta -Delta SacII, pET29a-C/EBPdelta -Delta NcoI, and pET29a-C/EBPbeta 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-beta -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/EBPdelta protein (C/EBPdelta -Delta 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 [alpha -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.

                              
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Table I
Oligonucleotides used in gel mobility shift experiments

Quantitative DNA-protein binding studies were performed with a constant amount of protein (50 ng of bacterial recombinant C/EBPdelta 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/EBPdelta 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/EBPdelta 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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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/EBPdelta 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/EBPdelta and the HS3D site and to determine whether C/EBPdelta 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/EBPdelta -- Quantitative gel-mobility shift assays were used to determine the affinity of C/EBPdelta 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/EBPdelta (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/EBPdelta in COS-7 cells. Antibodies to bacterially expressed C/EBPdelta or C/EBPbeta were raised in chickens and affinity-purified as described under "Experimental Procedures." Left panel, Western immunoblots of bacterially expressed C/EBPbeta (lanes 1 and 3) and C/EBPdelta (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/EBPbeta (lanes 5 and 7) or for rat C/EBPdelta (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/EBPdelta . Quantitative gel-mobility shift experiments were performed with nuclear extracts from COS-7 cells transiently transfected with a C/EBPdelta 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.

Analogous studies were performed using full-length recombinant C/EBPdelta 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/EBPdelta 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/EBPdelta . Photograph of purified fusion proteins containing an NH2-terminal S-tag derived from bacteria transformed with expression plasmids pET29a-C/EBPdelta (lane 1), pET29a-C/EBPdelta -Delta NcoI (lane 2), and pET29a-C/EBPdelta -Delta 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/EBPdelta . 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/EBPdelta 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).

Further evidence that HS3D functioned as a high-affinity binding site for C/EBPdelta was obtained from a series of cross-competition gel-mobility shift experiments, using nuclear extracts from COS-7 cells expressing C/EBPdelta . 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/EBPdelta 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/EBPdelta .


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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/EBPdelta 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.

C/EBPdelta Binds to HS3D as a Dimer-- Previous studies had shown that other members of the C/EBP family, including C/EBPalpha and C/EBPbeta , 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/EBPdelta and the nonpalindromic HS3D sequence. Bacterial fusion proteins were purified containing full-length and internally truncated rat C/EBPdelta fused to an NH2-terminal S-tag (Fig. 3, lanes 1 and 3). The truncated recombinant protein, C/EBPdelta -Delta SacII, lacked amino acids 23 through 152 of C/EBPdelta , 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/EBPdelta (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/EBPdelta 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/EBPdelta species. Thus, C/EBPdelta interacted with HS3D as a dimer.


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Fig. 6.   C/EBPdelta 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/EBPdelta 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/EBPdelta (C/EBPdelta -Delta SacII); F, free probe. A schematic diagram of full-length and truncated C/EBPdelta proteins appears below the autoradiogram.

C/EBPalpha and C/EBPbeta 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/EBPdelta to self-associate, protein cross-linking studies were performed with the purified truncated recombinant protein, C/EBPdelta -Delta 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/EBPdelta . Thus, like C/EBPalpha and C/EBPbeta , C/EBPdelta 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/EBPdelta dimerizes in the absence of DNA. Bacterially expressed C/EBPdelta -Delta 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.

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/EBPdelta 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/EBPdelta expressed and purified from bacteria. As seen in Fig. 8, recombinant C/EBPdelta 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/EBPdelta 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/EBPdelta (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).

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/EBPdelta . 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/EBPdelta 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/EBPdelta are in bold type and are marked by asterisks on the autoradiograph and on the DNA sequence.

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/EBPdelta 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/EBPdelta with either 32P-labeled DNA sequence. Thus the human and chicken HS3D sequences behave as high-affinity C/EBPdelta 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/EBPdelta . 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/EBPdelta 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).

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/EBPdelta 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/EBPdelta 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/EBPdelta . 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 beta -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/EBPdelta . 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 beta -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).

To establish a specific role for the human/chicken HS3D site in mediating PGE2-activated and C/EBPdelta -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/EBPdelta 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).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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/EBPdelta , the key transcription factor responsible for cAMP-activated IGF-I expression in these cells (17). We show that C/EBPdelta , 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/EBPdelta . C/EBPdelta 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/EBPdelta 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/EBPdelta 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/EBPdelta 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/EBPdelta assessed here resemble those of the related factors, C/EBPalpha and C/EBPbeta . 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/EBPalpha , beta , and delta  (18). Dimerization appears to be a prerequisite for DNA binding, since in previous studies with C/EBPalpha , 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/EBPalpha 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/EBPdelta 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/EBPalpha previously (34) versus full-length C/EBPdelta 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/EBPdelta -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/EBPdelta also is conserved and postulate that C/EBPdelta may be a critical intermediate in the hormonal control of IGF-I synthesis in osteoblasts in several species. Detailed analysis of recently generated C/EBPdelta -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/EBPbeta 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/EBPalpha and C/EBPbeta 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/EBPdelta 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/EBPdelta 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/EBPdelta activity in these cells and to determine the pathways through which C/EBPdelta stimulates IGF-I gene transcription.

    FOOTNOTES

* These studies were supported by National Institutes of Health Research Grants 5-RO1-DK37449 and 5-PO1-HD20805 (to P. R.) and by NASA Grant NAG5-6054 (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.

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., NRC3, Portland, OR 97201-3098. Tel.: 503-494-0536; Fax: 503-494-7368; E-mail: rotweinp{at}ohsu.edu.

2 Y. Umayahara, J. Billiard, C. Ji, M. Centrella, T. L. McCarthy, and Peter Rotwein, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: IGF-I, insulin-like growth factor-I; PGE2, prostaglandin E2; C/EBP, CCAAT/enhancer-binding protein; bp, base pair(s); RSV, Rous sarcoma virus.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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