INTRODUCTION

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-untranslated region of IGF-I exon 1 within a previously footprinted site termed HS3D (21) and showed that this segment of DNA was required for full hormonal responsiveness of IGF-I promoter 1 in osteoblasts (22). We also demonstrated by gel mobility shift assays that PGE₂ treatment induced nuclear protein binding to an HS3D oligonucleotide probe by a mechanism that did not require ongoing protein synthesis (22). Based on these observations and on the absence of a typical CRE DNA sequence within the hormonally responsive segment of IGF-I promoter 1 (22), we postulated that the IGF-I gene was regulated by PGE₂ by a potentially novel transcriptional mechanism.

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, a member of the CCAAT/enhancer-binding protein family, appears in osteoblast nuclear extracts after treatment of cells with PGE₂, that it interacts with an HS3D oligonucleotide probe with an affinity similar to that of a well characterized C/EBP site, and that it transactivates IGF-I promoter 1 and another neutral promoter only when a native HS3D sequence is present. Our results define C/EBP as a cAMP-regulated activator of IGF-I gene transcription in osteoblasts and show that the HS3D element within IGF-I promoter 1 is a functionally important binding site for this protein.

EXPERIMENTAL PROCEDURES

Antibodies to C/EBP (C-18 and 198) and to C/EBP (C-22) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Other antibodies to C/EBP (c76) and C/EBP (c150) were a gift from Dr. S. L. McKnight (University of Texas Southwestern Medical School, Dallas, TX).

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-AGAGCCTGCGCAATCGAAA-3) or four copies of a 19-bp mutated HS3D site, M6 (22) (5-AGAGCCTGTATGATCGAAA-3), plus cohesive ends for the restriction enzymes SacI and MluI, and were subcloned by standard procedures into SacI and MluI-digested RSV-LUC. The recombinants were designated +4 × WT HS3D and +4 × mut HS3D, respectively.

C/EBP expression vectors were constructed from a rat C/EBP cDNA in pHDIL6DBD (26). A segment containing the coding region was subcloned into plasmid Bluescript (Stratagene, La Jolla, CA) to make pBS-C/EBP. An EcoRI fragment from pBS-C/EBP was then inserted into EcoRI-digested pSV7d (27) and pcDNA3 (Invitrogen, Carlsbad, CA) in the appropriate orientation to generate pSV7d-C/EBP and pcDNA3-C/EBP, respectively.

C/EBP expression vectors were constructed as follows. First, the coding region of the intronless rat C/EBP gene was isolated from genomic DNA by nested PCR, using the oligonucleotide primers listed in Table I. The amplified fragment was purified, cloned between BamHI and EcoRI sites of plasmid Bluescript to make pBS-C/EBP, and sequenced in its entirety. Appropriate restriction fragments were then subcloned into expression vectors to generate pSV7d-C/EBP and pcDNA3-C/EBP.

Table I. Oligonucleotides for isolating the rat C/EBP gene by PCR Oligonucleotide Orientation Sequencea Locationb First PCR Sense 5-TGCGCGTCAGCTGGGGCTAG  87 to 68 Antisense 5-CAGTGCCCAAGAAACTGTAG 906 to 887 Second PCR Sense 5-GCGGATCCGAGGTGACAGCCCAACTTG  45 to 26 Antisense 5-GGAATTCGGTCGTTCGGAGTCTCTAAG 841 to 822 a Restriction sites used for cloning are underlined. b Relative to translation initiation site (38).

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 -galactosidase gene under SV40 promoter control (Promega Corp.) to normalize for transfection efficiency. Cultures at 50% confluent density were rinsed in serum-free medium and exposed to plasmids in the presence of Lipofectin^TM (Life Technologies, Inc.) for 3 h. The solution was then replaced with growth medium containing 5% fetal bovine serum, and the cells were incubated for 48 h until reaching confluent density. The cells then were rinsed with serum-free medium and treated for 6 h with vehicle (ethanol diluted 1:1000 or greater in serum-free medium) or 1 µM PGE₂. After incubation, the medium was aspirated, the cultures were rinsed with phosphate-buffered saline and lysed in cell lysis buffer (Promega Corp.), and luciferase activity was measured as described previously (20, 22).

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, or pcDNA3-C/EBP) and 1 ng of a vector containing the Renilla luciferase gene under control of the cytomegalovirus immediate early enhancer/promoter (pRL-CMV, Promega Corp.) to normalize for transfection efficiency. The medium was changed at 24 h after the addition of DNA, and 24 h later, following aspiration of medium and rinsing in phosphate-buffered saline, the cells were lysed and extracts were assayed, using the Dual-Luciferase Reporter Assay System (Promega Corp.). Light emission was measured with a Monolight 2010 luminometer (Analytical Luminescence, Ann Arbor, MI) by integration over 10 s of the reaction.

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 and C/EBP proteins were generated using a coupled in vitro transcription-translation system (TNT Coupled Reticulocyte Lysate Systems (Promega)) by following the manufacturer's directions. Plasmid DNA was incubated with amino acid mix minus methionine, [³⁵S]methionine (1000 Ci/mmol, Amersham Corp.), rabbit reticulocyte lysate, and T3 RNA polymerase in reaction buffer at 30 °C for 2 h. Proteins were separated by electrophoresis through 10% SDS-PAGE gels, and the dried gels were exposed to x-ray film.

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, or 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 for osteoblast cultures.

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 [-³²P]dATP (800 Ci/mmol, NEN Life Science Products), using the Klenow fragment of DNA polymerase I. Nuclear protein extracts (5-20 µg) were preincubated for 20 min on ice with up to 2 µg of poly(dI-dC) with or without unlabeled specific or nonspecific DNA competitor or antibodies in 25 mM HEPES, pH 7.6, 60 mM KCl, 7.5% glycerol, 0.1 mM EDTA, 5 mM dithiothreitol, and 0.025% bovine serum albumin. After the addition of 5 × 10⁴ cpm of DNA probe for 30 min on ice, samples were applied to a 4-20% nondenaturing polyacrylamide gradient gel (Novex, San Diego, CA) that had been pre-electrophoresed for 30 min at 12.5 V/cm at 25 °C in 45 mM Tris, 45 mM boric acid, 1 mM EDTA. Electrophoresis proceeded for 2.5 h under identical conditions. The dried gels were exposed to x-ray film at 80 °C with an intensifying screen.

Table II. Oligonucleotides used in gel mobility shift experiments Oligonucleotidea Sequence Reference HS3D wild type 5-TTCAGAGCAGATAGAGCCTGCGCAATCGAA 22 HS3D M6b 5-TTCAGAGCAGATAGAGCCTGTATGATCGAA 22 Rat albumin D site 5-GGTATGATTTTGTAATGGGGTAGG 36 Oct-1 5-TTTTAGAGGATCATGCAAAGGACGTACGAAA 48 a Top DNA strand is shown. b Mutation is underlined.

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 80 °C with intensifying screens.

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 80 °C with an intensifying screen.

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 (c150; diluted 1:1000 in blocking buffer) for 1 h at 25 °C. Subsequent steps were performed as described (32). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL Western blotting system, Amersham), followed by exposure to x-ray film.

RESULTS

In previous studies, we identified HS3D as an atypical cAMP response element in the 5-untranslated region of rat IGF-I exon 1 that mediated hormonally stimulated IGF-I gene transcription in primary rat osteoblasts (20, 22). By gel mobility shift assay, nuclear protein binding to this element was absent under basal conditions but was induced rapidly in osteoblast cultures exposed to PGE₂ (22). This DNA fragment contained a core octameric sequence, 5-CGCAATCG-3, which was required for both nuclear protein binding and transcriptional activation (22). We thus performed competition gel-mobility shift experiments to define the key nucleotides within the HS3D octameric core that were necessary for binding of PGE₂-induced nuclear proteins. A series of double-stranded oligomers 31 bp in length were generated with single nucleotide substitution mutations of purines for purines and pyrimidines for pyrimidines at eight different positions (Fig. 1A). Results with these competitor oligonucleotides indicated that only three mutants, 6B, 6D, and 7A, failed to inhibit binding of the ³²P-HS3D probe when used at a 200-fold molar excess (Fig. 2B), although mutant 6B was partially effective. Therefore, only three nucleotides within the HS3D core, 5-CGCAATCG-3 (underlined and in boldface type) appear to be critical for the binding of PGE₂-stimulated nuclear factors. The octameric HS3D core contains an optimal half-site for transcription factors of the C/EBP family, GCAAT (33), and the nucleotides within the half-site that we identified as essential for DNA-protein interactions with osteoblast nuclear proteins were recently found by others to be absolutely conserved for binding recombinant C/EBP proteins in a PCR-based binding site selection assay (34).

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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 and C/EBP mRNAs were detected in these cells. By antibody supershift experiments, we determined that an antiserum to C/EBP attenuated the DNA-protein complexes formed with Ob cell nuclear extracts, while two antibodies to C/EBP and an irrelevant antiserum to myogenin were ineffective (Fig. 3). In contrast to the closely spaced doublet formed between osteoblast nuclear proteins and ³²P-HS3D, at least three distinct DNA-protein complexes were seen when hepatic nuclear extracts were used in a parallel experiment (Fig. 3). Antibodies to C/EBP disrupted the most rapidly migrating of these bands, while antisera to C/EBP or myogenin had no effect. These results support the idea that a protein antigenically related to C/EBP comprises a major part of the HS3D-nuclear protein complex in Ob cells. By contrast, other factors, including C/EBP, are able to bind to the same DNA fragment in rat liver, where C/EBP is minimally expressed under basal physiological conditions (35).

An antibody to C/EBP interferes with the osteoblast PGE₂-induced HS3D gel shift complex.

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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 and C/EBP in different tissues.

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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 in nuclear extracts of these cells. The ~35-kDa immunoreactive protein comigrated with C/EBP generated in a linked in vitro transcription-translation system using the cloned gene as a template. The detected protein also was consistent in size with the bands reacting with a ³²P-labeled HS3D oligonucleotide probe after UV-mediated DNA-protein cross-linking and Southwestern blotting shown in Fig. 2.

PGE₂ induces the appearance of C/EBP in nuclear protein extracts.

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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 or C/EBP. Transfection with C/EBP produced a 5-fold increase in basal luciferase activity compared with no change over control values in cells receiving either the parental (empty) expression plasmid or a C/EBP expression plasmid. Treatment with PGE₂ of similarly transfected cells enhanced the activity of the IGF1711b-luciferase fusion gene in all cases (Fig. 6), in agreement with our previous results (20, 22). In addition, forced expression of C/EBP or C/EBP significantly increased the magnitude of the response to PGE₂ treatment. By contrast, co-transfection of each C/EBP expression plasmid or the empty parental vector with IGF1711c, a promoter-reporter gene that lacked the HS3D site (20), did not lead to stimulated luciferase activity in the absence or presence of PGE₂. C/EBP expression plasmids also had no effect on a promoterless reporter gene, pOLuc. These results demonstrate that C/EBP can transactivate IGF-I promoter 1 in osteoblasts through the HS3D region.

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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 in osteoblasts. Co-transfection with a C/EBP expression plasmid induced activity of a luciferase reporter gene containing four tandem copies of a 19-nucleotide HS3D oligonucleotide cloned 5 to a minimal RSV promoter (25) but did not stimulate a fusion gene with four copies of the mutant HS3D oligomer, M6, that was unable to bind to osteoblast nuclear extracts (Fig. 7). Treatment with PGE₂ further increased expression of the wild-type reporter gene. By contrast, co-transfection with either an empty vector (parental) or an expression plasmid for C/EBP did not enhance basal reporter activity; however, forced expression of C/EBP did increase the response to PGE₂ (Fig. 7). These studies show that a multimer of HS3D provides a sufficient target for hormonal activation of gene expression in osteoblasts and indicates that C/EBP and, to a lesser extent, C/EBP induce gene activation through this response element.

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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 was overexpressed contained enhanced binding activity toward a ³²P-HS3D oligonucleotide probe (Fig. 8). Treatment with PGE₂ further stimulated protein-DNA complex formation. Although forced expression of C/EBP did not alter the gel shift in nuclear extracts from control cultures, it did increase the response to PGE₂, as was observed in the functional assays.

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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 expression plasmid in COS-7 cells led to the appearance of a DNA-protein complex that co-migrated with binding activity detected in nontransfected, PGE₂-treated primary rat osteoblasts. This complex was disrupted by antisera to C/EBP but not by an antibody to C/EBP (Fig. 9). In a reciprocal experiment, nuclear extracts from COS-7 cells transfected with a C/EBP expression vector also gained DNA binding activity toward a ³²P-HS3D probe. This protein-DNA complex migrated faster on gel electrophoresis than the bands seen using C/EBP-transfected cells and was inhibited only by an antibody to C/EBP (Fig. 9).

COS

COS

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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 to a minimal RSV promoter, were co-transfected with C/EBP expression plasmids. Co-transfection of the wild-type 4 × HS3d-RSV-luciferase fusion gene with C/EBP led to a 131 ± 76-fold (mean ± S.E.) increase in enzymatic activity compared with cells co-transfected with the empty parental vector, pcDNA3 (Fig. 10A). Luciferase activity was minimally enhanced in cells receiving the C/EBP plasmid and a reporter gene containing either just the minimal RSV promoter or in addition four copies of the mutated HS3D sequence (Fig. 10A). Thus, C/EBP is necessary and sufficient to mediate transcriptional activation of a gene containing tandem HS3D sites in a nonosteoblast cell.

C/EBP activates a neutral promoter in COS-7 cells through the HS3D site.

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The stimulatory effect of C/EBP expression also was tested in COS-7 cells using increasing amounts of an expression plasmid and a fixed amount of a reporter gene containing four copies of the wild-type HS3D sequence. As little as 1 ng of a C/EBP plasmid mediated a significant increase in luciferase expression compared with cells transfected with 100 ng of the empty expression vector, and 100 ng of the C/EBP plasmid stimulated a 99 ± 23-fold (mean ± S.E.) rise in reporter gene activity. By contrast, co-transfection with 100 ng of the C/EBP expression vector had no effect (Fig. 10B). Taken together, the results in Fig. 10 demonstrate that HS3D can function as an enhancer for C/EBP in a nonosteoblast cell, and they support the role of C/EBP in regulating IGF-I gene expression in primary rat osteoblasts.

DISCUSSION

The studies presented in this report identify C/EBP as a PGE₂-activated transcriptional regulator of the IGF-I gene in primary rat osteoblast cultures and demonstrate that the HS3D element within the 5-untranslated region of exon 1 is a functionally important binding site for this protein. Osteoblast nuclear proteins of appropriate mobility on SDS-PAGE for C/EBP bound to a labeled HS3D oligonucleotide, as determined by Southwestern blotting and UV-mediated protein-DNA cross-linking. Mutational analysis of a C/EBP half-site within HS3D confirmed that it was required for osteoblast nuclear protein binding and for transcriptional activation by PGE₂ and by co-transfected C/EBP. Antibodies to C/EBP but not to C/EBP disrupted nuclear protein-HS3D DNA complexes as assessed by gel mobility shift assays. An antibody to C/EBP also recognized an appropriately sized protein by Western immunoblot of osteoblast nuclear extracts only after treatment of cells with PGE₂. Overexpression of C/EBP in primary rat osteoblast cultures led to elevated basal transcription of an IGF-I promoter-reporter gene containing the HS3D site and to enhanced stimulation after PGE₂ but had no effect on a fusion gene lacking this sequence. Forced expression of C/EBP also potentiated the response to PGE₂, but to a lesser extent than C/EBP. Hormonal responsiveness and stimulation of gene activity by C/EBP additionally were seen with a minimal promoter linked to four copies of wild-type HS3D but not to a mutated element that lacked the ability to bind nuclear proteins. Transcriptional activation of an HS3D-containing heterologous gene by C/EBP but not C/EBP also was observed in COS-7 cells. Taken together, these results show that C/EBP functions as a hormonally stimulated transcriptional activator of the IGF-I gene in osteoblasts and that these actions of C/EBP require the HS3D site located within IGF-I exon 1.

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 as the nuclear protein binding to HS3D. Previously, a rat C/EBP cDNA was isolated by expression cloning using a CRE oligonucleotide probe (38), indicating that C/EBP, like several other members of the C/EBP family (37), can bind to a CRE with high affinity. Conversely, CREB shows minimal affinity for HS3D, but C/EBP binds with an affinity similar to that of an albumin D site probe.

In some cell types, C/EBP has been shown to function as a cAMP-activated transcription factor (39). In these cells, C/EBP becomes phosphorylated by protein kinase A and is translocated to the nucleus, where it binds to target sites on specific genes, such as c-fos (39). Unlike C/EBP, C/EBP appears to lack a consensus phosphorylation site for protein kinase A and has been found not to be phosphorylated by the enzyme (38). Thus, the mechanisms of activation of C/EBP by PGE₂ or cAMP remain unclear. However, since the stimulation of IGF-I gene expression and the induction of nuclear protein binding toward HS3D by PGE₂ in osteoblasts does not require concurrent protein synthesis (22), it seems likely that a post-transcriptional mechanism is responsible. Although translocation of C/EBP to the nucleus previously has not been observed after cAMP activation in any cell type, it has been seen in hepatocytes in response to the cytokine, tumor necrosis factor- (40). In preliminary studies, we have observed a disappearance of C/EBP from the cytoplasm of PGE₂-treated osteoblasts and its rapid accumulation in nuclear extracts.² Further experiments are required to define the mechanisms of cyclic AMP-dependent protein kinase-dependent activation of C/EBP in these cells.

It is surprising that overexpression of C/EBP failed to stimulate IGF-I promoter activity under basal conditions in osteoblasts, although it enhanced the response to PGE₂ (but to a lesser extent than C/EBP). Previously published studies have indicated that expression of the highly homologous human IGF-I promoter can be induced by this protein after transient co-transfection into Hep3B cells (41). In these published experiments, a C/EBP binding site was mapped to the proximal part of promoter 1 in a location 5 to the HS3D site but also within a region of the promoter that is conserved between species (41) and present in both IGF1711b and IGF1711c luciferase plasmids used in Fig. 6. It is possible that osteoblasts have limiting amounts of an essential co-factor for gene activation by C/EBP, that C/EBP requires post-translational activation by cyclic AMP-dependent protein kinase or by another kinase in these cells, or that overexpression of C/EBP in osteoblasts leads to high level production of LIP, a transcriptional inhibitor that represents a truncated form of C/EBP derived by translation from an internal AUG codon (42). It is also surprising that overexpression of C/EBP in COS-7 cells did not activate a promoter with four copies of HS3D, since nuclear proteins from these cells contained predominantly full-length C/EBP² that did bind to an HS3D oligonucleotide in gel shift experiments. Again, perhaps a critical co-activator or activation step is absent from these cells.

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 is the nuclear factor in osteoblasts that enhances IGF-I gene transcription in response to a signal transduction pathway regulated by activation of cAMP. Our results provide an outline of the critical components of this pathway, from the cell-surface parathyroid hormone and PGE₂ receptors to the genomic HS3D target site for C/EBP on IGF-I promoter 1. Challenges for the future will be to unravel the specific intracellular mechanisms responsible for induction of C/EBP in these cells and to understand how different hormones interactively regulate expression of the IGF-I gene in bone.