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Endocrinology Vol. 141, No. 1 127-137
Copyright © 2000 by The Endocrine Society

ARTICLES

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 Osteoblasts1

Thomas L. McCarthy, Changhua Ji, Yun Chen, Kenneth Kim and Michael Centrella

Department of Surgery, Plastic Surgery Section, Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Thomas L. McCarthy, Ph.D., Department of Surgery, Plastic Surgery Section, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208041, New Haven, Connecticut 06520-8041. E-mail: thomas.mccarthy{at}yale.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoid has complex effects on osteoblasts. Several of these changes appear to be related to steroid concentration, duration of exposure, or specific effects on growth factor expression or activity within bone. One important bone growth factor, insulin-like growth factor I (IGF-I), is induced in osteoblasts by hormones such as PGE2 that increase intracellular cAMP levels. In this way, PGE2 activates transcription factor CCAAT/enhancer-binding protein-{delta} (C/EBP{delta}) and enhances its binding to a specific control element found in exon 1 in the IGF-I gene. Our current studies show that preexposure to glucocorticoid enhanced C/EBP{delta} and C/EBPß expression by osteoblasts and thereby potentiated IGF-I gene promoter activation in response to PGE2. Importantly, this directly contrasts with inhibitory effects on IGF-I expression that result from sustained or pharmacologically high levels of glucocorticoid exposure. Consistent with the stimulatory effect of IGF-I on bone protein synthesis, pretreatment with glucocorticoid sensitized osteoblasts to PGE2, and in this context significantly enhanced new collagen and noncollagen protein synthesis. Therefore, pharmacological levels of glucocorticoid may reduce IGF-I expression by osteoblasts and cause osteopenic disease, whereas physiological transient increases in glucocorticoid may permit or amplify the effectiveness of hormones that regulate skeletal tissue integrity. These events appear to converge on the important role of C/EBP{delta} and C/EBPß on IGF-I expression by osteoblasts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor I (IGF-I) is synthesized by many tissues, including bone, where it is a key autocrine and paracrine regulator of osteoblast activity (1, 2, 3). PTH and PGE2, which both increase bone resorption, also stimulate IGF-I synthesis by osteoblasts (4, 5). The effect of these hormones depends upon their ability to increase intracellular cAMP (6, 7, 8). Thus, the process of coupled bone remodeling may depend in part on new IGF-I synthesis by osteoblasts to replace previously resorbed bone (9).

With the availability of information and tools to examine the IGF-I gene promoter, it became possible to assess cis- and trans-acting molecular elements that account for the stimulatory effect of cAMP on IGF-I expression. In mammals the IGF-I gene is greater than 90 kb in length, and its expression originates from either of two transcriptional promoters (10). In liver, a primary source of circulating IGF-I, both gene promoters are used. In contrast, promoter 1 is the sole or dominant regulatory region of gene transcription in osteoblasts, as it is in other nonhepatic cell types (11). The cAMP responsive cis-acting region of the IGF-I gene, bp +202 to +209 (5'-CGCAATCG-3'), was initially termed HS3D by footprint analysis. This sequence occurs within a 5'-untranslated region (UTR) of exon 1 (7, 12) and appears to be the most evolutionarily conserved portion of the IGF-I gene, retaining 95% of sequence information between rat and chicken (13). More recent studies identified C/EBP{delta} as the principal cAMP-activated regulator of IGF-I gene transcription in osteoblasts, and that activated C/EBP{delta} binds specifically to HS3D to activate IGF-I gene transcription through promoter 1 (14). In the basal state, osteoblasts constitutively express C/EBP{delta}, and activation of preexisting C/EBP{delta} through a cAMP-dependent protein kinase A (PKA) pathway is sufficient to stimulate IGF-I gene expression (12).

In other tissues the C/EBP{delta} and C/EBPß isoforms are thought to be integral components of the acute phase response, where they regulate gene expression during infection, inflammation, and other conditions that engender tissue remodeling (15, 16, 17, 18, 19, 20, 21, 22, 23). C/EBPs regulate the expression of interleukin-1ß, tumor necrosis factor-{alpha}, and interleukin-6, which in bone can have important effects on osteoclast formation or activation. C/EBPs can therefore influence the expression of cytokines associated with both bone resorption as well as bone formation. Because IGF-I expression is stimulated by PTH and PGE2, which also activate bone resorption, IGF-I may have an integral, if not essential, role as a coupling factor in the bone remodeling sequence (9). Consequently, with the identification of C/EBP{delta} as the cAMP-dependent trans-activator of IGF-I expression in osteoblasts, it may also be relevant to consider C/EBPs as transcriptional coupling factors that coordinate bone remodeling in response to osteotropic hormones.

Cortisol and synthetic glucocorticoids such as dexamethasone (DEX) can have both permissive and suppressive effects on bone remodeling. These effects vary with dose and duration of treatment. For example, exposure to high doses of glucocorticoid or glucocorticoid exposure for extended periods produces clinically significant osteopenia in vivo (24, 25). In this regard, our studies and those from other laboratories showed that exposure to high doses of glucocorticoid suppresses new IGF-I gene expression by cultured osteoblasts (26, 27, 28). In contrast, intermittent exposure to glucocorticoid can enhance mineralized nodule formation by cultured osteoblasts and augment the anabolic response to PGs and PTH in cultured bone explants and isolated osteoblasts in vitro (29, 30, 31, 32, 33). The permissive effect of glucocorticoid on PG-activated osteoblasts also appears to include the IGF-I system, in part through changes in IGF-I expression and specific IGF-binding proteins (33, 34). However, molecular evidence to reconcile these opposing effects of glucocorticoid on IGF-I expression or activity has not been established. Therefore, our current studies were initiated to examine interactions between glucocorticoid and cAMP on IGF-I gene expression by osteoblasts and to assess whether they converge on changes in C/EBP expression or activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
Cells were prepared from parietal bones of 22-day-old Sprague Dawley rat fetuses (Charles River Laboratories, Inc., Raleigh, NC) using methods approved by the Yale animal care and use committee. Sutures were eliminated by dissection, and cells were released from parietal bones by five sequential 20-min collagenase digestions, as described previously (35, 36). Cells released during the last three digestions exhibit biochemical characteristics associated with differentiated osteoblasts, including high levels of PTH receptors and type I collagen synthesis, and a rise in osteocalcin expression in response to 1,25-dihydroxyvitamin D3 (36, 37). Histochemical staining demonstrates that approximately 80% of the cells express alkaline phosphatase (McCarthy, T. L., and M. Centrella, unpublished data). By these criteria, differential sensitivity to transforming growth factor-ß, bone morphogenetic protein-2, and various PGs and the ability to form mineralized nodules in vitro (38, 39, 40, 41, 42), osteoblast-enriched cultures are well distinguished from less differentiated periosteal cells. Cells pooled from the last three digestions were plated at 4800/cm2 in DMEM containing 20 mM HEPES (pH 7.2), 100 µg/ml ascorbic acid, penicillin and streptomycin (Life Technologies, Inc., Grand Island, NY), and 10% FBS (Sigma, St. Louis, MO). All treatments were carried out in serum-free medium. DEX and PGE2 (Sigma) were dissolved in 95% ethanol. Control treatments included the highest concentration of ethanol (vehicle) present in the treatment reagents. To assess IGF-I messenger RNA (mRNA) stability, cells were pretreated with vehicle or hormones and transcriptionally arrested with the RNA polymerase II inhibitor 5,6-dichloro-ß-D-ribofuranosylbenzimidizole, and the amount of mRNA remaining at various times during the next 24 h was measured (11).

mRNA analysis
Confluent cultures (~50,000 cells/cm2) were dissolved in 5 M guanidine monothiocyanate, 25 mM trisodium citrate, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol and extracted with phenol-chloroform-isoamyl alcohol (75:25:1) with 0.2 M sodium acetate (43). Total RNA was precipitated, ethanol-washed, dried, and resuspended in diethyl pyrocarbonate-treated water. mRNA levels were assessed by ribonuclease (RNase) protection assay. Antisense 32P-labeled RNA probes were prepared from cloned rat IGF-I, C/EBP{delta}, and C/EBPß constructs. A genomic probe for rat IGF-I exon 3 that contained its upstream intron was used to detect IGF-I pre-mRNA (369 nucleotides) and mature mRNA (143 nucleotides; coding sequence) present in total RNA extracts, as previously described (11). Expression plasmids encoding C/EBP{delta} and C/EBPß cloned into pcDNA3 were used to prepare [32P]complementary RNA with a Maxiscript kit (Ambion, Inc., Austin, TX). An 18S ribosomal RNA probe was 32P labeled with the T7 MEGAshortscript kit (Ambion, Inc.). The probes were gel-purified, and 8 x 104 cpm IGF-I, C/EBP{delta}, or C/EBPß [32P]complementary RNA probe were combined with 300 ng 18S probe at low specific activity (1 x 103 cpm, due to the high abundance of 18S transcripts) and 5 µg total cell RNA in hybridization buffer (80% formamide, 1 mM EDTA, 100 mM sodium citrate, and 300 mM sodium acetate, pH 6.4) at 45 C for 16 h. The samples were digested for 30 min at 37 C with RNase A (0.3 U) and RNase T1 (14 U), and then supplemented with 120 µg proteinase K and 0.5% SDS to inactivate RNases (11). Protected fragments (IGF-I pre-mRNA, 369 nt; IGF-I exon 3, 143 nt; C/EBP{delta}, 190 nt; C/EBPß, 260 nt; 18S, 80 nt) were precipitated with 1 vol isopropyl alcohol, collected by centrifugation, resolved on a 5% denaturing polyacrylamide gel, and visualized by autoradiography with Amersham Pharmacia Biotech Hyperfilm (Arlington Heights, IL) and a DuPont Cronex intensifying screen (Wilmington, DE).

Plasmids
A rat IGF-I promoter 1-luciferase fusion construct termed IGF1711b/Luc, a recombinant construct of IGF1711b/Luc with specific mutations within HS3D (HS3DmutAAA), a recombinant construct containing four tandem copies of the 19-bp wild-type HS3D sequence (5'AGAGCCTGCGCAATCGAAA-3') (italics indicate C/EBP element) termed 4X HS3D/Luc, and mutated 4X HS3D (4X HS3Dmut6, 5'AGAGCCTGTATGATCGAAA-3') (underline indicates mutated bases) containing four tandem copies of a nonfunctional HS3D element have all been previously described (7, 12, 14). HS3D contains the cAMP response element of the rat IGF-I promoter, recently identified as a functional C/EBP-binding sequence (12, 14). HS3Dmut6 fails to bind C/EBP{delta} or C/EBPß by electrophoretic mobility shift assay, and reporter construct 4X HS3Dmut6 is not activated by cAMP (12, 14). Rat C/EBP{delta} and C/EBPß complementary DNA clones (provided by Dr. Peter Rotwein, Oregon Health Sciences University), were subcloned into eukaryotic expression vector pSV7d, as previously described (14, 44). Plasmid pMMTV-Luc, which contains promoter DNA derived from the mouse mammary tumor virus (MMTV) fused upstream of luciferase, was provided by Dr. Ronald Evans (The Salk Institute, La Jolla, CA). A C/EBP{delta} dominant negative (C/EBP{delta} DN) construct was created by a SacII deletion of its activation domain. All plasmids were propagated in Escherichia coli strain DH5{alpha} with ampicillin selection and were prepared using a QIAGEN Midiprep Kit (Chatsworth, CA) and the manufacturer’s recommended protocol.

Transfections
Promoter/luciferase reporter plasmids were transfected using Lipofectin (Life Technologies, Inc., Gaithersburg, MD) at 0.6–0.75 µg DNA/4.8-cm2 culture well as previously reported (7, 12, 14). Transfection efficiency was assessed by cotransfection with a vector carrying the ß-galactosidase gene under the control of the simian virus 40 promoter. Cultures at 50% confluence were rinsed in serum-free DMEM, transfected for 3 h, and then refed with DMEM containing 5% FBS. After 24–48 h, the cells were rinsed with serum-free medium and treated for the indicated time with vehicle (ethanol diluted 1:1,000 or greater), dexamethasone (1–100 nM), or PGE2 (0.01–1 µM), as indicated. After treatment, cells were rinsed with PBS and lysed in 100 µl 25 mM Tris-phosphate (pH 7.8), 2 mM dithiothreitol (DTT), 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, and 1% Triton X-100 (cell lysis reagent, Promega Corp.). Lysates were collected, nuclei were pelleted at 12,000 x g for 5 min, and enzyme activity in supernatants was measured with commercial kits for luciferase (Promega Corp.) or ß-galactosidase (Tropix). Data were corrected for transfection efficiency and protein content (45).

Nuclear extracts
Osteoblast nuclear extracts were prepared as previously described (7, 12, 14, 46). Briefly, cell layers were rinsed twice with PBS at 4 C, and all subsequent steps were performed on ice or at 4 C. Cells were harvested, gently pelleted, and lysed in hypotonic buffer (10 mM HEPES, pH 7.4; 1.5 mM MgCl2; 10 mM KCl; and 0.5 mM DTT) with phosphatase inhibitors (1 mM sodium orthovanadate and 10 mM sodium fluoride), protease inhibitors (0.5 mM phenylmethylsulfonylfluoride, 1 µg/ml pepstatin A, 2 µg/ml leupeptin, and 2 µg/ml aprotinin; all from Sigma), and 1% Triton X-100. Nuclei were pelleted, and the cytoplasmic supernatant was collected. Nuclei were resuspended in hypertonic buffer containing 0.42 M NaCl, 0.2 mM Na2EDTA, 25% glycerol, and the phosphatase and protease inhibitors indicated above. Soluble nuclear proteins released after 30-min incubation were collected by centrifugation at 12,000 x g for 5 min, and stored in aliquots at -75 C.

Electrophoretic mobility shift assay (EMSA)
EMSA assays were conducted as previously described (12, 14, 47). Briefly, radiolabeled double stranded probe was prepared by annealing complementary oligonucleotides, followed by fill-in of single stranded overhangs with deoxy (d)-CTP, dGTP, dTTP, and [{alpha}-32P]dATP with the Klenow fragment of DNA polymerase I. Five to 10 µg nuclear protein were preincubated for 20 min on ice with 2 µg poly(dI:dC) with or without unlabeled specific or nonspecific competitor DNAs in 60 mM KCl, 25 mM HEPES (pH 7.6), 7.5% glycerol, 0.1 mM EDTA, 5 mM DTT, and 0.025% BSA. After the addition of 5 x 104 cpm DNA probe (0.1–0.2 ng) for 30 min on ice, samples were applied to a 5% nondenaturing polyacrylamide gel that had been preelectrophoresed for 30 min at 12.5 V/cm at 25 C in 45 mM Tris, 45 mM boric acid, and 1 mM EDTA and then run for 2.5 h at 12.5 V/cm. Dried gels were exposed to x-ray film at -75 C with an intensifying screen.

The probe containing the C/EBP-binding site that occurs in the IGF-I gene promoter, designated HS3D, used for gel shifts assays was 5'-GAGCAGATAGAGCCTGCGCAATCGAAATAAAGTC-3' (the C/EBP half-site is underlined). A canonical Sp1 probe (5'-ATTCGATCGGGCGGGGCGAGC-3'; the Sp1 binding motif is underlined) was used as an internal standard for select gel shift studies. Oligonucleotides were produced by Life Technologies, Inc. For supershift analyses, nuclear extracts were incubated with 1 µl specific antisera (anti-C/EBP{delta} and anti-C/EBPß antisera were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 C for 30 min before incubation with 32P-labeled oligonucleotide.

Protein synthesis
To measure protein synthesis rates, cells were pulsed with 12.5 µCi/ml [2,3-3H]proline (2.5 Ci/mmol; NEN Life Science Products, Boston, MA) for the last 2 h of culture. The cell layer was lysed by freeze-thawing, extracted in 0.5% Triton X-100 (Sigma), precipitated with 10% trichloroacetic acid, and chilled, and the acid-precipitated material was collected by centrifugation. Precipitates were acetone extracted, dried, resolubilized in 0.5 N acetic acid, and neutralized with NaOH. [3H]Proline incorporation into collagen (collagenase-digestible) and noncollagen (collagenase-resistant) protein was measured using bacterial collagenase (Worthington Biochemical Corp., Freehold, NJ) free of nonspecific protease activity and is shown as the total amount of [3H]proline incorporation into the cell layer in each culture (2). The percent collagen synthesis was calculated after correcting for the 5.4-fold greater relative abundance of proline in collagen vs. noncollagen protein.

Reagents
PGE2, DEX, and cortisol were obtained from Sigma. Antisera to C/EBP{delta} and C/EBPß were purchased from Santa Cruz Biotechnology, Inc.

Statistical analysis
Data were assessed by one-way ANOVA, with Kruskal-Wallis or Bonferonni methods for post-hoc analysis, using SigmaStat software (SPSS, Inc., Chicago, IL)


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of glucocorticoid and PGE2 on IGF-I mRNA
Our earlier studies revealed that glucocorticoid inhibits IGF-I mRNA and polypeptide expression by osteoblasts and suppresses the stimulatory effect of hormones such as PTH that increase intracellular cAMP (26). Consistent with this, when osteoblasts were treated for 16 h with 100 nM DEX and then supplemented for 6 h with 0.001–1 µM PGE2, the stimulatory effect of PGE2 on IGF-I pre-mRNA expression was significantly reduced; densitometry revealed a nearly 60% suppression of the maximally effective response to PGE2 (Fig. 1AGo, compare left and right panels). In contrast, when cells were pretreated with DEX, and the hormone was removed before stimulation with a moderate dose of PGE2 (0.1 µM), there was a synergistic, nearly 2-fold increase in PGE2 effectiveness. This effect peaked at 1 nM DEX, a dose that alone had no detectable influence on IGF-I pre-mRNA expression (Fig. 1BGo). In agreement with our earlier studies (11), the half-life of IGF-I mRNA in PGE2-treated cells was 15 h, without or with previous exposure to DEX, indicating no significant changes in IGF-I mRNA stability.



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  		Figure 1. Varied effects of glucocorticoid treatment on IGF-I mRNA. A (Left panel), Inhibitory effect of continuous high dose glucocorticoid treatment on IGF-I mRNA. Osteoblast cultures were treated for 16 h with vehicle or 100 nM DEX, and then treated for 6 h with control medium or the amounts of PGE2 indicated. Where used, DEX remained in the cultures for the full treatment interval. Relative transcript levels were determined by RNase protection assay and densitometry. B (Right panel), Permissive effect of low dose glucocorticoid pretreatment on IGF-I mRNA. Osteoblast cultures were pretreated for 16 h with vehicle or the amounts of DEX indicated, and then treated for 6 h with control medium or 0.1 µM PGE2. Where used, DEX was removed before PGE2 treatment. Due to differences in relative transcript abundance, IGF-I pre-mRNA in the top panels were assessed after a longer exposure interval by comparison to mature IGF-I mRNA and the 18S ribosomal RNA internal control in the lower panels.Data represent the results of two or three independent experiments.

 
Requirement for the C/EBP binding sequence in the IGF-I gene
To address mechanisms by which these effects occurred, osteoblasts were transfected with the cAMP-responsive IGF-I promoter/reporter gene construct, IGF1711b/Luc. Cells were pretreated with DEX and then stimulated for 6 h with PGE2. At the concentrations tested, DEX alone had no significant effect on IGF-I promoter activity even while it induced a nearly 10-fold increase in reporter gene expression driven by the glucocorticoid response elements in pMMTV-Luc (Fig. 2Go, right panel). In contrast, a 16-h preexposure to DEX dose-dependently increased the stimulatory effect of PGE2 on reporter gene expression driven by IGF1711b/Luc by 4-fold (Fig. 2Go, left panel). Consistent with its effect in PGE2-treated cells, DEX pretreatment enhanced the effect of forskolin on IGF-I promoter activity by 4.5 ± 0.1-fold, whereas the effect of a 6-h cotreatment of DEX and forskolin was essentially equivalent to that of treatment with forskolin alone (data not shown).



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  		Figure 2. Effect of glucocorticoid pretreatment on IGF-I promoter and C/EBP-dependent gene activation. Left panel, Osteoblast cultures were transfected with the IGF-I promoter construct IGF1711b/Luc and then treated for 16 h with vehicle or the amounts of DEX indicated. Cultures were subsequently treated for 6 h with control medium or with 1 µM PGE2. In the center panel osteoblast cultures were transfected with 4X HS3D/Luc, containing four tandem copies of the C/EBP-sensitive element from the IGF-I gene promoter, then treated for 16 h with vehicle or the amounts of DEX indicated. Cultures were subsequently treated for 6 h with control medium or 1 µM PGE2. Where used, DEX was removed before PGE2 treatment, although similar results occurred when DEX remained in the medium during the short PGE2 treatment interval. As a positive control for DEX treatment, parallel cultures were transfected with pMMTV-Luc and treated with the concentration of DEX indicated in the right panel. Data are the mean ± SEM and represent three independent experiments, with n = 3/experiment.

 
We earlier localized the cAMP-responsive element within the IGF-I gene to nucleotides +202 to +209 of exon 1, termed HS3D by footprint analysis, and identified it as a functional binding site for nuclear transcription factor C/EBP (7, 12, 14, 47). To assess whether this element accounted for the synergy between DEX and PGE2 on IGF-I expression, we examined changes in reporter gene activity driven by four copies of HS3D fused to a minimal viral promoter in construct 4X HS3D/Luc (14, 47). Consistent with results from studies with construct IGF1711b/Luc, pretreatment with DEX dose dependently enhanced the stimulatory effect of PGE2 on 4X HS3D/Luc (Fig. 2Go, center panel). In contrast, 6 h of cotreatment with DEX and PGE2 failed to activate 4X HS3D/Luc more than the effect of PGE2 alone (Fig. 3Go, left). Synergy between preexposure to DEX and subsequent treatment with PGE2 was undetectable when the HS3D elements were mutated to eliminate C/EBP binding within the synthetic reporter construct, 4X HS3Dmut6 (Fig. 3Go, center) or when the HS3D mutation was within the native IGF-I promoter (data not shown). No effect was evident in cells transfected with the promoterless parental construct (Fig. 3Go, right). Therefore, DEX cotreatment did not enhance the effect of cAMP on activation of preexisting C/EBP (12, 14), but the C/EBP-binding site was essential for the synergistic effect of DEX pretreatment. Notably, however, pretreatment with DEX sensitized osteoblasts to previously ineffective (0.01 µM) or marginally effective (0.1 µM) concentrations of PGE2 (Fig. 4Go, left panel). Again, no effect on IGF-I promoter activity was evident by cotreatment with DEX and PGE2, even with lower amounts of PGE2 treatment. Sensitization by DEX pretreatment also occurred with the C/EBP-dependent reporter construct 4X HS3D (Fig. 4Go, right panel).



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  		Figure 3. Effect of glucocorticoid pretreatment or cotreatment on C/EBP-dependent gene expression. Osteoblasts were transfected with empty parental vector, 4X HS3D/Luc, or 4X HS3Dmut6, which contains mutations in all four of the tandem C/EBP binding sequences. For pretreatment studies, cells were treated for 16 h with vehicle or 100 nM DEX, and then treated for 6 h with control medium or 1 µM PGE2. In these instances, DEX was removed before PGE2 treatment, although similar results occurred when DEX remained in the medium during the short PGE2 treatment interval. For cotreatment studies, cells were cotreated with DEX and PGE2 for 6 h. Data are the mean ± SEM and represent two independent experiments, with n = 3/experiment.

 


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  		Figure 4. Sensitizing effect of glucocorticoid on IGF-I gene promoter activity and C/EBP-dependent gene expression. Osteoblast cultures were transfected with either IGF-I promoter construct IGF1711b/Luc or 4X HS3D/Luc. For pretreatment studies, cells were treated for 16 h with vehicle or 100 nM DEX, and then treated for 6 h with control medium or 1 µM PGE2. In these instances, DEX was removed before PGE2 treatment, although similar results occurred when DEX remained in the medium during the short PGE2 treatment interval. For cotreatment studies, cells were cotreated with DEX and PGE2 for 6 h. Data are the mean ± SEM and represent two independent experiments, with n = 3/experiment.

 
Effects on C/EBP binding to DNA
EMSA was then used to determine the effect of DEX pretreatment on C/EBP binding, using 32P-labeled HS3D oligonucleotide and nuclear extracts from control and hormone-treated osteoblasts. As in previous studies (12, 14), nuclear extract from untreated cells produced no detectable level of nuclear factor binding, whereas as little as 4 h of treatment with PGE2 greatly enhanced DNA:protein complex formation. The two prominent complexes that formed were supershifted or eliminated by antiserum to C/EBP{delta}, whereas antiserum to C/EBPß had a lesser effect (Fig. 5AGo, first four lanes). Nuclear extract from osteoblasts pretreated with DEX formed a low, but detectable, level of complex that was sensitive to anti-C/EBP{delta} antiserum (Fig. 5AGo, lanes 5–7). Pretreatment with DEX before stimulation with PGE2 produced a 3-fold greater level of complex compared with that after treatment with PGE2 alone, similar to the effects of these agents on gene promoter activity (shown above in Figs. 2–4GoGoGo). Nuclear factor derived from cells treated in this way reacted strongly with antiserum to both C/EBP{delta} (supershifted) and C/EBPß (reduced binding; Fig. 5AGo, last three lanes). Complex formation with nuclear extract from cultures cotreated for 6 h with DEX and PGE2 was not significantly different from that with extract from cells treated with PGE2 alone (Fig. 5BGo, center panel). Furthermore, DEX and PGE2, alone or in a pretreatment protocol, did not affect the binding of transcription factor Sp1 (Fig. 5CGo, right panel), suggesting selective effects by both PGE2 and DEX on nuclear factor function.



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  		Figure 5. Effect of glucocorticoid pretreatment on C/EBP-dependent EMSA. A, Osteoblast cultures were pretreated for 16 h with vehicle or 100 nM DEX, and then treated for 4 h with control medium or 1 µM PGE2. Nuclear extracts were analyzed by EMSA with an oligonucleotide probe containing the HS3D (C/EBP)-binding sequence, without or with anti-C/EBP{delta} or anti-C/EBPß antiserum, as indicated. B, Osteoblast cultures were treated with 100 nM DEX or 1 µM PGE2, alone or in combination, for 4 h. C, Extracts used in A were tested with an independent oligonucleotide probe containing a consensus Sp1 binding sequence. Data are representative of three independent experiments.

 
Effects on C/EBP{delta} and C/EBPß expression
Western immunoblots showed that C/EBP{delta} and C/EBPß were both present in osteoblast-derived nuclear extracts. Earlier studies showed that PGE2 rapidly increased the amount of C/EBP in nuclear extracts, at least in part through translocation from preexisting cytoplasmic pools (14). Pretreatment with DEX enhanced the levels of both C/EBP{delta} and C/EBPß in nuclear extracts. This effect appeared maximal by 16 h, as no further accumulation occurred after 32 h of DEX treatment. By densitometry, the combined effect of pretreatment with DEX and subsequent stimulation with PGE2 was additive. In contrast, cotreatment with both hormones increased the level of each isoform of C/EBP to no more than that caused by stimulation with PGE2 alone (Fig. 6Go).



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  		Figure 6. Effect of glucocorticoid pretreatment on C/EBP by Western immunoblot analysis. Osteoblast cultures were pretreated for 16 h with vehicle or 100 nM DEX, and then treated for 4 h with control medium or 1 µM PGE2. DEX was removed before PGE2 treatment, although similar results occurred when DEX remained in the medium during the short PGE2 treatment interval. Alternately, cultures were cotreated with DEX and PGE2 for 4 h. Nuclear extracts were fractionated by SDS-PAGE, electroblotted, and probed with anti-C/EBP{delta} (A) or anti-C/EBPß (B) antiserum, as indicated. Arrowheads indicate the immunoreactive bands consistent with the known mol wt of C/EBP{delta} or C/EBPß. Results are representative of two or three independent experiments.

 
Based on these results, we examined the effect of DEX on steady-state mRNA levels for C/EBP{delta} and C/EBPß by RNase protection assay. Pretreatment with DEX caused significant and dose-dependent increases in C/EBP{delta} and C/EBPß mRNAs. Densitometry indicated that at 100 nM DEX, there was a 3-fold increase in C/EBP{delta} mRNA and a 4.5-fold increase in C/EBPß mRNA (Fig. 7Go). Therefore, the synergistic effects of DEX pretreatment and PGE2 stimulation on IGF-I expression, IGF-I gene promoter activity, and C/EBP-dependent reporter gene expression were consistent with the ability of glucocorticoid to increase new C/EBP{delta} and C/EBPß expression and their subsequent activation by PGE2.



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  		Figure 7. Effects of glucocorticoid on C/EBP{delta} and C/EBPß mRNAs. Osteoblast cultures were treated for 16 h with vehicle or with 1–100 nM DEX, and total RNA was assessed by RNase protection assay with probes specific for rat C/EBP{delta} (190 nucelotides) or C/EBPß (260 nucleotides). An 18S ribosomal RNA probe (90 nucleotides) was used as an internal control. Subsequent autoradiography and densitometry were used to determine relative transcript levels. Results are representative of three independent experiments.

 
To examine the modulatory effect of DEX on C/EBP-dependent IGF-I promoter activation further, we created a C/EBP{delta} dominant negative (C/EBP{delta} DN) expression construct that lacked the trans-activation domain sequence, but retained dimerization and DNA-binding domains. C/EBP{delta} DN effectively suppressed both C/EBP{delta}- and C/EBPß-dependent gene expression in COS-7 cells cotransfected with 4X HS3D/Luc (data not shown). With the IGF-I promoter-derived reporter construct, C/EBP{delta} DN dose dependently suppressed the effect of PGE2 by up to 94% in both control and DEX-pretreated osteoblasts (Fig. 8Go). This effect could not be attributed to nonspecific squelching of reporter gene expression by C/EBP{delta} DN, because expression plasmid loading was corrected by complementation with parental vector. Therefore, C/EBPs appear to be essential components for PGE2- and DEX-regulated IGF-I promoter activity.



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  		Figure 8. Expression of a dominant-negative C/EBP{delta} suppressed the ability of DEX to potentiate PGE2-activated IGF-I promoter function. Osteoblast cultures were transfected with the IGF-I promoter construct IGF1711b/Luc along with increasing amounts of the C/EBP{delta} dominant negative (DN) construct (complemented with parental vector to achieve an equal plasmid load). Cultures were subsequently treated for 16 h with vehicle or 100 nM DEX as indicated. Cultures were then treated for 6 h with control medium or 1 µM PGE2. DEX was removed before PGE2 treatment, although similar results occurred when DEX remained in the medium during the short PGE2 treatment interval. Data are the mean ± SEM and represent two independent experiments, with n = 3/experiment.

 
Effects of glucocorticoid and PGE2 on osteoblast protein synthesis
Finally, our earlier studies showed that an increase in IGF-I expression was responsible in part for the stimulatory effect of PTH or PGE2 on matrix protein synthesis in intact bone explants (9, 33). Because preexposure to DEX enhanced the effect of PGE2 on IGF-I expression, we examined the consequence of these effects on collagen and noncollagen protein synthesis in isolated osteoblasts. By itself, PGE2 treatment had little effect on collagen synthesis, whereas it produced a small increase in noncollagen protein synthesis (Fig. 9Go, closed circles). In these experiments, pretreatment with DEX alone decreased new collagen synthesis by 70 ± 3% and noncollagen protein synthesis by 30 ± 4%. However, pretreatment with DEX caused a dose-dependent 2- to 2.5-fold increase in new collagen synthesis in PGE2-treated cells that was maximal at 0.1 µM PGE2 (Fig. 9Go, left panel, closed squares). Similarly, a 3-fold increase in noncollagen protein synthesis occurred under these same conditions (Fig. 9Go, right panel, closed squares).



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  		Figure 9. Permissive effect of glucocorticoid pretreatment on bone cell protein synthesis in response to PGE2. Osteoblast cultures were pretreated for 16 h with vehicle (closed circles) or 100 nM DEX (closed squares), treated for a total of 24 h with vehicle or the amounts of PGE2 indicated, and pulse labeled with [3H]proline for the last 2 h of culture to assess effects on collagen and noncollagen protein synthesis. DEX was removed before PGE2 treatment. Consistent with earlier studies in bone, collagen (left panel) and noncollagen (right panel) protein syntheses were partially suppressed by pretreatment with glucocorticoid, as reported previously (33 ). The relative effects of PGE2 are expressed by direct comparison to vehicle or DEX-pretreated control values, indicated as 1 on the ordinates of each graph. Data are the mean ± SEM and represent three independent experiments, with n = 4/experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Glucocorticoids are widely used for treatment of asthma, rheumatoid arthritis, inflammatory bowel disease, autoimmune disease, and immunosuppression of organ transplant recipients. Circulating cortisol levels also increase with aging, vigorous exercise, traumatic injury, severe burns, invasive surgery, microgravity, or Cushing’s disease (25, 48, 49, 50, 51, 52). Sustained high levels of glucocorticoids, from pharmacological or endogenous sources, are well known to inhibit skeletal growth and bone remodeling. Importantly, the risk of low trauma fractures increases in nearly one third of all patients receiving long-term glucocorticoid therapy (25).

IGF-I synthesis by osteoblasts is significantly enhanced by cAMP-inducing factors such as PTH and PGE2 (4, 5), and many studies now substantiate that activation of cAMP-dependent PKA accounts for the anabolic effects of these hormones on bone (9, 53, 54). Activation of PKA increases C/EBP{delta} activation and binding at +202 to +209 bp within the 5'-UTR of exon 1 of the IGF-I gene (7, 12, 14, 47). This C/EBP-binding element appears evolutionarily conserved among fish, frogs, rats, and humans. Moreover, the entire 5'-UTR of exon 1 exhibits a remarkable 95% homology between rats and chickens (12, 13), suggesting its biological importance for IGF-I gene expression.

Our current studies show that preexposure to glucocorticoid increased the stimulatory effect of PGE2 on IGF-I synthesis through an increase in new C/EBP{delta} and C/EBPß expression. The effect of glucocorticoid on C/EBP expression alone was unable to produce large effects on DNA binding or IGF-I promoter activation. Rather, both C/EBP isoforms effectively formed DNA binding complexes and increased gene promoter activity only after treatment with PGE2, demonstrating that the higher amount of C/EBPs induced in this way still required activation. A C/EBP{delta} DN expression construct virtually eliminated the effects of DEX and PGE2, revealing a critical role for C/EBPs on regulation of IGF-I promoter activity. Furthermore, the effectiveness of PGE2 at activating the IGF-I promoter occurred at a much lower concentration after glucocorticoid pretreatment, suggesting that the normal in vivo spikes in glucocorticoid release that occur throughout the day may have permissive effects on local or circulating osteotropic factors such as PGE2 and PTH on IGF-I expression by osteoblasts. In contrast, high or sustained levels of glucocorticoid may mask these permissive effects by suppressing new IGF-I expression (26). Preexposure to glucocorticoid also increased collagen and noncollagen protein synthesis by osteoblasts in response to PGE2, consistent with changes previously observed in calvarial organ cultures (33), demonstrating a physiological consequence from these events.

Interestingly, unlike the IGF-I mRNA studies in Fig. 1Go, preexposure to a high dose of DEX increased the effect of PGE2 on IGF-I promoter/reporter gene expression. This effect persisted regardless of whether DEX remained in the medium during a subsequent, shorter treatment period with PGE2. In addition, in transfection studies, low doses of DEX only weakly activated the positive control reporter construct, pMMTV/Luc, whereas it potentiated cAMP-activated endogenous IGF-I gene expression. These findings may reflect different effects by glucocorticoid on native chromatin vs. episomal plasmid DNA, or differences between the half-lives of IGF-I mRNA and the rapidly turning over reporter enzyme luciferase. Moreover, our results suggest that the stimulatory effect of DEX on IGF-I promoter activity depends on an increase in C/EBP expression, which does not itself appear to enhance pMMTV/Luc expression directly. The weaknesses of some assay systems may therefore limit our understanding of results from individual studies, whereas information from several experimental approaches may better represent biological regulatory mechanisms.

C/EBPs regulate the expression of many cytokines and indirectly increase PG expression through an increase in cyclooxygenase-2 expression (55). Activation of C/EBP initiates a biochemical cascade associated with inflammation, wound healing, tissue remodeling, and control of cell proliferation and metabolism. In addition, expression of individual C/EBP isoforms varies with tissue differentiation (21, 56, 57, 58, 59, 60) and can be modulated by the endotoxin lipopolysaccharide, thermal injury, hypoxia, and inflammation itself (15, 16, 17, 18, 19, 20, 22, 61, 62). Acquisition of the adipocyte phenotype by 3T3-L1 fibroblasts parallels a progressive variation in C/EBP isoform expression from C/EBP{delta} to C/EBPß and finally to C/EBP{alpha}, where C/EBP{alpha} regulates terminal adipocyte differentiation. This process is initiated by several factors, including glucocorticoid and phosphodiesterase inhibitors that increase cAMP, whereas terminal adipocyte differentiation requires insulin or IGF-I (21, 58, 59, 60). Similarly, C/EBP{delta} is developmentally regulated in fetal lung, and its expression is further induced by cAMP and glucocorticoid (57). Because of the significant interacting effects that we noted among glucocorticoids, cAMP-inducing agents, and IGF-I on the control of bone cell function, a progression in C/EBP expression and activation also may be important for bone cell differentiation and skeletal integrity (1, 2, 6, 9, 26, 33).

Glucocorticoid-dependent alterations in hormone-induced adenylate cyclase activity or cAMP accumulation can occur in specific tissues or at specific times during development (29, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76). Whereas glucocorticoid diminishes PGE2-induced cAMP in MC3T3-E1 preosteoblasts (70), it enhances the stimulatory effect of PTH on cAMP synthesis in highly differentiated ROS 17/2.8 osteoblastic cells, in part through an increase in PTH receptor expression (75). Notably, short term exposure to glucocorticoid in vivo enhances PTH-induced cAMP in perfused bones of rats or dogs, whereas chronic glucocorticoid treatment attenuates PTH activity (29, 63). Other interacting processes may also be involved. For example, glucocorticoid can reduce protein kinase C activity in rat liver parenchymal cells (77), and increased levels of cAMP and/or activation of protein kinase A can potentiate glucocorticoid-dependent gene expression by increasing glucocorticoid receptor affinity (78, 79) and consequently its catabolic effects on connective tissue after long term glucocorticoid exposure (80).

Based on our current results, a working model for the influence of glucocorticoid on IGF-I induction and its actions in osteoblasts is shown in Fig. 10Go. At physiological levels, glucocorticoid has a synergistic effect with cAMP-inducing hormones and transiently induces C/EBPß and C/EBP{delta} expression in osteoblasts. When cAMP rises in response to hormone stimulation, higher endogenous levels of C/EBPs are quickly activated, translocate to the nucleus, and enhance IGF-I promoter activity. In addition, glucocorticoid may modulate PKA, which is stimulatory, and PKC, which suppresses IGF-I expression. Therefore, with transient glucocorticoid pretreatment, PGE2 or PTH may more effectively activate adenylate cyclase and favor PKA-dependent activation of C/EBP. In contrast, high sustained levels of glucocorticoid may diminish these cooperative events by interfering with C/EBP function and ultimately suppress IGF-I expression. This effect may be C/EBP isoform selective and may result from a physical association between C/EBPs and the glucocorticoid receptor (81, 82; our unpublished observation). However, other findings not presented here indicate no apparent inhibition of C/EBP nuclear translocation by DEX. These and related studies are the subject of ongoing investigations. Finally, transient or sustained exposure to glucocorticoid may influence the biological effects of newly synthesized IGF-I through the downstream signaling events that occur in response to IGF-I itself.



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  		Figure 10. Model of interactions among glucocorticoids and cAMP-inducing hormones on IGF-I gene expression and downstream biological activity in osteoblasts.

 
In conclusion, our current studies continue to reveal novel interactions between PKA-activating hormones and glucocorticoids and potential mechanisms for the permissive effects of glucocorticoids on IGF-I expression and protein synthesis by osteoblasts. Future studies will be necessary to define in more detail the complex molecular events that distinguish the permissive and suppressive effects of glucocorticoids on osteoblast function and skeletal integrity.


   Acknowledgments
 
We are grateful to Dr. Peter Rotwein (Oregon Health Sciences University, Portland, OR) for C/EBPß and C/EBP{delta} genomic clones and native and synthetic IGF-I promoter/reporter plasmid constructs, to Dr. Ronald Evans (The Salk Institute, La Jolla, CA) for plasmid pMMTV-Luc, and to Ms. Sandra Casinghino for technical assistance.


   Footnotes
 
1 This work was supported by NASA Grant NAG5–6054 (to T.L.M.), NIH Grant DK-47421 (to T.L.M.), and a grant from the Arthritis Foundation (to M.C. and T.L.M.). Back

Received June 2, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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