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Endocrinology Vol. 140, No. 10 4564-4572
Copyright © 1999 by The Endocrine Society

ARTICLES

Activation of the Insulin-Like Growth Factor-Binding Protein-5 Promoter in Osteoblasts by Cooperative E Box, CCAAT Enhancer-Binding Protein, and Nuclear Factor-1 Deoxyribonucleic Acid-Binding Sequences1

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

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

Address all correspondence and requests for reprints to: Thomas L. McCarthy, Ph.D., Section of Plastic Surgery, 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
 
Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) has IGF-dependent and -independent actions. PGE2 rapidly increases IGFBP-5 expression by osteoblasts through cAMP-dependent processes. A minimal DNA sequence required for basal and PGE2-stimulated IGFBP-5 promoter activity spans -69 to -35 bp. This region adjoins a functional TATA box and contains E box, CCAAT enhancer-binding protein (C/EBP), nuclear factor-1 (NF-1), and activator protein-2 (AP-2) transcription factor related binding motifs. In this study we compared minimal promoter sequences of -74 to +120 bp, without or with mutations in each potential regulatory element, by reporter gene expression and electrophoretic mobility shift assays. Mutation of the E box-related element reduced basal promoter activity by 50% and eliminated the 2-fold stimulatory effect of PGE2. In contrast, mutations in the C/EBP- or NF-1-related elements also reduced basal promoter activity without fully eliminating the PGE2 effect. Overexpression of C/EBP{delta} stimulated basal IGFBP-5 promoter activity, and this effect was eliminated by mutating the C/EBP-binding site. However, mutation of the AP-2-binding site or overexpression of AP-2 did not correlate with basal or PGE2-induced promoter activation. By electrophoretic mobility shift assay, prominent gel shift complexes occurred with osteoblast nuclear extracts and 32P-labeled probes spanning the E box-, C/EBP-, and NF-1-related motifs. These gel shift complexes were depleted by specific binding site mutations and were enhanced by PGE2. Increased binding by extracts from PGE2-treated cultures was blocked by cycloheximide treatment. These results identify several elements as integral binding sequences for both basal and PGE2-stimulated IGFBP-5 promoter activity. They further reveal that multiple sequences within this cluster form a basic transcription unit where nuclear factors can accumulate in a protein synthesis-dependent way and enhance IGFBP-5 expression by osteoblasts in response to PGE2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LIKE MOST TISSUES that synthesize and respond to the insulin-like growth factors (IGFs), osteoblasts also secrete an array of IGF-binding proteins (IGFBPs), including IGFBP-5 (1, 2, 3, 4, 5, 6). IGFBPs regulate IGF activity through their high affinity for IGF-I and IGF-II (7). Binding of IGFs to various IGFBPs may sequester the growth factors from the signal-transducing type 1 IGF receptor. However, select IGFBPs also bind cell matrix and membrane components that may concentrate or help to present IGFs to cell surface receptors (8, 9, 10).

IGFBP-5 was first discovered as a product of osteoblasts and was initially distinguished from other IGFBPs by its ability to enhance IGF activity (11, 12, 13, 14, 15). Selective proteases are produced by osteoblasts as well as other cells and appear to target IGFBP-5 for partial degradation (13, 16, 17, 18, 19). Proteolytic fragments of IGFBP-5 have reduced affinity for IGFs, and partial proteolysis may release IGFs to allow their association with IGF receptors. IGFBP-5 contains heparin- and hydroxyapatite-binding domains, perhaps allowing it to bind organic and inorganic components for storage in the extracellular skeletal matrix (15, 20, 21, 22, 23). A reservoir of IGFs associated with IGFBP-5 in this way may be released during the bone resorption phase of remodeling. In addition, IGFBP-5 associates directly with the membrane components of many cells, including osteoblasts (10, 23). Membrane association appears to reduce its binding affinity for IGFs, allowing IGFs to be concentrated and then released within the pericellular environment, and facilitating access to their own receptors. Phosphorylation of IGFBP-5 may also influence its function (24). Finally, IGFBP-5 may have some IGF-independent actions. IGFBP-5 stimulates the expression of GH receptors in UMR-106 osteosarcoma-derived cells, and carboxy-truncated IGFBP-5 itself appears mitogenic for osteoblasts (13, 25).

PGE2 and PTH stimulate the synthesis of IGFBP-5 by osteoblasts (3, 26). We previously reported promoter-dependent and promoter-independent mechanisms by which PGE2 stimulated IGFBP-5 expression (27), which was independently confirmed by other investigators (28). Within 6 h of exposure to PGE2, we noted a 2-fold increase in IGFBP-5 promoter activity. This effect was augmented by a 2-fold increase in IGFBP-5 messenger RNA (mRNA) half-life within 24 h of PGE2 treatment. Similar effects occurred in forskolin-treated cell cultures, indicating a cAMP-dependent mechanism of IGFBP-5 gene activation. Moreover, ongoing protein synthesis was required for PGE2 to elevate steady state IGFBP-5 mRNA levels (27).

The stimulatory effect of cAMP on IGFBP-5 promoter activation required only a small 5'-proximal region of the promoter. Studies with progressively truncated IGFBP-5 promoter/reporter transfection constructs revealed that a region spanning from -74 to +120 bp of the IGFBP-5 gene was necessary for optimal gene expression in response to PGE2. DNA between -74 bp and the start site of transcription contains several potential cis-acting elements, including E box-, CCAAT/enhancer-binding protein (C/EBP)-, nuclear factor-1 (NF-1)-, and activator protein-2 (AP-2)-related sequences (29). Our current study identified essential and cooperative elements within this cluster of regulatory sequences that can increase IGFBP-5 promoter activity in PGE2-activated osteoblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
Primary osteoblast-enriched cell cultures were prepared from the parietal bones of 22-day-old Sprague Dawley rat fetuses (30). Animals were housed and euthanized by methods approved by the Yale University animal care and use committee. Cranial sutures were eliminated during dissection, and parietal bones were digested with collagenase for five sequential 20-min intervals. Cells released during the last three digestions exhibit biochemical characteristics associated with differentiated osteoblasts. Cells were plated at 3000/cm2 in DMEM containing 20 mM HEPES (pH 7.2), 1 mg/ml ascorbic acid, penicillin, streptomycin (Life Technologies, Inc., Gaithersburg, MD), and 10% FBS (Sigma Chemical Co., St. Louis, MO). Cultures were expanded in standard DMEM containing 10% FBS before transfection.

Plasmids
Murine IGFBP-5 promoter constructs in pGL2Basic were described previously (27, 29). The rat C/EBP{delta} complementary DNA clone from Dr. Peter Rotwein (Oregon Health Sciences University, Portland, OR) was subcloned into eukaryotic expression vector pSV7d (31). AP-2 wild-type and mutant constructs were gifts from Dr. Trevor Williams (Yale University, New Haven, CT) (32). Site-directed mutations in the IGFBP-5 promoter were prepared by PCR using oligonucleotides containing mutated sequences, and mutated constructs were verified by sequence analysis. Plasmids were propagated in Escherichia coli strain DH5{alpha} with ampicillin selection and were prepared with a QIAGEN Midiprep Kit (Chatsworth, CA) by the manufacturer’s recommended protocol.

Transfection studies
IGFBP-5 promoter/reporter plasmids (0.6 µg/4.8-cm2 culture well) were cotransfected with a vector carrying the ß-galactosidase gene under the control of the Simian virus 40 promoter (0.6 µg/culture well) (27). Cultures at 50% confluent density were rinsed in serum-free DMEM and exposed to plasmids in the presence of lipofectin (Life Technologies, Inc.) for 3 h. The solution was then replaced with DMEM containing 5% FBS, and the cultures were allowed to replicate for 48 h. Cultures were rinsed with serum-free medium and treated for the indicated times with vehicle (ethanol diluted 1:10,000) or 1 µM PGE2 (Sigma Chemical Co.). After treatment, the medium was aspirated, and the cultures were rinsed with PBS, and then lysed in 100 µl cell lysis reagent (Promega Corp.). Nuclei were pelleted at 12,000 x g for 5 min, and the supernatants were stored at -75 C until assay. A commercial kit was used to measure luciferase (Promega Corp.). Luciferase reporter enzyme activity was determined by correcting for ß-galactosidase and cell extract protein content, determined by the Bradford assay.

Nuclear protein extracts
Osteoblast nuclear extracts were prepared as previously described (33, 34). Briefly, cells were rinsed with serum-free medium and the next day were treated with vehicle or PGE2 for 4 h. Medium was aspirated, and cultures were rinsed twice with PBS at 4 C. All subsequent steps were performed on ice or at 4 C. Cells were harvested and gently pelleted, and the pellets were washed with PBS. Cells were lysed in hypotonic buffer [10 mM HEPES (pH 7.4), 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM dithiothreitol] 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 Chemical Co.)] and 1% Triton X-100. Nuclei were pelleted, and the supernatant (cytoplasmic extract) 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 proteins released by a 30-min incubation at 4 C were collected by centrifugation at 12,000 x g for 5 min, and the supernatant was collected and aliquoted for storage at -75 C.

Electrophoretic mobility shift assay (EMSA)
EMSA experiments followed previously published methods (33, 35). 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, using the Klenow fragment of DNA polymerase I. Five to 10 µg nuclear extract protein were preincubated for 20 min on ice with 2 µg poly(dI:dC) without or with 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 dithiothreitol, and 0.025% BSA. After 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 was preelectrophoresed for 30 min at 12.5 V/cm at 25 C in 45 mM Tris, 45 mM boric acid, and 1 mM EDTA. Electrophoresis proceeded for 2.5 h under identical conditions. Dried gels were exposed to x-ray film at -75 C with an intensifying screen.

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Functional C/EBP-, E box-, and NF-1-related binding elements in the IGFBP-5 promoter and sensitivity to PGE2
We previously showed that a 6-h treatment with 1 µM PGE2, which potently induced cAMP in fetal rat osteoblasts, enhanced IGFBP-5 promoter activity by 1.6- to 2.3-fold (27). Using IGFBP-5 promoter constructs with as much as 4100 to as little as 31 bp of DNA upstream from the site of transcription initiation, we identified a cAMP-responsive region between -74 to -32 bp. This region contains potential binding sites for AP-2 (-35 to -43 bp), NF-1 (-40 to -52 bp), E box-binding proteins (-51 to -56 bp), and C/EBPs (-58 to -66 bp). Mutations were produced within the background of the IGFBP5-Luc6 construct (-74 to +120 bp) for each of these binding sequences. Wild-type and mutant sequences are shown in Fig. 1Go. Reporter gene expression was assessed in control and PGE2-treated cells transfected with wild-type and mutated constructs. As shown in Fig. 2Go, mutation of the E box element (LUC6M3) decreased basal promoter activity by 50% and completely suppressed the effect of PGE2. Mutations in the adjacent C/EBP (LUC6M2) and NF-1 (LUC6M4) elements also decreased basal promoter activity and reduced, but did not eliminate, the response to PGE2. In contrast, mutations of either a single upstream nucleotide in the C/EBP-binding sequence (LUC6M1) or three nucleotides in the AP-2 (LUC6M5) element did not modify the effect of PGE2. Lastly, truncation of the transcribed region (from +120 to +16 bp; LUC6M6) did not affect basal promoter activity or the stimulatory effect of PGE2.



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  		Figure 1. Wild-type and mutated IGFBP-5 promoter/reporter constructs. Relevant sequences of wild-type LUC4 and LUC6 (IGFBP5-Luc4, IGFBP5-Luc6) reporter constructs are shown on the top, with the transcription factor-binding sites boxed and denoted below. The position of the luciferase gene (LUC) is shown in the open box at the right of each construct. Mutated nucleotides in each construct are underlined and designated in bold. The 5'-end of IGFBP-5 promoter sequence in construct LUC4 extends to -1004 bp and the 3'-end of LUC6M6 truncates at +15 bp, as indicated.

 


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  		Figure 2. Expression of wild-type and mutated IGFBP-5 promoter/reporter constructs: effects on basal and PGE2-induced promoter activity. Site-directed mutations (detailed in Fig. 1Go) were incorporated in LUC6, a minimal IGFBP-5 promoter construct. The names and the nature of the alteration in each construct are indicated on the left. Constructs were transfected into cultured osteoblasts. Reporter gene expression was assessed in vehicle-treated control cells and in cells treated for 6 h with 1 µM PGE2. Results are from 2–5 experiments, with 6–15 replicates/condition. In untreated cells (left panel), promoter activity was significantly reduced compared to the wild-type LUC6 sequence (P < 0.05) by mutations present in all but LUC6M5, where only the AP-2-binding element was disrupted. Treatment with PGE2 (right panel) significantly increased promoter activity in cells transfected with all but parental vector pGL2Basic (Basic) and LUC6M3, containing a mutation in the E box-binding element (P < 0.05). Mutations in the C/EBP-binding element in LUC6M2, the E box-binding element in LUC6M3, and the NF-1-binding element in LUC6M4 each significantly suppressed promoter activity compared to the wild-type LUC6 promoter construct (P < 0.05).

 
C/EBP{delta} preexists in primary cultures of fetal rat osteoblasts, and its activity is enhanced in a cAMP-dependent way by PGE2 (35). Overexpression of C/EBP{delta} enhanced promoter activity directed by the wild-type LUC6 construct in untreated and PGE2-stimulated cultures (Fig. 3Go). Mutation of the C/EBP-binding site eliminated the stimulatory effect of C/EBP overexpression and reduced the effect of PGE2. Therefore, the C/EBP-related element within this short sequence of the IGFBP-5 promoter was functional, responded to C/EBP, and was sensitive to regulators of intracellular cAMP.



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  		Figure 3. Functional C/EBP-binding site influences PGE2-induced IGFBP-5 promoter activity: effects of C/EBP overexpression and C/EBP-binding site mutation. Osteoblasts were cotransfected with empty expression vector or C/EBP{delta} expression constructs and reporter constructs pGL2Basic, LUC6, and LUC6M2 (mutated in the C/EBP-binding site, detailed in Fig. 1Go). Reporter gene expression was assessed in control cells and in cells treated for 6 h with 1 µM PGE2. Results are from 2–4 experiments, with 6–12 replicates/condition. Overexpression of C/EBP{delta} significantly increased promoter activity in control and PGE2-induced cells expressing the wild-type LUC6 promoter construct (P < 0.05), but was without effect in cells transfected with the parental pGL2Basic or the LUC6M2 construct.

 
Although mutation of the AP-2-binding site did not affect IGFBP-5 promoter activity (Fig. 2Go), overexpression of wild-type AP-2 (but not truncated AP-2 containing only the DNA-binding domain), reduced the stimulatory effect of PGE2 (Fig. 4Go). Therefore, endogenous levels of AP-2 do not appear to enhance basal IGFBP-5 promoter activity or to mediate stimulation by PGE2 in osteoblasts. Nonetheless, in situations where AP-2 levels may rise significantly in osteoblasts, cAMP-dependent control of IGFBP-5 expression may be suppressed.



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  		Figure 4. AP-2 does not account for PGE2-induced IGFBP-5 promoter function. Osteoblasts were cotransfected with empty expression vector, wild-type (WT) or dominant-negative (DN) AP-2, and full-length IGFBP-5 reporter construct LUC4 (detailed in Fig. 1Go) (27 ). Reporter gene expression was assessed in control cells and in cells treated for 6 h with 1 µM PGE2. Results are from three experiments, with nine replicates per condition. Treatment with PGE2 significantly enhanced promoter activity, whereas overexpression of wild-type AP-2 (AP-2 WT) caused a statistically significant (P < 0.05) reduction in its stimulatory effect.

 
NF binding to NF-1-binding element in the IGFBP-5 promoter
EMSA was used to characterize further the cis-acting elements within this region of the IGFBP-5 promoter. Wild-type and mutated, double stranded oligonucleotides (ds oligo) spanning specific portions of the IGFBP-5 promoter where these control elements cluster (Fig. 5Go) were combined with nuclear extract from control or PGE2-induced cells. PGE2 caused a time-dependent increase in NF binding to a short 32P-labeled ds oligo, SLUC7 that contains NF-1- and AP-2-binding sequences (Fig. 6AGo). Unlabeled SLUC7 and ds oligo with a consensus NF-1 binding motif effectively competed with [32P]SLUC7 for binding to nuclear protein. In contrast, unlabeled ds oligos with C/EBP-binding elements (C/EBP and HS3D) did not affect nuclear factor complex formation with this probe (Fig. 6Go, B and C).



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  		Figure 5. Oligonucleotide probes used in EMSA. The relevant sequence of the wild-type IGFBP-5 promoter region is shown on the top, with the transcription factor-binding sites boxed and denoted below. Names assigned to probes are shown on the right of each sequence, and mutations are underlined and designated in bold. Lower case, italicized nucleotides are extended, nonspecific sequences added to oligonucleotide probes to facilitate radiolabeling. Boxed nucleotides in probes C/EBP, HS3D, and NF-1 represent specific NF-binding sequences. The sequences of consensus element probes are shown below.

 


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  		Figure 6. EMSA with IGFBP-5 promoter DNA containing NF-1- and AP-2-binding sites. Nuclear extract (5 µg) from osteoblasts treated with vehicle or 1 µM PGE2 for 0, 1, or 4 h (A) or for 4 h (B and C) was combined with ds oligo [32P]SLUC7, which spans -54 to -32 bp from the IGFBP-5 promoter and contains NF-1- and AP-2-binding sites. In B and C, [32P]SLUC7 was combined with the unlabeled competitor oligos indicated, as detailed in Fig. 5Go. Complexes were resolved on a 5% nondenaturing PAGE and displayed by autoradiography. Results are representative of two or three analogous studies.

 
NF binding to C/EBP- and E box-related binding elements in the IGFBP-5 promoter
A ds oligo containing both the C/EBP- and E-box-related elements (LUC6) formed several complexes with osteoblast-derived nuclear proteins. Of these, two complexes, termed C1 and C2, were principally enhanced by PGE2 treatment (shown by arrows in Fig. 7AGo). NF from PGE2-treated cells bound less well to [32P]LUC6M2, a ds oligo with a mutated C/EBP-related element. Binding to complex C2 was partially reduced by this mutation, and virtually nothing comigrated with complex C1 (Fig. 7BGo). [32P]E box, a shorter LUC6-derived ds oligo in which the only intact NF-binding site was the E box-related element, formed a slow migrating complex similar to the C2 band produced with the [32P]LUC6 probe. Formation of complex C2 was enhanced by PGE2 and partially or wholly suppressed by the mutations in the E box-related element introduced in oligos [32P]E boxM1 and [32P]E boxM3 (Fig. 7CGo). Therefore, LUC6 bound nuclear proteins at both C/EBP (C1)- and E box (C2)-related binding sites. Moreover, formation of these complexes was enhanced by PGE2, and each complex appeared to contain multiple nuclear factor components (discussed below). Cycloheximide rapidly blocked the stimulatory effect of PGE2 on nuclear factor binding within complexes C1 and C2 (Fig. 7DGo), consistent with its effect on IGFBP-5 mRNA (27). Competition with a consensus C/EBP oligo reduced binding within complex C1 to control levels, but had little effect on complex C2 (Fig. 7EGo).



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  		Figure 7. EMSA with IGFBP-5 promoter DNA containing C/EBP- and E box-binding sites. Nuclear extracts (5 µg) from osteoblasts treated with vehicle or 1 µM PGE2 for 0, 2, or 4 h (A) or 4 h (B–E) were combined with [32P]ds oligo (see Fig. 5Go), which spans all or part of -80 to -44 bp from the IGFBP-5 promoter and contains CCAAT, C/EBP, and/or E box protein-binding sites. In B, [32P]LUC6M2, with the C/EBP-binding site mutation, was combined with extract from PGE2-treated cells. Note the complete loss of complex C1 and the reduction of complex C2. In C, [32P]E box, [32P]E boxM1, or [32P]E boxM3 was combined with nuclear extracts from control (C) or 4 h 1 µM PGE2 (P)-treated cells. In D, [32P]LUC6 was combined with nuclear extract from control (C) or 4 h 1 µM PGE2 (P)-treated cells that had been pretreated for 20 min in control medium or with 2 µM cycloheximide (Chx). In E, [32P]LUC6 was combined with nuclear extract from control (C) or 4 h 1 µM PGE2 (P)-treated cells in the absence or presence of homologous oligo LUC6 (25X) or increasing amounts (20-, 50-, and 150-fold) of a ds oligo containing a consensus binding site for C/EBP. Complexes were resolved on a 5% nondenaturing PAGE and displayed by autoradiography. C1 and C2 indicate C/EBP and E box binding complexes. Results are representative of two or three analogous studies.

 
Cooperative effects on NF binding to C/EBP-related complex
Consistent with results in Fig. 7Go, the E box-related complex C2 remained in part responsive to PGE2 when the C/EBP-binding sequence was mutated in probe LUC6M2. However, disruption of the E box-related motif in LUC6M3 also eliminated the stimulatory effect of PGE2 within the C/EBP-related complex C1 (Fig. 8AGo). These results were confirmed by competition binding studies with unlabeled wild-type and mutated ds oligos. Unlabeled wild-type ds oligo LUC6 reduced all complex formation with NF from PGE2-treated cells. Essentially all of complex C1 and part of C2 were retained with unlabeled LUC6M2, containing the C/EBP-binding site mutation. Similarly, only a part of complex C2 was retained by competition with unlabeled LUC6M3, containing the E box mutation. For control, unlabeled oligonucleotide with only a consensus NF-1-binding sequence had no effect (Fig. 8BGo). Therefore, although basal binding in complex C1, associated with the C/EBP-binding element, could occur without E box protein binding, stimulation by PGE2 within this complex appeared to cooperate with nuclear factor binding to the E box element associated with complex C2.



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  		Figure 8. EMSA further defining PGE2-dependent C/EBP and E box binding complexes in the IGFBP-5 promoter. In A, nuclear extract (5 µg) from osteoblasts treated for 4 h with vehicle (C) or 1 µM PGE2 (P) was combined with wild-type [32P]LUC6 or with [32P]LUC6M2 or [32P]LUC6M3 containing mutations in the C/EBP or E box motifs, respectively, as detailed in Fig. 5Go. Note the reduction in E box protein binding in complex C2 when C/EBP-binding site complex C1 is eliminated by mutation. In B, nuclear extract from PGE2-treated cells was combined with [32P]LUC6 without (0) or with a 50-fold molar excess of unlabeled ds oligos LUC6 (wild-type), LUC6M2 (C/EBP mutation), LUC6M3 (E box mutation), or consensus oligonucleotide-binding sequence for NF-1. In C, nuclear extract from PGE2-treated cells was combined with [32P]LUC6 without (0) or with unlabeled ds oligos containing wild-type (E box) or mutated (E boxM3) E box binding motif present in 50-, 100-, or 200-fold molar excess. Note partitioning of PGE2-dependent and -independent complexes in C2 by various mutations.

 
PGE2-sensitive and insensitive components in complex C2
As shown in Fig. 8Go, A and B, ds oligos LUC6M2 and LUC6M3 distinguished complex C2 into two entities of slightly different molecular mass. The faster migrating, PGE2-sensitive complex was retained when only the C/EBP-related sequence was mutated. Furthermore, it was not competed for by unlabeled ds oligos that contained mutated E box-related sequence, but readily displaced by wild-type E box ds oligos, either within the context of the whole LUC6 sequence or when the C/EBP-binding sequence was eliminated (Fig. 8Go, B and C). Even so, the stimulatory effect of PGE2 on NF binding to the smaller complex in C2 was significantly reduced with [32P]LUC6M2, which contained a mutated C/EBP-binding site (Fig. 8AGo). Therefore, complex C2 contained protein that associated with an E box-related element, that was sensitive to PGE2, and that cooperated with other proteins associated with the C/EBP-binding sequence. It also contained a slightly higher molecular mass complex that was associated with the C/EBP-related binding element and appeared independent of the stimulatory effect of PGE2, although it may directly or indirectly cooperate with protein in complex C1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGFBP-5 is synthesized by osteoblasts. In vitro, its expression is induced by physiological hormones such as PGE2 and PTH as well as other inducers of cAMP (3, 26, 27, 28). Whereas many possible cAMP response regions are dispersed throughout at least 3 kb of the mouse IGFBP-5 promoter DNA, we earlier reported that a short 5'-proximal segment of 50–75 bp supported a cAMP-dependent increase in gene expression (27). In this study we showed that PGE2 increased the association of several NFs within a tight cluster of DNA-binding sites in this region comprising sequences that can associate with AP-2-, E box-binding proteins, NF-1, and C/EBP. Duan and Clemmons (36) previously demonstrated that AP-2 regulates basal and cAMP-dependent IGFBP-5 transcription in human dermal fibroblasts. However, mutation of the AP-2 site in this region of the IGFBP-5 promoter did not diminish the stimulatory effect of PGE2 in osteoblasts. Rather, overexpression of AP-2 inhibits PGE2-dependent gene activation in osteoblasts, perhaps resulting from its forced association within this cluster of elements and thereby blocking the effect of other NFs.

Effects on factors other than AP-2, therefore, appear to account for cAMP-dependent increases in IGFBP-5 expression in osteoblasts. In this regard, mutation of the E box-related element significantly decreased basal promoter activity and eliminated the stimulatory effect of PGE2. Oligonucleotides encompassing the E box sequence formed a large molecular mass complex with NFs that were enriched by PGE2 treatment. We have not yet identified the osteoblast-derived nuclear proteins that associate with these elements in the IGFBP-5 promoter. E box-binding proteins include tissue-restricted transcription factors [MyoD, myogenin, Myf-5, Myf-6, adipocyte determination and differentiation factor-1, scleraxis, and twist] and ubiquitous transcription factors (myc, myb, E2A gene products E47 and E12, and upstream stimulatory factor-1 and -2) that can heterodimerize in complex ways (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48). Our initial studies to identify E-box binding proteins associated with IGFBP-5 expression showed that nuclear extract from PGE2-activated osteoblasts failed to react with antibodies to adipocyte determination and differentiation factor-1, c-myc, c-myb, E47, E12, twist, upstream transcription factor, or AP-4. E box elements occur in promoters for many developmentally regulated genes (49, 50, 51, 52, 53), suggesting complex and perhaps tissue-specific regulation. Proteins that associate with this sequence to regulate cAMP-dependent IGFBP-5 expression in osteoblasts may, therefore, be restricted to only a small number of cell types and await further characterization.

Interestingly, the reported sequence of the human IGFBP-5 promoter (54) indicates the presence of an additional adenosine that might disrupt the function of the 5'-proximal E box element that occurs in both the murine and rat promoters (29, 55). However, the C/EBP, NF-1, and AP-2 elements in this tight cluster, shown in Fig. 1Go, remain intact in the human, rat, and murine genomes, suggesting their functional significance. Therefore, the importance of AP-2 in human dermal fibroblasts and the E box-binding protein, C/EBP, and NF-1 in osteoblasts may relate to species or tissue differences within key regulatory elements that control IGFBP-5 expression.

PGE2 also increased NF binding to both the NF-1- and C/EBP-related elements in this region of the IGFBP-5 promoter. Mutation of either site-reduced basal promoter-dependent gene activation. However, unlike the effect of the E box mutation, a response to PGE2 treatment was partially retained in these instances. By gel shift analysis, disruption of the C/EBP-related sequence potently suppressed basal NF binding, but did not fully eliminate a protein that associated with the E box-like element in extracts from cAMP-induced cells. In contrast, disruption of the E box element still permitted basal NF binding to a short segment of the promoter, but suppressed the general stimulatory effect of PGE2. Interactions between E box-binding proteins and C/EBP have been described in several gene promoters (56, 57, 58, 59). PGE2 not only activates preexist C/EBP{delta} in rat osteoblasts (35), it also induces new C/EBP{delta} expression in these cells (our unpublished studies). In this regard, overexpression of C/EBP{delta} enhanced basal and PGE2-stimulated IGFBP-5 promoter activity. Moreover, our earlier studies showed that protein synthesis was needed to increase IGFBP-5 mRNA in PGE2-treated osteoblasts (27), and we currently find that suppression of new protein synthesis limited basal and PGE2-induced NF binding. Consequently, proteins that associate with the E box may directly or indirectly enhance or stabilize its own binding and that by other NFs, such as C/EBP{delta}, which are induced or activated by PGE2 in osteoblasts, within this region of the IGFBP-5 promoter.

In summary, our current data predict that a 5'-proximal E box-related element is important for basal and PGE2-stimulated IGFBP-5 gene promoter activity in fetal rat osteoblasts, and that it participates at an early or critical step in gene activation. Our current and earlier studies further indicate that PGE2-dependent activation of IGFBP-5 mRNA transcription requires de novo synthesis of cooperative E box-binding protein, C/EBP, or related factors. NF-1, C/EBP, or related factors and adapter protein(s) may facilitate complex formation among the E box-binding protein and components of the transcription apparatus. In this way, they enhance IGFBP-5 expression by stimulated bone cells, as shown by a preliminary model described in Fig. 9Go.



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  		Figure 9. Model of cAMP-dependent (PGE2) activation of the IGFBP-5 promoter. PGE2 rapidly and potently increases intracellular cAMP in osteoblasts, followed by an increase in IGFBP-5 transcription initiation and mRNA accumulation (26 34 ). The increase in IGFBP-5 mRNA requires new protein synthesis, indicating new transcription factor expression. Our current studies indicate that 1) an E box-binding protein, C/EBP{delta}, and NF-1 preexist in rat osteoblasts. However, cycloheximide blocks NF binding to the IGFBP-5 promoter, suggesting that these NFs or other components needed for their activation or assembly into transcription complexes must be newly or continuously synthesized. Our current reporter gene expression and EMSA further suggest that 2) association of the E box-binding protein is an initial or central step in the assembly of PGE2-activated factors on the IGFBP-5 promoter. This is followed by 3) NF binding to C/EBP and NF-1 elements, and finally, 4) formation of a transcription preinitiation complex.

 
IGFBP-5 was first identified in osteoblast cultures based on its ability to potentiate IGF activity and the stimulatory effects of truncated IGFBP-5. IGFBP-5 also may have an important role in storing IGFs in the calcified skeletal matrix (15). IGF-I is a key anabolic growth factor for bone. Its own expression is induced by PGE2 and other cAMP inducers by way of a C/EBP-binding element (33, 35, 60), suggesting that C/EBP may be an important transcription regulator of multiple components of the IGF axis in osteoblasts. Understanding mechanisms that regulate IGFBP-5 expression within bone may provide a way to exploit its unique ability to enhance IGF-I activity and thereby improve bone mass when skeletal tissue integrity is compromised. Future studies will focus on identifying with certainty the trans-acting nuclear factors that are activated or newly synthesized in response to cAMP-inducing agents in osteoblasts.


   Acknowledgments
 
We are grateful to Dr. Peter Rotwein (Oregon Health Sciences University) for parental IGFBP-5 promoter constructs and C/EBP{delta} and C/EBPß expression constructs, to Dr. Trevor Williams (Yale University) for AP-2 wild-type and mutant expression constructs, to Dr. Richard Gaynor (University of Texas Southwestern Medical Center) for antiserum to AP-4, and to Sandra Casinghino for technical assistance.


   Footnotes
 
1 This work was supported by NIH Grant DK-47421 (to T.L.M.). Back

Received February 4, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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