Insulin-like growth factors-I (IGF-I) ()and -II are abundant locally produced growth regulators in skeletal tissue(1) . While the synthesis of IGFs is hormonally controlled, their actions are ultimately determined by way of signal-transducing receptors. IGF binding proteins (IGFBPs) are a family of secreted proteins that also avidly bind IGFs and modify their actions by altering their access to cell surface receptors(2, 3) . Of the six known IGFBPs, fetal rat osteoblasts synthesize five, IGFBP-2, -3, -4, -5, and -6(4) . However, IGFBP-5 is the only IGFBP with a demonstrated ability to potentiate IGF actions in bone cells(5) . Therefore, agents that stimulate IGFBP-5 synthesis may have an important anabolic role in skeletal growth and the maintenance of skeletal integrity.

The initial identification of IGFBP-5 was based upon its ability to augment IGF-I activity in bone cell cultures, as well as its structural and sequence similarities to other IGFBPs(6, 7, 8, 9) . The mechanism of potentiation of IGF action by IGFBP-5 has been attributed to its association within pericellular compartments (cell membrane and matrix components), resulting in a high local concentration of IGFs in close proximity to cell surface IGF receptors(5, 10, 11) . Recent studies suggest the ability of IGFBP-5 to bind ligand decreases after its association with matrix or select polysaccharides and the actions of IGFBP-5-selective proteases(12, 13, 14, 15) . The subsequent release of highly concentrated IGF in the pericellular environment thus enhances IGF receptor binding and its biological effects. IGFBP-5 has the additional attribute of high affinity binding to the calcium phosphate component of bone, which may serve to concentrate IGFs in inorganic bone matrix for storage and subsequent activation during periods of localized bone resorption(8) .

In bone cell cultures, both IGFBP-5 and IGF-I synthesis are regulated by prostaglandin E (PGE), parathyroid hormone (PTH), other agents that stimulate cAMP synthesis, or by cAMP itself (4, 16) . PGE is produced by osteoblasts in response to PTH, to cytokines such as interleukin-1 and transforming growth factor-, and to mechanical strain, and PGE has been shown to mediate various biological actions on osteoblasts initiated by these stimuli(17, 18, 19, 20, 21, 22) . PGE may thus serve as a local analog of PTH. Unlike PTH, however, its influence may be more highly focused due to its synthesis within the skeleton. Furthermore, PGE has demonstrated anabolic actions in bone that depend on the cellular state of differentiation and on dose and duration of treatment(23, 24, 25) .

While the molecular mechanisms by which PGE regulates IGF-I synthesis in osteoblasts are only currently being elucidated(26, 27) , even less is known about IGFBP-5 synthesis in skeletal tissue. Previous observations reveal that PGE enhances steady-state levels of IGFBP-5 mRNA(4) . We now demonstrate that transcriptional and post-transcriptional pathways are both activated by PGE to stimulate IGFBP-5 gene expression. These steps appear to require ongoing protein synthesis, indicating that promoter-dependent and -independent processes each regulate IGFBP-5 production in response to PGE in primary fetal rat osteoblast-enriched cultures.

EXPERIMENTAL PROCEDURES

Cell Cultures

()

Plasmids

RNA Analysis

Transfection Studies

Statistical Analysis

RESULTS

PGE increases the level of IGFBP-5 mRNA and protein in primary fetal rat osteoblast-enriched cultures(4) . The magnitude of the rise in transcript abundance is time-dependent. In agreement with earlier studies(4) , IGFBP-5 mRNA levels increased by 1.4-7-fold following 4-32 h of PGE (1 µM) treatment (Fig. 1). A subsequent analysis by RNase protection assay confirmed these observations and additionally demonstrated a time-dependent rise in abundance of nascent transcripts, first observed at 3 h after PGE treatment, with a 3-fold increase seen at 8 h (data not shown). These results suggest that PGE may stimulate IGFBP-5 transcription, although this effect appears delayed when compared with another PGE-stimulated gene, IGF-I, for which nascent transcripts increase within 30 min of PGE treatment(26) .

Figure 1: Time course of IGFBP-5 mRNA induction by PGE in primary osteoblast-enriched cultures. Confluent, serum-deprived cultures of osteoblasts were treated with vehicle (ethanol) or PGE (1 µM) for 4, 6, 8, 24, or 32 h, as indicated in each panel). RNA blots were prepared as described under ``Experimental Procedures,'' hybridized with P-labeled rat IGFBP-5 cDNA and antisense 18 S rRNA, washed, and visualized by autoradiography. On the left are pooled data for four independent Northern blots. On the right is a representative blot with IGFBP-5 transcripts shown in the upper panel and the 18 S rRNA pattern shown below. RNA standards (Life Technologies, Inc.) were used to determine the length (in kilobases (kb)) of IGFBP-5 transcripts, shown in the right panel.

To investigate potential transcriptional mechanisms influenced by PGE treatment, gene transfer experiments were conducted. In initial studies, segments of the murine IGFBP-5 promoter containing various lengths of 5`-flanking DNA and 120 base pairs (bp) of exon 1 were fused to the luciferase reporter gene, transiently transfected into osteoblast-enriched cultures, and analyzed for reporter gene expression 48 h later. As shown in Fig. 2, IGFBP-5 promoter constructs with 1406, 1004, and 156 bp (IGFBP5-Luc3, -Luc4, and -Luc5) of 5`-flanking DNA directed comparably high luciferase activity (100, 95.6, and 84.3%, relative to IGFBP5-Luc3). In contrast, fusion plasmids with 75 bp or less of 5`-flanking DNA (IGFBP5-Luc6, -Luc7, and -Luc8) had progressively diminished activity (22.9 to 5.9%, relative to IGFBP5-Luc3). Luciferase expression for a positive control viral promoter-driven construct, pGL2-Control, was included for comparison. These data indicate that regions between -156 and -51 bp contain cis-acting regulatory element(s) needed for high level basal promoter activity in osteoblast cultures, analogous to the areas that we defined in Hep G2 (hepatocyte) and C2I (myoblast) cell lines(32, 37) . Two recombinant plasmids with longer 5`-flanking segments of 4100 and 3000 bp (IGFBP5-Luc1 and -Luc2) directed lower luciferase activity, suggesting the presence of inhibitory elements in the 5`-flanking region of the promoter. Promoter activity was orientation-specific. When the 1004-bp promoter fragment was inserted into the luciferase vector in the reversed orientation (IGFBP5-Luc4 rev, Fig. 2), reporter expression was minimal, being comparable with the promoterless pGL2-Basic parental plasmid.

Figure 2: Identifying regions controlling basal IGFBP-5 promoter function in osteoblast-enriched cultures. Various IGFBP-5 promoter-luciferase reporter plasmids (depicted in the left panel) were co-transfected with pSV--galactosidase control vector into osteoblast-enriched cultures (9.6 cm) using Lipofectin. Cultures were grown to confluence (48 h), the growth medium was aspirated, and the cultures were rinsed with serum-free Dulbecco's modified Eagle's medium. Cultures were exposed to control medium (containing ethanol vehicle) for 6 h. Cytoplasmic extracts were prepared, and luciferase activity was determined as described under ``Experimental Procedures.'' Data were corrected for transfection efficiency (-galactosidase expression) and for protein content of cytoplasmic extracts. Transfections were performed in duplicate or triplicate, and results are pooled data for three or more separate experiments for a total of eight or more replicate cultures. The mean ± S.E. for pooled experiments is shown. Luciferase activity was determined by single channel photon counting, and background levels were 930 cpm/µg of protein. Control transfections included an SV40 promoter/enhancer-luciferase reporter plasmid (pGL2-Control) and promoterless pGL-2 Basic (both from Promega Corp.). Numbers on the far right in parentheses indicate the percent maximal luciferase expression determined from the mean value for each construct, as compared with the mean value for IGFBP5-Luc3, which has been set at 100%.

Recombinant IGFBP-5 promoter-luciferase fusion constructs were next used to identify promoter elements that participated in PGE-stimulated IGFBP-5 expression. While basal luciferase activity for IGFBP5-Luc2 through -Luc7 varied up to 14-fold (Fig. 2), 6 h of PGE treatment enhanced their ability to drive luciferase expression to a similar extent, ranging from 2.3- to 1.6-fold (Fig. 3). The shortest construct responsive to PGE, IGFBP5-Luc 7, contained only 51 bp of 5`-flanking DNA. However, deletion of the next 20 bp (IGFBP5-Luc8) eliminated the effect of PGE on reporter gene expression. Using IGFBP5-Luc 4, a comparable increase was seen after 24 h of PGE treatment, and these effects were duplicated by treatment with forskolin (10 µM), a strong inducer of adenylate cyclase activity (Fig. 4). Plasmids with internal deletions spanning -69 to -51 bp and -52 to -32 bp in the background of the very active IGFBP5-Luc 4 construct had diminished basal activity as shown previously(32) . Importantly, however, each of these two deletion constructs clearly responded to PGE treatment, although at modestly reduced levels (1.4- and 1.7-fold, respectively; Fig. 5).

Figure 3: Effect of PGE on IGFBP-5 promoter activity following transient transfection into osteoblast-enriched cultures. Various IGFBP-5 promoter-luciferase reporter plasmids (depicted in Fig. 2) were co-transfected with a pSV--galactosidase control vector into osteoblast-enriched cultures (9.6 cm) using Lipofectin. Cultures were grown to confluence (48 h), the growth medium was aspirated, and the cultures were rinsed with serum-free Dulbecco's modified Eagle's medium. Cultures were exposed to control medium (containing vehicle) or PGE (1 µM) for 6 h. Cytoplasmic extracts were prepared and luciferase activity was determined. Data are corrected for transfection efficiency (-galactosidase expression) and for protein content of cytoplasmic extracts. Transfections were performed in duplicate or triplicate, and results are pooled data for three or more separate experiments and for a total of eight or more replicate cultures. The mean ± S.E. for luciferase expression compared with vehicle-treated cultures (-fold stimulation) for pooled experiments is shown. PGE caused a statistically significant elevation in luciferase expression (p < 0.05 versus pGL-2 Basic) for IGFBP-5 promoter reporter plasmids IGFBP5-Luc2, -Luc3, -Luc4, -Luc5, -Luc6, and -Luc7.

Figure 4: Forskolin and PGE induce comparable increases in IGFBP-5 promoter activity in osteoblast-enriched cultures. The IGFBP-5 promoter-luciferase reporter plasmid, IGFBP5-Luc4, was co-transfected with a pSV--galactosidase control vector into osteoblast-enriched cultures using Lipofectin, as described in Fig. 3and under ``Experimental Procedures.'' Cultures were grown to confluence (48 h), the growth medium was aspirated, and the cultures were rinsed with serum-free Dulbecco's modified Eagle's medium. Cultures were exposed to control medium (containing vehicle), forskolin at 10 µM, or PGE at 1 µM for 6 or 24 h, as indicated. Cytoplasmic extracts were prepared and luciferase activity was determined. Data are corrected for -galactosidase expression and for protein content of cytoplasmic extracts. Transfections were performed in duplicate or triplicate, and results are representative data for three separate experiments and for a total of eight or more replicate cultures. The mean ± S.E. for luciferase expression is shown.

Figure 5: Effect of PGE on the activity of mutant IGFBP-5 promoter following transient transfection into osteoblast-enriched cultures. IGFBP-5 promoter-luciferase reporter plasmids having deletion mutations spanning -69 to -51 bp, or -52 to -32 bp, in the IGFBP5-Luc 4 background (IGFBP5-Luc4a and IGFBP5-Luc4b, respectively) (32) were co-transfected with an pSV--galactosidase control vector into osteoblast-enriched cultures using Lipofectin, as described under ``Experimental Procedures'' and in the legend to Fig. 2. Cultures were exposed to control medium (containing vehicle) or PGE (1 µM) for 6 h. Cytoplasmic extracts were prepared and luciferase activity was determined. Data are corrected for transfection efficiency (-galactosidase expression) and for protein content of cytoplasmic extracts. Transfections were performed in duplicate or triplicate, and results are pooled data for three or more separate experiments and for a total of eight or more replicate cultures. The relative effect of PGE compared to vehicle-treated cultures (-fold stimulation) is shown for pooled experiments.

In aggregate, these results show that the IGFBP-5 promoter is very active in primary osteoblast cultures but demonstrate that PGE stimulated only a 2-fold increase in IGFBP-5 gene transcription, even after a 24-h incubation. This contrasts sharply with Northern blot data showing up to a 6-fold rise in steady-state IGFBP-5 mRNA levels at 24 h (Fig. 1). Thus, the discrepancy between maximal promoter activity and steady-state transcripts encoding IGFBP-5 in response to PGE indicates the participation of transcriptional and post-transcriptional mechanisms in regulating IGFBP-5 gene expression. Therefore, the RNA polymerase II selective inhibitor DRB was used to examine the influence of PGE on IGFBP-5 mRNA stability. Cultures were first treated with vehicle alone or PGE (1 µM) for 24 h, followed by DRB (75 µM) to arrest gene transcription. PGE treatment caused a 2-fold rise in the half-life of IGFBP-5 mRNAs; vehicle-treated control cultures had a t = 9 h, while cultures pretreated with PGE had t = 18 h (Fig. 6). Consequently, PGE enhanced the stability of IGFBP-5 transcripts. To explore further the mechanisms involved in induction of IGFBP-5 mRNA after PGE treatment, we examined the effect of the protein synthesis inhibitor, cycloheximide. At a dose of 2 µM, cycloheximide blocked >90% of ongoing protein synthesis, as measured by incorporation of [H]proline into trichloroacetic acid-precipitable material in PGE-treated cultures (-cycloheximide, 12.6 ± 0.9 10 cpm versus +cycloheximide, 1.3 ± 0.1 10 cpm). Cycloheximide also completely inhibited the induction of IGFBP-5 mRNA after PGE treatment (Fig. 7). These results contrast with the lack of an effect of cycloheximide on the rise in IGF-I transcript abundance following PGE treatment, ()and demonstrate that inducible protein(s) contribute to the transcriptional and post-transcriptional effects of PGE on IGFBP-5 gene expression in osteoblast-enriched cultures.

Figure 6: Effect of PGE on the stability of IGFBP-5 mRNA in osteoblasts. Osteoblast-enriched cultures were treated for 24 h with control (vehicle) or PGE (1 µM). Cultures were then supplemented with 75 µM DRB for the additional time intervals indicated. RNA was isolated, and 15 µg from each sample were fractionated by electrophoresis, blotted, and hybridized to a P-labeled rat IGFBP-5 cDNA, as described under ``Experimental Procedures.'' Data are relative IGFBP-5 abundance, as compared with transcript levels at the time of DRB addition (percent initial). Panel A, control treated cultures are shown in closed circles and those for the PGE are shown in closed squares. Data are from three separate experiments. Panel B, a representative Northern blot is shown with IGFBP-5 transcripts in the upper panel and the 18 S rRNA pattern below. RNA standards (Life Technologies, Inc.) were used to determine the length (in kilobases (kb)) of IGFBP-5 transcripts, shown at the left of panel B.

Figure 7: Effect of cycloheximide on IGFBP-5 transcript levels in control and PGE-treated osteoblast-enriched cultures. Confluent, serum-deprived cultures of osteoblasts were treated with control (C, vehicle) or PGE (P, 1 µM) for 6 h, in the absence or presence of 2 µM cycloheximide. RNA blots were prepared as described under ``Experimental Procedures,'' hybridized with P-labeled rat IGFBP-5 cDNA, washed, and visualized by autoradiography. A representative Northern blot probing is shown in the upper panel. The hybridized probe was eluted, and the blot was hybridized with P-labeled antisense 18 S ribosomal RNA riboprobe (Ambion, Houston, TX; shown below). RNA standards (Life Technologies, Inc.) were used to determine the length (in kilobases (kb)) of IGFBP-5 transcripts, shown on the left. Parallel cultures were treated with the same cycloheximide solution and cultures pulsed with [H]proline to assess effectiveness of the protein synthesis inhibitor; 90-95% inhibition of protein synthesis was observed. The Northern blot is representative of three independent experiments.

DISCUSSION

IGFBP-5 expression is activated through cAMP-dependent pathways in osteoblasts and in other cell culture models(4, 16, 39) . While IGFBP-5 transcripts accumulate in response to PGE and other agents that elevate cAMP, little is known about the mechanisms of hormone-induced IGFBP-5 synthesis in osteoblasts. We now present data demonstrating stimulation of IGFBP-5 promoter activity by PGE and show that PGE also enhances IGFBP-5 mRNA stability. These results indicate that promoter-dependent and -independent mechanisms function together to regulate IGFBP-5 gene expression.

Unstimulated fetal rat osteoblasts synthesize IGFBP-5 mRNA and protein(4, 40, 41) . Results from transient transfection experiments confirm our earlier studies that near-maximal basal promoter activity resides within the first 156 bp of 5`-flanking DNA and that over 20% of basal activity is controlled by the proximal 75 bp of the promoter (32) . Similar to our earlier evidence, promoter function is attenuated by internal deletions that eliminate nucleotides -69 to -51 or -52 to -32, encoding segments that span a DNase I-footprinted region identified with Hep G2 nuclear protein extracts(32) .

In transient transfections of osteoblasts, PGE treatment for 6 or 24 h increased luciferase activity driven by the IGFBP-5 promoter 1.6-2.3-fold in constructs containing as little as 51 bp or as much as 3000 bp of 5`-flanking DNA. The shortest promoter fragments mediating PGE-induced gene transcription do not contain a consensus cAMP response element. However, a potential binding site for transcription factor AP-2 is present between nucleotides -44 and -36, and at least six AP-2 sites are dispersed throughout the 3000 bp of the IGFBP-5 promoter. The apparent decline in response to PGE treatment seen with recombinant plasmids having progressively shorter promoter segments may reflect loss of individual AP-2 binding sites or other potential cAMP-responsive cis-elements.

While this paper was in preparation, Duan and Clemmons (42) reported the involvement of AP-2 in basal and cAMP-mediated IGFBP-5 transcription in human dermal fibroblast cell lines. In their study, forskolin stimulated IGFBP-5 promoter activity 2.8-fold, a result similar in magnitude to our observation in osteoblasts. They identified an AP-2 site within nucleotides -55 to -36 in the human IGFBP-5 promoter as the key hormone response element(42) . An identical AP-2 site is present in a comparable location in the murine promoter, as noted above. While deletion of this site in construct IGFBP5-Luc4b reduced basal promoter activity, it caused only a modest decline in the effect of PGE. Therefore, additional AP-2 sites or alternative cAMP response elements may be functional in osteoblasts.

The modest 2-fold effect of PGE on IGFBP-5 transcription does not account for the 6-fold increase in steady-state IGFBP mRNA seen following a 24-h incubation. As demonstrated here, PGE also caused a doubling of IGFBP-5 transcript half-life, from 9 to 18 h. Of note, the t for IGFBP-5 mRNA in osteoblast-enriched cultures under basal conditions, 9 h, is similar to the 11-12-h transcript half-life measured by us in C2I myoblasts (37) but differs somewhat from reported values of 14 and 20 h obtained under basal conditions in a similar osteoblast culture model(40, 41) , which may be accounted for by small differences in experimental design. Thus, both transcriptional and post-transcriptional effects of PGE contribute to the induction of IGFBP-5 mRNA following hormone treatment. These dual actions on IGFBP-5 gene expression can be distinguished from transcriptional effects of PGE on the IGF-I gene, which appear to be mediated through an element found in the proximal part of promoter 1, the major IGF-I gene promoter(27) . In addition, the actions of PGE to enhance IGFBP-5 gene expression require ongoing protein synthesis, since they were obliterated by preincubation with cycloheximide, while PGE-stimulated IGF-I gene transcription occurs even in the absence of new protein synthesis. ()

Levels of IGFBP-5 in extracellular compartments are modulated not only by rates of gene expression and protein biosynthesis but also by post-translational mechanisms. The existence of IGFBP-5-selective proteases has been documented in a variety of cultured cells, including normal human osteoblasts(12, 13, 14, 43) , and IGF-mediated stabilization of IGFBP-5 abundance has been described in culture models derived from bone and other cell types(44) . Since IGFBP-5 enhances the anabolic actions of IGF-I in bone cells(5) , analysis of the multiple mechanisms involved in modifying IGFBP-5 availability within the skeleton should have direct impact in understanding how growth factors regulate skeletal cell metabolism.

FOOTNOTES

*

This work was supported by National Institutes of Health Grants DK47421 (to T. L. M.) and DK42748 (to P. R.) and by NASA Grant NAGW-4550 (to T. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence and reprint requests should be addressed: Section of Plastic Surgery, P. O. Box 208041, Yale University School of Medicine, New Haven, CT 06520-8041. Tel.: 203-785-4927; Fax: 203-737-1311 or 203-785-5714.

()

The abbreviations used are: IGF, insulin-like growth factor; bp, base pair(s); DRB, 5,6-dichloro-1--D-ribofuranosylbenzimidazole; IGFBP, IGF binding protein; PGE, prostaglandin E; PTH, parathyroid hormone.

()

T. L. McCarthy and M. Centrella, unpublished data.

()

T. L. McCarthy, M. J. Thomas, Y. Umayahara, H. Shu, M. Centrella, and P. Rotwein, manuscript in preparation.

()

T. L. McCarthy, M. J. Thomas, Y. Umayahara, H. Shu, M. Centrella, and P. Rotwein, unpublished observation.

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