Skeletal Growth Factors Regulate the Synthesis of Insulin-like Growth Factor Binding Protein-5 in Bone Cell Cultures

doi:10.1074/jbc.270.18.10771

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Volume 270, Number 18, Issue of May 5, pp. 10771-10776, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

Skeletal Growth Factors Regulate the Synthesis of Insulin-like Growth Factor Binding Protein-5 in Bone Cell Cultures (*)

Ernesto Canalis (1) (2)(§), Bari Gabbitas (1)

From the (1) Departments of Research and Medicine, Saint Francis Hospital and Medical Center, Hartford, Connecticut 06105 and the (2) University of Connecticut School of Medicine, Farmington, Connecticut 06030

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

ABSTRACT

Skeletal cells secrete insulin-like growth factors (IGFs) I and II and six known IGF binding proteins (IGFBPs). IGFBP-5 stimulates bone formation, and its synthesis correlates with changes in osteoblast cell growth. We tested the effects of basic fibroblast growth factor (bFGF), transforming growth factor 1 (TGF 1), and platelet-derived growth factor (PDGF) BB on IGFBP-5 expression in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Treatment of Ob cells with bFGF, TGF 1, and PDGF BB caused a time- and dose-dependent decrease in IGFBP-5 mRNA levels and inhibited IGFBP-5 polypeptide levels in the extracellular matrix. The effects of bFGF, TGF 1, and PDGF BB on IGFBP-5 transcripts were independent of cell division and were observed in the presence and absence of hydroxyurea. bFGF, TGF 1, and PDGF BB did not modify the decay of IGFBP-5 mRNA in transcriptionally arrested Ob cells, and they inhibited IGFBP-5 heterogeneous nuclear RNA and the rate of IGFBP-5 transcription. In conclusion, bFGF, TGF 1, and PDGF BB inhibit IGFBP-5 expression in Ob cells independently of their mitogenic activity and through mechanisms that involve decreased transcription.

INTRODUCTION

Skeletal cells are known to secrete insulin-like growth factors (IGFs)() I and II as well as the six known IGF binding proteins (IGFBPs) (1, 2, 3, 4, 5, 6) . IGFs I and II are among the most important local regulators of bone cell function because of their abundance in bone tissue as well as their stimulatory actions on multiple aspects of bone formation (1, 3, 7) . The synthesis and activity of IGFs are regulated by systemic hormones, by other skeletal growth factors, and by IGFBPs (8, 9, 10) . Whereas the exact function of IGFBPs in bone is not known, most of the IGFBPs have been reported to have inhibitory activities on bone formation (11, 12, 13) . However, IGFBP-5 has been consistently shown to increase bone cell growth and enhance the actions of IGF I on this process (14) . Furthermore, the expression of IGFBP-5 appears to be related to the stage of osteoblast growth and differentiation (15) . Therefore, studies to define agents that regulate the synthesis and activity of IGFBP-5 in bone cells are critical to our understanding of its role in bone physiology.

Investigations from this and other laboratories have revealed that skeletal growth factors inhibit the synthesis of IGFs I and II in cells of the osteoblastic lineage and that the synthesis of IGF I and IGFBP-5 is coordinated (9, 10, 16, 17) . Studies using osteoblast cultures have revealed that hormones that stimulate or inhibit IGF I synthesis have similar effects on IGFBP-5 synthesis (16, 17) . Furthermore, IGF I enhances the synthesis and stability of IGFBP-5 (17, 18) . We postulated that skeletal growth factors with potent mitogenic activity not only decrease skeletal IGFs I and II synthesis but may have analogous effects on IGFBP-5 expression, and these changes are relevant to the growth and differentiated function of the osteoblast.

The present studies were undertaken to examine the effects of basic fibroblast growth factor (bFGF), transforming growth factor 1 (TGF 1), and platelet-derived growth factor (PDGF) BB on IGFBP-5 synthesis in cultures of osteoblast-enriched cells from fetal rat parietal bone (Ob cells) and to determine possible mechanisms involved.

MATERIALS AND METHODS

Culture Technique

The culture method used was described in detail previously (16) . Parietal bones were obtained from 22-day-old fetal rats immediately after the mothers were sacrificed by blunt trauma to the nuchal area. This project was approved by the Institutional Animal Care and Use Committee of Saint Francis Hospital and Medical Center. Cells were obtained by five sequential digestions of the parietal bone using bacterial collagenase (CLS II, Worthington). Cell populations harvested from the third to the fifth digestions were cultured as a pool and were previously shown to have osteoblastic characteristics (19) . Ob cells were plated at a density of 8,000-12,000 cells/cm

and cultured in a humidified 5% CO

incubator at 37 °C until reaching confluence (about 50,000 cells/cm

). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with nonessential amino acids, 100 µg/ml L-ascorbic acid, penicillin, streptomycin, and 20 mM HEPES (all from Life Technologies, Inc.), and 10% fetal bovine serum (HyClone, Logan, UT). At confluence the cells were rinsed and transferred to serum-free medium for 20-24 h, when they were again rinsed with serum-free medium, and then exposed to test or control medium in the absence of serum for 2-48 h. The medium of cultures lasting 48 h was replaced with fresh solutions after 24 h. Cycloheximide, hydroxyurea (both from Sigma), and recombinant human PDGF BB (Austral, San Ramon, CA) were added directly to the culture medium. Basic FGF (Austral) was dissolved in 20 mM sodium citrate and diluted 1:5,000 or greater in culture medium, and recombinant human TGF

1 (Austral and a gift from Genentech, South San Francisco, CA) was either added directly to the medium or dissolved in 5 mM HCl and diluted 1:3,000 or greater in DMEM. 5,6-dichlorobenzimidazole (DRB) (Sigma) was dissolved in absolute ethanol and diluted 1:200 in DMEM; in experiments where DRB was used, all experimental groups were exposed to an equal amount of ethanol. For the nuclear run on experiment, subconfluent cultures of Ob cells were trypsinized, subcultured at a 1:6 dilution, and grown to confluence in DMEM supplemented with 10% fetal bovine serum. Cells were serum-deprived for 24 h and treated for 2-24 h in serum-free DMEM. For RNA analysis, the cell layer was extracted with guanidine thiocyanate at the end of the incubation and stored at -80 °C. For protein analysis, the conditioned medium was obtained, the extracellular matrix was extracted, and both were processed for Western blots. For the nuclear run on assay, nuclei were isolated by Dounce homogenization.

Northern Blot Analysis

Total cellular RNA was isolated with guanidine thiocyanate, at acid pH, followed by phenol-chloroform (Sigma) extraction (20) . RNA was precipitated with isopropyl alcohol, resuspended, and reprecipitated with ethanol. The RNA recovered was quantitated by spectrometry, and equal amounts of RNA from control or test samples were loaded on a formaldehyde-agarose gel following denaturation. RNA standards (BDH Ltd., Poole, U. K.) were used to determine transcript size. The gel was stained with ethidium bromide to visualize RNA standards and ribosomal RNA, documenting equal RNA loading of the various experimental samples. The RNA was then blotted onto Gene Screen Plus charged nylon (DuPont). A 300-base pair HindIII restriction fragment of the rat IGFBP-5 cDNA (kindly provided by Dr. S. Shimasaki, La Jolla, CA) was purified by agarose gel electrophoresis (21) . IGFBP-5 cDNA was labeled with [

P]dCTP and [

P]dATP (50 µCi of each at a specific activity of 3,000 Ci/mmol; (DuPont) using the random hexanucleotide-primed second strand synthesis method (22) . Hybridizations were carried out at 42 °C for 16-72 h, and post-hybridization washes were performed at 65 °C in 0.1

saline-sodium citrate. The bound radioactive material was visualized by autoradiography on Kodak X-AR5 film, employing Cronex Lightning Plus intensifying screens (DuPont). Relative hybridization levels were determined by densitometry. Northern analyses shown are representative of three or more cultures.

Heterogeneous Nuclear RNA (hnRNA) Analysis

To examine changes in hnRNA, rat IGFBP-5 intron 2-specific primers were designed following partial sequence analysis of intron 2. For this purpose, intron 2 of IGFBP-5 was amplified from rat genomic DNA (Promega, Madison, WI), using the sense exon 2 primer 5`-GACTCTCGGGAGCATGAGGAAC-3` and the antisense exon 3 primer 5`-AGCTTCCATGTGTCTGCGGCAG-3`, by polymerase chain reaction (PCR) using Taq DNA polymerase in accordance with manufacturer's instructions (Perkin Elmer). The PCR amplification was performed for 30 cycles of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min. A single PCR product of approximately 780 base pairs was purified by agarose gel electrophoresis and subcloned into pCR

II (Invitrogen, San Diego, CA), and its identity was confirmed by partial DNA sequence analysis.

To determine changes in hnRNA, total RNA from control and test samples was prepared as described for Northern analysis. One µg of RNA was treated with DNase and reverse-transcribed in the presence of the rat IGFBP-5 intron 2-specific antisense primer 5`-CTTAGGATGCACGTGGTT-3` at 42 °C for 30 min with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) The newly transcribed cDNA was amplified by 25 PCR cycles of 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min, following the addition of the IGFBP-5 intron 2-specific sense primer 5`-GCACCATCCCAATCTGAT-3`, Taq DNA polymerase, and 5 µCi of [-P]dCTP (3,000 Ci/mmol, DuPont) as described (23, 24) . The PCR products were fractionated by electrophoresis on a 6% urea-polyacrylamide denaturing gel, visualized by autoradiography, and quantitated by densitometry. An internal DNA standard was included in the PCR and used to correct for variations in amplification. The standard was obtained by amplification of SV40 promoter sequences in the pGL2-P plasmid DNA using the composite sense primer 5`-GCACCATCCCAATCTGATattagtcagcaaccatagtc-3`, and antisense primer 5`-CTTAGGATGCACGTGGTTggttccatcctctagaggat-3`. The capital letters indicate IGFBP-5 intron 2 sequences, while the lowercase letters represent pGL2-P vector sequences.

Nuclear Run On Assay

To examine changes in the rate of transcription, nuclei were isolated by Dounce homogenization in a Tris buffer containing 0.5% Nonidet P-40. Nascent transcripts were labeled by incubation of nuclei in a reaction buffer containing 500 µM each of ATP, CTP, and guanidine triphosphate, 150 units of RNAsin (Promega), and 250 µCi of [

P]UTP (3,000 Ci/mmol, DuPont) (25) . RNA was isolated by treatment with DNase I and proteinase K, followed by phenol-chloroform extraction and ethanol precipitation. Linearized plasmid DNA containing about 1 µg of cDNA was immobilized onto GeneScreen Plus by slot blotting according to the manufacturer's directions (DuPont). The plasmid vector pGL2-Basic (Promega) was used as a control for nonspecific hybridization, and a mouse 18 S cDNA clone (ATCC, Rockville, MD) was used to estimate loading of the gel. Equal counts/min of [

P]RNA from each sample were hybridized to cDNAs using the same conditions as for Northern blot analysis and were visualized by autoradiography.

Western Blots

Extracellular matrix was prepared as described (26, 27) . Briefly, Ob cells were rinsed in phosphate-buffered saline, cell membranes were removed with 0.5% Triton X-100 (Sigma), pH 7.4, and nuclei and cytoskeleton were removed by incubation with 25 mM ammonium acetate, pH 9.0, for 5 min. The extracellular matrix was rinsed with phosphate-buffered saline and scraped from the culture plates. Aliquots from the conditioned medium or the extracellular matrix were fractionated by polyacrylamide gel electrophoresis on 10-18% denaturing gradient gels (28) . For Western immunoblots, proteins were transferred to Immobilon P membranes (Millipore, Bedford, MA), blocked with 2% bovine serum albumin, and exposed to a 1:500 dilution of rabbit antisera raised against native human IGFBP-5 (UBI, Lake Placid, NY), in 1% bovine serum albumin overnight. Blots were exposed to goat anti-rabbit IgG antisera conjugated to horseradish peroxidase, washed, and developed with a horseradish peroxidase chemiluminescence detection reagent (DuPont). For Western ligand blots, transferred proteins were incubated with

I-IGF II, and the blot was developed by autoradiography as described (29) . IGFBP-5 was identified by co-migration with recombinant human IGFBP-5 (Austral).

RESULTS

Northern blot analysis of total RNA extracted from confluent cultures of Ob cells revealed a predominant IGFBP-5 transcript of 6.0 kilobases, although in accordance with observations in other cell systems, smaller size transcripts were also detected (30, 31, 32, 33) . Continuous treatment of Ob cells with bFGF, TGF 1, and PDGF BB caused a time-dependent decrease in IGFBP-5 steady state mRNA levels. Treatment of Ob cells with bFGF at 6 nM for 6 h caused no change in IGFBP-5 mRNA, whereas TGF 1 at 0.4 nM and PDGF BB at 3.3 nM for 6 h caused a slight decrease in IGFBP-5 transcripts (Fig. 1). After 24 h of treatment with bFGF, TGF 1, and PDGF BB at the indicated doses, densitometric analysis revealed a 75% or greater decrease in IGFBP-5 mRNA, and the inhibition was sustained for 48 h. Because the growth factors tested increase -actin and glyceraldehyde-3-phosphate dehydrogenase mRNA, IGFBP-5 mRNA levels were not corrected for changes in -actin or glyceraldehyde-3-phosphate dehydrogenase mRNA, and uniformity of RNA loading of the gels was estimated by ethidium bromide staining of ribosomal RNA (9) .() Continuous treatment of Ob cells with bFGF, TGF 1, and PDGF BB for 48 h caused a dose-dependent inhibition of IGFBP-5 transcript levels (Fig. 2). Densitometric analysis revealed that bFGF at 6 nM and PDGF BB at 3.3 nM decreased IGFBP-5 mRNA by 70% and that TGF 1 at 1.2 nM decreased IGFBP-5 transcripts to virtually undetectable levels.

Figure 1: Effect of basic fibroblast growth factor ( FG) at 6 nM, transforming growth factor

1 ( T

) at 0.4 nM, and platelet-derived growth factor ( BB) at 3.3 nM on IGFBP-5 mRNA expression in cultures of Ob cells treated for 6, 24, or 48 h. Total RNA from control ( C) or treated cultures was subjected to Northern blot analysis and hybridized with a

P-labeled rat IGFBP-5 cDNA. Visualization of the 18 and 28 S ribosomal RNA by ethidium bromide staining demonstrates RNA loading of the gel. IGFBP-5 mRNA was visualized by autoradiography and is shown in the upperpanel, while ribosomal RNA is shown below.

Figure 2: Effect of bFGF, transforming growth factor

1 ( TGF

), and PDGF BB, in nanomolar concentrations, on IGFBP-5 mRNA expression in cultures of Ob cells treated for 48 h. Total RNA from control (0) or treated cultures was subjected to Northern blot analysis and hybridized with a

Western ligand blot analysis of the extracellular matrix of untreated Ob cells revealed that they expressed a predominant IGFBP, which co-migrated with an IGFBP-5 standard and had a molecular mass of 31 kDa (Fig. 3 A). Western immunoblots confirmed the presence of a major form of immunoreactive IGFBP-5 of 31 kDa and two minor forms in the 29-30-kDa range (Fig. 3 B). These probably represent different degrees of IGFBP-5 glycosylation (18, 30) . bFGF at 6 nM, TGF

1 at 1.2 nM, and PDGF BB at 3.3 nM for 24 h decreased the 31-kDa form of IGFBP-5 as determined by Western ligand and immunoblots (Fig. 3, A and B). The Western immunoblot revealed immunoreactive protein bands of 45-50 kDa, which increased with TGF

1 and PDGF BB treatment. These proteins were not detected in the Western ligand blot shown in Fig. 3A. Stripping of the immunoblot shown and incubation with

I-IGF II as ligand visualized IGFBP-5 but not the 45-50-kDa proteins, confirming that they were not IGFBPs (not shown). The levels of IGFBP-5 in the culture medium were low, making the detection of an inhibitory effect impractical (not shown).

Figure 3: Effect of basic fibroblast growth factor ( FG) at 6 nM, transforming growth factor

1 ( T

) at 1.2 nM, and platelet-derived growth factor ( BB) at 3.3 nM on IGFBP-5 polypeptide levels in cultures of Ob cells treated for 24 h. A, extracts from extracellular matrix of control ( C) and treated cultures were subjected to Western ligand blot and IGFBPs detected by incubation with

I-IGF II and autoradiography. The arrow indicates the migration of a human IGFBP-5 standard (not shown). B, extracts from extracellular matrix were subjected to Western immunoblot analysis, and IGFBP-5 was detected using an anti-IGFBP-5 antibody and a chemiluminescence detection system.

To determine whether or not the effects observed on IGFBP-5 mRNA levels were dependent on protein synthesis, serum-deprived confluent cultures of Ob cells were treated with the various growth factors in the presence or absence of cycloheximide at 3.6 µM. In earlier experiments cycloheximide at doses of 2 µM and higher was found to inhibit protein synthesis in Ob cell cultures by 80-85% (34) . Northern blot analysis revealed that treatment with cycloheximide for 24-48 h caused a comparable and marked decrease in IGFBP-5 transcript levels so that further inhibitory effects of bFGF, TGF

1, and PDGF BB could not be detected (Fig. 4). To determine if the changes in IGFBP-5 mRNA levels observed were related to the mitogenic activity of the growth factors studied, serum-deprived Ob cell cultures were treated with the various factors in the presence or absence of hydroxyurea at 1 mM, a dose previously shown to block DNA synthesis in Ob cells (35) . Northern blot analysis revealed that treatment with hydroxyurea for 48 h did not modify IGFBP-5 transcripts and did not prevent the inhibitory effect of bFGF, TGF

1, or PDGF BB (Fig. 5).

Figure 4: Effect of basic fibroblast growth factor ( FG) at 6 nM, transforming growth factor

1 ( T

) at 1.2 nM, and platelet-derived growth factor ( BB) at 3.3 nM in the presence (+) and absence (-) of cycloheximide at 3.6 µM on IGFBP-5 mRNA expression in cultures of Ob cells treated for 48 h. Total RNA from control ( C) or treated cultures was subjected to Northern blot analysis and hybridized with a

Figure 5: Effect of basic fibroblast growth factor ( FG) at 6 nM, transforming growth factor

1 ( T

) at 1.2 nM, and platelet-derived growth factor ( BB) at 3.3 nM in the presence (+) or absence (-) of hydroxyurea at 1 mM on IGFBP-5 mRNA expression in cultures of Ob cells treated for 48 h. Total RNA from control ( C) or treated cultures was subjected to Northern blot analysis and hybridized with a

To examine whether or not the effects of the growth factors studied on IGFBP-5 were due to changes in transcript stability, confluent cultures of Ob cells were exposed to control or growth factor-containing medium for 60 min. and then treated with the RNA polymerase II inhibitor DRB in the absence or presence of bFGF at 6 nM, TGF

1 at 1.2 nM, and PDGF BB at 3.3 nM for 6, 16, or 24 h (36) . The half-life of IGFBP-5 mRNA in transcriptionally arrested Ob cells was estimated at 20 h, and it was not changed by treatment with any of the growth factors tested (Fig. 6). Treatment of Ob cells with bFGF at 6 nM, TGF

1 at 1.2 nM, and PDGF BB at 3.3 nM for 2, 6, or 24 h decreased IGFBP-5 hnRNA expression as estimated by reverse transcription PCR (Fig. 7). This would suggest a change in RNA transcription or processing. After 2 and 6 h of treatment, bFGF and TGF

1 decreased IGFBP-5 hnRNA by at least 90% as determined by densitometry, whereas PDGF BB inhibited IGFBP-5 hnRNA by 30-50%. The inhibitory effect of PDGF BB on hnRNA was not observed after 24 h of treatment, whereas bFGF and TGF

1 were effective for up to 24 h. The growth factors tested did not change the signal of the internal standard, indicating uniform PCR. Furthermore, no signal of the hnRNA product was detected in any of the samples tested when the reverse transcription step was omitted prior to the PCR, eliminating the possibility of DNA contamination. To confirm whether the growth factors tested modified the transcription of the IGFBP-5 gene, nuclear run on assays were performed on nuclei from Ob cells treated for 2-24 h. bFGF at 6 nM, TGF

1 at 1.2 nM, and PDGF BB at 3.3 nM inhibited the rate of IGFBP-5 transcription after 14 h by 20-60% and after 24 h by 70-80% (Fig. 8). The factors were not effective after 2 h, and after 6 h of treatment only a modest decrease in the rate of IGFBP-5 transcription was observed (not shown).

Figure 6: Effect of bFGF at 6 nM, TGF

1 at 1.2 nM, and PDGF BB at 3.3 nM on IGFBP-5 mRNA decay in transcriptionally blocked Ob cells. Cultures were treated with bFGF, TGF

1, or PDGF BB 1 h before and 6, 16, or 24 h after the addition of DRB. RNA was subjected to Northern blot analysis and hybridized with a

P-labeled rat IGFBP-5 cDNA, visualized by autoradiography, and quantitated by densitometry. Values are means ± S.E. for three or more cultures, except for TGF

1 24 h after DRB, which represents two cultures.

Figure 7: Effect of basic fibroblast growth factor ( FG) at 6 nM, transforming growth factor

1 ( T

) at 1.2 nM, and platelet-derived growth factor ( BB) at 3.3 nM on heterogeneous nuclear RNA expression in cultures of Ob cells treated for 2, 6, or 24 h. Total RNA from control ( C) or treated cultures was extracted, and 1 µg was subjected to competitive reverse transcription-PCR in the presence of IGFBP-5 intron 2-specific 5`-sense and 3`-antisense primers and of 5 µCi of [

P]dCTP. The reverse transcriptase-PCR products were fractionated by polyacrylamide gel electrophoresis and visualized by autoradiography. IGFBP-5 hnRNA is shown in the upperpanel, and the internal standard, prepared as described under ``Materials and Methods,'' is shown below.

Figure 8: Effect of basic fibroblast growth factor ( FG) at 6 nM, transforming growth factor

1 ( T

) at 1.2 nM, and platelet-derived growth factor ( BB) at 3.3 nM on IGFBP-5 transcription rates in cultures of Ob cells treated for 14 ( left panel) or 24 h ( right panel). Nascent transcripts were labeled in vitro with [

P]UTP, and the labeled RNA was hybridized to immobilized cDNA for IGFBP-5. Murine 18 S cDNA was used to demonstrate loading, and pGL2-Basic vector DNA ( pGL2-b) was used as a control for nonspecific hybridization.

DISCUSSION

Recent studies have demonstrated that skeletal cells synthesize IGFs I and II and six known IGFBPs. The present investigation was undertaken to determine whether growth factors known to be synthesized by skeletal cells modify IGFBP-5 expression in calvarial derived Ob cells. We demonstrated that bFGF, TGF 1, and PDGF BB decrease IGFBP-5 mRNA levels in Ob cells in a time- and dose-dependent manner and that de novo protein synthesis is required for basal IGFBP-5 transcript expression. The growth factors tested are known to stimulate bone cell replication, but the decrease in IGFBP-5 synthesis in Ob cells was not modified by the DNA synthesis inhibitor hydroxyurea, suggesting that this effect was independent from their mitogenic activity. Experiments in transcriptionally blocked Ob cells, using the RNA polymerase II inhibitor DRB, revealed that bFGF, TGF 1, and PDGF BB did not modify IGFBP-5 mRNA stability. This, in conjunction with a decrease in hnRNA levels and in the rate of IGFBP-5 gene transcription caused by the three growth factors studied, suggests that bFGF, TGF 1, and PDGF BB inhibit IGFBP-5 expression at the level of RNA transcription. Our studies revealed that changes in the rate of IGFBP-5 transcription were delayed and somewhat less intense than those in hnRNA. This could be due to technical differences. However, since changes in hnRNA levels may be due to effects on transcription or RNA processing, it is possible that the growth factors studied had an early effect on RNA processing in addition to their effects on IGFBP-5 transcription.

Intact IGFBP-5 is primarily present in the extracellular matrix of skeletal and nonskeletal cells (27) , and the three growth factors studied decreased IGFBP-5 in this compartment. PDGF BB and TGF 1 also increased proteins migrating with a M of 45,000-50,000. The nature of the protein reacting with the IGFBP-5 antibody is presently unknown. However, this protein(s) does not appear to be an IGFBP since it did not bind radiolabeled IGF II. The amount of IGFBP-5 secreted to the culture medium of Ob cells under the described culture conditions is small, and peptide degradation occurs, so inhibitory changes are difficult to detect. Modifications in IGFBP-5 protease levels or activity is another level of regulation by which skeletal growth factors may modify IGFBP-5 polypeptide in bone cells (37, 38) . It was recently shown that matrix metalloproteinase I or interstitial collagenase degrades IGFBP-5, and bFGF, TGF 1, and PDGF BB regulate interstitial collagenase synthesis in skeletal and nonskeletal cells (38, 39) .() This would suggest that the growth factors studied have the capability of regulating IGFBP-5 by transcriptional and post-translational mechanisms.

While there are uncertainties about the physiological concentrations of bFGF, TGF 1, and PDGF BB in bone cultures, their effects on IGFBP-5 synthesis were observed at doses that modify other parameters of metabolic function in Ob cells and doses that are known to inhibit IGF I and II synthesis in bone (8, 9, 10) . This suggests that the inhibition of IGFBP-5 synthesis may be physiologically relevant. A decrease in IGFs I and II as well as IGFBP-5 synthesis might mediate selected effects of growth factors on bone cell function. Recently, it was shown that bFGF and PDGF BB increase the synthesis of skeletal IGFBP-4, a binding protein with inhibitory properties in bone (5) . This effect as well as an inhibition of IGFBP-5, IGF I, and IGF II synthesis may be relevant to the actions of selected growth factors on bone cell function. On the other hand, bFGF, TGF 1, and PDGF BB have complex effects in bone, and it is likely that they have a number of actions on bone metabolism, which are independent of their effects on the IGF-IGFBP axis (8) .

IGFBP-5 has significant effects on bone cell growth, and its expression is coordinated with stages of osteoblast cell growth. In addition, its expression in myoblasts is correlated with cell differentiation and, as in bone cells, it is inhibited by growth factors with mitogenic properties (40, 41) . These observations suggest a coordinated expression of IGFBP-5 in bone and muscle and a possible relevance to the expression of the differentiated phenotype in the musculoskeletal system. In fact, bFGF, PDGF BB, and (to a somewhat more variable extent) TGF 1 have been shown to inhibit the differentiated expression of the osteoblastic phenotype, and they also inhibit IGFBP-5 synthesis (8, 42, 43) . Furthermore, factors such as IGFs I and II, which stimulate osteoblastic differentiated function, increase IGFBP-5 synthesis and stability in bone cells (17, 18, 30) . The stimulatory effects of IGFBP-5 on bone cell function are unique since IGFBP-2, -3, and -4 inhibit various parameters of bone formation (11, 12, 13) . IGFBP-1 plays a role in glucose homeostasis, and it has not been reported to have a specific function in skeletal metabolism (44, 45) . IGFBP-6 inhibits IGF II-induced differentiation of myoblasts, but its effects in bone cells are not known (46) .

In conclusion, the present studies demonstrate that bFGF, TGF 1, and PDGF BB inhibit IGFBP-5 transcripts and polypeptide levels in skeletal cells through mechanisms that may involve diminished transcription. The reduced level of IGFBP-5 by local growth factors in the bone microenvironment may constitute an important level of control of the autocrine and paracrine actions of IGF in bone via IGFBPs.

FOOTNOTES

*: This work was supported by Grant DK42424 from NIDDKD, National Institutes of Health, and Grant AR21707 from NIAMSD, National Institutes of Health. 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: Dept. of Research, Saint Francis Hospital and Medical Center, 114 Woodland St., Hartford, CT 06105-1299. Tel.: 203-548-4068; Fax: 203-548-5415.
: The abbreviations used are: IGF, insulin-like growth factor; IGFBP, IGF binding protein; bFGF, basic fibroblast growth factor; TGF 1, transforming factor 1; PDGF, platelet-derived growth factor; DMEM, Dulbecco's modified Eagle's medium; DRB, 5,6-dichlorobenzimidazole; hnRNA, heterogeneous nuclear RNA; PCR, polymerase chain reaction.
: E. Canalis, unpublished observations.
: E. Canalis and B. Gabbitas, unpublished observations.
: S. Varghese and E. Canalis, unpublished observations.

ACKNOWLEDGEMENTS

We thank Dr. S. Shimasaki for the rat IGFBP-5 cDNA clone, Dr. Yu Dong for assistance with the design of IGFBP-5 intronic primers, Cathy Boucher and Deena Kjeldsen for technical assistance, and Beverly Faulds for expert secretarial help.

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