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Osteogenic Protein-1 and Insulin-Like Growth Factor I Synergistically Stimulate Rat Osteoblastic Cell Differentiation and Proliferation¹

Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760

Address all correspondence and requests for reprints to: Dr. Lee-Chuan C. Yeh, Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies have shown that osteogenic protein-1 (OP-1; also known as BMP-7) alters the steady state levels of messenger RNA (mRNA) encoding insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding proteins (IGFBPs) in primary cultures of fetal rat calvaria (FRC) cells. In the present study, the effects of exogenous IGF-I on bone cell differentiation and mineralized bone nodule formation induced by OP-1 were examined. Exogenous IGF-I synergistically and dose dependently enhanced OP-1 action in stimulating [³H]thymidine incorporation, alkaline phosphatase activity, PTH-dependent cAMP level, and bone nodule formation. Maximal synergism between OP-1 and IGF-I was observed when both factors were added simultaneously. Synergism was not observed when FRC cells were pretreated with IGF-I for 24 h, followed by OP-1 treatment. These findings suggest that IGF-I acted on OP-1-sensitized FRC cells. To examine the mechanism(s) by which this sensitization may occur, levels of mRNA encoding OP-1 receptor, IGF-I receptor, and IGFBPs were measured. The mRNA levels of both type I and II OP-1 receptors were elevated by OP-1, but were not changed further by combined OP-1 and IGF-I treatment. IGF-I receptor gene expression was not changed by OP-1, IGF-I, or a combination of both factors. OP-1 alone or together with IGF-I increased the steady state IGFBP-3 mRNA level and reduced the steady state mRNA levels of IGFBP-4, -5, and -6. IGF-I alone did not change the steady state mRNA levels of IGFBP-3, -4, and -6, but elevated that of IGFBP-5. Des(1–3)-IGF-I, which has a lower affinity for IGFBPs, was more effective than the full-length IGF-I in enhancing the OP-1-induced alkaline phosphatase activity. Exogenous IGFBP-5 inhibited the OP-1-induced alkaline phosphatase activity and reduced the synergistic stimulatory effect of IGF-I and OP-1. These findings strongly suggest that the OP-1-induced down-regulation of IGFBPs, especially that of IGFBP-5, is an important mechanism by which OP-1 and IGF-I synergize to stimulate FRC cell differentiation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
OSTEOGENIC protein-1 (OP-1; also known as BMP-7) is a member of the bone morphogenetic protein (BMP) subfamily of the transforming growth factor-ß (TGFß) superfamily (1, 2, 3). Recombinant human OP-1 induces new bone formation in vivo (4, 5, 6) and stimulates the synthesis of various biochemical markers characteristic of in vitro osteoblast differentiation. These markers include, but are not limited to, alkaline phosphatase (AP), osteocalcin, osteopontin, and PTH receptors (7, 8, 9, 10, 11, 12, 13). In primary cultures of fetal rat calvaria (FRC) cells, OP-1 stimulates cell proliferation and promotes differentiation of confluent FRC cells into an osteoblastic phenotype (9). OP-1 not only stimulates the proliferation and differentiation of osteoblasts in vitro, but also participates in the recruitment of osteoclasts (7, 14).

OP-1 has been shown to change the expression of the different components of the insulin-like growth factor (IGF) system (15, 16, 17, 18). For example, OP-1 increased the IGF-II level in the conditioned medium (CM) of SaOS-2 and TE85 human osteosarcoma cell lines, but did not change the very low level of IGF-I secreted into the CM or the level of the cell surface IGF-I receptor (15). OP-1 also increased the levels of IGF-binding protein-3 (IGFBP-3) and IGFBP-5 and decreased that of IGFBP-4 in the CM (15, 16, 17). In primary cultures of FRC cells, OP-1 stimulated the steady state messenger RNA (mRNA) levels of IGF-I, IGF-II, and IGFBP-3, and decreased the mRNA levels of IGFBP-4, -5, and -6, without changing the IGF-I receptor mRNA level (18). Inhibition of IGF-I expression by an antisense oligonucleotide in FRC cells partially blocked OP-1-induced AP activity. These data have led to the hypothesis that the effects of OP-1 on FRC cells are mediated at least in part through changes in gene expression of IGF-I, IGF-II, and the IGFBPs. The present study was pursued to assess whether OP-1 and IGF-I act independently to regulate osteoblast development or whether these two growth factors interact to influence osteoblastic cell proliferation and/or differentiation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
All reagents were of molecular biology grade. All buffers were prepared with diethyl pyrocarbonate-treated water. SeaKem ME and GTG agarose were purchased from FMC BioProducts (Rockland, ME). Restriction enzymes were purchased from New England Biolabs (Beverly, MA). FBS, Hanks’ Balanced Salt Solution (HBSS), serum-free MEM medium, penicillin/streptomycin stock, trypsin-EDTA (0.05% trypsin-0.53 mM EDTA), and collagenase were obtained from Life Technologies (Grand Island, NY). Radioisotopes were purchased from ICN Pharmaceuticals (Irvine, CA). Recombinant human OP-1 was provided by Stryker Biotech (Natick, MA) and was dissolved in 50% acetonitrile-0.1% trifluoroacetic acid. Human IGF-I (Life Technologies) was dissolved in 0.1 M acetic acid and stored as aliquots (100 ng/µl) at -20 C. Human TGFß1 (R&D Systems, Minneapolis, MN) was reconstituted in sterile 4 mM HCl-0.1% BSA and stored as a 1 ng/ml stock solution at -20 C. Human PTH [PTH-(1–34)] and 3-isobutyl-1-methylxanthine were obtained from Sigma Chemical Co. (St. Louis, MO). Human PTH was reconstituted in sterile 0.01% acetic acid-0.1% BSA and stored at -20 C as a 1-mM stock. The cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, was dissolved in dimethylsulfoxide at a concentration of 500 mM and stored as aliquots at -20 C. Des(1, 2, 3)-IGF-I was purchased from Peninsula Laboratories (Belmont, CA) and was solubilized and stored as described for IGF-I.

Cell culture
Primary osteoblast cell cultures were prepared from calvaria of day 19 or 20 fetal rats using previously described procedures (18, 19, 20). Briefly, the calvarium was stripped of the periosteum and digested with a mixture of trypsin and collagenase for five 20-min intervals. The digestion mixture after each 20-min interval was removed, and fresh digestion mixture was added to the calvarium. Digestions 1 and 2 were discarded. FRC cells were harvested from digestions 3–5. Cells were plated in T-75 flasks at a density of 2.5 x 10⁴/cm² in complete MEM medium containing 10% FBS, vitamin C (100 µg/ml), and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin sulfate). Cells were incubated at 37 C with 5% CO₂ for 3–4 days until they reached confluence. Cells [passage 1 (P1)] were replated at a density of 1–2 x 10⁶/T-75 flask, allowed to grow 3–4 days until confluent, and then frozen in liquid N₂ at the second passage. For experimentation, frozen cells (P2) were thawed and cultured in T-75 flasks for 4 days until confluent, then subcultured into 48-well plates (Corning Glass Works, Corning, NY) or T-150 flasks (Corning) at a density of 2.5 x 10⁴/cm². Cells (P3) were allowed to grow for 4–5 days until confluent. Confluent FRC cells were rinsed once with HBSS to remove CM and then incubated in serum-free MEM medium (with 0.1% BSA) in the presence (100–500 ng/ml) or absence of OP-1 and with (10–100 ng/ml) or without exogenous IGF-I as described in the legend of each figure.

Thymidine incorporation
The extent of thymidine incorporation into DNA was determined essentially as previously described (11). Briefly, confluent FRC cells grown in 48-well plates were treated with varying concentrations (100–500 ng/ml) of OP-1 with (10–100 ng/ml) or without IGF-I in serum-free MEM medium for 18 h. Cells were incubated with [³H]thymidine (5 µCi/ml) for an additional 6 h. After removal of the medium containing the unincorporated thymidine, cells were rinsed with cold 1 x PBS. The radiolabeled DNA was precipitated by cold 10% trichloroacetic acid for 15 min, solubilized in 0.1 N NaOH at 37 C for 10 min, and neutralized with 0.1 N HCl. The amount of radioactivity was determined by scintillation spectrometry in the presence of UniverSol cocktail (ICN, Costa Mesa, CA).

AP activity assay
For measurements of AP activity, confluent FRC cells were grown and treated in 48-well plates as described above. The medium was replenished with fresh medium after 24 h. After 48 h of treatment, cells were rinsed with PBS and lysed by sonication in 0.05% Triton X-100 in PBS (100 µl/well) for 60 sec at room temperature. Total cellular AP activity was measured with p-nitrophenyl phosphate as a substrate in 2-amino-2-methyl-1-propanol buffer, pH 10.3, at 37 C using a commercial assay kit (Sigma Chemical Co.). Reactions were terminated by the addition of 0.5 N NaOH. Absorbance of the reaction mixture was measured at 405 nm using a Genenchem automatic plate reader (Hewlett-Packard, Palo Alto, CA). Protein was measured according to the method of Bradford (21), using BSA as a standard. AP activity was expressed as nanomoles of p-nitrophenol liberated per µg total cellular protein.

cAMP assay
Confluent FRC cells grown in 48-well plates were treated with varying concentrations of OP-1 with or without IGF-I in serum-free MEM. After 24 h, the medium was replenished with fresh medium. After 48 h of treatment, cells were rinsed with HBSS and incubated in fresh serum-free medium containing 3-isobutyl-1-methylxanthine (1 mM) for 15 min. Cells were treated with 0.01% acetic acid (HAc)-0.1% BSA or 100 nM PTH for 10 min. The level of cAMP in the cell lysate was determined using a Biotrak cAMP enzyme immunoassay (Amersham, Arlington Heights, IL) following the manufacturer’s instructions. cAMP levels were normalized to total cellular protein.

Bone nodule formation
Confluent FRC cells in six-well plates were treated in serum-free MEM containing ascorbic acid (100 µg/ml) with solvent vehicle or OP-1 (200 ng/ml) in the absence or presence of varying concentrations of IGF-I (10, 25, and 50 ng/ml). Treatments were refreshed every other day. From day 11 on, 10 mM ß-glycerol phosphate was included in each treatment to stimulate mineralization (22). On day 15, cells were rinsed with 1 x PBS, fixed in 10% neutral buffered formalin, and stained using a modified von Kossa method (23). The total nodule area in each culture well was quantified using Visage 110 (BioImage, Ann Arbor, MI).

RNA isolation
Confluent FRC cells in T-150 flasks were treated with solvent vehicle or OP-1 (200 ng/ml) in the absence or presence of IGF-I (25 ng/ml) for 48 h. At the end of treatment intervals, cells in T-150 flasks were rinsed with ice-cold 1 x PBS solution to remove serum-free MEM. Total RNA was isolated using the RNeasy kit from Qiagen (Chatsworth, CA) following the manufacturer’s instructions. RNA was dissolved in diethyl pyrocarbonate-treated H₂O, and the concentration of RNA was measured by its absorbance at 260 nm. The intactness of the RNA sample was examined by gel electrophoresis on 1% agarose after ethidium bromide staining. Only RNA preparations showing intact species were used for subsequent analyses.

Labeling of complementary DNA (cDNA)
All cDNA fragments used for Northern analyses were produced by digestion of the parent plasmids with the appropriate pairs of restriction endonucleases as previously described (18). The resultant DNA fragments were purified by agarose gel electrophoresis and Geneclean II (BIO 101, La Jolla, CA). The 400-bp IGFBP-2 probe was obtained by digestion of pRBP2–501 with EcoRI/HindIII. The 700-bp IGFBP-3 probe was obtained by digestion of pRBP3-AR with KpnI/BamHI. The 444-bp IGFBP-4 probe was obtained by digestion of pRBP4-SH with SmaI/HindIII. The 270-bp IGFBP-5 probe was obtained by digestion of pRBP5-SH with SacI/HindIII. The 246-bp IGFBP-6 probe was obtained by digestion of pRBP6-PP with PstI/PstI. The 265-bp IGF receptor probe was obtained by EcoRI/BamHI digestion of the IGF-I receptor gene sequence cloned in pGEM-3. The cDNA probes for ActR-I (Activin receptor I; ALK-2), BMP receptor IA (BMPR-IA; ALK-3), and BMPR-IB (ALK-6) were generated by reverse transcription-PCR using the specific primer sets described below: for ActR-I: sense primer, 5'-ACG CCT CTT GAA TTC TCC GAG-3'; antisense primer, 5'-CTC CAC GTC TCG GGG ATT GAG-3'; for BMPR-IA: sense primer, 5'-CAG TAC ACA GGA AAG CTT ACA-3'; antisense primer, 5'-GTA ACA AAA GCA GCT GGA GAA-3'; and for BMPR-IB: sense primer, 5'-AAG CGG CGG CGG GTT AAC TTC-3'; antisense primer, 5'-GAT CCA CTT CCC GAG CTC TGA-3'. The PCR-generated fragments were subcloned and propagated in Escherichia coli. The 580-bp ActR-I insert was obtained by digestion with EcoRI/AvaII. The 530-bp BMPR-IA insert was obtained by digestion with HindIII/PvuII. The 660-bp BMPR-IB insert was obtained by digestion with HpaI/SacI. The 800-bp BMPR-II insert was obtained by PstI digestion of human BMPR-II cloned in pCMV5. Purified cDNA fragments were labeled with [-³²P]deoxy-CTP using the DECAprime II DNA labeling system (Ambion Co., Austin, TX). The labeled cDNA probes were purified through a Midi-SELECT G-25 spin column (5 Prime-3 Prime, Boulder, CO) to remove the unincorporated nucleotides. The 18S ribosomal RNA (rRNA) was probed with a ³²P-labeled 18S-specific oligonucleotide (5'-GCCGTGCGTACTTAGACATGCATG-3').

Northern blot analysis
The mRNA levels for IGF-I receptor, type I and II OP-1 receptor, and IGFBPs were determined by Northern analysis as previously described (18). Denatured total RNAs (20 µg) were analyzed on 2.2 M formaldehyde-1% Seakem Genetic Technology Grade agarose gels. RNA standards (0.24–9.5 kilobases) from Life Technologies were used as size markers. The fractionated RNA was transferred onto a Nytran Plus membrane using a Turboblot apparatus (Schleicher and Schuell, Keene, NH). After cutting the lane containing the standards from the blot, the RNA was covalently linked to the membrane using the UV cross-linker (Stratagene, La Jolla, CA). The membranes were incubated overnight at 42 C with cDNA probes. The blots were washed and exposed to a screen for the PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and analyzed as previously described (18). Results represent the average from probing three or four blots with different RNA preparations for each cDNA probe. Blots were also probed with an 18S rRNA oligonucleotide probe to correct for loading variations.

Statistical analysis
Multiple means were compared with one-way ANOVA, followed by Student’s t test for paired comparisons with the control. The ANOVA and Student’s t test programs in the PSI-Plot (Poly Software International, Salt Lake City, UT) for personal computers were used for the analyses.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of exogenous IGF-I on DNA synthesis in OP-1-treated FRC cells
Figure 1A shows that OP-1 treatment of confluent FRC cells resulted in a dose-dependent stimulation of [³H]thymidine incorporation, with a maximum 2-fold stimulation. Half-maximal and maximal stimulation of [³H]thymidine incorporation occurred at OP-1 concentrations of approximately 150 and 500 ng/ml, respectively. The effect of IGF-I on DNA synthesis in OP-1-treated FRC cells was also examined. IGF-I alone stimulated DNA synthesis slightly (1.3-fold), but significantly (P < 0.04; Fig. 1B). This observation is in agreement with the previous finding that IGF-I has a weak mitogenic activity in FRC cells (24). The combined OP-1 and IGF-I treatment of FRC cells produced a statistically significant (P < 0.05) enhancement of DNA synthesis beyond that caused by OP-1 or IGF-I alone (Fig. 1B). Maximum enhancement was observed at 200 ng/ml OP-1 and 25 ng/ml IGF-I (1.3-fold stimulation compared with OP-1 alone and 1.8-fold compared with IGF-I alone).

View larger version (16K): [in this window] [in a new window]   Figure 1. A, Dose response of OP-1 on [3H]thymidine incorporation in FRC cells. Confluent FRC cells in 48-well plates were incubated in serum-free MEM containing vehicle or varying concentrations of OP-1 (100, 200, or 500 ng/ml) for 18 h. The treatments (6 wells/treatment) were then pulsed with [3H]thymidine (5 µCi/ml) for 6 h, as described in Materials and Methods. After a total of 24 h of incubation, the extent of [3H]thymidine incorporation into DNA was determined and expressed as disintegrations per min/well. Values are the mean ± SE of four independent experiments using different preparations of FRC cells. B, The synergistic effect of exogenous IGF-I on OP-1-induced [3H]thymidine incorporation in FRC cells. Confluent FRC cells in 48-well plates were incubated with OP-1 in the presence of exogenous IGF-I (10, 25, and 50 ng/ml) for 18 h and pulsed with [3H]thymidine for an additional 6 h, as described above. , Control (no OP-1), only vehicle was added in serum-free medium; •, 100 ng/ml OP-1; , 200 ng/ml OP-1; , 500 ng/ml OP-1. Values are the mean ± SE of four independent experiments using different preparations of FRC cells. *, P < 0.01 compared with vehicle control (A) and with OP-1 alone control (B).

View larger version (17K): [in this window] [in a new window]   Figure 2. A, Dose response of OP-1 on AP activity in FRC cells. Confluent FRC cells in 48-well plates were incubated in serum-free MEM containing vehicle or varying concentrations of OP-1 (100, 200, or 500 ng/ml). After 24 h of incubation, medium was replaced with fresh medium containing the appropriate additives. After a total of 48-h incubation, cells were lysed with 0.05% Triton X-100/PBS as described in Materials and Methods. The activity of AP was expressed as nanomoles of p-nitrophenol per µg total protein. The relative activity of AP was normalized to that of the solvent vehicle-treated control (100%). Values are the means of four independent experiments (with 6 wells/treatment condition) of different preparations of FRC cells. *, P < 0.01 compared with the control. B, The synergistic effect of exogenous IGF-I on OP-1-induced AP activity in FRC cells. Confluent FRC cells in 48-well plates were incubated with OP-1 in the presence of exogenous IGF-I (10, 25, 50, and 100 ng/ml) for 48 h as described above. , Control (no OP-1), only vehicle was added in serum-free medium; •, 100 ng/ml OP-1; , 200 ng/ml OP-1; , 500 ng/ml OP-1. *, P < 0.01; , P < 0.05 (compared with each OP-1 concentration alone).

View larger version (133K): [in this window] [in a new window]   Figure 3. Formation of bone nodules in FRC cells as a function of OP-1 and IGF-I/OP-1. Confluent FRC cells in six-well plates were treated with vehicle (well 1), IGF-I (25 ng/ml; well 2), OP-1 (200 ng/ml; well 3), and OP-1 (200 ng/ml) in the presence of increasing concentrations of IGF-I (10, 25, and 50 ng/ml; wells 4, 5, and 6, respectively). Media were changed every 2 days. On days 11 and 13, ß-glycerol phosphate (10 mM) was added to all cultures. On day 15, the cells were processed for visualization and quantitation of nodule formation. Nodules were visible under the phase contrast microscope after staining by the von Kossa technique.

View larger version (16K): [in this window] [in a new window]   Figure 4. Effect on AP activity of IGF-I added at different times to FRC cells treated with OP-1. Confluent cells in 48-well plates were incubated with serum-free MEM containing solvent vehicle, IGF-I (25 ng/ml), or OP-1 (200 ng/ml). IGF-I (25 ng/ml) was added to the cultures 0, 2, 4, 6, or 24 h after OP-1 treatment. After 48 h of incubation, cells were lysed, and the level of AP activity was measured and normalized to the control, which is expressed as 1. Shown are the mean ± SE from six culture wells from each experiment and are representative of four independent FRC cell preparations. *, P < 0.01 compared with solvent control. , P < 0.01 compared with OP-1 alone.

View larger version (59K): [in this window] [in a new window]   Figure 5. Northern analysis of OP-1 type I and type II receptor gene expression in FRC cells. Total RNA was isolated from FRC cells treated with solvent vehicle (lane 1), OP-1 (200 ng/ml; lane 2), OP-1 (200 ng/ml) plus IGF-I (25 ng/ml; lane 3), or IGF-I (25 ng/ml; lane 4) for 48 h. The RNA was denatured, resolved on 1% agarose gel containing formaldehyde, and transferred onto a Nytran Plus membrane. The blot was hybridized with the cDNA probe for the individual receptor type or with an oligonucleotide for 18S rRNA. After washings, the blot was exposed to a PhosphorImager screen.

View larger version (49K): [in this window] [in a new window]   Figure 6. Northern analysis of IGFBP-2 to -6 expression in FRC cells. Total RNA was isolated from FRC cells treated as described in Fig. 5. The blot was hybridized with the cDNA probes for the individual IGFBPs or with an oligonucleotide for 18S rRNA. The blot was exposed to a PhosphorImager screen.

View larger version (14K): [in this window] [in a new window]   Figure 7. Quantitative analysis of the steady state mRNA levels of IGFBPs. The intensity of the hybridized RNA species on Northern blots, as shown in Fig. 6, was quantified by phosphorimaging. The relative mRNA level was normalized to the control value (100%). Results are the mean ± SE from four independent experiments using four different FRC cell preparations and are corrected for 18S rRNA. *, P < 0.01 compared with the control.

View larger version (15K): [in this window] [in a new window]   Figure 8. Effects of des(1–3)-IGF-I on OP-1-stimulated AP activity in FRC cells. The level of AP activity in FRC cells treated with 200 ng/ml OP-1 and varying concentrations of IGF-I (•) or des(1–3)-IGF-I () was measured. Controls were treated with solvent vehicle only. Treatments were performed for 48 h, with a change of fresh medium at 24 h. Results are normalized to the AP activity in FRC cells treated with OP-1 alone, which is 5- to 7-fold higher than that in the solvent vehicle-treated culture. *, P < 0.05 compared with the control.

View larger version (20K): [in this window] [in a new window]   Figure 9. Effects of exogenous IGFBP-5 on the OP-1-induced AP activity in FRC cells. Confluent FRC cells grown in 48-well plates were treated with vehicle (control), IGF-I alone (25 ng/ml), IGFBP-5 alone (50 ng/ml), IGF-I (25 ng/ml) plus IGFBP-5 (50 ng/ml), OP-1 (200 ng/ml) alone, OP-1 (200 ng/ml) plus IGFBP-5 (50 ng/ml), OP-1 (200 ng/ml) plus IGF-I (25 ng/ml), and OP-1 (200 ng/ml), IGF-I (25 ng/ml), and IGFBP-5 (50 ng/ml). All test substances were added simultaneously, and treatment was conducted for 48 h, with a change of fresh medium at 24 h. Results are normalized to the AP activity in solvent vehicle-treated controls. Values are the means of six replicates of each condition of two independent experiments using different FRC cell preparations. **, P < 0.01 compared with OP-1-treated culture; *, P < 0.05 compared with OP-1- and IGF-I-treated culture.

View larger version (16K): [in this window] [in a new window]   Figure 10. Inhibition of basal AP activity by TGFß1 in FRC cells. Confluent FRC cells in 48-well plates were incubated in serum-free MEM containing vehicle (lane 1) or 200 ng/ml OP-1 (lane 2); in 0.05, 0.5, and 2 ng/ml TGFß1 alone (lanes 3–5); or in a combination of varying concentrations of TGFß1 and 10, 25, or 50 ng/ml IGF-I (lanes 6–14). The treatments (six wells per treatment) were refreshed after 24-h incubation. After a total of 48-h incubation, cells were lysed as described in Fig. 2. The activity of AP was expressed as nanomoles of p-nitrophenol per µg total protein. Values are the mean of three independent experiments of different preparations of FRC cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Although the synergistic effect could be observed when FRC cells were treated with OP-1 for up to 24 h before IGF-I addition, maximum synergism was observed when cells were treated with OP-1 and IGF-I simultaneously or when IGF-I was added within 2 h of OP-1. Published kinetic studies in OP-1-treated FRC cells demonstrate that the IGF-I mRNA levels do not increase significantly until 24 h posttreatment (18). It is possible that the observed synergism between OP-1 and exogenous IGF-I may be the function of an accelerated sequence of events induced after OP-1 stimulation of FRC cells. We previously showed that OP-1 caused a 2-fold increase in IGF-I mRNA level. A previous study in which measurements were made at both the mRNA and protein levels in osteoblastic cells under the same experimental conditions shows that the levels were correlated (35). Extrapolating from those results, we expect that the bioavailable IGF-I level in OP-1- plus IGF-I-treated cultures is higher than that in cultures treated with IGF-I alone. It is noteworthy that IGF-I alone elevated the IGFBP-5 mRNA level compared with vehicle control value, suggesting that OP-1 might exert a dominant effect on IGFBP-5 expression relative to that of IGF-I. These changes in IGFBPs, in turn, impact upon the response to endogenous and exogenous IGF-I (see below). In agreement with this supposition is the current observation that the cellular response to OP-1 was unchanged after IGF-I pretreatment. These observations suggest that sensitization of FRC cells to IGF-I action by OP-1 is a prerequisite for synergism between OP-1 and IGF-I. These protein factors influence cellular activities differentially and, consequently, affect the timing of changes in the cellular events that culminate in important effects on bone formation.

The data on the mRNA level of the IGFBPs in OP-1-treated FRC cells imply involvement of IGFBPs in the observed synergism between OP-1 and IGF-I. Two current results provide experimental evidence supporting this hypothesis: 1) des(1, 2, 3)-IGF-I, the IGF-I analog that shows a decreased binding affinity for the IGFBPs, and 2) effects of exogenous IGFBP-5 on OP-1 action. Des(1, 2, 3)-IGF-I, the truncated form of IGF-I, like the full-length IGF-I molecule, exhibited a synergistic effect with OP-1 on AP activity in FRC cells. However, des(1, 2, 3)-IGF-I was more potent than the full-length IGF-I at low concentrations. The present observation agrees with the previous postulate that a decrease in the affinity of des(1, 2, 3)-IGF-I for IGFBPs resulted in an elevated level of unbound growth factor that interacted with the IGF-I receptor (34). The stimulation suggests that the mechanism by which OP-1 and the native, full-length IGF-I synergize to stimulate AP activity is through the decrease in the levels of inhibitory IGFBPs by OP-1. IGFBP-5, in particular, appears to be a target for down-regulation by OP-1. At higher concentrations, the levels of stimulation by both forms of IGF-I were similar. Presumably, at these high concentrations of des(1, 2, 3)-IGF-I and IGF-I, the IGF-I receptors were saturated, and the role of the IGFBPs in regulating the bioavailability of IGF-I was minimized. The data support the hypothesis that IGFBPs play a role in the action of OP-1 in FRC cells. The finding further implies that the synergism between OP-1 and IGF-I observed with the full-length IGF-I molecule was at least partially the result of an IGF-I receptor-mediated event. The observation that exogenous IGFBP-5 blocked the OP-1-induced AP activity and the combined OP-1 and IGF-I synergism supports the hypothesis that OP-1 sensitizes cells to IGF-I by reducing the level of IGF-I inhibitory binding proteins. As a result, OP-1 and IGF-I are able to synergistically stimulate FRC cell differentiation.

The observed synergism exhibited by OP-1 and IGF-I in FRC cells is the first reported between OP-1 and another growth factor. The actions of BMP-2 and BMP-3, which share 60% and 42% amino acid sequence homology with OP-1, have been reported to be potentiated by nonpeptide factors, such as vitamin D and retinoic acid in clonal cell lines (36, 37, 38). It is noteworthy that our results demonstrate that TGFß1, which shares 35% amino acid sequence identity with OP-1, exhibited an antagonistic effect on AP activity in FRC cells, with half-maximum inhibition at about 0.5 ng/ml. Exogenous IGF-I did not change the TGFß1 inhibitory effect. This observation agrees with earlier reports that TGFß1 inhibits AP gene expression in FRC cells (39) and AP activity in MC3T3-E1 murine osteoblast-like cells (40) and mature MBA-15.6 human osteoblastic cell (38). However, TGFß1 very slightly stimulates enzymatic activity in the human preosteoblastic cell MBA-15.4 (38). In osteoblastic cells, it is clear that the actions of OP-1 and TGFß1 are diverse, thus emphasizing the physiological significance of OP-1 in osteoblastic cell development.

In summary, we have demonstrated a synergism between OP-1 and IGF-I in the stimulation of biochemical and morphological markers characteristic of bone cell differentiation in primary cultures of fetal rat calvaria cells. IGFBPs and IGFBP-5, in particular, are among the factors involved in the synergism between OP-1 and IGF-I.

	Acknowledgments

 
The authors thank Stryker Biotech for the support. Plasmids used for the rat IGFBPs studies were kindly provided by Drs. S. Shimisaki and N. Ling, Whittier Institute (La Jolla, CA). We thank Dr. David R. Clemmons, University of North Carolina (Chapel Hill, NC), for providing purified human IGFBP-5; Dr. Joan Massague, Memorial Sloan-Kettering Cancer Center (New York, NY), for providing the human BMPR-II cDNA; and Dr. Lynda F. Bonewald, Department of Medicine, University of Texas Health Science Center (San Antonio, TX), for her valuable advice concerning the bone nodule formation assay. We also thank Megan Asher and Dr. Allison Kitten for preparation of initial supplies of frozen FRC cell stocks, and Karen P. Betchel for technical assistance.

	Footnotes

 
¹ Presented in part at the 10th International Congress of Endocrinology, San Francisco, CA, 1996, p 463 (Abstract P2–233).

Received February 24, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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