Potentiating role of IGFBP-2 on IGF-II-stimulated alkaline phosphatase activity in differentiating osteoblasts

Claudia Palermo,1 Paola Manduca,3 Elisabetta Gazzerro,1 Luca Foppiani,1 Daniela Segat,4 and Antonina Barreca1,2

1Department of Endocrinology and Metabolism, 2Center of Excellence for Biomedical Research, 3Department of Oncology, Biology and Genetics, and 4Department of Experimental Medicine, University of Genova; I-16132 Genoa, Italy

Submitted 4 February 2003 ; accepted in final form 4 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The insulin-like growth factor (IGF) system plays an important role in the autocrine and paracrine regulation of bone formation and remodeling. The aim of this study was to evaluate the role of the autocrine IGF system during osteogenic differentiation in rat tibial osteoblasts (ROB) in culture. In this in vitro model, the stages of osteogenesis studied were S1, corresponding to the onset of alkaline phosphatase (AP) expression (days 0-3); S2, coincident with the peak of AP expression in differentiation culture conditions (days 4-6), and S3, corresponding to the onset of mineral deposition in the extracellular matrix (days 7-9). The results showed that conditioned medium of ROB contains greater amounts of IGF-II than IGF-I at all differentiation stages. Both peptides showed the highest concentrations on day 3 of differentiation (end of S1). All IGF-binding proteins (IGFBPs), except IGFBP-1 and -6, were detected, and IGFBP-2 was the most abundant IGFBP present in the conditioned media, and its degradation increased from S1 to S3. By semiquantitative RT-PCR, IGF-I and IGF-II were highly expressed on days 3 and 6, whereas IGFBP-2 was constantly expressed. We focused our study on the role of IGF-II and IGFBP-2 on the synthesis of AP, an early marker of osteoblast maturation. The results showed that a significant increase in AP expression was induced by IGF-II added to the differentiating osteoblasts continuously or in S1 but not in S2 or S3. IGFBP-2 was able to potentiate endogenous and exogenous IGF-II-dependent stimulation of AP activity, and its proteolytic degradation in late stages of osteogenesis (S2 and S3) was highly correlated with the increase of active matrix metalloproteinase-2 in the CM and with the decreased efficacy of IGF-II action. These data suggest that IGFBP-2, at nearly equimolar concentration with IGF-II, plays a potentiating role in IGF-II action on ROB differentiation in vitro.

insulin-like growth factor II; insulin-like growth factor-binding protein 2; insulin-like growth factors; insulin-like growth factor-binding proteins; alkaline phosphatase induction; osteogenesis in vitro


A BODY OF EVIDENCE SUGGESTS that the complex system of the insulin-like growth factors (IGFs), their binding proteins (IGFBPs), and their type I receptor plays an important role in the autocrine and paracrine regulation of bone formation and remodeling (7, 11, 25, 26, 44, 52). Indeed, IGF-I and IGF-II have been found in bone extracts of several different species, and IGF-II is the most abundant growth factor stored in bone (10, 44). In vitro studies have shown that the synthesis of both IGF-I and -II is differentially expressed in relation to the osteogenic lineage and bone tissue in culture and that it is modulated by numerous circulating hormones and local growth factors (3, 7, 11, 21, 25, 52). It has been thoroughly demonstrated that, through the type I IGF receptor, IGFs induce both proliferation and differentiation of osteoblasts as well as of chondrocytes (32, 54). Furthermore, besides its mitogenic effects, the IGF peptide increases the production of several bone matrix proteins, stimulates collagen type I expression, and inhibits collagen degradation, possibly through downregulation of collagenase expression, and induces alkaline phosphatase (AP) expression (32, 39, 53, 58, 59). Moreover, mice lacking functional IGF-I genes exhibit severe impairment of bone growth (34), whereas targeted overexpression of IGF-I to osteoblasts of transgenic mice increases bone mineral density and the formation rate and volume of cancellous bone (62). IGFs are therefore critically involved in osteoblast differentiation, matrix production and mineralization, as well as osteoclastic activity.

As in other tissues, autocrine/paracrine actions of IGFs in bone are modulated by a family of six structurally related binding proteins (10, 11, 17, 29, 30, 43, 51). A positive and negative modulation, depending on the experimental conditions and on the cell system utilized, is attributed to IGFBPs. On the whole, the inhibitory effect, exerted through competition with the receptors for binding with the IGF peptide, is what is most frequently described. The potentiating effect may occur with those binding proteins, such as IGFBP-1, -2, -3, and -5, that bind to the cell membrane or to the extracellular matrix, thus holding the peptide, protecting the IGF against proteolytic degradation, and favoring its slow release, which avoids down-regulation of the receptor. In vitro studies on the actions of IGF-I have revealed that IGFBP-3 and IGFBP-5 can enhance IGF action in rat osteoblasts (10, 11, 17, 30). Moreover, recent studies support the concept that IGFBP-5 functions as a growth factor that also stimulates bone formation via an IGF-independent mechanism (42). In contrast, IGFBP-4, which is secreted by a variety of osteoblastic cells, including normal human osteoblast-like cells, appears to be a potent inhibitor of IGF-II actions in bone cells and to be under the critical control of an IGF-dependent IGFBP-4 protease (14, 15, 33, 45, 47, 49, 50). The IGFBP proteases also play an important role in IGF release mechanisms at the tissue and plasma levels by reducing the affinity of the IGFBPs not only for the IGFs but also for the extracellular matrix and the cell membrane (29). Among the various kinds of proteases specific for IGFBP, the matrix metalloproteinases (MMP-1, -2, and -3), known as extracellular matrix-degrading enzymes, can exert effects on cellular growth and differentiation through degradation of IGFBPs (19, 20, 56).

The interaction between IGFs and the various IGFBPs is further complicated by evidence that the secretion of some components of the IGF system is hormonally regulated and modulated according to the stage of differentiation achieved by the cells (9, 24, 35, 38, 57).

The present study was conducted to characterize the role of autocrine IGF system components during osteogenic differentiation in rat tibial osteoblasts (ROB) in culture, a model system of osteogenesis in vitro already characterized (18, 36, 48, 55).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

Recombinant human IGF-II, the IGF-II analog des(1-6)IGF-II (desIGF-II), and IGFBP-2 were purchased from GroPep (North Adelaide, Australia), and recombinant tissue inhibitor of metalloproteinases (TIMP-1) was from Chemicon International (Temecula, CA). Tissue culture media and reagents to make up differentiating medium were purchased from either Sigma-Aldrich (Milan, Italy) or Life Technologies (Grand Island, NY). L-Ascorbic acid phosphate and {beta}-glycerolphosphate were purchased from Wako Chemicals (Richmond, VA). Fetal calf serum (FCS) was purchased from Euro Clone (Pero, Italy). Tissue culture plasticware was purchased from Corning (Corning, NY) and Iwaki (Bibbi Sterlin, Straffordshire, UK). Other reagents were purchased from Sigma (Saponin, gelatin type A, proteinase K, Hoechst 33258, Kit 104 LL for the measurement of AP, and Kit 587-A for calcium determination).

For the radioimmunoassays (RIA), IGFBP-2 RIA and IGFBP-1 and IGFBP-3 immunoradiometric assay (IRMA) reagents were purchased from Diagnostic System Laboratories (Webster, TX), IGF-I RIA was from Medgenix (Fleurus, Belgium), Anti-IGF-II MoAb was from Sera-Lab Techno-genetics, (Trezzano, Italy), and 125I-labeled IGF-II (2,000 Ci/mM) was purchased from Amersham (Aylesbury, UK).

IGFBP polyclonal antibodies were obtained from Upstate Biotechnology (Lake Placid, NY; IGFBP-1, -2, and -4) or GroPep (polyclonal antibody against IGFBP-5 and -6); antiserum to IGFBP-3 was kindly provided by Celtrix Pharmaceuticals (Santa Clara, CA). Mouse monoclonal IgG anti-type I IGF receptor (for immunofluorescence) was from GroPep, and monoclonal anti IGF-I receptor ({alpha}-IR3 clone) for flow cytometry analysis was purchased from Oncogene Research Products (Cambridge, MA).

For Western blot, Hybond C-extra nitrocellulose membranes, Enhanced Chemiluminescence (ECL) Detection Solution, and 125I IGF-II were from Amersham, nonfat dried milk was from Bio-Rad (Hercules, CA), and horseradish peroxidase-linked anti-rabbit IgG was from Upstate Biotechnology.

Band intensity was quantified using a scanner (Artiscan 6000 C) and the program UN-SCAN-IT (Silk Scientific, Orem, UT).

For immunofluorescence, goat serum, ACyTM3-conjugated IgG anti-rabbit (for IGFBP-2, -3, -4, and -5) or anti-mouse (for IGF receptor) secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. For flow cytometry analysis, the secondary donkey anti-mouse fluorescein isothiocyanate (FITC)-conjugated antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

For RNA extraction and RT-PCR, Tri Pure Isolation Reagent kits were purchased from Roche Molecular Biochemicals, (Indianapolis, IN), MMLV reverse transcriptase was from Eurobio (Les Ulis, France), and DyNAzyme II DNA polymerase was from Finnzymes (Oy, Finland). Oligonucleotide primers designed for the amplification were purchased from TIB-Molbiol (CBA, Genoa, Italy).

Cell Cultures

ROB were obtained from 7-day-old Wistar rat tibial diaphyses, and primary cultures were prepared from diaphysial chips, as in Stringa et al. (55). Long-term-cultured cells were obtained and characterized as described in Manduca et al. (36); cells between 65 and 75 cumulative population doublings were utilized for all the experiments, routinely cultured in Coon's modified F-12 supplemented with 10% FCS. Differentiation culture conditions are in the aforesaid medium with the addition of ascorbic acid (100 µg/ml) and {beta}-glycerolphosphate (10 mM).

Confluent cells were transferred to a differentiation medium supplemented with 1% FCS (day 0) in the absence or presence of the test substances (IGF-II, desIGF-II, IGFBP-2, and TIMP-1) in concentrations described in the text and figures. ROB were treated with test agents continuously (for 9 days) or for intervals of time during the progression of differentiation. These were chosen to coincide with the first stage of osteogenesis (S1), corresponding to the onset of the expression of AP (days 0-3), with the second stage (S2) coincident with the peak of AP expression in differentiation culture conditions and with the initial organization of cells in multistrata to subsequently form nodular structures (days 4-6), and with the third stage (S3) corresponding to the onset of mineral deposition in the extracellular matrix (days 7-9).

Analysis of IGFs and IGFBPs Released into Medium

RIA analyses. After extensive rinses with warm PBS, ROB cells (106 cells/4 ml) at subsequent stages of differentiation (days 0, 3, 6, and 9) were plated in serum-free differentiation medium supplemented with 0.5% BSA for 24 h. At the end of the incubation, the conditioned media (CM) were acidified by 18-h dialysis at 4°C against 1 M acetic acid in Spectra-Por 6 membranes (mol wt cut-off, 1,000), lyophilized, and resuspended with 0.1 M acetic acid-0.15 M NaCl (pH of the mixture <3). IGF-I and IGF-II were separated from IGFBPs by gel filtration on FPLC Superdex-75 column equilibrated with 0.1 M acetic acid-0.15 M NaCl, pH 2.75. Fractions were pooled at 0.1 Kav intervals, lyophilized, reconstituted in PBS, and analyzed for IGF-I and -II and IGFBP-1, -2, and -3 immunoreactivity. IGF-I and -II contents of media were evaluated in the fractions eluted from the Superdex-75 column in the molecular weight range of the free peptide, whereas IGFBP contents were evaluated in the fractions eluted in the molecular mass range of >20 kDa. Results are expressed as nanomoles per liter 106 cells. The sensitivity of the IGF-I RIA was 0.9 nM/l; the intra- and interassay coefficients of variation were 6 and 7.5%, respectively. The sensitivity of the RIA for IGF-II was 0.72 nM/l; the intra- and interassay coefficients of variation were 6 and 9%, respectively. No cross-reactivity could be evidenced between IGF-I and IGF-II with the respective antibodies used in the assays up to concentrations of 500 ng/ml of both peptides. The sensitivity of the IRMA for IGFBP-1 was 0.05 nM/l; the intra- and interassay coefficients of variation were 2.5 and 4.6%, respectively. The sensitivity of the RIA for IGFBP-2 was 0.02 nM/l; the intra- and interassay coefficients of variation were 3.7 and 6.5%, respectively. The sensitivity of the RIA for IGFBP-3 was 0.04 nM/l; the intra- and interassay coefficients of variation were 3.25 and 5.6%, respectively.

Western ligand blot analyses. Aliquots of CM, corresponding to 0.5 ml of original medium (~125 x 103 cells), were denatured and fractionated under nonreducing conditions on 10% SDS-PAGE and then transferred electrophoretically to Hybond C-extra nitrocellulose membranes. After transfer, membranes were dried and then washed consecutively in 0.01 M Tris·HCl, 0.15 M NaCl, and 0.05% sodium azide (saline solution) containing 3% Nonidet P-40 (buffer A), 1% BSA (buffer B), or 0.1% Tween 20 (buffer C) and finally with 50 ml of buffer containing 1% BSA, 0.1% Tween 20, and 40,000 cpm/ml of 125I-labeled IGF-II. After incubation with labeled IGF-II for 24 h at 4°C, membranes were washed twice with buffer C and three times with saline solution, dried, and subjected to autoradiography.

Western immunoblot analyses. Aliquots of CM, corresponding to 0.5 ml of original medium, were denatured, fractionated, and transferred electrophoretically to Hybond C-extra nitrocellulose membranes as described for ligand blot. After transfer, nonspecific binding sites were blocked by treating membranes with Tris-buffered saline-Tween (TBS-T: 0.02 M Tris base, 0.137 M NaCl, 0.5% Tween 20; pH 7.6, with 1 M HCl) containing 5% nonfat dried milk for 1 h at 22°C on a rotating shaker. After five washes with TBS-T, membranes were incubated for 16 h at 4°C with a 1:2,500 dilution of anti-human IGFBP-2, -3, -4, -5, and -6 antibodies (different from those utilized for immunoassays) in TBS-T containing 1% BSA. Membranes were washed five times with TBS-T and then incubated for 1 h at 22°C with a 1:3,000 dilution of horseradish peroxidase-linked anti-rabbit IgG, washed as before, and immersed for 0.5-1 min in the chemiluminescence detection solution. Subsequently, membranes were exposed for ~0.5 min to generate immunoblots.

Analyses of IGFBP-2 Proteolytic Activity and of MMPs in CM

IGFBP-2 proteolytic activity. To evaluate the proteolytic activity for IGFBP-2 present in the CM at different stages of osteogenesis, 0.5 ml of medium conditioned by ROB in differentiation stages S1, S2, and S3 were lyophilized, reconstituted with 100 µl of distilled H2O, and incubated with IGFBP-2 in a 37°C water bath for different time periods (30, 120, and 300 min). Because the results demonstrated that proteolysis of IGFBP-2 was time dependent (data not shown), we chose to incubate for 5 h increasing concentrations (0.2, 0.5, and 1 nM) of IGFBP-2 with CM before Western immunoblot. In a second set of experiments, 0.2 nM IGFBP-2 was incubated with CM in the presence of 40 nM TIMP-1. The amounts of intact IGFBP-2 and of the generated fragments were visualized by immunoblotting, as described above, and quantified by densitometry.

Analyses of MMPs. Secretion of MMPs was evaluated in media conditioned by ROB cells at different stages of differentiation by zymogram, as in Filanti et al. (18).

Immunofluorescence Analyses

For flow cytometry, ROB (105cell/100 µl PBS) were incubated with mouse monoclonal anti-IGF-I receptor at a 1:10 dilution for 60 min on ice. Subsequently, samples were resuspended in PBS and centrifuged at 1,300 rpm at 4°C for 5 min. After the supernatant was discarded, a secondary donkey anti-mouse FITC-conjugated antibody at a 1:25 dilution was added, and samples were incubated for 60 min on ice in the dark. The samples were then washed in PBS and the resuspended cells fixed with 500 µl of 1% paraformaldehyde and stored in the dark at 4°C until analysis. Autofluorescence was assessed by using the secondary antibody alone. Sample analysis was performed by FACS Calibur (Becton Dickinson). The mean fluorescence intensity was expressed as molecules of equivalent soluble fluorochrome (MESF), calculated using FITC-labeled microbead standards (Flow Cytometry Standards, San Juan, PR). Results were expressed as MESF x 103/cell.

AP Activity

AP activity was quantitated in cell lysates (obtained with 0.01% SDS) by spectrophotometric measurement of p-nitrophenol 20-min release at 37°C. DNA content was determined in the cell lysates (digested with 50 mg/ml proteinase K at 50°C overnight) by fluorimetry using 0.1 mg/ml Hoechst 33258 dye. AP activity was expressed as International Units per milliliter per 100 micrograms of DNA.

Quantitative Determination of Calcium Incorporation

The rate of calcium incorporation was quantified in cell lysates by spectrophotometric measurement of the complex calcium-o-cresolphthalein. Calcium concentration was expressed as milligrams per 100 micrograms of DNA.

RNA Extraction and RT-PCR

Total RNA was isolated from cells by using the Tri Pure Isolation Reagent kit according to the manufacturer's protocol. Reverse transcription was performed during 60 min at 42°C in a total volume of 20 µl containing 5 µg of oligo(dT)-primed RNA, 1 mM dNTP, 10 U RNase inhibitor, reverse transcriptase buffer 5X, and 100 U of MMLV reverse transcriptase. PCR was performed in a total volume of 50 µl containing 1 U of DyNAzyme II DNA polymerase, 2 mM MgCl2, 0.1 mM dNTP, 5 pM of 3' and 5' oligoprimers, DyNAzyme II DNA polymerase buffer 10X, and 5 µl of cDNA. Samples were subjected to 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C [osteocalcin (OC) and the core-binding factor Cbfa-1], 55°C [IGF-I, collagen type I (Col I), AP, and GAPDH], 53°C (IGF-II) and 45°C (IGFBP-2) for 30 s, and extension at 72°C for 30 s in 0.2-ml thin-walled tubes. PCR products were size-fractionated by agarose electrophoresis [2% agarose gel containing 0.01% ethidium bromide and 1x Tris-borate-EDTA (TBE) buffer]. Primer sequences used were as follows: 1) IGF-I sense 5'-ATA CAC ATC ATG TCG TCT TC-3'; antisense 5'-GAA TCT TGT TTC CTG CAC TTC-3'; 2) IGF-II sense 5'-GCC CCA GCG AGA CTC TGT GCG-3'; antisense 5'-GCC CAC GGG GTA TCT GGG GAA-3'; 3) IGFBP-2 sense 5'-TGG AGG AGC CCA AGA AGC T-3', antisense 5'-GGT TCA CAC ACC AGC AAC TC-3'; 4) OC sense 5'-CGT GTT GGT TAA TGC CAC TG-3', antisense 5'-CCA CGT GTC AGC AAC TCT GT-3'; 5) Cbfa-1 sense 5'-ATT TAG GGC GCA TTC CTC ATC-3', antisense 5'-TGT AAT CTG ACT CTG TCC TTG TGG AT-3'; 6) Col I sense 5'-GTG CTA AAG GTG CCA ATG GT-3', antisense 5'-CTT TCC AGG TTC TCC AGC AG-3'; 7) AP sense 5'-GCA CAA CAT CAA GGA CAT CG-3', antisense 5'-GCT GTG AAG GGC TTC TTG TC-3'; and 8) GAPDH sense 5'-CGA TCC CGC TAA CAT CAA AT-3', antisense 5'-GGA TGA AGG GAT GAT GTT CT-3'.

Statistical Analysis

Statistical analysis was performed by nonparametric test on paired (Wilcoxon signed rank test) and unpaired (Mann-Whitney test) observations. P < 0.05 was considered significant. Results are expressed as the means ± SE. To minimize variation among different experiments, the AP results are expressed as relative variation from untreated control value ({Delta} value).


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Modifications of IGF System During in Vitro Osteogenic Differentiation

Because some IGFBPs bind to the cell membrane and to the extracellular matrix, the modifications of the components of the IGF system in ROB cultures throughout all the stages of osteogenesis in vitro were studied by different methods.

Chromatographic analysis, performed after acid treatment of medium conditioned for 24 h (days 0, 3, 6, and 9), demonstrated the presence of IGF-I, IGF-II, IGFBP-2, and IGFBP-3 immunoreactivity, but not of IGFBP-1. By immunoassay, all detectable peptides showed the highest concentrations on day 3 of differentiation (end of S1). The IGF peptide most represented at all differentiation stages was IGF-II, whereas only small amounts of IGF-I were present (Fig. 1A). Likewise, IGFBP-2 was the most abundantly detected binding protein in the CM. By semiquantitative RT-PCR, IGF-I and IGF-II showed highly expressed on days 3 and 6, whereas IGFBP-2 showed constantly expressed (Fig. 1B). By indirect immunofluorescence, IGFBP-3, -4, and -5 and type I IGF receptor were evaluated during osteogenesis in vitro (data not shown). IGFBP-3, -4, and -5 were present in all cells. IGF type I receptor was slightly expressed and did not show any variation during osteogenesis. The low number of IGF-I receptors was demonstrated by flow cytometry analysis (net MESF 163 ± 10 x 103/cell).



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Fig. 1. Modifications of IGF and IGF-binding protein (IGFBP) levels during rat tibial osteoblast (ROB) osteogenesis in vitro. A: ROB cells were cultured in serum-free differentiating medium supplemented with 0.5% BSA for 24 h on days 0, 3, 6, and 9 (day 0 = confluence). IGF-I and -II and IGFBP-2 and -3 were separated by gel filtration on a FPLC Superdex-75, and fractions were analyzed for specific immunoreactivity. IGF-II and IGFBP-2 were the peptides most abundantly present in the conditioned media of ROB; all peptides showed their highest concentration on day 3 of differentiation [end of stage 1 (S1)]. B: confluent ROB were cultured in differentiating medium supplemented with 1% FCS, and relative differences in mRNA abundance for IGF-I, IGF-II, and IGFBP-2 were evaluated by semiquantitative RT-PCR (see MATERIALS AND METHODS); IGF-I and -II showed highly expressed on days 3 and 6, whereas IGFBP-2 did not show modifications. Expression of IGFs and IGFBP-2 mRNA was normalized for GAPDH expression; base pairs are indicated on right.

 

To further investigate the time-modulated behavior of the IGF system, we collected media conditioned for 3 days at different time windows during osteogenesis [S1 (days 0-3), corresponding to the onset of the expression of AP; S2 (days 4-6), coincident with the peak of AP expression in differentiation culture conditions; and S3 (days 7-9), corresponding to the onset of mineral deposition in the extracellular matrix]. These CM were analyzed by Western immuno- and ligand blotting and by gelatin zymography. The results showed that the concentrations of IGFBP-3, -5, and -6 in CM were nearly below detection at any stage of differentiation, whereas IGFBP-4 was present and showed a degradation pattern similar to that of IGFBP-2 (data not shown). Immunoblot analysis of IGFBP-2 showed a doublet at 30-32 kDa present in CM collected at all times during osteogenesis. In addition, proteolytic fragments of ~16 kDa were detected and were seen to increase from S2 to S3, in accordance with the gradual decrease of the intact form (Fig. 2A). Densitometric analysis revealed that the sum of the intact form plus the proteolytic fragment did not differ in the three differentiation stages. Modulation of IGFBP-2 expression during osteogenesis was thus apparently due to a posttranslation mechanism. Ligand blot analysis, using 125I-IGF-II, demonstrated a band of only ~30 kDa, consistent with the known molecular mass of IGFBP-2 by this method, which decreased from S1 to S3, whereas proteolytic fragments did not bind IGF-II (Fig. 2B). Along with the appearance of IGFBP-2 proteolytic fragments, zymography on gelatin showed upregulation and activation of MMP-2 in the CM of ROB at the S2 and S3 differentiation stages (Fig. 2C).



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Fig. 2. IGFBP-2 protein levels and functional forms in conditioned media (CM) collected at different stages of ROB osteogenesis in vitro. CM were collected at 3-day intervals: 1st stage of osteogenesis (S1, days 0-3), 2nd stage (S2, days 4-6), and 3rd stage (S3, days 7-9). Autoradiographs representative of different experiments are shown. A: Western immunoblot analysis. Anti-human IGFBP-2 polyclonal antibody detected a 30- to 32-kDa doublet (molecular size of the intact protein) and a lower band of ~16 kDa (known proteolytic fragment of IGFBP-2) increasing from S2 to S3, in accordance with the gradual decrease of the intact form. B: Western ligand blot analysis. Membranes were incubated with 125I-labeled IGF-II for 24 h at 4°C. The only band detected shows a molecular mass ~30 kDa, consistent with the known molecular mass of IGFBP-2, which decreases from S1 to S3, whereas proteolytic fragments did not bind IGF-II. C: zymogram on a gelatin substrate illustrating metalloproteinase synthesis and activity during osteogenesis. Increasing amounts of pro-matrix metalloproteinase (MMP)-2 (72 kDa), of its 69-kDa form, and of the lower molecular size forms are shown. Higher enzymatic activity is detected with progression from S1 to S3 and are evidenced as areas of lysis of the gelatin. D: degradation of IGFBP-2 upon incubation with CM collected in S1, S2, and S3. Different concentrations (lane 1, 1 nM; lane 2, 0.5 nM; lane 3, 0.2 nM) of human IGFBP-2 were incubated for 5 h at 37°C with CM before analysis by Western blot. Results show that CM from S1 has low proteolytic activity, whereas those from S2 and S3 degrade IGFBP-2 to a fragment of 16 kDa. E: degradation of IGFBP-2 (0.2 nM) upon incubation with CM collected in S2 and S3 in the presence or absence of 40 nM tissue inhibitor of metalloproteinase (TIMP)-1. Presence of TIMP-1 inhibits the degradation of IGFBP-2 blocking the proteolytic activity of CM.

 

To ascertain whether the CM collected at different times during osteogenesis differed in terms of its proteolytic activity on IGFBP-2, CM were incubated for 5 h in a water bath at 37°C with increasing concentrations of IGFBP-2. The results confirmed that CM from S1 had low proteolytic activity, whereas those from S2 and S3 degraded IGFBP-2 to a fragment of 16 kDa (Fig. 2D). This suggests that proteinases specific for IGFBP-2 are produced by ROB during osteogenesis in vitro. The presence of TIMP-1 was able to inhibit the degradation of IGFBP-2 by blocking the proteolytic activity of CM from S2 and S3 (Fig. 2E).

Effect of Treatment with IGF-II and IGFBP-2 on AP

The modulation of IGF-II and of IGFBP-2 during osteogenic progression suggested that these factors might play a role in the progression of the phenotypic maturation of osteoblasts. We chose to monitor the expression of AP, an early marker of osteoblast maturation in vitro, on treatment with IGF-II and IGFBP-2, added to the cultures at concentrations near the physiological range for these cells.

When exogenous IGF-II was added to the differentiating osteoblasts continuously, the treatment caused a significant increase in AP expression (Fig. 3). The induction was detectable on day 3 and maximal on day 6 (101 ± 11% over untreated controls, P < 0.0001), reaching the level obtained with cells differentiating in the presence of 10% FCS (143 ± 30%). In contrast, treatment with the same concentration of desIGF-II, an analog of IGF-II characterized by a very reduced binding affinity for IGFBPs, did not induce a statistically significant modification in AP expression compared with untreated controls (Fig. 3).



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Fig. 3. Effect of treatment with IGF-II on alkaline phosphatase (AP). Confluent cells were transferred to a differentiation medium supplemented with 1% FCS (day 0) in the absence or presence of IGF-II and des(1-6)IGF-II (desIGFII). Cell lysates were collected for AP determination every 3rd day during osteogenesis. Continuous treatment (for 9 days) with exogenous IGF-II, but not with desIGF-II, caused a significant (P = 0.01 and P < 0.0001 vs. untreated controls on days 3 and 6, respectively) increase in AP expression, comparable to that obtained with cells differentiating in the presence of 10% FCS. Values are presented as {Delta}% of control for 4 different cultures.

 

To evaluate whether cells at subsequent stages of differentiation would present a different responsiveness to IGF-II, we performed a pulse treatment. Exposure of ROB to IGF-II limited to S1, S2, or S3 induced a significant stimulation of AP synthesis only in cells treated in S1 (73 ± 11%, P < 0.0001; Fig. 4).



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Fig. 4. Effect of treatment during S1 with IGF-II and IGFBP-2 on AP. Cconfluent cells were transferred to a differentiation medium supplemented with 1% FCS (day 0) in the absence or presence of IGF-II, IGFBP-2, and 3 nM IGF-II preincubated with increasing concentrations of IGFBP-2. Shaded area indicates period during which test substances were added. Pulse treatment during S1 with exogenous IGF-II or IGFBP-2 alone (at 0.5 nM) induced a significant stimulation of AP synthesis on day 6 (P < 0.0001 and P = 0.003 vs. untreated controls, respectively). Treatment with IGF-II after preincubation with increasing concentrations of IGFBP-2 caused a significant increase in AP expression (P < 0.001 at a concentration of IGFBP-2 of 0.5 and 1 nM; P < 0.0001 at 3 nM), comparable to that obtained with IGF-II alone. Values are presented as {Delta}% of control for 4 different cultures.

 

As the IGFBP-2/IGF-II, as well as the IGFBP-2/IGFs' molar ratio in CM appeared downmodulated during osteogenesis, being highest in the first stage of differentiation, we investigated the role of IGFBP-2 during osteogenesis in our in vitro model. Differentiating cultures were therefore treated with increasing concentrations of IGFBP-2, alone or after preincubation at 22°C for 2 h with 3 nM IGF-II, to allow the formation of binary complexes. The results demonstrated that IGFBP-2 alone was able to induce a significant (P < 0.001) dose-dependent stimulation of AP when added during S2 (Fig. 5), whereas it was active only at the lowest concentration (P = 0.003) when added during S1 (Fig. 4). Moreover, treatment of ROB with IGF-II and IGFBP-2 combined during differentiating stage S2, i.e., the stage in which cells were unresponsive to IGF-II alone, caused a statistically significant increase in AP with a 3-day shift (P < 0.001; Fig. 5). This potentiating effect of IGFBP-2 on IGF-II action was less pronounced at S1 (Fig. 4). In agreement with the effect on AP secretion, treatment in S2 with IGF-II and IGFBP-2 combined increased gene expression of AP (Fig. 6).



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Fig. 5. Effect of treatment during S2 with IGF-II and IGFBP-2 on AP. Confluent cells were transferred to differentiation medium supplemented with 1% FCS (day 0) in the absence or presence of IGF-II, IGFBP-2, and 3 nM IGF-II preincubated with increasing concentrations of IGFBP-2. Shaded area indicates period during which test substances were added. Pulse treatment of ROB during S2 with IGF-II and IGFBP-2 combined caused a statistically significant increase in AP on day 9 (P = 0.001 at 0.5 nM and P < 0.0001 at 1 and 3 nM IGFBP-2 vs. IGF-II alone), whereas IGF-II alone proved stimulatory only in ROB cultures pretreated with TIMP-1 (P < 0.0001). IGFBP-2 alone was able to significantly (P < 0.001 vs. untreated controls) increase AP. Values are presented as {Delta}% of control for 4 different cultures.

 


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Fig. 6. Spontaneous and IGF-II, IGFBP-2 modulated expression of core-binding factor-1 (Cbfa-1), AP, collagen type I (Col I), and osteocalcin (OC). ROB cells expressed mRNA transcripts of all proteins studied. Confluent ROB were cultured in differentiating medium supplemented with 1% FCS in the absence (control) or presence of test substances added (+) in S1 or S2. mRNA transcripts were evaluated by semiquantitative RT-PCR (see MATERIALS AND METHODS) at different times during differentiation in vitro (S1, S2, and S3). IGF-II and IGFBP-2, alone or in combination, at near-physiological dose (3 nM) did not have a significant effect on Cbfa-1 and Col I expression. In contrast, IGF-II added in S1 and, particularly, the IGF-II·IGFBP-2 complex in S2 were effective in stimulating AP and, with a delay period, OC expression. Expression of mRNAs was normalized for GAPDH expression; base pairs are indicated on left.

 

Finally, before treatment with IGF-II, ROB were preincubated with 20 nM recombinant TIMP-1, a tissue inhibitor of metalloproteinases. Administration of TIMP-1 restored the cell responsiveness to IGF-II in S2, showing that the inhibition of IGFBP-2 proteolysis is important and that the intact form of IGFBP-2 enhances IGF-II action in our model (Fig. 5).

Effect of Treatment with IGF-II and IGFBP-2 on Other Phenotype Markers

In Fig. 6, a representative RT-PCR experiment is shown: gene expression of Cbfa-1, a transcription factor involved in commitment to the osteoblast lineage, and of Col I was not affected by treatment with 3 nM IGF-II and IGFBP-2, alone or combined, whereas treatment with IGF-II and IGF-II·IGFBP-2 complex was effective in stimulating OC expression (Fig. 6). Calcium incorporation was not significantly affected by the treatment (Fig. 7).



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Fig. 7. Effect of treatment with IGF-II or IGFBP-2 on calcium incorporation during differentiation in vitro. Confluent cells were transferred to differentiation medium supplemented with 1% FCS (day 0)in the absence or presence of IGF-II (3 nM), IGFBP-2 (3 nM), and IGF-II preincubated with IGFBP-2; the rate of calcium incorporation was quantitated in cell lysates by spectrophotometric measurement. Treatment with IGF-II and IGFBP-2, alone or in combination, did not show a significant effect on this marker of osteoblast differentiation. Shaded area indicates period during which test substances were added.

 


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
In this study, we evaluated the modification and the role of the IGF system in an already characterized model of osteogenic differentiation obtained from ROB in culture (18, 36, 48, 55). In agreement with previous data on bone tissues and osteoblastic cells (3, 7, 8, 10, 21, 25, 26, 35, 44, 52), in our rat model the components of the IGF system most represented were IGF-II and IGFBP-2, -3, -4, -and -5 either in the CM or bound to the cell membrane and to the extracellular matrix. However, the components of the IGF system most represented in the CM during ROB differentiation in vitro were IGF-II and IGFBP-2, two peptides known to be closely interrelated in that IGF-II stimulates IGFBP-2 synthesis and IGFBP-2 shows greater affinity for IGF-II than for IGF-I. When the endogenously produced IGF-II is considered, the reduction of the peptide on day 6, despite a sustained expression similar to that of day 3, is consistent with reduced protection from degradation by the proteolyzed IGFBP-2 in this stage. Unlike what was reported previously in MC3T3-E1 murine osteoblasts (57) and rat calvaria preosteoblasts (4), low levels of the other IGFBPs, except for IGFBP-4, were found in the CM of ROB. Nonetheless, differences in the expression of the components of the IGF system among osteogenic cells from different species and, in the same species, from different skeletal sites have been reported (9, 24, 35), and in our ROB cultures cell-associated IGFBP-3 and -5 were present, as in other osteoblastic cell systems. In contrast, IGFBP-1 and -6 could not be detected by any method used.

The IGF system is complex and well regulated. Indeed, some proteases of IGFBP that play a potentiating role when degraded, like IGFBP-3 and -5, are inhibited by the IGFs, as if through a feedback mechanism, whereas the protease of IGFBP-4, which almost invariably exerts an inhibitory effect, is stimulated by the IGFs, which thus accelerate its removal. Exhaustive studies have been done to characterize the critical role played in bone by IGFBP-4 and -5, two of the main IGFBPs produced by bone cells (14, 15, 33, 42, 45, 49, 50), whereas little is known about the behavior and the modulatory role of IGFBP-2 in bone tissue. In the osteoblastic model studied, this peptide showed a decrease throughout the S2 and S3 differentiation stages of osteogenesis, which coincides with the peak of AP expression and with the onset of mineral deposition in the extracellular matrix, respectively. The findings that the proteolytic activity of the CM of ROB, on both endogenous and exogenous IGFBP-2, was increased during S2 and S3, that the sum of intact IGFBP-2 plus proteolytic fragments in the CM did not differ in the three differentiation stages, and that there is no variation of IGFBP-2 expression indicate that the modulation of IGFBP-2 during osteogenesis was mostly due to a posttranslation mechanism. In line with the notion that proteinases specific for IGFBP-2 are produced by ROB during osteogenesis in vitro, and with the relatively lower levels of the other IGFBPs in the CM, binding with IGF-II occurred exclusively in the molecular weight range of the intact form of IGFBP-2, which decreased from S1 to S3. These data also show that IGFBP-2 proteolytic fragments are not biologically active and that the association of IGF-II in the differentiating ROB milieu occurs with the intact form of IGFBP-2. In parallel with the appearance of IGFBP-2 proteolytic fragments, upregulation and activation of MMP-2 were also apparent, and the proteolytic activity of CM of ROB was greatly reduced in the presence of TIMP-1, suggesting that the IGFBP-2-degrading proteinases may, at least in part, be MMPs.

The modification of IGF-II and IGFBP-2 and -2 proteases suggested that these factors might play a role in the progression of the phenotypic maturation of osteoblasts. We chose to monitor AP expression, an early marker of osteoblast maturation in vitro, on treatment with IGF-II and IGFBP-2 added to the cultures at concentrations near the physiological range for these cells.

When exogenous IGF-II was added continuously to the differentiating osteoblasts, the treatment caused an increase in AP expression comparable to that obtained with cells differentiating in the presence of 10% FCS. Surprisingly, treatment with the same concentration of desIGF-II, an analog of IGF-II characterized by a very reduced binding affinity for IGFBPs, did not induce a statistically significant modification in AP expression compared with untreated control cells. However, the relatively slight effect of desIGF-II, compared with that of the natural IGF-IGFBP complex in long-term cell culture, may be due to the relatively greater potency of the uncomplexed peptide in downregulating type I IGF receptors, thus attenuating IGF action (61). Indeed, it has been shown that the addition of multiple small doses of IGF-I stimulates DNA synthesis in hamster kidney fibroblasts, whereas high doses are less effective or ineffective (5). Moreover, it has been demonstrated in vivo that the application of IGF-I·IGFBP-3 complex to wounds promotes healing more effectively than equivalent doses of IGF-I alone (23, 46). Given that the type I IGF receptor on most cells typically approaches saturation at IGF concentrations of 5 nM or lower, it is clear that regulation of IGF bioavailability is a key function of the IGFBPs (2). In our cell cultures, which were characterized by low IGF-I receptor expression, the IGF-II·IGFBP-2 complex may constitute a slow-release form of IGF peptide that, besides protecting the IGF peptide from degradation, attenuates the downregulation of type I IGF receptors.

In agreement with the involvement of IGFBP-2 in the different responsiveness of the cells to IGF-II, exposure of ROB to IGF-II alone in S1, S2, or S3 induced a significant stimulation of AP synthesis only in cells treated at S1, when soluble IGFBP-2 was higher. As the IGFBP-2/IGF-II molar ratio appeared downmodulated during osteogenesis, whereas the IGF type I receptor did not show any variation, we investigated the effect of IGFBP-2 during osteogenesis in our in vitro model. The findings that IGFBP-2 was able to induce a significant dose-dependent stimulation of AP when added during S2, and only at the lowest concentration when added during S1, support the notion that the potentiating effect of IGFBP-2 on IGF-II-induced AP activity is exerted via an IGF-dependent mechanism at physiological concentrations. Interestingly, Arai et al. (1) have shown that the IGF·IGFBP-2 complex, at a molar ratio similar to that found in the CM of ROB, has a particular affinity for glycosaminoglycans, which are abundant in the bone matrix in later stages than S1. In this way, the IGF peptide is stored within the target tissue for interaction with the receptor. Indeed, treatment of ROB with equimolar concentrations of IGF-II and IGFBP-2 combined caused a statistically significant increase in AP during differentiating stage S2, when cells presenting with reduction of intact bioactive IGFBP-2 were unresponsive to IGF-II alone. To verify the potentiating modulatory effect of intact IGFBP-2 on IGF-stimulated AP activity, ROB cultures were preincubated with recombinant TIMP-1, which has been demonstrated to inhibit proteolytic degradation by MMPs of IGFBP-1, -2, -3, and -5 but not of IGFBP-4 (37), before treatment with IGF-II. The results showed that, at S2, TIMP-1 restored cell responsiveness to IGF-II. As IGFBP-3 and -1 appear relatively low to undetectable in our system, and IGFBP-5 exerts a potentiating effect on IGF-dependent actions when proteolyzed (28) whereas IGFBP-4 is inhibitory in its intact form, we regard the stimulatory effect of TIMP-1 in our system mainly as a result of the protection of IGFBP-2 from degradation (Fig. 2E).

Both stimulatory and inhibitory effects of IGFBP-2 on IGF activity have been reported, depending on the model examined and on the concentration of IGFBP-2 relative to the IGF peptide (16, 29, 40, 51). In vitro and in vivo overexpression of IGFBP-2 showed that this binding protein can exert inhibitory effects on IGF actions (16, 27, 60). In contrast, it has been demonstrated that the IGF-II·IGFBP-2 complex was as effective as IGF-II alone in stimulating human osteoblast proliferation in vitro (31). In vivo, data obtained in patients with hepatitis C-associated osteosclerosis (31) and in prepubertal children with constitutionally tall stature (22) suggest that IGFBP-2 may facilitate the targeting of IGFs, and in particular of IGF-II, to skeletal tissues with a subsequent stimulation of osteoblast proliferation and activity. Moreover, subcutaneous administration of IGF-II·IGFBP-2 complex stimulates bone formation and prevents loss of bone mineral density in a rat model of disuse osteoporosis (12). Finally, the involvement of IGFBP-2 in a number of tumors, including colonic neoplasia and malignant adrenocortical tumors, is consistent with a stimulatory role of IGFBP-2 (6, 41) found in our system of osteogenesis.

In conclusion, IGFBP-2 potentiates IGF-II effects in early stages of differentiation of rat osteoblasts. This observation further underlines the importance of the balance between stimulatory and inhibitory IGFBPs and their specific and nonspecific proteases in bone tissue.


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 ABSTRACT
 MATERIALS AND METHODS
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This work was supported by research grants 9906153187-005 and KH03025623 from Ministero dell'Università e della Ricerca Scientifica (Rome, Italy) and Fondo per gli Investimenti della Ricerca di Base (FIRB RBAU019TMF_001).


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Barreca, Cattedra di Endocrinologia, DiSEM, Univ. of Genova, Viale Benedetto XV, no 6, I-16132 Genoa, Italy (E-mail: barreca{at}unige.it).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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