Insulin-like growth factor (IGF)-binding protein-5-(201---218) region regulates hydroxyapatite and IGF-I binding

Phil G. Campbell and Dennis L. Andress

Orthopaedic Research Laboratory, Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 15212; and Departments of Medicine, Veterans Affairs Medical Center and University of Washington, Seattle, Washington 98108

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Insulin-like growth factor-binding protein-5 (IGFBP-5), the major bone IGFBP, modifies the biological activity of IGFs within the osteoblastic pericellular environment. Because glycosaminoglycans modulate IGFBP-5 binding to osteoblast organic extracellular matrix (ECM), we assessed whether the heparin binding domain of IGFBP-5, IGFBP-5-(102---218), modifies the interaction of IGFBP-5 with the inorganic bone ECM hydroxyapatite (HA). Synthetic IGFBP-5-(201---218) peptide increased the binding of IGFBP-5 to HA as well as the binding of IGF-I to HA-bound IGFBP-5. This action was specific for the heparin-binding domain, because IGFBP-5-(130---138), IGFBP-5-(138---152), and IGFBP-5-(1---169) were without effect. IGFBP-5-(201---218) was found to bind directly to IGFBP-5 and cause a threefold enhancement of the IGF-I binding affinity for IGFBP-5, whether IGFBP-5 was bound to HA or was in a matrix-free fluid phase. Heparin inhibited the binding of IGFBP-5 to HA and blocked the interaction of IGFBP-5 with IGFBP-5-(201---218) in the fluid phase, suggesting that the primary heparin-binding domain of IGFBP-5 specifically enhances the binding of IGFBP-5 to HA and increases IGF-I binding to IGFBP-5.

bone; extracellular matrix; osteoblast

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

THE INSULIN-LIKE GROWTH FACTORS (IGFs), as well as other growth factors, are present in mineralized bone matrix (8, 14, 16, 18, 22) and are thought to regulate osteoblast function as part of an autocrine/paracrine mechanism of bone remodeling (23). The IGFs are the most abundant of the growth factors to be isolated from human bone (8, 14), likely because of the high concentration of osteoblast-derived IGF-binding protein (BP)-5 bound to bone matrix, including hydroxyapatite (HA) (7). Despite knowledge of abundant IGFBP-5 within HA, the mechanism(s) of IGFBP-5 binding to this inorganic extracellular matrix (ECM) component is yet to be determined. One possible mechanism would be for the heparin-binding region of IGFBP-5 (1) to directly interact with HA, as IGFBP-3 binds to pericellular ECM (9).

IGFBP-5 also binds to several proteins that are common constituents of the organic ECM (19). Glycosaminoglycan (GAG)-containing proteins do not appear to specifically mediate IGFBP-5 binding to fibroblastic (27), osteoblastic (1), or endothelial (9) types of ECM. However, where pericellular IGFBP-5 interaction does occur, GAG binding to the heparin-binding domain within the carboxy-terminal region of IGFBP-5 [IGFBP-5-(201---218)] may be important (6, 9). In addition to mediating the binding of IGFBP-5 to GAGs (1, 5, 6) and to other ECM proteins (9), the IGFBP-5-(201---218) region also mediates the binding of IGFBP-5 to plasminogen and/or plasmin (10a), the latter of which regulates proteolytic degradation of intact IGFBP-5 (10a).

Because the IGFBP-5-(201---218) sequence appears to have multiple functions in mediating the bioactivity and binding of IGFBP-5 to the pericellular environment, we sought to determine whether this heparin-binding region may also be important in mediating the interaction of IGFBP-5 and IGF-I with the bone inorganic ECM, HA. Our results indicate that IGFBP-5-(201---218) not only enhances the binding of IGFBP-5 to HA but that it also binds directly to IGFBP-5 in such a way as to increase IGF-I binding to IGFBP-5.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials. Recombinant human insulin-like growth factor I (IGF-I) was purchased from GroPep (Adelaide, Australia). Recombinant intact IGFBP-5 (IGFBP-5I) and IGFBP-5-(1---169) were produced in a baculovirus expression system and purified by affinity chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) (3). The IGFBP-5 peptides IGFBP-5-(201---218) (amino acids RKGFYKRKQCKPSRGRKR), IGFBP-5-(138---152) (amino acids KLTQSKFVGGAENTA), IGFBP-5-(130---142) (amino acids EAVKKDRRKKLTQ), and IGFBP-5-(235---252) (amino acids VDGDGFQCHTFDSSNVE) were synthesized and purified by RP-HPLC. IGF-I, IGFBP-5, and IGFBP-5-(201---218) were iodinated by the chloramine T method to specific activities of 150 µCi/µg, 11 µCi/µg protein, and 25 µCi/µg, respectively (12). HA beads were obtained from Calbiochem (La Jolla, CA). Insulin radioimmunoassay (RIA)-grade bovine serum albumin (BSA) was purchased from Sigma Chemical (St. Louis, MO). NUNC 96-well medium binding immunological plates were from Fisher Scientific (Pittsburgh, PA).

Charcoal IGFBP Assay. A basic assay consisted of equilibrating increasing concentrations of IGFBP-5 peptides with ~20,000 counts/min (cpm) 125I-labeled IGF-I in 50 mM tris(hydroxymethyl)aminomethane (Tris) and 2.5% BSA, pH 7.4, for 1 h at 37°C in a total reaction volume of 200 µl in 1.5-ml screw-cap Eppendorf plastic tubes. Complexes of 125I-IGF-I and IGFBP-5 were determined by the addition of 400 µl 1% activated charcoal, 0.2 mg/ml protamine sulfate, and 1% BSA in phosphate-buffered saline (PBS), pH 7.4, to the 200-µl incubations. Reactants (600 µl total volume) were vortexed and incubated in a 4°C ice bath for 15 min. Incubations were terminated by centrifugation for 5 min at 14,000 g, and 500-µl supernatants, containing 125I-IGF-I/IGFBP-5 complexes, were counted for radioactivity. Uncomplexed 125I-IGF-I was absorbed by charcoal and removed in the pellet fraction. Nonspecific binding was determined by the inclusion of 100 ng of unlabeled IGF-I, and all binding results were corrected for this value. Preliminary experiments determined that, under these conditions, binding equilibrium had been achieved. To determine the effect of IGFBP-5 peptides [IGFBP-5-(201---218), IGFBP-5-(130---142), or IGFBP-5-(138---152)] on 125I-IGF-I association with IGFBP-5I, ~20,000 counts/minute (cpm) 125I-IGF-I and 6.25 ng/ml IGFBP-5I, unless otherwise stated, were equilibrated in the absence or presence of IGFBP-5 peptides as indicated in the appropriate figure legends. Bound 125I-IGF-I was determined as described above. Heparin (HE) at 100 µg/ml was included where specifically stated. In some experiments, increasing concentrations of unlabeled IGF-I were added in the presence or absence of 1 µg/ml IGFBP-5-(201---218). Binding kinetics were determined by the method of Scatchard with the LIGAND program originally written by Munson and Rodbard as modified for microcomputers by McPherson (21).

IGFBP-5 plate binding assay. Binding of IGFBP-5 peptides to IGFBP-5I was characterized by use of an immobilized IGFBP-5-based assay system. Optimal binding conditions, IGFBP-5I coating concentration, plate type, and incubation parameters were determined in preliminary experiments, and the optimized conditions make up the following assay protocol. Ninety-six-well immunological plates were coated with 100 µl of 0.1 M Na2CO3, pH 9.8, containing 5 µg/ml of IGFBP-5I overnight at 4°C. The plates were rinsed with 200 µl PBS and blocked with 200 µl 10 mM Tris · HCl, 150 mM NaCl, 0.05% Tween 80, 1% BSA, and 0.02% NaAz, pH 7.5, for 1 h at 37°C. Plates were rinsed, and ~30,000 cpm 125I-IGFBP-5-(201---218) were incubated with increasing concentrations of IGFBP-5-(201---218), IGFBP-5-(130---142), IGFBP-5-(138---152), or HE in 200 µl assay buffer for 1 h at 37°C. Unbound radioactivity was removed by rinsing the wells twice with 200 µl of ice-cold assay buffer. Bound radioactivity was solubilized with 200 µl 1 N NaOH, transferred to 12 × 75-mm glass test tubes, and counted for radioactivity. Background binding was assessed by determining the binding of 125I-IGFBP-5-(201---218) in wells coated with 0.1 M Na2CO3 in the absence of IGFBP-5I.

125I-IGFBP-5 binding to HA. Stock slurry solutions of 10% HA beads (wt/vol) were maintained at 4°C in 30 mM Tris acetate, 10 mM sodium phosphate, 0.05% Tween 20, and 0.2% NaAz, pH 7.4 (HA assay buffer). For an assay, beads were suspended by gently swirling and were further diluted, and 50 µl of a 1% HA bead slurry were placed in 1.5-ml screw-cap plastic Eppendorf tubes and equilibrated with 125I-IGFBP-5I and competitors, as indicated in Figs. 1 and 2, in a total volume of 200 µl for 30 min at 23°C, with occasional vortexing. Unbound 125I-IGFBP-5I was removed by centrifuging at 10,000 g, aspirating, and removing the supernatant, followed by washing the bead pellet twice with 1 ml HA assay buffer. After the final rinse, 1 ml Milli-Q filtered water (MQH2O) was added and the beads were resuspended and transferred to 12 × 75-mm glass test tubes to count bound radioactivity.

HA bead IGFBP assay. A basic assay consisted of equilibrating increasing concentrations of IGFBP-5 peptides with ~20,000 cpm 125I-IGF-I in HA assay buffer and 50 µl of 10% HA beads for 1 h at 37°C in a total reaction volume of 200 µl in 1.5-ml screw-cap Eppendorf plastic tubes. Tubes were vortexed approximately every 15 min. Preliminary experiments determined that under these conditions binding equilibrium had been achieved. Incubations were terminated by centrifuging tubes at 10,000 g for 1 min, discarding the supernatant, and rinsing the pellets with two additional 1-ml washes of HA assay buffer. After the final rinse, 1 ml MQH2O was added and the beads were resuspended and transferred to 12 × 75-mm tubes for determination of radioactivity. Nonspecific binding of 125I-IGF-I was determined by excluding IGFBP-5 peptides during the equilibration period. To determine the effect of IGFBP-5 peptides [IGFBP-5-(201---218), IGFBP-5-(130---142), or IGFBP-5-(138---152)] on 125I-IGF-I association with IGFBP-5I, 20,000 cpm 125I-IGF-I and 6.25 ng/ml IGFBP-5I, unless otherwise stated, were equilibrated in the absence or presence of IGFBP-5 peptides (see Figs. 4-7). Bound 125I-IGF-I was determined as described above. In some experiments, increasing concentrations of unlabeled IGF-I were added in the presence or absence of 1 µg/ml IGFBP-5-(201---218). Binding kinetics were determined by the method of Scatchard with the LIGAND program originally written by Munson and Rodbard as modified for microcomputers by McPherson (21).

Affinity cross-linking of IGF-I to 125I-IGFBP-5I. 125I-IGFBP-5I (100,000 cpm) was incubated alone and with IGFBP-5-(201---218) (10 µg/ml) and/or IGF-I (500 ng/ml) for 1 h at 37°C in a total volume of 50 µl 50 mM phosphate buffer, pH 7.4. Bound reactants were cross-linked with 0.5 mM disuccinimidyl suberate, final concentration, on ice for 15 min. The cross-linking reaction was terminated by adding 0.5 M Tris · HCl, pH 7.4, to a final concentration of 50 mM for 15 min on ice. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, 3×, was added and samples were separated on a 17.5% nonreducing gel (20), with the gel dried and autoradiographed by use of Kodak X-Omat film overnight at 23°C.

Statistical analysis. Statistical analysis was performed by analysis of variance followed by Tukey's honestly significant difference test with multivariate general linear hypothesis (26). Where appropriate, the Student's t-test was used to compare two means. Probability values <0.05 were considered significant.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

125I-IGFBP-5 binding to HA. Equilibration of 125I-IGFBP-5I with HA for 30 min at 23°C resulted in a dose-dependent increase in the specific binding of 125I-IGFBP-5I to HA (Fig. 1A) with an association binding constant (Ka) of 4.6 × 106 l/mol. Binding of 125I-IGFBP-5I to HA was actively competed by HE with an approximate 50% inhibitory concentration (IC50) of 1 µg/ml (Fig. 1B). At maximal inhibition of binding, control binding of 82,500 of 420,000 cpm added was reduced to nonspecific 11,000 cpm. This effect of HE is similar to that in other studies investigating IGFBP-5 binding to organic ECM (1, 9, 19) and suggests that the HE-binding region of IGFBP-5 may be important in the interaction with HA. To evaluate this further, the effect of the HE-binding peptide IGFBP-5-(201---218) on 125I-IGFBP-5I binding to HA was determined (Table 1). Contrary to the expected inhibitory response, coincubation of IGFBP-5-(201---218) with 125I-IGFBP-5I and HA resulted in increased 125I-IGFBP-5I binding to HA (P < 0.001). To determine whether the enhancing effect of IGFBP-5-(201---218) involved binding of IGFBP-5-(201---218) to HA, IGFBP-5-(201---218) was preequilibrated with HA before the addition of 125I-IGFBP-5I. The resulting increase in 125I-IGFBP-5I binding (P < 0.001) further suggested that IGFBP-5-(201---218) bound to IGFBP-5I as well as to HA. The response was specific for IGFBP-5-(201---218) because another basic peptide of IGFBP-5, IGFBP-5-(130---142), which does not contain the primary HE-binding domain, failed to elicit a similar response. We interpret the minor inhibitory ability of IGFBP-5-(130---142) (P = 0.007), under coincubation but not preincubation conditions, to represent a possible low-specificity charge competition for IGFBP-5I binding. Direct binding of IGFBP-5-(201---218) to HA was confirmed by HE-dependent binding of 125I-IGFBP-5-(201---218) to HA (total control specific binding was 53% of 150,000 cpm added; nonspecific binding was 4% of total cpm added).


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Fig. 1.   125I-labeled intact human insulin-like growth factor 5 (125I-IGFBP-5I) binding to hydroxyapatite (HA) is inhibited by heparin (HE). A: increasing amounts of 125I-IGFBP-5I were incubated with HA beads (1:500 final dilution) in a total volume of 100 µl for 30 min at 23°C. When HE (100 µg/ml) was used, the mean nonspecific binding (NSB) was 1.1% of total counts/min (cpm) added. Bound IGFBP-5I was analyzed by Scatchard. B: increasing concentrations of HE were incubated with 125I-IGFBP-5I (~400,000 cpm) in the presence of HA beads (1:400 final dilution) for 30 min at 23°C in a final volume of 200 µl. Beads were rinsed and bound 125I-IGFBP-5I was determined. B/Bo, ratio of bound radioactivity in the presence of competitor over the bound radioactivity in the absence of any competitor; Ka, association binding constant. HE at 625 µg/ml was used for NSB estimate. Values are means of duplicate determinations.

                              
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Table 1.   IGFBP-5-(201---218) increases binding of 125I-IGFBP-5I to HA

To further evaluate the interaction of IGFBP-5 and IGFBP-5-(201---218) to HA, the NH2-terminal IGFBP-5 fragment IGFBP-5-(1---169), which does not contain the IGFBP-5-(201---218) sequence, was used to further establish specificity of the IGFBP-5-(201---218) region. The acidic carboxy-terminus peptide of IGFBP-5, IGFBP-5-(235---252), was also tested because the relatively abundant negatively charged groups might represent a potential binding site on the IGFBP-5I molecule for IGFBP-5-(201---218). Neither of these peptides affected IGFBP-5I binding to HA (Fig. 2). Furthermore, IGFBP-5-(201---218) stimulation of IGFBP-5I binding to HA (P < 0.001) was also unaffected by either peptide [IGFBP-5-(201---218) + IGFBP-5-(235---252), P = 0.70; IGFBP-5-(201---218) + IGFBP-5-(1---169), P = 0.88]. This is additional confirmation for the specificity of the IGFBP-5-(201---218) response and suggests that the IGFBP-5-(1---169) and IGFBP-5-(235---252) regions are unlikely binding sites on IGFBP-5 for the IGFBP-5-(201---218) peptide and that IGFBP-5-(1---169) and IGFBP-5-(235---252) regions are likely not involved in the binding of IGFBP-5I to HA.


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Fig. 2.   Lack of effect of IGFBP-5-(1---169) and IGFBP-5-(235---252) on 125I-IGFBP-5I binding to HA. 125I-IGFBP-5I was incubated with HA beads (1:400 final dilution) for 30 min at 23°C in a final volume of 200 µl with the indicated additions of IGFBP-5 peptides IGFBP-5-(201---218), IGFBP-5-(1---169), and IGFBP-5-(235---252). Bound 125I-IGFBP-5I and NSB were determined as described in Fig. 1. Bars, means ± SE of 2 separate experiments conducted in triplicate. Differences between means were determined by analysis of variance (ANOVA) and Tukey's honestly significant difference (HSD) test; * significant (P < 0.05) difference from control group.

Because a direct interaction between IGFBP-5I and IGFBP-5-(201---218) was implied by the above experiments, an HA-independent immobilized IGFBP-5I binding assay was used to determine the direct binding of IGFBP-5-(201---218) to IGFBP-5I. In this assay, 125I-IGFBP-5-(201---218) bound specifically to immobilized IGFBP-5I, as shown by the competition binding curve in Fig. 3, confirming the HA binding data. The IC50 for competing unbound IGFBP-5-(201---218) was ~25 ng/ml. Other basic amino acid-containing IGFBP-5 peptides not containing the HE-binding domain, IGFBP-5-(130---142) and IGFBP-5-(138---152), each exhibited an ~1,000-fold lower IC50, further confirming the specificity of IGFBP-5-(201---218) binding to IGFBP-5I. HE actively competed for 125I-IGFBP-5-(201---218) binding to IGFBP-5I (data not shown), confirming the requirement for an active HE-binding domain. Taken together with the above results, these data indicate that IGFBP-5-(201---218) binds both to HA and to IGFBP-5I and that this is responsible for the increase in IGFBP-5I binding.


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Fig. 3.   125I-IGFBP-5-(201---218) binds directly to IGFBP-5I. Ninety- six-well plates were coated with IGFBP-5I overnight (Plate binding assay), and 125I-IGFBP-5-(201---218) (~30,000 cpm/well) was added in the presence of increasing concentrations of IGFBP-5 peptides. bullet , IGFBP-5-(201---218); black-triangle, IGFBP-5-(138---152); black-square, IGFBP-5-(130---142). Values are means of duplicate determinations.

Influence of HA and IGFBP-5-(201---218) on IGFBP-5 function. With use of 125I-IGF-I as the reporter of IGFBP-5I binding to HA, IGFBP-5I was found to specifically bind to HA in a concentration-dependent manner (Fig. 4A), with maximal recorded binding occurring at a ratio of bound IGF-I to total cpms added of 0.62, which represents 15,419 cpm specifically bound of 24,842 cpm added. Nonspecific binding was 1,500 cpm or 6% of cpm added. In contrast, the carboxy-truncated IGFBP-5 peptide IGFBP-5-(1---169) showed markedly reduced binding to HA, and the other IGFBP-5 fragments did not exhibit binding in this assay. Because 125I-IGF-I was used as the reporter, some of these binding differences could be secondary to different binding affinities for IGF-I. To evaluate this further, the charcoal IGFBP binding assay was used to quantitate fluid phase 125I-IGF-I binding to IGFBP-5I (Fig. 4B). As expected, IGFBP-5I bound more 125I-IGF-I than IGFBP-5-(1---169). At the maximal recorded binding point for IGFBP-5I, 16,000 cpm were specifically bound of 29,307 cpm added, with nonspecific binding at 2,000 cpm or 6.8%. None of the other IGFBP-5 fragments were capable of binding to 125I-IGF-I in this assay. Therefore, the likely explanation for those fragments failing to demonstrate activity in Fig. 4A was due to their failure to bind IGF-I.


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Fig. 4.   125I-IGF-I binding to IGFBP-5I and to IGFBP-5 peptides in fluid and solid phases. A: with HA assay (solid phase), increasing concentrations of various IGFBP-5 peptides were bound to HA beads in the presence of 125I-IGF-I (~20,000 cpm). After 1 h at 37°C, HA beads were washed and bound radioactivity was determined. Bound 125I-IGF-I in the absence of IGFBP-5I was used as the NSB. Values are means of 2 separate experiments performed in duplicate. B: with charcoal assay (fluid phase), increasing concentrations of IGFBP-5 peptides were incubated with 125I-IGF-I (~20,000 cpm). After 1 h at 37°C, charcoal was added for 15 min at 4°C. Tubes were centrifuged and supernatants were sampled to determine bound radioactivity. 125I-IGF-I in the absence of IGFBP-5I was used as the NSB. Values are means ± SE of 2 separate experiments performed in duplicate. Individual IGFBP-5 peptides shown in A and B: bullet , IGFBP-5I; black-square, IGFBP-5-(1---169); black-triangle, IGFBP-5-(201---218); black-down-triangle , IGFBP-5-(130---142); black-lozenge ,IGFBP-5-(138---152).

Although IGFBP-5-(201---218) itself does not bind IGF-I, its ability to bind directly to IGFBP-5 could alter the IGF-I-binding capacity of IGFBP-5I. To evaluate this possibility, IGFBP-5I was incubated with and without IGFBP-5 (10 µg/ml) in both the charcoal and the HA assays by use of 125I-IGF-I as the reporter. IGFBP-5-(201---218) specifically increased 125I-IGF-I binding to IGFBP-5I (P < 0.001) by approximately threefold in both assays, whereas neither IGFBP-5-(130---142) nor IGFBP-5-(138---152) altered 125I-IGF-I binding (Fig. 5). Because IGFBP-5-(201---218) does not bind 125I-IGF-I, all of the increased binding was attributable to IGFBP-5I.


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Fig. 5.   IGFBP-5-(201---218) specifically stimulates 125I-IGF-I binding to fluid- and solid-phase IGFBP-5I. IGFBP-5I (6.25 ng/ml) and 125I-IGF-I (~20,000 cpm) were incubated in charcoal (fluid phase) or HA assay (solid phase) with 10 µg/ml IGFBP-5-(201---218), IGFBP-5-(130---142), or IGFBP-5-(138---152). After 1 h at 37°C, bound 125I-IGF-I was determined. Bars, means ± SE of 4 experiments performed in triplicate for each fragment. IGFBP-5-(1---169) did not affect 125I-IGF-I binding to IGFBP-5I. Differences between means were determined by ANOVA and Tukey's HSD test; * significant (P < 0.05) difference from control group.

To further evaluate the effects of IGFBP-5-(201---218) on the kinetics of IGFBP-5I binding to 125I-IGF-I, increasing concentrations of IGFBP-5I were tested in the presence or absence of 1 µg/ml IGFBP-5-(201---218) by use of the charcoal assay (Fig. 6A). As shown, IGFBP-5-(201---218) increased 125I-IGF-I binding at all concentrations tested, with a plateau being reached at 60 ng/ml IGFBP-5I. Nearly identical results were also obtained using the HA assay (data not shown). To determine whether there was a change in the 125I-IGF-I binding affinity in response to IGFBP-5-(201---218), competitive binding studies with increasing concentrations of unlabeled IGF-I were performed. As shown in Fig. 6B, Scatchard analysis revealed a threefold increase in the binding affinity, from a Ka 7.1 ± 2.2 × 109 l/mol (means ± SE of 3 experiments) without IGFBP-5-(201---218) to a Ka 21.4 ± 4.7 × 109 l/mol (means ± SE of 2 experiments) with IGFBP-5-(201---218) (P = 0.05), and no change in the total number of binding sites. Similar results were found when the study was performed in the HA assay; the Ka increased from 0.7 to 3 × 109 l/M after the addition of IGFBP-5-(201---218). The ~10-fold difference between the Ka values of soluble IGFBP-5I and HA-bound IGFBP-5I for IGF-I is similar to the ~7-fold difference observed between soluble IGFBP-5I and organic ECM-bound IGFBP-5I (19). This suggests a constant change in IGFBP interaction, with IGF-I independent of the nature of the ECM component. In contrast to these effects, IGFBP-5-(201---218) did not increase the IGF-I binding affinity for IGFBP-5-(1---169), suggesting that the enhancing effect of IGFBP-5-(201---218) is localized to the carboxy-terminal portion of IGFBP-5. The stimulatory effect of IGFBP-5-(201---218) on the binding of IGFBP-5I to 125I-IGF-1 was concentration dependent in both the fluid- and solid-phase assays (Fig. 7), with maximum fluid-phase binding ~40% less than in the solid-phase assay.


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Fig. 6.   IGFBP-5-(201---218) increases binding affinity of IGFBP-5I for IGF-I. A: with charcoal assay, increasing concentrations of IGFBP-5I were incubated without (bullet ) and with (black-square) 1 µg/ml IGFBP-5-(201---218). Values are means ± SE of 2 separate experiments performed in duplicate. B: IGFBP-5I (6.25 ng/ml) was incubated in charcoal assay with 1 µg/ml IGFBP-5-(201---218). An increasing concentration of unlabeled IGF-I was used in competition. Scatchard plots were developed to determine binding parameters. Values are means of duplicate determinations.


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Fig. 7.   IGFBP-5-(201---218) stimulation of 125I-IGF-I binding to fluid- and solid-phase IGFBP-5I is concentration dependent. Increasing concentrations of IGFBP-5-(201---218) were incubated with 6.25 ng/ml IGFBP-5I in either charcoal (fluid phase, bullet ) or HA (solid phase, black-square) assay for 1 h at 37°C with ~20,000 cpm 125I-IGF-I. Values are means ± SE of duplicate determinations. No effect of IGFBP-5-(201---218) was observed with IGFBP-5-(1---169) in fluid phase, whereas in solid phase 5-10 µg/ml produced a 30-40% increase in 125I-IGF-I binding.

Affinity cross-linking was used to further confirm that IGFBP-5-(201---218) increased the binding of 125I-IGFBP-5 to IGF-I (Fig. 8). There was no obvious mobility shift when IGFBP-5-(201---218) was incubated with 125I-IGFBP-5I, although a 2-kDa change would not be easily detected (lane 2). Importantly, IGFBP-5-(201---218) increased IGF-I binding to intact IGFBP-5 (lane 3). This contrasts with results of Arai et al. (5), who reported that IGFBP-5-(201---218) did not affect IGFBP-5I binding of IGF-I in a fluid-phase assay; however, the authors used 100 ng/ml IGFBP-5I. Under our experimental conditions, this concentration places the binding of IGF-I toward the maximum binding plateau of IGFBP-5I. We used 6.25 ng/ml of IGFBP-5I, which yields ~50% maximal binding, providing a sufficient binding range in which to observe any binding enhancement.


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Fig. 8.   IGFBP-5-(201---218) increases the 40-kDa 125I-IGFBP-5I/IGF-I complex. 125I-IGFBP-5I (100,000 cpm) was incubated with IGFBP-5-(201---218) (10 µg/ml) and/or IGF-I (500 ng/ml) for 1 h at 37°C. Bound reactants were cross-linked with dicusate calcium and then separated over 17.5% SDS-polyacrylamide gel electrophoresis, dried, and autoradiographed.

Because the action of IGFBP-5-(201---218) to increase IGF-I binding may be occurring at or near the HE-binding domain within the carboxy-terminal region, binding experiments with and without HE were performed. As shown in Table 2, HE decreased 125I-IGF-I binding to IGFBP-5I in the charcoal assay by 40% (P < 0.001) and completely inhibited the enhanced 125I-IGF-I binding of IGFBP-5I induced by IGFBP-5-(201---218) [IGFBP-5-(201---218) to IGFBP-5-(201---218) + HE, P < 0.001; HE to IGFBP-5-(201---218) + HE, P = 0.159]. The present results and those of others (6) confirm that the HE-binding domain of IGFBP-5 does not contain the IGF-I-binding domain. The inhibitory effect of HE on IGFBP-5 binding of IGF-I is likely due to an altered IGFBP-5 confirmation resulting from a 17-fold loss in IGF-I binding affinity (5). That HE blocked the ability of IGFBP-5-(201---218) to stimulate 125I-IGF-I binding of IGFBP-5I in the present study is likely a result of both reduced binding affinities of IGF-I to IGFBP-5I and inhibition of IGFBP-5-(201---218) binding to IGFBP-5I.

                              
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Table 2.   Influence of heparin and IGFBP-5-(201---218) on IGFBP-5I binding of 125I-IGF-I

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

By showing that intact IGFBP-5 can bind directly to HA and that this interaction is inhibited by HE, we proposed that the HE-binding domain contained within IGFBP-5-(201---218) could mediate the binding of IGFBP-5 to HA. This hypothesis was further strengthened by finding that HA bound to the amino-terminal fragment, IGFBP-5-(1---169), with much less avidity than the intact molecule. In search for a putative role of IGFBP-5-(201---218) in mediating HA binding, we discovered that IGFBP-5-(201---218) enhanced HA binding of intact 125I-IGFBP-5, an effect that was accentuated by its preincubation with HA. These data suggested that not only was IGFBP-5-(201---218) binding to HA, but that it may also be binding to IGFBP-5 itself, because the peptide was not acting as a competitive inhibitor. To evaluate this further, we performed binding studies of 125I-IGFBP-5-(201---218) with immobilized IGFBP-5I and found that the labeled peptide bound to intact IGFBP-5 with high affinity. Thus, in addition to establishing that some portion of the 201-218 amino acid region of IGFBP-5 is responsible for its binding to HA, we have shown that this peptide, which contains basic amino acids in a specific sequence, is capable of binding to IGFBP-5I. Presumably, one or more acidic regions within IGFBP-5 are the putative sites for this interaction, with the exception of the amino-terminal acidic region, IGFBP-5-(235---252), which did not inhibit the interaction of IGFBP-5-(201---218) with IGFBP-5I.

The ability of IGFBP-5 to bind unto itself suggests the possibility of dimer formation. We sometimes observe an ~70-kDa protein band in pure 125I-IGFBP-5 preparations, which likely represents IGFBP-5 dimers. Presuming that this ~70 kDa protein is indeed a dimer of IGFBP-5, we would expect that the protein would not bind to HE and be resistant to plasmin proteolysis (10a). We in fact observe these very characteristics for the ~70-kDa protein (data not shown), which suggest, but do not prove, the existence of an IGFBP-5 dimer. Additional experiments, such as immunoblotting, will be required to prove the existence of IGFBP-5 dimers.

We used both solid- and fluid-phase binding assays to assess conditions that may alter IGF-I binding to IGFBP-5. During the course of these studies, it became apparent that the binding of 125I-IGF-I to IGFBP-5I was enhanced by the presence of IGFBP-5-(201---218). This effect was especially prominent at low IGFBP-5 concentrations and was due to an increased binding affinity, as evidenced by a three- to fourfold increase in Ka. Importantly, this increase in binding affinity occurred regardless of whether IGFBP-5 was bound to HA or was present in an unbound state (fluid phase). The mechanism for the increased IGF binding affinity was not apparent from these studies. However, because the peptide does not bind to IGF-I, we propose that it binds to IGFBP-5 and alters the structural conformation of the intact molecule to increase its affinity for IGF-I.

The question of the physiological relevance of IGFBP-5 binding to HA remains to be addressed. Although many proteins are known to bind to HA, a property commonly used in protein purification (17), in bone the interaction of specific proteins with HA is physiologically relevant. The most classic of these HA-binding proteins in bone is osteocalcin, which binds to HA with a dissociation constant (Kd) of 10-6 M (15). We report here that IGFBP-5 binds to HA with a higher affinity, a Kd of 10-7 M. Although all species of IGFBP readily bind HA (unpublished results from our laboratory), differences in respective binding affinities may explain the differential localization of IGFBPs in bone. The relatively high binding affinity of IGFBP-5 to HA may explain the six- to eightfold greater concentration of IGFBP-5 found in cortical bone than in IGFBP-3 (24) and may strongly suggest that the IGFBP-5 interaction with HA is physiologically relevant.

These studies add to a growing database indicating that IGFBP-related peptides may have a physiological role. For example, it was recently shown that synthetic peptides of the HE-binding domain of IGFBP-3 inhibit the binding of IGFBP-3 and IGFBP-5 to endothelial cells (9). These same peptides also bind to endothelial cell-derived ECM and are capable of directly stimulating glucose uptake in endothelial cells (9). Whether the cell-associated effects of these IGFBP fragments are mediated by peptide binding to surface receptors has not been determined. However, surface binding sites have been identified for IGFBP-3 (25) and IGFBP-5 (1) that may be important in altering cellular activity. In the present study, the HE-binding domain of IGFBP-5 modifies the localization of IGFBP-5 and IGF-I on the HA surface, which suggests a mechanism for the modification of IGFBP-5/IGF-I immobilization into the inorganic matrix of bone.

HE and other GAGs compete for pericellular binding of IGFBP-5 to non-GAG proteins (e.g., fibronectin, collagen type III, vitronectin), which are likely to be important in localizing it to specific pericellular compartments, depending on the type of GAG present (1, 2, 9, 19, 27). Although HE clearly inhibited HA binding of IGFBP-5 in our studies, the effect of endogeneous GAGs in modulating the binding of IGFBP-5 in the periosteoblast environment is unclear. Little is known about the role of GAG-containing proteoglycans in the unmineralized ECM after its deposition onto mineralized HA. It is clear, however, that in studies of osteoblast-derived ECM, the enzymatic treatment to remove GAG side chains actually enhances the binding of IGFBP-5 (2). Thus some GAGs may interfere with IGFBP-5 binding to other ECM proteins that are capable of binding IGFBP-5 (19).

The present studies suggest possible functions for IGFBP-5 in the osteoblastic pericellular environment. Degradation of IGFBP-5I likely occurs at the osteoblast surface through its interaction with the plasmin system (10, 10a, 12, 13, 24). IGFBP-5-(201---218) and plasmin-generated HE-binding fragments function to inhibit the activation of plasminogen to active plasmin, and IGFBP-5-(201---218) inhibits IGFBP-5 interaction with plasminogen (10a). The net effect of these processes is the downregulation of plasminogen-associated proteolysis, suggesting a possible regulatory role for plasminogen-produced IGFBP-5 degradation products. In contrast, the NH2-terminal fragment IGFBP-5-(1---169), presumably also a proteolytic product, does not affect plasmin proteolysis (10a) but does bind to the osteoblast surface (1) and stimulates mitogenic activity independent of IGF stimulation (3). The extent by which plasminogen-produced natural IGFBP-5 peptides impact IGF-I and osteoblast function is currently being investigated. Our preliminary evidence demonstrates that plasminogen-treated IGFBP-5-(201---218) retains its ability to enhance IGFBP-5I binding to IGF-I, suggesting that the IGFBP-5-(201---218) region of IGFBP-5 survives plasminogen proteolysis functionally intact. Thus proteolysis of IGFBP-5I may not only provide a mechanism to alter IGFBP-5 subsequent proteolytic events but may also enhance deposition of intact IGFBP-5 and IGF within mineralized bone.

    ACKNOWLEDGEMENTS

This work was supported by National Cancer Institute Grant 5R29-CA-54363, the Allegheny-Singer Research Institute, and the Research Service of the Department of Veterans Affairs.

    FOOTNOTES

A preliminary report of this work was presented at the 10th International Congress of Endocrinology, San Fransciso, CA, June 12-15, 1995.

Address for reprint requests: P. G. Campbell, Orthopaedic Research Laboratory, Dept. of Orthopaedic Surgery, 9th Floor South Tower, Allegheny Univ. of the Health Sciences, 320 E. North Ave., Pittsburgh, PA 15212-4756.

Received 26 March 1997; accepted in final form 23 July 1997.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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