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
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ABSTRACT |
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
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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS |
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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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.
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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. , IGFBP-5-(201 218); , IGFBP-5-(138 152);
, IGFBP-5-(130 142). Values are means of duplicate
determinations.
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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: ,
IGFBP-5I; , IGFBP-5-(1 169);
, IGFBP-5-(201 218); , IGFBP-5-(130 142);
,IGFBP-5-(138 152).
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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.
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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 ( ) and with ( ) 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, ) or HA (solid phase, ) 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.
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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.
 |
DISCUSSION |
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 |
1.
Andress, D. L.
Heparin modulates the binding of insulin-like growth factor (IGF) binding protein-5 to a membrane protein in osteoblastic cells.
J. Biol. Chem.
270:
28289-28296,
1995[Abstract/Free Full Text].
2.
Andress, D. L.
Comparison studies of IGFBP-5 binding to osteoblasts and osteoblast-derived extracellular matrix.
Prog. Growth Factor Res.
6:
337-344,
1995[Medline].
3.
Andress, D. L.,
and
R. S. Birnbaum.
Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action.
J. Biol. Chem.
267:
11467-11472,
1992.
4.
Andress, D. L.,
S. M. Loop,
J. Zapf,
and
M. C. Kiefer.
Carboxy-truncated insulin-like growth factor binding protein-5 stimulates mitogenesis in osteoblast-like cells.
Biochem. Biophys. Res. Commun.
195:
25-30,
1993[Medline].
5.
Arai, T.,
A. Arai,
W. H. Busby,
and
D. R. Clemmons.
Glycosaminoglycans inhibit degradation of insulin-like growth factor-binding protein-5.
Endocrinology
135:
2358-2363,
1994[Abstract].
6.
Arai, T.,
J. Clarke,
A. Parker,
W. Busby, Jr.,
T. Nam,
and
D. R. Clemmons.
Substitution of specific amino acids in insulin-like growth factor (IGF) binding protein-5 alters heparin binding and its change in affinity for IGF-I in response to heparin.
J. Biol. Chem.
271:
6099-6106,
1996[Abstract/Free Full Text].
7.
Bautista, C. M.,
D. L. Baylink,
and
S. Mohan.
Isolation of a novel insulin-like growth factor (IGF) binding protein from human bone: a potential candidate for fixing IGF-II in human bone.
Biochem. Biophys. Res. Commun.
176:
765-763,
1991.
8.
Bautista, C.,
S. Mohan,
and
D. J. Baylink.
Insulin-like growth factors I and II are present in the skeletal tissues of ten vertebrates.
Metabolism
39:
96-100,
1990[Medline].
9.
Booth, B. A.,
M. Boes,
D. L. Andress,
B. L. Dake,
M. C. Kiefer,
C. Maack,
R. J Linhardt,
K. Bar,
E. E. O. Caldwell,
J. Weiler,
and
R. S. Bar.
IGFBP-3 and IGFBP-5 association with endothelial cells: role of C-terminal heparin binding domain.
Growth Regul.
5:
1-7,
1995[Medline].
10.
Campbell, P. G.
Localization of the insulin-like growth factor (IGF) and plasmin systems in osteosarcoma cells: mechanism for the utilization of bone matrix IGFs (Abstract).
Mol. Biol. Cell.
4:
S1694,
1993.
10a.
Campbell, P. G.,
and
D. L. Andress.
Plasmin degradation of insulin-like growth factor-binding protein-5 (IGFBP-5): regulation by IGFBP-5-(201
218).
Am. J. Physiol.
273 (Endocrinol. Metab. 36):
E996-E1004,
1997[Abstract/Free Full Text].
11.
Campbell, P. G.,
J. F. Novak,
K. Wines,
and
P. E. Walton.
Localization of plasmin activity on osteosarcoma cells: cell surface protolysis of insulin-like growth factor binding proteins.
Growth Regul.
3:
95-98,
1993[Medline].
12.
Campbell, P. G.,
J. F. Novak,
T. B. Yanosick,
and
J. H. McMaster.
Involvement of the plasmin system in dissociation of the insulin-like growth factor-binding protein complex.
Endocrinology
130:
1401-1412,
1992[Abstract].
13.
Campbell, P. G.,
K. Wines,
T. B. Yanosick,
and
J. F. Novak.
Binding and activation of plasminogen on the surface of osteosarcoma cells.
J. Cell. Physiol.
159:
1-10,
1994[Medline].
14.
Canalis, E.,
T. McCarthy,
and
M. Centrella.
Isolation of growth factors from adult bovine bone.
Calcif. Tissue Int.
43:
346-351,
1988[Medline].
15.
Cole, D. E.,
and
D. A. Hanley.
Osteocalcin.
In: Bone Volume 3, Bone Matrix and Bone Specific Products, edited by B. K. Hall. Boca Raton, FL: CRC, 1991, p. 240-294.
16.
Frolick, C. A.,
L. F. Ellis,
and
D. C. Williams.
Isolation and characterization of insulin-like growth factor II from human bone.
Biochem. Biophys. Res. Commun.
151:
1001-1018,
1988.
17.
Gorbunoff, M. J.
Protein chromatography on hydroxyapatite columns.
Methods Enzymol.
182:
329-339,
1985.
18.
Hauschka, P. V.,
T. L. Chen,
and
A. E. Mavrakos.
Polypeptide growth factors in bone matrix.
Ciba Found. Symp.
136:
207-225,
1988[Medline].
19.
Jones, J. I.,
A. Gockerman,
W. H. Busby, Jr.,
C. Camacho-Hubner,
and
D. R. Clemmons.
Extracellular matrix contains insulin-like growth factor binding protein-5 potentiation of the effects of IGF-I.
J. Cell Biol.
121:
679-687,
1993[Abstract].
20.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
21.
McPherson, G. A.
KINETIC, EBDA, LIGAND, LOWRY: A Collection of Radioligand Binding Analysis Programs. Cambridge, UK: Elsevier-BIOSOFT, 1985.
22.
Mohan, S.,
and
D. J. Baylink.
The role of insulin-like growth factor-I in the coupling of bone formation to resorption.
In: Modern Concepts of Insulin-Like Growth Factors, edited by E. M. Spencer. New York: Elsevier Science, 1991, p. 169-184.
23.
Mohan, S.,
J. C. Jennings,
T. A. Linkhart,
and
D. J. Baylink.
Primary structure of human skeletal growth factor: sequence homology with human insulin-like growth factor-II.
Biochim. Biophys. Acta
966:
44-55,
1988[Medline].
24.
Nicolas, V.,
S. Mohan,
Y. Honda,
A. Prewett,
R. D. Finkelman,
D. J. Baylink,
and
J. R. Farley.
An age-related decrease in the concentration of insulin-like growth factor binding protein-5 in human cortical bone.
Calcif. Tissue Int.
57:
206-212,
1995[Medline].
25.
Oh, Y.,
H. Muller,
H. Pham,
and
R. G. Rosenfeld.
Demonstration of receptors for insulin-like growth factor binding protein-3 on Hs578T human breast cancer cells.
J. Biol. Chem.
268:
26045-26048,
1993[Abstract/Free Full Text].
26.
Wilkinson, L.
SYSTAT: The System for Statistics. Evanston, IL: SYSTAT, 1990.
27.
Yang, W.-H.,
M. Yanahgishita,
and
M. M. Rechler.
Heparin inhibition of insulin-like growth factor-binding protein-3 to human fibroblasts and rat glioma cells: role of heparan sulfate proteolglycans.
Endocrinology
137:
4363-4371,
1996[Abstract].
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