Plasmin degradation of insulin-like growth factor-binding
protein-5 (IGFBP-5): regulation by IGFBP-5-(201
218)
Phil G.
Campbell and
Dennis L.
Andress
Orthopaedic Research Laboratory, Allegheny University of the Health
Sciences, Pittsburgh, Pennsylvania 15212; and the Departments of
Medicine, Veterans Affairs Medical Center and University of
Washington, Seattle, Washington 98108
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ABSTRACT |
Using the major
bone insulin-like growth factor-binding protein (IGFBP) IGFBP-5, we
took a mechanistic approach in evaluating the role of the
heparin-binding domain of IGFBP-5 in regulating plasmin (Pm)
proteolysis of IGFBP-5. Using synthetic IGFBP-5 peptide fragments, we
determined that the heparin-binding domain, IGFBP-5-(208
218), inhibits Pm proteolysis of intact IGFBP-5. The mechanism of action of
IGFBP-5-(201
218) was by inhibiting Pm binding to substrate IGFBP-5.
IGFBP-5-(201
218) action was independent of site of proteolysis, fluid, or solid phase interaction. In addition, IGFBP-5-(201
218) was
found to inhibit plasminogen (Pg) activation to Pm. IGFBP-5-(201
218) did not directly inhibit the activity of Pm, urokinase Pg activator (PA), or tissue-type PA but acted as a competitive inhibitor of Pg
activation by PA, which is in contrast to the stimulating effect of
heparin on Pg activation. These data indicate that the heparin-binding domain contains the serine protease (Pg-to-Pm) binding site region of
IGFBP-5, and that this region, which is presumed to represent a
Pm-induced proteolytic product of IGFBP-5, is capable of regulating Pm
action.
osteoblast physiology; hydroxyapatite; insulin-like growth factor
bioavailability
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INTRODUCTION |
THE PLASMIN (Pm) system is involved in
normal bone remodeling through a variety of effects, including
initiating bone resorption through the activation of latent
collagenases (30) and through the activation of osteoblastic regulatory
peptides transforming growth factor-
(TGF-
) (21, 27) and
insulin-like growth factors I (IGF-I) and II (11). The effects of the
Pm system on osteoblast function are highly regulated and are localized
to the cell surface and underlying extracellular matrix (ECM) (8, 12).
Pm activation of IGFs comprises the proteolysis of specific IGF-binding
proteins (IGFBPs), thus affecting the bioactivity of IGFs (11). In
vivo, IGFs are complexed to IGFBPs in the circulation (15) and within
the osteoblastic pericellular environment (1, 2, 18). Pm preferentially
cleaves IGFBPs, dissociating IGFs and thus allowing IGFs to interact
with their specific cell surface receptors (11). In addition, IGFBPs
are known to both stimulate and inhibit IGF function in bone (2, 9,
23). For example, the major bone IGFBP, IGFBP-5, augments IGF function
in osteoblasts (2, 23) and targets IGFs to the osteoblastic cell
surface (1, 8), where an active Pm system mediates IGF access to the
IGF-I receptor (8, 12).
Specific regions within the IGFBP-5 molecule are likely to mediate
these various processes. The heparin-binding domain of IGFBP-5,
IGFBP-5-(201
218), modulates the binding of IGFBP-5 to cellular and
extracellular pericellular domains (7). The IGFBP-5-(1
169) fragment,
which contains only minor heparin-binding domain regions, modifies
osteoblast function independently of IGFs by directly stimulating
mitogenesis (3). This effect is most likely mediated through its
binding to a specific membrane protein (1). However, in either case,
endogenous glycosaminoglycan moities do not mediate pericellular
binding of IGFBP-5 (1, 7).
The same region of IGFBP-5, IGFBP-5-(201
218), has also been reported
to inhibit the proteolysis of intact IGFBP-5 in fibroblast-conditioned media (4, 25) and to inhibit metalloproteinase-dependent proteolysis of
IGFBP-4 in osteoblast-conditioned media (14). Moreover, heparin itself
is also known to inhibit IGFBP-5 proteolysis (4, 25). Whether heparin
or other glycosaminoglycans are involved in stabilizing
Pm-associated IGFBP-5 proteolytic events in the osteoblastic
pericellular environment is unknown. The role of the IGFBP-5-(201
218)
domain in the proteolysis of IGFBP-5 by Pm remains to be determined.
The purpose of the present study was to evaluate the potential effect
of the heparin-binding synthetic peptide IGFBP-5-(201
218) on
Pm-mediated IGFBP-5 proteolysis, including direct inhibition of Pm
action and modification of the activation of the zymogen plasminogen
(Pg) to active Pm.
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EXPERIMENTAL PROCEDURES |
Materials.
Recombinant human IGF-I was purchased from GroPep, (Adelaide,
Australia). Recombinant intact human 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). Glucose Pm was purified from fresh frozen
plasma using lysine-Sepharose in the presence of 1 mM benzamidine, 50 µg/ml trypsin inhibitor, and 20 kallikrein-inhibitor units/ml
aprotinin (12). Human Pm and tissue-type Pg activator (tPA) was
purchased from American Diagnostica (Greenwich, CT). Human single-chain
urokinase PA (scuPA) was the generous gift of Dr. A. Mazar (Abbott
Laboratories, Abbott Park, IL). scuPA was activated by Pm at a ratio of
20:1 (scuPA/Pm) for 30 min at 23°C. Pm was removed from activated
urokinase PA (uPA) by aprotinin-Sepharose. The IGFBP-5 peptides
IGFBP-5-(201
218), IGFBP-5-(138
152), and IGFBP-5-(130
142) were synthesized and purified by RP-HPLC. IGF-I, IGFBP-5I, and IGFBP-5-(201
218)
were iodinated by the chloramine T method to a specific activity of
~150 µCi/µg protein (11).
Charcoal IGFBP assay.
Pm, at 50 ng/ml, was incubated in 50 mM tris(hydroxymethyl)aminomethane
(Tris) and 2.5% bovine serum albumin (BSA), pH 7.4, with 6.25 ng/ml
IGFBP-5 in the presence of IGFBP-5 peptides
[IGFBP-5-(201
218), IGFBP-5-(130
142), or
IGFBP-5-(138
152)], as indicated in Figs. 1-10. After a 1-h
37°C incubation in a total reaction volume of 145 µl, proteolytic
reactions were terminated by the addition of 5 µg/5 µl aprotinin.
125I-labeled IGF-I was then added,
and incubations were continued in a total volume of 200 µl for 1 h at
37°C. 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. Reactants (600 µl total
volume) were vortexed and incubated in a 4°C icebath 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. Binding of
125I-IGF-I to
IGFBP-5I in the absence of Pm
represented control binding. Nonspecific binding was determined by the
inclusion of 100 ng of unlabeled IGF-I to non-Pm-treated
IGFBP-5I and
125I-IGF-I, and all binding
results were corrected for this value. Results are presented as
percentages of nonplasmin control.
Heparin bead and heparin-Sepharose IGFBP assays.
Stock slurry solutions of 10% hydroxyapatite (HA) (Calbiochem, La
Jolla, CA) and 1:5 heparin-Sepharose beads (Pharmacia Bioteck, Piscataway, NJ) were maintained at 4°C in 30 mM Tris-acetate, 10 mM
sodium phosphate, 0.05% Tween 20, and 0.2% sodium azide, pH 7.4 (assay buffer). For an assay, beads were suspended by gently swirling,
and 50 µl of the 10% HA bead slurry or 10 µl of the 1:5
heparin-Sepharose were placed in 1.5-ml screw-cap plastic Eppendorf
tubes and equilibrated with
125I-IGFBP-5I
[~100,000 counts/min (cpm)] in a total volume of 200 µl
for 30 min at 23°C, with occasional vortexing. Unbound
125I-IGFBP-5I
was removed by washing the bead pellet twice with 1 ml assay buffer. Pm
(250 ng/ml) and IGFBP-5-(201
218) reactants (10 µg/ml) were
incubated with immobilized
125I-IGFBP-5I
in a total volume of 200 µl at 37°C with occasional vortexing. At
30- and 60-min intervals, tubes were centrifuged (10,000 g for 30 s), and 25-µl aliquots of
supernatants were used to determine released radioactivity.
Radioactivity released from beads was corrected for total incubation
volume (200 µl) and is presented as the percentage of total IGFBP-5
originally bound to beads (%total IGFBP bound) or percentage of
Pm-mediated release (%Pm control).
Peptide protease fingerprinting assay.
Protease fingerprinting assays were carried out as previously described
(10), with the primary exception that the reaction buffer was changed
to 30 mM Tris acetate, 10 mM sodium phosphate, 0.1% Tween 20, and
0.2% sodium azide, pH 7.4 (assay buffer). For the assays,
125I-IGFBP-5I
(100,000 cpm), Pm, IGFBP-5 peptides, and other reactants were incubated
(as described in Figs. 1-10) in 50 µl assay buffer for 1 h at
37°C. Reactions were terminated by the addition of 1 µg
aprotinin, followed by 25 µl 3× nonreducing sample buffer and
boiling for 3 min. Reactants were eluted through a 17.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the
gel was dried and autoradiographed.
Assessment of Pm proteolysis by ligand blot assay.
Experiments to assess Pm treatment of IGFBP-5 and the effect on IGFBP-5
function as determined by ligand blot assay were performed as
previously described (11).
IGFBP-5I (100 ng/ml) was reacted with increasing concentrations of Pm in the presence or absence of 10 µg/ml IGFBP-5-(201
218) in 1.5-ml Eppendorf plastic screw-cap tubes
in 50 µl of 30 mM Tris acetate, 10 mM sodium phosphate, 0.1% Tween
20, and 0.2% sodium azide, pH 7.4. Incubations were terminated after 1 h at 37°C with the addition of 10 µg aprotinin. Twenty-five
microliters of 3× nonreducing SDS-PAGE sample buffer were added
and samples were boiled for 3 min. Reactants were eluted through an
11% SDS-PAGE and electroblotted onto Immobilon-P (Millipore, Bedford,
MA). Blots were blocked with 3% BSA in Tris-buffered saline and
equilibrated with 5 × 106
cpm 125I-IGF-I overnight at
4°C. Unbound 125I-IGF-I was
removed by extensive washing, and the blot was air dried and
autoradiographed.
Plate binding assays.
Binding of IGFBP-5 peptides to Pg was characterized using an
immobilized Pg-based assay system. Ninety-six-well immunological plates
(NUNC, Fisher Scientific, Pittsburgh, PA) were coated with 5 µg/ml of
Pg in 0.1 M
Na2CO3,
pH 9.8, 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% sodium azide, pH 7.5, for 1 h at 37°C. Plates were rinsed twice with 200 µl PBS and once with
200 µl of 30 mM Tris acetate, 10 mM sodium phosphate, 0.1% Tween 20, and 0.2% sodium azide, pH 7.4 (assay buffer).
125I-IGFBP-5I
(100,000 cpm) or
125I-IGFBP-5-(201
218) (20,000 cpm) was incubated with increasing concentrations of
IGFBP-5-(201
218), IGFBP-5-(130
142), IGFBP-5-(138
152), or heparin
in 200 µl assay buffer for 4 h at 4°C. Wells were rinsed 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.
Amidolytic assays.
The effect of IGFBP-5 peptides on Pg activation to Pm was determined
using Pm chromogenic substrate S-2551 (Pharmacia). IGFBP-5-(201
218) (5-40 µg/ml), IGFBP-5-(130
142) (20 µg/ml),
IGFBP-5-(138
152) (20 µg/ml), or heparin (500 nM) was preincubated
with 25-400 nM Pg (final concentration) for 3 min at 37°C in
the presence of 0.2 mM S-2551 (final concentration). Assay buffer was
30 mM Tris acetate, 10 mM sodium phosphate, 0.1% Tween 20, and 0.2%
sodium azide, pH 7.4. After preincubation, 5 nM of uPA was added to
initiate Pg activation, and the absorbance change at 405 nm was
monitored every 10 s for 5 min at 37°C by use of a Molecular
Devices ThermoMax microplate reader (Menlo Park, CA). The initial
reaction velocites were determined and, where noted (see Figs. 7 and
8), Lineweaver-Burk plots were calculated.
Natural IGFBP-5 Pm-induced IGFBP-5 fragments were also tested for their
ability to inhibit Pg activation to Pm.
IGFBP-5I (5 µg) was digested for
30 min at 37°C with 100 nM Pm. Pm was removed by
ultrafiltration (30,000 molecular-weight cutoff filter), and heparin-binding fragments of IGFBP-5 were bound to
heparin-Sepharose and eluted by a two-step elution with 0.3 M and 1 M
NaCl. NaCl was removed by trichloroacetic acid (TCA) precipitation. The
TCA pellet was rinsed twice with Milli-Q filtered water, solubilized in
100 µl of assay buffer, and 20-µl aliquots were reacted with 100-400 nM Pg and 5 nM uPA. A mock digestion excluding the
addition of IGFBP-5I was also
performed to confirm that no artifacts were introduced during the
IGFBP-5 fragment preparation process.
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RESULTS |
Pm degrades IGFBP-5 into several smaller fragments (Fig.
1A,
lane 2); IGF-I bound to IGFBP-5 does
not inhibit the Pm effect (lane 3).
Heparin prevented Pm degradation of IGFBP-5 (Fig.
1B), whereas IGFBP-5-(1
169) did
not. This suggested that heparin may inhibit proteolysis by its
interaction with the carboxy-terminal portion of IGFBP-5. Because
heparin binds predominantly to the 201-218 amino acid region of
IGFBP-5 (5, 16), we coincubated this peptide with Pm and found that it
was capable of inhibiting Pm-induced proteolysis. This response was
specific because other basic peptides within the IGFBP-5 molecule
[IGFBP-5-(130
132) and IGFBP-5-(138
152)] failed to alter
the Pm response.

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Fig. 1.
Plasmin degradation of
125I-labeled intact human
insulin-like growth factor-binding protein-5
(125I-IGFBP-5I) and
effect of insulin-like growth factor I (IGF-I), heparin, and IGFBP-5
peptides. A: plasmin degradation
of complexed and uncomplexed
125I-IGFBP-5I.
125I-IGFBP-5I
[100,000 counts/min (cpm)] was incubated with or
without 10 ng of IGF-I for 1 h at 37°C to form
IGF-I-complexed IGFBP-5 and uncomplexed IGFBP-5. Plasmin (Pm,
2 µg/ml) was added, and reactants were incubated for 1 h at 37°C
in a total volume of 50 µl. Incubations were terminated by the
addition of 25 µl 3× SDS-polyacrylamide gel electrophoresis
(PAGE) nonreducing sample buffer, boiled 3 min, and eluted over 17.5%
SDS-PAGE. Gel was dried and autoradiographed. Lane 1,
125IGFBP-5I alone; lane
2, 125I-IGFBP-5I + Pm; lane 3,
125I-IGFBP-5I/IGF-I
complex + Pm. B: protection of
125I-IGFBP-5I
from Pm degradation by heparin and IGFBP-5 peptides.
125I-IGFBP-5I
(100,000 cpm) was reacted with 50 ng/ml Pm, as indicated, for 1 h at
37°C. Pm proteolysis was terminated by the addition of 1 µg aprotinin. Reactants were separated by 17.5% SDS-PAGE.
Various factors were coreacted with Pm, as indicated: heparin, 1 mg/ml;
IGFBP-5-(130 142), 10 µg/ml; IGFBP-5-(138 142), 10 µg/ml;
IGFBP-5-(201 218), 10 µg/ml; or IGFBP-5-(1 169), 1 µg/ml.
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To quantitate the inhibitory effect of IGFBP-5-(201
218), we utilized
an IGF-I charcoal-binding assay and found that increasing concentrations of Pm resulted in a concentration-dependent loss of
IGF-I-binding activity, with a half-maximally effective concentration (EC50) of 20 ng/ml (Fig.
2A). The
presence of 10 µg/ml IGFBP-5-(201
218) resulted in a shift of the
EC50 to 100 ng/ml. The protective
effect of the peptide was concentration dependent, with maximal
protection achieved at 10 µg/ml IGFBP-5-(201
218) (Fig.
2B). The protective nature of
IGFBP-5-(201
218) was specific, because two other basic IGFBP-5
fragments, IGFBP-5-(138
152) and IGFBP-5-(130
142), failed to protect
IGFBP-5 from Pm proteolysis (Fig. 3).
Similar inhibitory results were observed with
125I-IGFBP-5 peptide mapping
assays (Fig. 4). Although the peptide was
protective under all conditions tested, the inhibition of proteolysis
could be overcome with higher concentrations of Pm, suggesting that the
peptide was acting as a competitive inhibitor of Pm action (Fig. 4).
Additionally, ligand blot analysis confirmed that the protective
effects of IGFBP-5-(201
218) were specifically affecting IGFBP-5 and
its ability to bind IGF-I (Fig. 5).

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Fig. 2.
IGFBP-5-(201 218) protects the IGF-I-binding ability of
IGFBP-5I from Pm degradation.
A: increasing concentrations of Pm
were incubated with 6.25 ng/ml
IGFBP-5I for 1 h at 37°C in
the presence or absence of 10 µg/ml IGFBP-5-(201 218).
125I-IGF-I/IGFBP-5I
complexes were determined by charcoal IGFBP assay. Values are means ± SE of 3 separate experiments performed in duplicate.
B: Pm at 50 ng/ml was incubated with
IGFBP-5 with increasing concentrations of IGFBP-5-(201 218) for 1 h at
37°C.
125I-IGF-I/IGFBP-5I
complexes were formed and determined as described above.
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Fig. 3.
Protection of IGFBP-5I from Pm
proteolysis is specific for IGFBP-5-(201 218). Pm, at 50 ng/ml, was
incubated with 6.25 ng/ml IGFBP-5I
in the absence or presence of IGFBP-5-(201 218), IGFBP-5-(130 142),
or IGFBP-5-(138 152). Incubations were for 1 h at 37°C and were
terminated by addition of 5 µg aprotinin.
125I-IGF-I was then added, and
incubations were continued for an additional 1 h at 37°C.
125I-IGF-I/IGFBP-5I
complexes were determined by charcoal IGFBP assay. Bars, means ± SE
of triplicate determinations.
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Fig. 4.
IGFBP-5-(201 218) protection of
125I-IGFBP-5I
from Pm: effect of increasing Pm concentration.
125I-IGFBP-5I
was incubated with increasing concentrations of Pm, with and without 10 µg/ml IGFBP-5-(201 218), for 1 h at 37°C in a total volume of 50 µl. Incubations were terminated by addition of 1 µg aprotinin.
Proteolysis was assessed by protease fingerprinting assay, as described
in EXPERIMENTAL PROCEDURES.
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Fig. 5.
Ligand blot analysis of IGFBP-5-(201 218) protection of
IGFBP-5I proteolysis.
IGFBP-5I (100 ng/ml) was reacted
with increasing concentrations of Pm in the presence or absence of 10 µg/ml IGFBP-5-(201 218). Incubations were for 1 h at 37°C. Pm
proteolysis was terminated by addition of 1 µg aprotinin. Reactants
were run over 11% SDS-PAGE under nonreducing conditions. Ligand blot
was performed with 125I-IGF-I
followed by autoradiography.
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We next examined whether a protective effect on Pm proteolysis could be
seen when intact IGFBP-5 was immobilized to a solid support. This is
physiologically relevant because IGFBP-5 is stored in bone matrix (6,
26). As shown in Table 1,
125I-IGFBP-5 that was bound to HA
(paradigm for the inorganic bone matrix) or to heparin-Sepharose beads
(paradigm for the organic bone matrix) could be proteolyzed by Pm, and
the degradation was partially inhibited by IGFBP-5-(201
218). Within a
given bead type (HA or heparin-Sepharose), IGFBP-5-(201
218) inhibited
Pm-induced release of IGFBP-5 radioactivity at each time point
(P < 0.002; Student's
t-test).
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Table 1.
IGFBP-5-(201 218) inhibits plasmin-mediated proteolysis of
125I-IGFBP-5I bound to HA beads and
heparin-Sepharose
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To further assess the mechanism of the IGFBP-5-(201
218) inhibitory
effect, we considered whether IGFBP-5-(201
218) interfered with the
binding to IGFBP-5 substrate to Pm. IGFBP-5-(201
218) effectively
inhibited the binding of
125I-IGFBP-5 to Pg with a
half-maximally inhibiting concentration (IC50) of 100 ng/ml (Fig.
6). In contrast, IGFBP-5-(130
142)
exhibited minimal competition, and IGFBP-5-(138
152) was slightly more
inhibitory with an IC50 of 20 µg/ml. Heparin inhibited the binding of
125I-IGFBP-5I
to Pg at an IC50 of 1 µg/ml
(data not shown). The heparin concentration required to inhibit IGFBP-5
binding may reflect the presence of two low-affinity heparin-binding
sites on IGFBP-5 in addition to the high-affinity heparin-binding site
at residues 205-212 (16). The question remained, Did
IGFBP-5-(201
218) directly bind to Pg, thus directly competing for
IGFBP-5 binding to Pg? To access this we used an assay that quantitates
the extent of 125I-IGFBP-5-(201
218) binding to
Pg. We found that saturable binding of the IGFBP-5-(201
218) to Pg
occurred in a specific manner with an
IC50 of 50 ng/ml (data not shown).
IGFBP-5-(130
142) did not compete for
125I-IGFBP-5-(201
218) binding to
Pg, and IGFBP-5-(138
152) was minimally effective as a competitor,
exhibiting only 40% inhibition of binding at 20 µg/ml. Heparin
exhibited a biphasic response, with <500 ng/ml heparin increasing
125I-IGFBP-5-(201
218) binding to
Pg (maximum of 155% of control) and >500 ng/ml heparin decreasing
125I-IGFBP-5-(201
218) binding
with an IC50 of 60 µg/ml (data
not shown). On the basis of these data, one possible mechanism whereby IGFBP-5-(201
218) inhibits the Pm-induced proteolysis of intact IGFBP-5 is by directly competing for binding to Pm, thus interfering with the binding of Pm to substrate IGFBP-5.

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Fig. 6.
IGFBP-5-(201 218) inhibits binding of
125I-IGFBP-5I
to plasminogen (Pg). Plates were coated with 5 µg/ml Pg overnight at
4°C.
125I-IGFBP-5I
(100,000 cpm/well) were incubated in the presence of increasing
concentrations of IGFBP-5-(201 218), IGFBP-5-(130 142), and
IGFBP-5-(138 152). Bovine serum albumin (BSA)-coated wells were used
to determine nonspecific binding. Incubations were in 200 µl assay
buffer for 4 h at 4°C. Unbound
125I-IGFBP-5I
was removed by rinsing the wells twice with ice-cold assay buffer.
Values are means of duplicate determinations expressed as a ratio of
bound radioactivity in the presence of competitor over the bound
radioactivity in the absence of any competitor (B/Bo)
125I-IGFBP-5I.
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Another possible mechanism whereby fragments of IGFBP-5 can affect Pm
proteolysis is through the activation of Pg to proteolytic active Pm by
PA. To evaluate this possibility, IGFBP-5-(201
218), IGBP-5-(130
142), and IGFBP-5-(138
142) were tested for their ability
to influence Pg activation by uPA by use of an amidolytic assay that measures Pm proteolysis. Only IGFBP-5-(201
218) reduced the
activation of Pg by uPA (Fig. 7).
To evaluate activation efficiency, Lineweaver-Burk plots were
determined for IGFBP-5-(201
218) and compared with those generated by
heparin, a known stimulator of Pg activation. As shown in Fig.
8, IGFBP-5-(201
218) decreased Pg
activation efficiency by more than twofold, whereas heparin increased
efficiency by more than threefold. IGFBP-5-(201
218) was also found to
inhibit tPA activation of Pg, similarly to uPA (data not shown).
IGFBP-5-(201
218) did not affect the specific amidolytic activities of
Pm, uPA, or tPA (data not shown), confirming specificity of
IGFBP-5-(201
218) to actual activation of Pg. Natural Pm fragments of
IGFBP-5 were also tested to confirm that Pm degradation products of
IGFBP-5 were capable of affecting the activation of Pg to Pm.
Pm-induced fragments of IGFBP-5 were bound to heparin-Sepharose, and
heparin-binding fragments eluting >300 mM NaCl were collected, precipitated to remove excess NaCl, and found to reduce the activation efficiency of Pg by uPA (Fig. 9). To
exclude the possibility that substances other than IGFBP-5 fragments
might be affecting Pg activation, mock protease digestions without
IGFBP-5 were performed and found not to change control activation (data
not shown).

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Fig. 7.
IGFBP-5-(201 218) inhibits urokinase Pg activator (uPA) activation of
Pg. Increasing concentrations of IGFBP-5-(201 218) ( ), 20 µg/ml
IGFBP-5-(130 142) ( ), or 20 µg/ml IGFBP-5-(138 152) ( ) were
preincubated with 200 nM Pg and 0.2 mM S-2551 for 3 min at 37°C.
Amidolytic activities were determined on addition of 5 nM uPA for 5 min
at 37°C. Initial reaction velocities
(V) were determined as described in
EXPERIMENTAL PROCEDURES.
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Fig. 8.
Activation efficiency of Pg by uPA: contrasting effects of
IGFBP-5-(201 218) and heparin. IGFBP-5-(201 218) (40 µg/ml,
A) or heparin (500 nM,
B) was preincubated with increasing
concentrations of Pg (25-400 nM) and S-2551 for 3 min at 37°C.
uPA (5 nM) was added to initiate catalytic reactions. Initial reaction
V values were determined, and results
are presented as Lineweaver-Burk plots. Symbols are means of 2 separate
experiments.
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Fig. 9.
Pm-generated IGFBP-5 heparin-binding fragment(s) inhibit activation of
Pg by uPA. Five micrograms of
IGFBP-5I were digested with 100 nM
Pm for 30 min at 37°C. Pm was removed by ultrafiltration (30,000 molecular-weight cutoff filter), and heparin-binding fragments of
IGFBP-5 were collected by heparin-Sepharose chromatography and
trichloroacetic acid (TCA) precipitation, as described in
EXPERIMENTAL PROCEDURES. TCA pellet
was solubilized in 100 µl of assay buffer, and 20-µl aliquots were
reacted with 100-400 nM Pg and 5 nM uPA to determine amidolytic
activity.
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DISCUSSION |
Pm activation at the osteoblast surface has the potential for
regulating growth factor responses by an action that involves its
separation from a carrier protein. Both TGF-
and the IGFs, which are
dependent on carrier protein interactions, become activated after Pm
proteolysis (11, 21, 27). In the case of the IGF system, IGFBP-5/IGF-I
complexes appear to be susceptible to the serine protease action of Pm
in a predictable fashion. For example, we found that IGF-I,
when bound to IGFBP-5I, does not
prevent Pm proteolysis of IGFBP-5 and that proteolytic
fragments of IGFBP-5 do not bind to IGF-I. Thus IGF-I should be free to
interact with surface receptors without interference from IGFBP-5
degradation products. We have previously demonstrated that cell surface
generation of Pm on osteoblastic cells results in proteolysis of
endogenous IGFBP species as well as specific IGFBP species IGFBP-1
(11), IGFBP-3 (10), and IGFBP-4 (10). As is demonstrated by
Pm-proteolyzed IGFBP-1 (11), proteolytically released IGF-I retains its
full biological potential. Using
U2OS osteoblastic cells as a
source of IGFBP-5, we demonstrated that cell surface-generated Pm
activity will reduce IGFBP-5 in the conditioned media by 78.6% (data
not shown). These data support a role for Pm in the regulation of IGFBP-5 function in the osteoblastic pericellular environment.
The interest that specific fragments of IGFBP-5 may possess biological
activity relates to previous work showing that truncated forms of
NH2-terminal (1, 3) and
COOH-terminal (25) peptides stimulate osteoblast activity (3) and
inhibit generalized IGFBP-5 proteolysis (25), respectively. Peptides
responsible for the latter effect have been shown to bind heparin while
lacking IGF-binding capability (1). In the current report, we have
extended the study of heparin-binding peptides of IGFBP-5 to evaluate
whether they affect the Pm system itself, assuming that if an effect
were present it would likely be inhibitory in nature. Our results, which confirm this hypothesis, show that the heparin-binding synthetic peptide IGFBP-5-(201
218) modifies Pm proteolysis of intact IGFBP-5 by
at least two independent mechanisms:
1) blocking the interaction of Pm
with IGFBP-5 and 2) inhibiting Pg
activation to Pm.
The dichotomy of the heparin effects in regulating Pm proteolysis of
IGFBP-5 illustrates the complexity of the regulatory mechanisms. The
ability of heparin and IGFBP-5-(201
218), but not
IGFBP-5-(1
169), IGFBP- 5-(130
142), or
IGFBP-5-(138
152), to block Pm proteolysis of IGFBP-5 suggests that at
least one important cleavage site may reside within the
carboxy-terminal region of the IGFBP-5 molecule. Whether the heparin
inhibitory action occurs by binding to and blocking the
IGFBP-5-(201
218) region or whether heparin induces a conformational
change in the tertiary structure of IGFBP-5 is not apparent from these
studies. However, because solid-phase heparin does not prevent Pm
proteolysis of IGFBP-5, it is likely that the proteolytic inhibitory
effect of heparin results from inhibition of Pm binding to IGFBP-5, as the ability of heparin to inhibit IGFBP-5 binding to Pg suggests. This
concept is further supported by our findings and those of others (20,
28) demonstrating that heparin stimulates the activation of Pg to Pm by
PAs (Fig. 8B).

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|
Fig. 10.
Model summarizing immediate effects of heparin and IGFBP-5-(201 218)
on Pm proteolysis of IGFBP-5. Arrows from heparin and
IGFBP-5-(201 218) to Pg or Pm denote direct binding interaction with
the respective zymogen or protease. +, Stimulation of activity;
, inhibition of activity.
|
|
The synthetic peptide IGFBP-5-(201
218) contains binding sites for
heparin and Pg, localizing the regulation of Pm proteolysis to
the carboxyl portion of IGFBP-5. Consistent with this
notion, we found that IGFBP-5-(201
218) binds directly to Pg and
blocks the binding of IGFBP-5 to Pg. This suggests that
IGFBP-(201
218) inhibits Pm proteolysis of IGFBP-5 by blocking the
interaction of Pm with IGFBP-5. This may occur through
IGFBP-5-(201
218) binding to Pm and directly blocking the Pm binding
site and/or through IGFBP-5-(201
218) binding to IGFBP-5 and
blocking the cleavage site for Pm. Additional new evidence now
indicates that IGFBP-5-(201
218) does bind to intact IGFBP-5 (8a).
Whether the binding sequences within IGFBP-5-(201
218) are the same
for heparin, Pg, and IGFBP-5 is not clear at this time. A recent report
by Arai et al. (5) indicated that Arg-201, Lys-202,
Lys-206, and Arg-214 are involved in heparin binding to
IGFBP-5, suggesting the likelihood of a definite binding site
overlap among heparin, Pg, and IGFBP-5 within the IGFBP-5-(201
218)
sequence. This would be similar to shared binding domains for Pg, tPA,
heparin, and collagen/gelatin, which are also present in two other
Pm-labile proteins, laminin and fibronectin (17, 22).
Although IGFBP-5-(201
218) clearly interferes with the Pm interaction
with IGFBP-5, it also inhibits Pg activation to Pm. The mechanism
appears to be mediated through decreased Pg activation efficiency. This
is in contrast to the effects of heparin, which increased Pg activation
efficiency. Peptide fragments of various Pm substrate proteins are
known to modify the activation of Pg to Pm (19, 20, 24, 28, 31).
Whereas fragments of fibrin (20, 31) and laminin (19) stimulate the
activation of Pg, vitronectin (19) inhibits Pg activation. Similar to
the effects of IGFBP-5-(201
218) reported here, the heparin-binding
domain of vitronectin (29) is the responsible element inhibiting Pg activation. Interestingly, peptide sequences within the heparin-binding domain of fibronectin can exhibit both inhibitory and stimulatory effects on Pg activation (24, 28). Because such bioactive fragments can
be included in proteolytic products, including IGFBP-5 proteolytic
products, these ECM-derived peptide fragments have the capacity to
modify subsequent proteolysis, both as an autocrine loop for a specific
protein substrate and as a general proteolytic capacity of the Pm
system.
The effects of heparin and IGFBP-5-(201
218) on Pg activation and Pm
degradation of IGFBP-5 are summarized in Fig. 10. The activation of Pg
to Pm by PA is differentially regulated by heparin and
IGFBP-5-(201
218), whereas both heparin and IGFBP-5-(201
218) inhibit
Pm proteolysis of IGFBP-5. The actions of heparin and IGFBP-5-(201
218) appear to require direct binding to Pg and Pm. This
direct binding results in a conformational change in Pg that alters its
activation efficiency and competitively blocks the binding of IGFBP-5
to Pm, which inhibits IGFBP-5 proteolysis.
In the osteoblastic pericellular environment, Pm regulation of IGF-I
interaction with IGFBP-5 is likely localized to the cell surface
interface with the fluid and ECM solid phases (1, 8, 10, 12), where Pm
is protected from abundant fluid phase protease inhibitors. IGF-I
inhibits uPA synthesis by the osteoblast (13) and reduces the binding
affinity of the Pg/Pm receptor (12). IGFBP-5-(201
218) downregulates
Pm-associated proteolysis by reducing the activation efficiency of Pg
and by blocking the interaction of Pm with IGFBP-5. The extent to which
feedback control of Pm activation occurs with natural IGFBP-5
peptides that are produced by Pm degradation is currently being
investigated.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Thomas Yanosick for technical assistance
and Dr. Andrew Mazar of Abbott Laboratories for the gift of scuPA.
 |
FOOTNOTES |
This work was supported by National Institutes of Health National
Cancer Institute Grant 5R29-CA-54363 and the Research Service of the
Department of Veterans Affairs (Seattle, WA).
A preliminary report of this work was presented at the 17th Annual
Meeting of the American Society for Bone and Mineral Research, Baltimore, MD, September 9-13, 1995.
Address for reprint requests: P. G. Campbell, Dept. of Orthopaedic
Surgery, Orthopaedic Research Laboratory, 9th Floor, South Tower,
Allegheny Univ. of the Health Sciences, 320 E. North Ave., Pittsburgh,
PA 15212.
Received 28 March 1997; accepted in final form 22 July 1997.
 |
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