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Department of Medicine, University of North Carolina School of
Medicine, Chapel Hill, North Carolina 27599
Department of Medicine, University of North Carolina School of
Medicine, Chapel Hill, North Carolina 27599
Address all correspondence and requests for reprints to: David R. Clemmons, M.D., Division of Endocrinology, Department of Medicine CB 7170, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7170.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that IGFBP-5 binds to heparin and heparan sulfate (4, 9). Similarly, glycosaminoglycan containing proteoglycans, such as tenascin, that are present in the ECM also bind IGFBP-5 (9). This binding occurs through highly charged residues in IGFBP-5 that are located between residues 201 and 218 (4, 9, 10). Similar to ECM binding, glycosaminoglycan binding of IGFBP-5 also results in a reduction in the affinity of IGFBP-5 for IGF-I (4) and in protection from proteolysis (8). However, competitive binding studies using intact ECM have shown that all of the ECM binding of IGFBP-5 could not be accounted for by binding to proteoglycans (5). Therefore, these studies were undertaken to determine if extracellular matrix proteins other than proteoglycans might bind IGFBP-5.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionMaterials and MethodsResultsDiscussionReferences |
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Purification of PAI-I
Human dermal fibroblasts (GM-10) were purchased from Coriell
Institute (Camden, NJ). They were grown in EMEM supplemented with 10%
calf serum as described previously (7). Confluent monolayers were
washed twice in the serum-free medium, then 50 cc was added to
175-cm2 flasks (Falcon Labware, Division of
Becton-Dickinson), and the medium was collected after 24 h. This
was repeated until 1.2 liters of serum-free medium had been collected.
After purification by heparin sepharose affinity chromatography and
1 antichymotryptic peptide affinity chromatography as
described previously (12), the fractions containing IGFBP-5 protease
activity were applied to an IGFBP-5 affinity column. This column was
prepared by conjugating a synthetic 18 amino acid peptide that
contained the IGFBP-5 sequence between amino acids 201–218 to Affigel
10 (Bio-Rad Richmond, CA). This sequence is also present in IGFBP-3,
but it is not present in any other form of IGFBP. The peptide was
conjugated according to manufacturer’s instructions by incubating 5.6
mg of peptide with 7.0 cc of activated sepharose in 0.1 M
Na HEPES buffer, pH 7.5, for 4 h at 8 C. Unreacted sites were
blocked by a further incubation for 1 h with 0.25 M
Tris. The gel was washed, and the pool of activity that had been
purified from fibroblast conditioned medium was applied. The purified
active fractions that were eluted with 0.5 M NaCl were
concentrated and further analyzed by SDS-PAGE (12.5% gel). After
electrophoresis, the proteins were transferred to a nitrocellulose
membrane that was stained with Ponceau-S. A 55-kDa band was excised and
subjected to amino acid sequencing. Amino terminal amino acid
sequencing was conducted as described previously using Edman
degradation (13).
Preparation of human IGFBP-5
The human IGFBP-5 that was used in these studies was obtained by
purifying the protein from Chinese hamster ovary (CHO) cell conditioned
medium. CHO cells were transfected with an IGFBP-5 complementary DNA
(cDNA) that contained the entire protein coding region and had been
prepared as previously described (7). The cells were transfected, then
maintained in methotrexate (50 µg/ml) because the plasmid that was
used (pNUT) contains the gene that contains dihydrofolate reductase
resistance. After selection with methotrexate, 50 µg/ml, the highest
secreting clones were further subcultured in DMEM containing 10%
dialyzed FCS. The cells were grown to confluency and 50 ml of
serum-free medium were added to 175-cm tissue culture flasks and
incubated for 48 h. Eight hundred cubic centimeters of conditioned
medium were purified using a three-step purification procedure of
phenyl sepharose chromatography, IGF-I affinity chromatography, and
HPLC (C4 column) (Vydac, Hesperia, CA), as previously described (10).
The purified protein was homogenous by SDS-PAGE with silver staining
(14). The protein content was determined by amino acid composition
analysis (7).
Coimmunoprecipitation, ligand blotting, and immunoblotting
The ability of IGFBP-5 to bind to the plasminogen activator
inhibitor-1 was analyzed by coimmunoprecipitation. It was determined
that the lowest nonspecific binding and the highest sensitivity of
detection could be obtained by immunoprecipitating with anti-PAI-1
antisera. The IGFBP-5 that had been coimmunoprecipitated was then
detected by immunoblotting (5) or by ligand blotting using
125I-IGF-I (15). PAI-1 and polyclonal anti-PAI-1 antiserum
were obtained from American Diagnostica (Greenwich, CT). In most
experiments, IGFBP-5 (100 ng/ml) was incubated with PAI-1 at 100 ng/ml,
and anti-PAI-1 was added at a 1:5000 dilution. The reagents were
allowed to incubate for 16 h at 4 C in 0.03 M sodium
phosphate, pH 7.4, containing 0.01 M EDTA, 0.1% Tween 80,
and 0.02% BSA. Protein A sepharose (3 mg) was added and incubated for
2 h at 4 C. The immune complexes were precipitated by centrifuging
at 3000 x g for 5 min. The pellets were rinsed with
1.0 ml of the buffer listed previously and 50 µl of 2x Laemmli
sample buffer was added. The sample was centrifuged (2000 x
g for 5 min) and the supernatant loaded onto a 12.5% SDS
polyacrylamide gel. The proteins were electrophoresed through a 12.5%
gel using nonreducing conditions, then transferred to Immobilon
membranes (Millipore, Bedford, MA) as previously described (15). After
transfer, the filters were immunoblotted using a 1:1000 dilution of
anti-IGFBP-5 antiserum. The immune complexes were detected using the
Protoblot system (Promega, Madison, WI) according to manufacturer’s
recommendation. The electrophoretic mobilities of the bands that were
detected were compared with known mol wt standards (Bethesda Research
Laboratories, Gaithersburg, MD). The photographic negatives were
scanned using a Hoffer GS-300 scanner (Hoffer Scientific Instruments,
San Francisco, CA), and the results are expressed in scanning units.
For more sensitive detection of IGFBP-5, ligand blotting was performed.
Five hundred thousand counts per minute of 125I-IGF-I [150
µCi/µg (16)] was added to the filters and incubated overnight
using conditions described previously (17). The filters were washed,
and autoradiography was used to detect bound 125I-IGF-I.
For some experiments, quantitation of the amount of IGFBP-5 was
determined by analysis autoradiography band intensity using a Phor
Imager and Image Quant SF software (Molecular Dynamics, Sunnyvale, CA).
The results are expressed as arbitrarily defined scanning units.
To enhance the sensitivity detection of IGFBP-5 after coimmunoprecipitation, 125I-IGFBP-5 (30 µCi/µg) (260,000 cpm/ml) that had been prepared as described previously (12), was incubated and was used with PAI-1 (200 ng/ml), and a 1:10,000 dilution of anti-PAI-1 antiserum in 0.25 ml of 0.03 M sodium phosphate containing 0.01 M EDTA, 0.1% Tween 80, 0.1% BSA, pH 7.0. After an overnight incubation at 4 C, the immune complexes were precipitated by adding rabbit IgG (1.0 µl) and protein-A sepharose (10 µl of a 20 mg/ml solution) then centrifuged. In some experiments, duplicate tubes contained unlabeled IGFBP-5 or IGFBP-5 mutants (100 ng/ml). Similarly, in some experiments unlabeled IGFBP-5 peptides that had been prepared as described previously (10) were used. Fifty microliters of Lammeli sample buffer were added, and the samples were heated to 65 C then electrophoresed through a 12.5% SDS gel and transferred to Immobilon filters. The precipitated 125I-IGFBP-5 was detected by autoradiography. In some experiments, vitronectin (200 ng/ml) was incubated with 125I-IGFBP-5 and PAI-1 then the PAI-1 immunoprecipitated as described previously. Similarly, in some experiments antivitronectin antiserum (1:5000 dilution) was added with PAI-1 or vitronectin and the complexes immunoprecipitated. The amount of 125I-IGFBP-5 that was immunoprecipitated was determined by SDS-PAGE with autoradiography.
Measurement of binding affinity
To determine the affinity of the PAI-1/IGFBP-5 interaction,
increasing concentrations of unlabeled IGFBP-5 (10–1000 ng/ml) were
mixed with 125I-IGFBP-5 (50,000 cpm/ml), and a constant
amount of unlabeled PAI-1 (100 ng/ml). After an overnight incubation,
the anti-PAI-1 antiserum was added (1:5000 dilution), and the bound
125I-IGFBP-5 was coimmunoprecipitated with protein A
sepharose as previously described, then counted in a gamma
spectrometer.
IGFBP-5 proteolysis
The effect of PAI-1 and vitronectin on IGFBP-5 proteolysis was
determined by incubating IGFBP-5 with an IGFBP-5 protease that had been
purified by heparin sepharose chromatography from fibroblast
conditioned medium (12). The partially purified protein was added to
0.25 ml of 0.05 M Tris, pH 7.0, with IGFBP-5 (50 ng/ml) and
vitronectin (10 µg/ml) or PAI-1 (10 µg/ml). After 14 h at 37
C, the products of the reaction were analyzed by immunoblotting.
Preparation of synthetic peptides and IGFBP-5 mutants
Synthetic peptides containing 11–20 amino acids from four
different regions of IGFBP-5 were synthesized and purified as described
previously (10). Their sequences are as follows: A,
DRKGFYKRKQCKPSRGRKR; B, AVKKDRRKKLT; C, ALLHGRGVCLNEKS; D,
RPKHTRISELKAE. To determine their capacity to interfere with IGFBP-5
binding to PAI-1, they were added using concentrations between 0.1 and
5.0 µg/ml. IGFBP-5 mutants containing the substitutions for basic
amino acids were prepared using in vitro mutagenesis as
previously described (9, 10). They were purified to homogeneity and
their protein content determined by comparing their HPLC peak areas to
an IGFBP-5 standard whose concentration had been determined by amino
acid composition analysis (9). They were each shown to have an affinity
for IGF-I that was similar to the wild-type, nonmutated protein
(10).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionMaterials and MethodsResultsDiscussionReferences |
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Coincubation of PAI-1 (100 ng/ml) and IGFBP-5 with anti-PAI-1 resulted
in coimmunoprecipitation of IGFBP-5 (Fig. 1A
). To prove
that coimmunoprecipitation of the IGFBP-5/PAI-1 complex was specific,
anti-PAI-1 antibody and protein-A sepharose were added to tubes that
received IGFBP-5 alone. This resulted in minimal precipitation of
IGFBP-5. To analyze this interaction by a more sensitive method,
125I-IGFBP-5 was used as a ligand. 125I-IGFBP-5
was incubated with unlabeled PAI-1 and anti-PAI-1 antibody. As shown in
Fig. 1B, the addition of unlabeled PAI-1 resulted in a large increase
in the amount of 125I-IGFBP-5 that was coimmunoprecipitated
when compared with the sample that did not contain PAI-1. The addition
of excess unlabeled IGFBP-5 greatly reduced the amount of
125I-IGFBP-5 that was coimmunoprecipitated, indicating that
the immunoprecipitation was specific. Similarly, if anti-PAI-1 was
omitted, minimal 125I-IGFBP-5 was precipitated by protein A
sepharose.
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).
Chondroitin sulfate A and B had some effect but were less potent.
Because charged glycosaminoglycans were shown to be important for this
interaction, the effect of increasing concentrations of heparin on the
IGFBP-5/PAI-1 interaction was determined. Heparin concentrations of 1
and 10 µg/ml resulted in complete inhibition of the IGFBP-5 binding
to PAI-1 (data not shown). Heparan sulfate and heparin contain O-linked
sulfate groups in the 2 or 3 position of the iduronic acid ring.
Chondroitin sulfate B (dermatan sulfate) that had an intermediate
effect also contains sulfate groups in the same positions, but it
contains fewer sulfate groups per molecule (Fig. 2). In contrast,
chondroitin sulfate A and C do not have O-linked sulfates in the 2 or 3
position and they had significantly reduced effects on the
PAI-1/IGFBP-5 binding. This suggests that the presence of O-linked
sulfate groups in these specific positions of the glycosaminoglycan
structure is required for maximal inhibition of binding.
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, peptide A,
which contained the IGFBP-5 region from amino acid 201 to 218, was an
effective competitive inhibitor. Peptide B, whose amino acid sequence
contains a similar number of charged residues from a different region
of the IGFBP-5 molecule (10), had a reduced effect. The effect of
peptide C was variable and in some experiments, it was a much less
effective inhibitor of coimmunoprecipitation (data not shown). Peptide
D had no effect on PAI-1 binding. Both peptides C and D contain fewer
charged residues than peptides A or B.
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). A form that
contained three substitutions, R202A, K206A, and R207A, also had
decreased binding. A form that contained four substitutions at
positions R201A, K20N, K206N, and K208N had a 71% reduction in
detectable PAI-1 binding. In contrast, two other mutants that contained
substitutions for charged residues in the region of IGFBP-5 between
positions 201 and 218 and K134A/R136A had nearly normal PAI-1 binding.
This suggests that both the positional location and the number of
charged residues that are altered determine binding affinity. In
general, the mutant forms that had reduced binding for
glycosaminoglycans (9) had some alteration in PAI-1 binding, but there
were distinct differences. For example, the R201A, K202N, K206N, K208N
mutant has only minimally reduced GAG binding (9). This suggests that
the GAG binding site and the PAI-1 binding site, while both charge
dependent, have different configurations.
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). The
addition of vitronectin (an ECM protein that stabilizes PAI-1) with
PAI-1 did not result in additional inhibition of IGFBP-5 proteolysis.
Neither tissue plasminogen activator (TPA) or urokinase can
significantly degrade IGFBP-5; therefore, it is unlikely that PAI-1 is
acting to directly inhibit a TPA-like protease. The binding of
125I-IGF-I to IGFBP-5 was determined in the presence and
absence of PAI-1 and the results analyzed by the method of Scatchard:
the addition of PAI-1 had no effect on the affinity of IGFBP-5 for
IGF-I (data not shown).
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, when coimmunoprecipitation was performed using
anti-PAI-1 antibodies, increasing concentrations of vitronectin
inhibited the amount of IGFBP-5 that could be coimmunoprecipitated with
PAI-1 by 33% (Fig. 6, compare lane 2 to lane 3). This suggests that
vitronectin is capable of binding to PAI-1 and preventing the
IGFBP-5/PAI-1 complex formation. When the experiment was repeated with
antivitronectin antibodies, vitronectin and 125I-IGFBP5
coprecipitated (Fig. 6, panel B). PAI-1 (100 ng/ml) interfered with the
interaction and reduced the amount of 125I-IGFBP-5 that
could be coprecipitated to basal levels.
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). In contrast, an equal concentration
nonimmune IgG had no effect. Approximately 27% of the total
125I-IGFBP-5 binding could be inhibited by this antibody.
This suggests that one of the components of ECM that binds to IGFBP-5
is PAI-1. However, because glycosaminoglycans inhibit IGFBP-5 binding
to PAI-1 and are abundant in ECM, they may be a more important binding
determinant. To determine the affinity of IGFBP-5 for PAI-1,
competitive binding assays using unlabeled and 125I-IGFBP-5
were performed. As shown in Fig. 7, the results are
consistent with a single site model with relatively high affinity. When
the data from three experiments were analyzed by the method of
Scatchard, the affinity (Kd) was determined to be 9.1
x 10-8 M.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionMaterials and MethodsResultsDiscussionReferences |
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Previously, we have shown that the amount of IGFBP-5 that is localized in the extracellular matrix positively modulates both the cell growth and DNA synthesis responses to IGF-I(5). Specifically, the mitogenic response of fibroblasts to IGF-I is enhanced if increased amounts of IGFBP-5 are present in the ECM. Furthermore, ECM localization protects IGFBP-5 from proteolysis, thus resulting in increased IGF-I being present at this site (7). A further understanding of why binding of IGF-I to IGFBP-5 in the ECM results in enhancement rather than inhibition of its action was made clearer by the observation that, after ECM localization of this binding protein, its affinity for IGF-I is reduced 8-fold (5, 9). Because the affinity of non-ECM associated IGFBP-5 is 20- to 30-fold greater than the affinity of the type I IGF receptor, this reduction in affinity would allow IGF-I that is localized bound to ECM to be in a more favorable equilibrium with receptors on cell surfaces. Therefore, ECM localization of IGFBP-5 and IGF-I results in protection from proteolysis and maintenance of a reservoir of IGF-I that can be released to receptors at critical time points during cell cycle progression. Because binding to PAI-1 does not lower IGFBP-5 affinity for IGF-I, this suggests that there may be differences in the affinity of ECM-associated IGFBP-5 and that the net reduction in affinity may be dependent upon whether binding is to predominantly glycosaminoglycans or to other proteins such as PAI-1. Therefore, the factors that regulate the distribution of IGFBP-5 among the various ECM binding proteins may indirectly alter IGFBP-5 affinity and thereby modulate IGF-I action.
The variables that regulate the synthesis of IGFBP-5 are also important determinants of its abundance in ECM. We have recently shown that IGF-I stimulates IGFBP-5 transcription by smooth muscle cells (19), and Dong and Canalis have confirmed this finding in osteoblasts (20). Furthermore, they demonstrated that retinoic acid is also a potent stimulant of IGFBP-5 synthesis by bone cells and results in increased amounts of IGFBP-5 in the ECM (20). Hakeda et al. (21) have shown that prostaglandin-E and PTH stimulate IGFBP-5 release from osteoblast ECM and this could modulate IGF-I levels in the pericellular environment.
In addition to the factors that increase IGFBP-5 synthesis, we have shown that several connective tissue cell types release an IGFBP-5 protease into their conditioned medium (7, 12, 19). The factors that regulate the activity of this protease may be important determinants of IGFBP-5 abundance in interstitial fluids and in the ECM. These studies show that PAI-1 binding to IGFBP-5 alters its susceptibility to proteolysis in the interstitial fluid. However, because multiple substances, such as PAI-1 (9) and vitronectin may bind to IGFBP-5 in ECM, other ECM proteins may function coordinately with PAI-1 or vitronectin to stabilize IGFBP-5. The synthesis of PAI-1 may also be increased coordinately with IGFBP-5. Specifically, Padaytly et al. and Afossa et al. have shown that IGF-I increases PAI-1 synthesis, both in vivo and in vitro; therefore, it is possible that PAI-1 and IGFBP-5 synthesis are coordinately regulated by IGF-I in selected cell types (22, 23). Conversely, factors such as thrombin (24) that cause release of PAI-1 from ECM might function to decrease ECM-associated IGFBP-5. Heparin functions to inhibit the IGFBP-5/PAI-1 interaction and to reduce the abundance of PAI-1 or IGFBP-5 within ECM (25, 26, 27); therefore, heparin inhibition of IGFBP-5 binding to ECM could be a component of the mechanism by which heparin functions to inhibit smooth muscle cell replication.
IGF-I synthesis and peptide abundance is also often increased at sites of injury (28, 29). Similarly PAI-1 expression is increased after injury. The 5' flanking sequences of both the IGFBP-5 and PAI-1 genes contain the Egr-1 injury response element (30), which mediates an increase in the transcription of specific genes after injury. Therefore, it is possible that PAI-1 and IGFBP-5 synthesis may be directly increased in response to injury. Because IGF-I synthesis also increases after injury, this increase in IGF-I could further enhance IGFBP-5 expression by a mechanism that is distinct from Egr-1 (19).
PAI-1 is a component of smooth muscle cell extracellular matrix. It is also synthesized by endothelium in response to injury; therefore, because it binds tightly to IGFBP-5, it could provide a mechanism for stabilizing large amounts of IGFBP-5 in the newly developing ECM during neointima formation. PAI-1 also has other interesting properties because it binds vitronectin (31, 32) and the PAI-1 binding domain is also the region of vitronectin that interacts with the vitronectin receptor (31). This suggests other potential mechanisms for enhancing IGF-I action. Proteases, such as thrombin, could release vitronectin from the ECM and allow more vitronectin to interact with its receptor. Similarly, if they degrade IGFBP-5, they would allow release of IGF-I to receptors. Simultaneous vitronectin receptor and IGF receptor occupancy has been shown to be a potent stimulant of smooth muscle cell migration (33), a process that is an important component of neointina formation.
The exact site of molecular interaction between PAI-1 and IGFBP-5 remains to be determined. However, it is clear that ionic residues are involved. Similarly, glycosaminoglycans may interfere with this interaction, and there may be a competitive equilibrium between proteoglycans that are present in the extracellular matrix and PAI-1. Because plasminogen is not the major protease in pSMC or fibroblast medium that degrades IGFBP-5, it is not surprising that PAI-1 binding of IGFBP-5 did not completely protect it from proteolysis. However, plasmin can degrade IGFBP-5, so PAI-1 could play an indirect role in maintaining intact IGFBP-5 by inhibiting plasmin formation (34). The charged residue motif that binds PAI-1 is clearly distinct from the heparin binding site (9), although overlapping residues are used. This suggests that the highly charged regions of IGFBP-5 that are surface exposed recognize distinct substrates differently and that there may be specificity for substrate binding. The exact factors that control the surface exposure of distinct sites in IGFBP-5 are unknown but will be interesting points for future study.
THE INSULIN-LIKE growth factors (IGFs) are present in physiological fluids bound to IGF binding proteins (IGFBPs) (1, 2). Six IGF binding proteins with high affinities for IGF-I and II have been identified (3). Although IGFBP-5 binds to the IGFs with high affinity in interstitial fluids, when it is associated with extracellular matrix its affinity is decreased 8- to 15-fold (4). This lower affinity results in better equilibration of IGF-I with its receptor and potentiation of IGF-I stimulated fibroblast (5) or smooth muscle cell growth (6). Extracellular fluids also contain proteases that degrade IGFBP-5 and cleave it into non-IGF binding fragments (7). However, when IGFBP-5 is associated with ECM, it is protected from proteolysis (5, 8); therefore, ECM binding may provide a mechanism for localizing IGF-I in focal areas and provide better access to receptors. This localization could be important in mediating the cellular response to injury when IGF-I expression is increased in the local microenvironment. Therefore, the factors in extracellular matrix that account for IGFBP-5 localization have the potential to indirectly regulate IGF-I actions.
We have previously shown that IGFBP-5 binds to heparin and heparan sulfate (4, 9). Similarly, glycosaminoglycan containing proteoglycans, such as tenascin, that are present in the ECM also bind IGFBP-5 (9). This binding occurs through highly charged residues in IGFBP-5 that are located between residues 201 and 218 (4, 9, 10). Similar to ECM binding, glycosaminoglycan binding of IGFBP-5 also results in a reduction in the affinity of IGFBP-5 for IGF-I (4) and in protection from proteolysis (8). However, competitive binding studies using intact ECM have shown that all of the ECM binding of IGFBP-5 could not be accounted for by binding to proteoglycans (5). Therefore, these studies were undertaken to determine if extracellular matrix proteins other than proteoglycans might bind IGFBP-5.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionMaterials and MethodsResultsDiscussionReferences |
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Purification of PAI-I
Human dermal fibroblasts (GM-10) were purchased from Coriell
Institute (Camden, NJ). They were grown in EMEM supplemented with 10%
calf serum as described previously (7). Confluent monolayers were
washed twice in the serum-free medium, then 50 cc was added to
175-cm2 flasks (Falcon Labware, Division of
Becton-Dickinson), and the medium was collected after 24 h. This
was repeated until 1.2 liters of serum-free medium had been collected.
After purification by heparin sepharose affinity chromatography and
1 antichymotryptic peptide affinity chromatography as
described previously (12), the fractions containing IGFBP-5 protease
activity were applied to an IGFBP-5 affinity column. This column was
prepared by conjugating a synthetic 18 amino acid peptide that
contained the IGFBP-5 sequence between amino acids 201–218 to Affigel
10 (Bio-Rad Richmond, CA). This sequence is also present in IGFBP-3,
but it is not present in any other form of IGFBP. The peptide was
conjugated according to manufacturer’s instructions by incubating 5.6
mg of peptide with 7.0 cc of activated sepharose in 0.1 M
Na HEPES buffer, pH 7.5, for 4 h at 8 C. Unreacted sites were
blocked by a further incubation for 1 h with 0.25 M
Tris. The gel was washed, and the pool of activity that had been
purified from fibroblast conditioned medium was applied. The purified
active fractions that were eluted with 0.5 M NaCl were
concentrated and further analyzed by SDS-PAGE (12.5% gel). After
electrophoresis, the proteins were transferred to a nitrocellulose
membrane that was stained with Ponceau-S. A 55-kDa band was excised and
subjected to amino acid sequencing. Amino terminal amino acid
sequencing was conducted as described previously using Edman
degradation (13).
Preparation of human IGFBP-5
The human IGFBP-5 that was used in these studies was obtained by
purifying the protein from Chinese hamster ovary (CHO) cell conditioned
medium. CHO cells were transfected with an IGFBP-5 complementary DNA
(cDNA) that contained the entire protein coding region and had been
prepared as previously described (7). The cells were transfected, then
maintained in methotrexate (50 µg/ml) because the plasmid that was
used (pNUT) contains the gene that contains dihydrofolate reductase
resistance. After selection with methotrexate, 50 µg/ml, the highest
secreting clones were further subcultured in DMEM containing 10%
dialyzed FCS. The cells were grown to confluency and 50 ml of
serum-free medium were added to 175-cm tissue culture flasks and
incubated for 48 h. Eight hundred cubic centimeters of conditioned
medium were purified using a three-step purification procedure of
phenyl sepharose chromatography, IGF-I affinity chromatography, and
HPLC (C4 column) (Vydac, Hesperia, CA), as previously described (10).
The purified protein was homogenous by SDS-PAGE with silver staining
(14). The protein content was determined by amino acid composition
analysis (7).
Coimmunoprecipitation, ligand blotting, and immunoblotting
The ability of IGFBP-5 to bind to the plasminogen activator
inhibitor-1 was analyzed by coimmunoprecipitation. It was determined
that the lowest nonspecific binding and the highest sensitivity of
detection could be obtained by immunoprecipitating with anti-PAI-1
antisera. The IGFBP-5 that had been coimmunoprecipitated was then
detected by immunoblotting (5) or by ligand blotting using
125I-IGF-I (15). PAI-1 and polyclonal anti-PAI-1 antiserum
were obtained from American Diagnostica (Greenwich, CT). In most
experiments, IGFBP-5 (100 ng/ml) was incubated with PAI-1 at 100 ng/ml,
and anti-PAI-1 was added at a 1:5000 dilution. The reagents were
allowed to incubate for 16 h at 4 C in 0.03 M sodium
phosphate, pH 7.4, containing 0.01 M EDTA, 0.1% Tween 80,
and 0.02% BSA. Protein A sepharose (3 mg) was added and incubated for
2 h at 4 C. The immune complexes were precipitated by centrifuging
at 3000 x g for 5 min. The pellets were rinsed with
1.0 ml of the buffer listed previously and 50 µl of 2x Laemmli
sample buffer was added. The sample was centrifuged (2000 x
g for 5 min) and the supernatant loaded onto a 12.5% SDS
polyacrylamide gel. The proteins were electrophoresed through a 12.5%
gel using nonreducing conditions, then transferred to Immobilon
membranes (Millipore, Bedford, MA) as previously described (15). After
transfer, the filters were immunoblotted using a 1:1000 dilution of
anti-IGFBP-5 antiserum. The immune complexes were detected using the
Protoblot system (Promega, Madison, WI) according to manufacturer’s
recommendation. The electrophoretic mobilities of the bands that were
detected were compared with known mol wt standards (Bethesda Research
Laboratories, Gaithersburg, MD). The photographic negatives were
scanned using a Hoffer GS-300 scanner (Hoffer Scientific Instruments,
San Francisco, CA), and the results are expressed in scanning units.
For more sensitive detection of IGFBP-5, ligand blotting was performed.
Five hundred thousand counts per minute of 125I-IGF-I [150
µCi/µg (16)] was added to the filters and incubated overnight
using conditions described previously (17). The filters were washed,
and autoradiography was used to detect bound 125I-IGF-I.
For some experiments, quantitation of the amount of IGFBP-5 was
determined by analysis autoradiography band intensity using a Phor
Imager and Image Quant SF software (Molecular Dynamics, Sunnyvale, CA).
The results are expressed as arbitrarily defined scanning units.
To enhance the sensitivity detection of IGFBP-5 after coimmunoprecipitation, 125I-IGFBP-5 (30 µCi/µg) (260,000 cpm/ml) that had been prepared as described previously (12), was incubated and was used with PAI-1 (200 ng/ml), and a 1:10,000 dilution of anti-PAI-1 antiserum in 0.25 ml of 0.03 M sodium phosphate containing 0.01 M EDTA, 0.1% Tween 80, 0.1% BSA, pH 7.0. After an overnight incubation at 4 C, the immune complexes were precipitated by adding rabbit IgG (1.0 µl) and protein-A sepharose (10 µl of a 20 mg/ml solution) then centrifuged. In some experiments, duplicate tubes contained unlabeled IGFBP-5 or IGFBP-5 mutants (100 ng/ml). Similarly, in some experiments unlabeled IGFBP-5 peptides that had been prepared as described previously (10) were used. Fifty microliters of Lammeli sample buffer were added, and the samples were heated to 65 C then electrophoresed through a 12.5% SDS gel and transferred to Immobilon filters. The precipitated 125I-IGFBP-5 was detected by autoradiography. In some experiments, vitronectin (200 ng/ml) was incubated with 125I-IGFBP-5 and PAI-1 then the PAI-1 immunoprecipitated as described previously. Similarly, in some experiments antivitronectin antiserum (1:5000 dilution) was added with PAI-1 or vitronectin and the complexes immunoprecipitated. The amount of 125I-IGFBP-5 that was immunoprecipitated was determined by SDS-PAGE with autoradiography.
Measurement of binding affinity
To determine the affinity of the PAI-1/IGFBP-5 interaction,
increasing concentrations of unlabeled IGFBP-5 (10–1000 ng/ml) were
mixed with 125I-IGFBP-5 (50,000 cpm/ml), and a constant
amount of unlabeled PAI-1 (100 ng/ml). After an overnight incubation,
the anti-PAI-1 antiserum was added (1:5000 dilution), and the bound
125I-IGFBP-5 was coimmunoprecipitated with protein A
sepharose as previously described, then counted in a gamma
spectrometer.
IGFBP-5 proteolysis
The effect of PAI-1 and vitronectin on IGFBP-5 proteolysis was
determined by incubating IGFBP-5 with an IGFBP-5 protease that had been
purified by heparin sepharose chromatography from fibroblast
conditioned medium (12). The partially purified protein was added to
0.25 ml of 0.05 M Tris, pH 7.0, with IGFBP-5 (50 ng/ml) and
vitronectin (10 µg/ml) or PAI-1 (10 µg/ml). After 14 h at 37
C, the products of the reaction were analyzed by immunoblotting.
Preparation of synthetic peptides and IGFBP-5 mutants
Synthetic peptides containing 11–20 amino acids from four
different regions of IGFBP-5 were synthesized and purified as described
previously (10). Their sequences are as follows: A,
DRKGFYKRKQCKPSRGRKR; B, AVKKDRRKKLT; C, ALLHGRGVCLNEKS; D,
RPKHTRISELKAE. To determine their capacity to interfere with IGFBP-5
binding to PAI-1, they were added using concentrations between 0.1 and
5.0 µg/ml. IGFBP-5 mutants containing the substitutions for basic
amino acids were prepared using in vitro mutagenesis as
previously described (9, 10). They were purified to homogeneity and
their protein content determined by comparing their HPLC peak areas to
an IGFBP-5 standard whose concentration had been determined by amino
acid composition analysis (9). They were each shown to have an affinity
for IGF-I that was similar to the wild-type, nonmutated protein
(10).
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Results |
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Coincubation of PAI-1 (100 ng/ml) and IGFBP-5 with anti-PAI-1 resulted
in coimmunoprecipitation of IGFBP-5 (Fig. 1A
). To prove that coimmunoprecipitation
of the IGFBP-5/PAI-1 complex was specific, anti-PAI-1 antibody and
protein-A sepharose were added to tubes that received IGFBP-5 alone.
This resulted in minimal precipitation of IGFBP-5. To analyze this
interaction by a more sensitive method, 125I-IGFBP-5 was
used as a ligand. 125I-IGFBP-5 was incubated with unlabeled
PAI-1 and anti-PAI-1 antibody. As shown in Fig. 1B, the addition of
unlabeled PAI-1 resulted in a large increase in the amount of
125I-IGFBP-5 that was coimmunoprecipitated when compared
with the sample that did not contain PAI-1. The addition of excess
unlabeled IGFBP-5 greatly reduced the amount of
125I-IGFBP-5 that was coimmunoprecipitated, indicating that
the immunoprecipitation was specific. Similarly, if anti-PAI-1 was
omitted, minimal 125I-IGFBP-5 was precipitated by protein A
sepharose.
To determine whether this interaction was influenced by
glycosaminoglycans, which are known to bind to IGFBP-5, soluble GAGs
were added with PAI-1 and 125I-IGFBP-5, then
coimmunoprecipitation performed. Heparin and heparan sulfate completely
inhibited the coimmunoprecipitation reaction (Fig. 2). Chondroitin sulfate A and B had some
effect but were less potent. Because charged glycosaminoglycans were
shown to be important for this interaction, the effect of increasing
concentrations of heparin on the IGFBP-5/PAI-1 interaction was
determined. Heparin concentrations of 1 and 10 µg/ml resulted in
complete inhibition of the IGFBP-5 binding to PAI-1 (data not shown).
Heparan sulfate and heparin contain O-linked sulfate groups in the 2 or
3 position of the iduronic acid ring. Chondroitin sulfate B (dermatan
sulfate) that had an intermediate effect also contains sulfate groups
in the same positions, but it contains fewer sulfate groups per
molecule (Fig. 2). In contrast, chondroitin sulfate A and C do not have
O-linked sulfates in the 2 or 3 position and they had significantly
reduced effects on the PAI-1/IGFBP-5 binding. This suggests that the
presence of O-linked sulfate groups in these specific positions of the
glycosaminoglycan structure is required for maximal inhibition of
binding.
Because the binding was inhibited by glycosaminoglycans, this suggested
that a region of charged amino acids within IGFBP-5 might be required
for binding. To test this hypothesis, synthetic peptides containing
regions of IGFBP-5 sequence with several charged amino acids were
incubated with 125I-IGFBP-5 and PAI-1 and
coimmunoprecipitated. As shown in Fig. 3, peptide A, which contained the IGFBP-5 region from amino acid 201 to
218, was an effective competitive inhibitor. Peptide B, whose amino
acid sequence contains a similar number of charged residues from a
different region of the IGFBP-5 molecule (10), had a reduced effect.
The effect of peptide C was variable and in some experiments, it was a
much less effective inhibitor of coimmunoprecipitation (data not
shown). Peptide D had no effect on PAI-1 binding. Both peptides C and D
contain fewer charged residues than peptides A or B.
To further determine the specific amino acids that were involved in the
PAI-1/IGFBP-5 interaction, coprecipitation was conducted using IGFBP-5
mutants that contain neutral substitutions for basic amino acids
between residues 201 and 218. A form of IGFBP-5 that contained four
substitutions K211N, R214A, K217A, and R218A had a 24% reduction in
its ability to bind to PAI-1 (Fig. 4). A
form that contained three substitutions, R202A, K206A, and R207A, also
had decreased binding. A form that contained four substitutions at
positions R201A, K20N, K206N, and K208N had a 71% reduction in
detectable PAI-1 binding. In contrast, two other mutants that contained
substitutions for charged residues in the region of IGFBP-5 between
positions 201 and 218 and K134A/R136A had nearly normal PAI-1 binding.
This suggests that both the positional location and the number of
charged residues that are altered determine binding affinity. In
general, the mutant forms that had reduced binding for
glycosaminoglycans (9) had some alteration in PAI-1 binding, but there
were distinct differences. For example, the R201A, K202N, K206N, K208N
mutant has only minimally reduced GAG binding (9). This suggests that
the GAG binding site and the PAI-1 binding site, while both charge
dependent, have different configurations.
The effect of PAI-1 binding to IGFBP-5 on its susceptibility to
proteolytic cleavage was determined. When IGFBP-5 was incubated with an
IGFBP-5 protease obtained from fibroblast conditioned medium, there was
time-dependent cleavage of the substrate into a 22-kDa band. The
addition of PAI-1 to the incubation buffer resulted in a 1.6-fold
increase in intact IGFBP-5 band intensity (Fig. 5). The addition of vitronectin (an ECM
protein that stabilizes PAI-1) with PAI-1 did not result in additional
inhibition of IGFBP-5 proteolysis. Neither tissue plasminogen activator
(TPA) or urokinase can significantly degrade IGFBP-5; therefore, it is
unlikely that PAI-1 is acting to directly inhibit a TPA-like protease.
The binding of 125I-IGF-I to IGFBP-5 was determined in the
presence and absence of PAI-1 and the results analyzed by the method of
Scatchard: the addition of PAI-1 had no effect on the affinity of
IGFBP-5 for IGF-I (data not shown).
Because multiple extracellular matrix proteins may bind to IGFBP-5
simultaneously, we determined if vitronectin, a known binding protein
for PAI-1, could alter the PAI-1/IGFBP-5 interaction. As shown in Fig. 6, when coimmunoprecipitation was
performed using anti-PAI-1 antibodies, increasing concentrations of
vitronectin inhibited the amount of IGFBP-5 that could be
coimmunoprecipitated with PAI-1 by 33% (Fig. 6, compare lane 2 to lane
3). This suggests that vitronectin is capable of binding to PAI-1 and
preventing the IGFBP-5/PAI-1 complex formation. When the experiment was
repeated with antivitronectin antibodies, vitronectin and
125I-IGFBP5 coprecipitated (Fig. 6, panel B). PAI-1 (100
ng/ml) interfered with the interaction and reduced the amount of
125I-IGFBP-5 that could be coprecipitated to basal
levels.
To assess the physiological significance of PAI-1/IGFBP-5 binding,
extracellular matrix was prepared from human fibroblast cultures as
previously described (18). 125I-IGFBP-5 was incubated
overnight with the ECM extracts and increasing concentrations of
anti-PAI-1 antibody. This antibody recognizes ECM bound PAI-1. The
addition of increasing amounts of anti-PAI-1 to the incubation mixture
partially blocked ability of 125I-IGFBP-5 to bind to the
ECM (Table 1). In contrast, an equal
concentration nonimmune IgG had no effect. Approximately 27% of the
total 125I-IGFBP-5 binding could be inhibited by this
antibody. This suggests that one of the components of ECM that binds to
IGFBP-5 is PAI-1. However, because glycosaminoglycans inhibit IGFBP-5
binding to PAI-1 and are abundant in ECM, they may be a more important
binding determinant. To determine the affinity of IGFBP-5 for PAI-1,
competitive binding assays using unlabeled and 125I-IGFBP-5
were performed. As shown in Fig. 7, the
results are consistent with a single site model with relatively high
affinity. When the data from three experiments were analyzed by the
method of Scatchard, the affinity (Kd) was determined to be
9.1 x 10-8 M.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionMaterials and MethodsResultsDiscussionReferences |
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Previously, we have shown that the amount of IGFBP-5 that is localized in the extracellular matrix positively modulates both the cell growth and DNA synthesis responses to IGF-I(5). Specifically, the mitogenic response of fibroblasts to IGF-I is enhanced if increased amounts of IGFBP-5 are present in the ECM. Furthermore, ECM localization protects IGFBP-5 from proteolysis, thus resulting in increased IGF-I being present at this site (7). A further understanding of why binding of IGF-I to IGFBP-5 in the ECM results in enhancement rather than inhibition of its action was made clearer by the observation that, after ECM localization of this binding protein, its affinity for IGF-I is reduced 8-fold (5, 9). Because the affinity of non-ECM associated IGFBP-5 is 20- to 30-fold greater than the affinity of the type I IGF receptor, this reduction in affinity would allow IGF-I that is localized bound to ECM to be in a more favorable equilibrium with receptors on cell surfaces. Therefore, ECM localization of IGFBP-5 and IGF-I results in protection from proteolysis and maintenance of a reservoir of IGF-I that can be released to receptors at critical time points during cell cycle progression. Because binding to PAI-1 does not lower IGFBP-5 affinity for IGF-I, this suggests that there may be differences in the affinity of ECM-associated IGFBP-5 and that the net reduction in affinity may be dependent upon whether binding is to predominantly glycosaminoglycans or to other proteins such as PAI-1. Therefore, the factors that regulate the distribution of IGFBP-5 among the various ECM binding proteins may indirectly alter IGFBP-5 affinity and thereby modulate IGF-I action.
The variables that regulate the synthesis of IGFBP-5 are also important determinants of its abundance in ECM. We have recently shown that IGF-I stimulates IGFBP-5 transcription by smooth muscle cells (19), and Dong and Canalis have confirmed this finding in osteoblasts (20). Furthermore, they demonstrated that retinoic acid is also a potent stimulant of IGFBP-5 synthesis by bone cells and results in increased amounts of IGFBP-5 in the ECM (20). Hakeda et al. (21) have shown that prostaglandin-E and PTH stimulate IGFBP-5 release from osteoblast ECM and this could modulate IGF-I levels in the pericellular environment.
In addition to the factors that increase IGFBP-5 synthesis, we have shown that several connective tissue cell types release an IGFBP-5 protease into their conditioned medium (7, 12, 19). The factors that regulate the activity of this protease may be important determinants of IGFBP-5 abundance in interstitial fluids and in the ECM. These studies show that PAI-1 binding to IGFBP-5 alters its susceptibility to proteolysis in the interstitial fluid. However, because multiple substances, such as PAI-1 (9) and vitronectin may bind to IGFBP-5 in ECM, other ECM proteins may function coordinately with PAI-1 or vitronectin to stabilize IGFBP-5. The synthesis of PAI-1 may also be increased coordinately with IGFBP-5. Specifically, Padaytly et al. and Afossa et al. have shown that IGF-I increases PAI-1 synthesis, both in vivo and in vitro; therefore, it is possible that PAI-1 and IGFBP-5 synthesis are coordinately regulated by IGF-I in selected cell types (22, 23). Conversely, factors such as thrombin (24) that cause release of PAI-1 from ECM might function to decrease ECM-associated IGFBP-5. Heparin functions to inhibit the IGFBP-5/PAI-1 interaction and to reduce the abundance of PAI-1 or IGFBP-5 within ECM (25, 26, 27); therefore, heparin inhibition of IGFBP-5 binding to ECM could be a component of the mechanism by which heparin functions to inhibit smooth muscle cell replication.
IGF-I synthesis and peptide abundance is also often increased at sites of injury (28, 29). Similarly PAI-1 expression is increased after injury. The 5' flanking sequences of both the IGFBP-5 and PAI-1 genes contain the Egr-1 injury response element (30), which mediates an increase in the transcription of specific genes after injury. Therefore, it is possible that PAI-1 and IGFBP-5 synthesis may be directly increased in response to injury. Because IGF-I synthesis also increases after injury, this increase in IGF-I could further enhance IGFBP-5 expression by a mechanism that is distinct from Egr-1 (19).
PAI-1 is a component of smooth muscle cell extracellular matrix. It is also synthesized by endothelium in response to injury; therefore, because it binds tightly to IGFBP-5, it could provide a mechanism for stabilizing large amounts of IGFBP-5 in the newly developing ECM during neointima formation. PAI-1 also has other interesting properties because it binds vitronectin (31, 32) and the PAI-1 binding domain is also the region of vitronectin that interacts with the vitronectin receptor (31). This suggests other potential mechanisms for enhancing IGF-I action. Proteases, such as thrombin, could release vitronectin from the ECM and allow more vitronectin to interact with its receptor. Similarly, if they degrade IGFBP-5, they would allow release of IGF-I to receptors. Simultaneous vitronectin receptor and IGF receptor occupancy has been shown to be a potent stimulant of smooth muscle cell migration (33), a process that is an important component of neointina formation.
The exact site of molecular interaction between PAI-1 and IGFBP-5 remains to be determined. However, it is clear that ionic residues are involved. Similarly, glycosaminoglycans may interfere with this interaction, and there may be a competitive equilibrium between proteoglycans that are present in the extracellular matrix and PAI-1. Because plasminogen is not the major protease in pSMC or fibroblast medium that degrades IGFBP-5, it is not surprising that PAI-1 binding of IGFBP-5 did not completely protect it from proteolysis. However, plasmin can degrade IGFBP-5, so PAI-1 could play an indirect role in maintaining intact IGFBP-5 by inhibiting plasmin formation (34). The charged residue motif that binds PAI-1 is clearly distinct from the heparin binding site (9), although overlapping residues are used. This suggests that the highly charged regions of IGFBP-5 that are surface exposed recognize distinct substrates differently and that there may be specificity for substrate binding. The exact factors that control the surface exposure of distinct sites in IGFBP-5 are unknown but will be interesting points for future study.
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2 This work was supported by NIH Grants HL-56850 and AG-02331.
Received September 12, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionMaterials and MethodsResultsDiscussionReferences |
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Vß3 integrin is necessary for smooth
muscle cells to migrate in response to IGF-I. Proc Natl Acad Sci 93:2462–2467
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