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GROWTH FACTORS-CYTOKINES-ONCOGENES |
University of North Carolina, Chapel Hill, North Carolina 27599
Address all correspondence and requests for reprints to: David R. Clemmons, M.D., CB 7170, 6111 Thurston-Bowles, Division of Endocrinology, University of North Carolina, Chapel Hill, North Carolina 275990-7170. E-mail: endo{at}med.unc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vß3 integrin alters signaling by platelet-derived
growth factor (3) fibroblast growth factor
(1) and IGF-I (5). Blocking ligand occupancy
of Vß3 has been shown to attenuate IGF-I stimulated cell migration
and DNA synthesis in cultured aortic smooth muscle cells (SMC)
(5, 6). Specifically blocking ligand occupancy with the
disintegrin antagonist, echistatin, has been shown to attenuate these
cellular processes. More specifically, blocking ligand occupancy of
Vß3 with disintegrins results in attenuation of IGF-I stimulated
receptor phosphorylation. It also inhibits the phosphorylation of
downstream signaling components such as, insulin receptor substrate-1
(IRS-1), and PI-3 kinase (5, 7). Because PI-3 kinase
activation is required for IGF-I stimulated cell migration
(8), this presumably is the signal transduction pathway
through which the inhibitory effect of disintegrin exposure is
mediated. Conversely, enhancing ligand occupancy of Vß3 with
ligands such as, vitronectin and thrombospondin-1, results in
enhancement of the ability of IGF-I to stimulate IGF-I receptor-linked
signaling (5). Because SMC synthesize vitronectin, this
provides an autoregulatory mechanism by which IGF-I responsiveness may
be increased (9). Smooth muscle cells also synthesize high
affinity IGF binding proteins that are secreted into the
microenvironment (10, 11). One form of IGF-I binding
protein, IGFBP-5, is deposited into the extracellular matrix and
functions as a reservoir for IGF-I within the matrix of connective
tissue cell types (11, 12). IGFBP-5 synthesis by SMC is
stimulated by IGF-I (10). In previous studies, we have
reported that IGFBP-5 can bind to several ECM-associated proteins
including plasminogen activation inhibitor-I (13),
osteopontin and thrombospondin-1 (14) with high affinity.
Because there is abundant vitronectin and IGFBP-5 within the ECM,
and both proteins can modulate IGF-I activity, these studies were
conducted to determine if IGFBP-5 could bind to vitronectin and if this
interaction caused any significant alteration in the ability of IGF-I
to stimulate cell migration or DNA synthesis. |
Materials and Methods |
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Coimmunoprecipitation of human IGFBP-5 and victronectin
The ability of IGFBP-5 to bind to vitronectin was analyzed by
coimmunoprecipitation. 125I-IGFBP-5 (specific
activity 15 and 21 µCi/µg) was prepared and purified as
described previously (14).
125I-IGFBP-5 (80,000 cpm) was incubated with
human vitronectin (100 ng/ml) in 0.25 ml of 30 mM sodium
phosphate (pH 7.4), 10 mM EDTA 0.1% BSA, and 0.5%
Tween-20 for 14 h at 4 C. At that time, a 1:1000 dilution of
rabbit antivitronectin antibody was added. Following an overnight
incubation, 900 µg of protein A was added to the same buffer and the
mixture was centrifuged. The pellet was washed 5 times, the mixture
dissolved in 1.5x Laemmli sample buffer and the proteins were
separated by SDS-PAGE using a 12.5% gel. The proteins were transferred
to Immobilon-PSQ membranes (Millipore Corp.,
Bedford, MA) and the amount of radiolabeled IGFBP-5 that had been
precipitated was determined by autoradiography and scanning
densitometry. The scanning data were analyzed using the NIH Image
Program. To determine the specific regions of IGFBP-5 that bound to
vitronectin, four synthetic peptides that contained sequences from four
distinct regions of IGFBP-5 (13) were coincubated with
125I-IGFBP-5 plus vitronectin and
immunoprecipitation performed in the manner described previously. The
peptides were added to concentration of 1 µg/ml. The method of
peptide synthesis and purification of these peptides has been reported
previously (13). The sequences are as follows:
Peptide A, RGRKGFYKRKQCKPSRGRKR; peptide B, AVKKDRRKKLT; peptide C, HALLHGRGVCLNEKS; peptide D, RPKHTRISELKAE.
In some experiments, IGFBP-5 mutants that had been prepared by previously described methods were used (17). These mutants were added to the incubation buffer at a concentration of 1 µg/ml. The mutants had had one or more basic amino acids between positions 201 and 218 within IGFBP-5 converted to neutral residues. Some of these mutants contained amino acid substitutions that had been shown to markedly alter their ability to bind to extracellular matrix (17). The mutants that were analyzed contained the following amino acid substitutions: R201A, K202N; K202A, K206A, R207A; K217A, R218A; K211N, R214A, K217N, R218A; R207A, K211N; K138N, K139N; K134A, R136A, K211N; K211N and R214A. The method of purification and characterization of these mutants has been reported previously (17). None had alterations in their affinity for IGF-I. An additional mutant that had had one charged residue changed to a neutral residual and four hydrophobic residues in the IGF binding domain changed to nonhydrophobic residues was also used, K68A, P69N, L70N, L73N, L74N. This mutant has a greater than 1,000-fold reduction in its affinity for IGF-I (18).
Measurement of biologic actions and binding affinity
To determine the affinity of vitronectin for
125I-IGFBP-5 (50,000 cpm per tube) was incubated
with vitronectin, 100 ng/ml, and increasing concentrations of
unlabelled IGFBP-5 (2–160 ng/ml). The composition of the incubation
buffer was as described previously. After an overnight incubation, the
bound IGFBP-5 was precipitated as described previously using
antivitronectin antiserum and the quantity precipitated determined by
gamma counting. The data were subjected to Scatchard analysis to
calculate the affinity constant of IGFBP-5 for vitronectin. To
determine whether vitronectin binding to IGFBP-5 would alter its
affinity for IGF-I, 25,000 cpm/tube of 125I-IGF-I
(specific activity 150 µCi/µg) was incubated with IGFBP-5 (200
ng/ml) plus vitronectin (100 ng/ml) and increasing concentrations of
unlabelled IGF-I (0.5–100 ng/ml). After an overnight incubation,
IGFBP-5 was precipitated with 6.25% polyethylene glycol and bound
125I-IGF-I was determined as described previously
(19). The results were analyzed by the method of
Scatchard.
To determine the effect of ECM-associated vitronectin on the capacity of IGFBP-5 to alter IGF-I stimulated cell migration, six-well plates (Falcon no. 3046) were incubated with vitronectin (1 µg/ml) for 14 h at 37 C and washed twice with serum-free DMEM. Some culture dishes were then exposed to IGFBP-5, 1.0 µg/ml, or one of the IGFBP-5 mutants for 4 h at 37 C; then porcine aortic SMC that had been isolated and maintained in culture as described previously (20) were plated on the vitronectin coated plates at a density of 50,000 cells/cm2 in DMEM supplemented with 0.2% FBS and IGFBP-5 or one of the mutants. After 24 h, when the monolayers had reached confluent density, they were washed twice then wounded (21) and exposed to IGF-I alone (100 µg/ml) or IGF-I (100 µg/ml) plus IGFBP-5 or one of its mutants (1.0 µg/ml) in 1.0 ml of serum-free DMEM. The number of cells migrating across the wound margin during the subsequent 48 h was determined directly by counting. For each wound, ten separate areas of migrating cells were counted as described previously (6). Using these conditions, approximately 11% of the total number of cells that are counted in the area that is distal to the wound margin are due to increased cell division and not to migration. Control cultures were plated on plastic dishes in DMEM supplemented with 10% FBS at 50,000 cells/cm2. After allowing 4 h for attachment, the medium was replaced with DMEM containing 0.2% FBS, and the cultures were exposed to native or mutant forms of IGFBP-5 for 24 h. The confluent monolayers were washed, then wounded and exposed to IGF-I or IGF-I plus native or mutant IGFBP-5 as described for the cultures that had been plated as vitronectin.
To determine the ability of wild-type and mutant forms of IGFBP-5 to alter IGF-I stimulated DNA synthesis, SMC were plated on vitronectin-coated 96-well plastic plates at a density of 10,000 cells/well in serum-free DMEM and allowed to grow for 2 d to reach confluence. During this time period, some cultures were also exposed to 1.0 µg/ml of native or mutant forms of IGFBP-5. After 48 h, the cultures were rinsed in serum-free medium, then exposed to increasing concentrations of IGF-I (0–50 ng/ml) in 0.2 ml of DMEM containing 0.2% platelet poor plasma and 0.5 µCi of 3H-thymidine (25 µCi/umol Amersham Pharmacia Biotech, Piscataway, NJ). After 36 h, the amount of 3H-thymidine incorporation into DNA was assessed as described previously (22). Control cultures were plated on plastic plates at 10,000 cells/cm2 in DMEM containing 10% FBS. After 4 h, this medium was aspirated, the cultures were rinsed with serum-free DMEM and 0.2 ml DMEM with 0.2% FBS containing native IGFBP-5 or the IGFBP-5 mutants (1.0 µg/ml) were added for 48 h. At that time, increasing concentrations of IGF-I and 3H-thymidine were directly added as described previously. The incubation was continued for 36 h, and 3H-thymidine incorporation into DNA was quantified. Statistical analysis was performed using the paired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the
addition of vitronectin caused an increase in the amount of IGFBP-5
that could be coimmunoprecipitated. Increasing concentrations of
heparin reduced the amount of IGFBP-5 that was precipitated as did
heparan sulfate. To assess the regions of IGFBP-5 that were important
for this interaction each of four peptides with the sequences noted in
Materials and Methods was coincubated with
125I-IGFBP-5 and vitronectin. As shown in Fig. 2, the addition of both peptides A and B
resulted in competitive inhibition of the interaction between these two
proteins. In contrast, peptides C and D had no effect. An excess of
native unlabeled IGFBP-5 also significantly reduced binding. Scanning
densitometry showed that peptides A and B reduced binding by 77 and
78%, respectively, of the maximum reduction that was obtained with
native IGFBP-5. To determine if glycosaminoglycans might be inhibiting
the binding of IGFBP-5 to vitronectin by inhibiting charge-charge
interactions in regions of the IGFBP-5 molecule that were not located
in the region encompassed by peptide A, the effect of increasing
concentrations of heparin in the presence of peptide A was compared
with peptide A alone. As shown in Fig. 3, peptide A resulted in a 76% reduction in IGFBP-5 band intensity.
Heparin inhibited binding by 91% and the combination of heparin plus
peptide A resulted in further significant reduction (99% inhibition),
suggesting that at least part of this interaction was due to charge
interactions that were not contained in the peptide A sequence.
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native IGFBP-5 reduced the amount of
125I-IGFBP-5 that bound to vitronectin to
background level. The K211N mutant had equal competing
activity. In contrast, several of the mutants had reduced
competing activity and the greatest loss of binding activity was noted
with the R201A, K202N, R206A, R208A mutant but several other mutants
also had reduced activity. The effects of the other mutants that were
tested is shown in Fig. 5. The R201A,
K202A, K206N, R208A mutant had reduced competing activity as did the
R201A, K202N; K217N, R218A; K211N, R214A, K217N, R218A and K138, K139N
mutants but the greatest reduction was noted with the K134A, K136A,
K211N mutant. The scanning densitometry units that were derived from
three separate experiments that were comparable to the experiments
shown in Figs. 4 and 5 and are presented in Table 1. Of note is the fact that two mutants
that contained substitutions for amino acids with the peptide B region,
K134A, K136A, K211N and the K138, K139N mutants had significantly
attenuated competing activity.
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To determine the ability of IGFBP-5 to modulate the SMC response to
IGF-I and vitronectin, IGF-I (100 ng/ml) and IGFBP-5 (1 µg/ml) were
incubated with cells that had been plated on vitronectin then the
ability of the cells to migrate in response to IGF-I following wounding
was determined. As shown in Table 2,
vitronectin enhanced the cell migration response to IGF-I. Exposure to
native IGFBP-5 enhanced the ability of IGF-I plus vitronectin to
stimulate cell migration compared with cultures exposed to IGF-I and
vitronectin. In contrast the R201A, K202N, K206A, R208A and K134A,
K136A, K211N mutants had no enhancing activity compared with IGF-I plus
vitronectin. The addition of the mutant form that had a greater than
1,000-fold reduction in its affinity for IGF-I resulted in no change in
the response to IGF-I plus vitronectin. When these results were
compared with cells plated on plastic that had not had their ECM
enriched in vitronectin two significant differences were noted. First
the absolute number of cells that migrated in response to IGF-I plus
IGFBP-5 was significantly less compared with the response to IGF-I +
IGFBP-5 in the presence of vitronectin (e.g. 110 ± 28
compared with 42 ± 11 cells, P < 0.01).
Similarly, the absolute difference between the number of cells
migrating in response to IGF-I plus IGFBP-5 and IGF-I alone was
significantly greater for cells plated on a vitronectin-enriched matrix
compared with cells plated on plastic (e.g. 36 ± 5
compared with 9 ± 3 cells P < 0.05). Second for
the cells plated on plastic the response to IGF-I plus the K134A,
R136A, K211N mutant that bound poorly to vitronectin was not
significantly different compared with the response of the cells to
IGF-I plus native IGFBP-5, whereas when this combination was tested
using cells plated on vitronectin, SMC migration was significantly less
than the response of the cells to IGF-I plus native IGFBP-5. This
suggests that this difference is due to the inability of this mutant to
bind to vitronectin. To determine if these results were also noted when
DNA synthesis was measured, the ability of cells plated on vitronectin
to increase their incorporation of 3H-thymidine
into DNA in response to IGF-I and IGFBP-5 or to IGF-I plus the IGFBP-5
mutants was compared with cells plated on plastic. If the cells were
plated on vitronectin, the combination of IGF-I plus native IGFBP-5
resulted in significant enhancement of the ability of these cells to
respond to IGF-I (Fig. 6). The addition
of the two mutants that did not bind to vitronectin showed that their
ability to enhance the response to IGF-I plus vitronectin was
significantly attenuated compared with wild-type IGFBP-5. In contrast,
if the cells were plated on plastic the response to IGF-I plus
IGFBP-5 was significantly less (P < 0.01) compared
with cells plated on vitronectin when IGF-I was added at concentrations
of 5 or 20 ng/ml. In addition, if the cells were plated on plastic the
response to the IGF-I plus the K134A, R 136A, K211N mutant was
significantly increased compared with the response to IGF-I alone
although it was significantly less than the response to IGF-I plus
IGFBP-5 (P < 0.05). The response to IGF-I plus the
R210A, K202A, R206A, R208A mutant was no greater than the response to
IGF-I alone whether cells were plated on plastic or on vitronectin.
Therefore, native IGFBP-5 appeared to enhance the cellular migration
and DNA synthesis responses to IGF-I for cells plated on plastic or
vitronectin, but if cells were plated on a vitronectin enriched matrix
the degree of enhancement was increased.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In previous studies, we concluded that the ability of IGFBP-5 to
enhance IGF-I actions is likely due to sequestering increased amounts
of IGF-I within the ECM (12, 23). These increased amounts
of IGF-I are then available to equilibrate with IGF-I receptors.
However, vitronectin can enhance IGF-I actions by binding to the
Vß3 integrin (5, 6). To exclude the possibility that
the effects observed in these studies were due to a direct effect of
IGFBP-5 on the ability of vitronectin to signal through Vß3 rather
than being mediated through changes in IGF-I binding, we used an
IGFBP-5 mutant that we had prepared previously that does not bind
to IGF-I. When this mutant was added under identical conditions, there
was no change in the cell migration or DNA synthesis responses compared
with those observed with IGF-I plus vitronectin. Because this mutant
had no reduction in its ability to bind to vitronectin this finding
strongly suggests that unlike other published reports of IGF-I
independent effects of IGFBP-5 (24, 25, 26); ECM associated
IGFBP-5 has no positive modulatory effects on arterial SMC
responses that are independent of its ability to bind to this growth
factor.
We have previously reported that the IGFBP-5 binds with high affinity to osteopontin, thrombospondin-1, and plasminogen activator inhibitor-I (13, 14). These binding interactions are all mediated through specific basic amino acids between positions 201–218 within IGFBP-5. To determine the regions of IGFBP-5 that were required for binding to vitronectin, we used four peptides that contained IGFBP-5 sequences. These results showed that the region between amino acids 201 and 218 was important. However, unlike the interaction between osteopontin and thrombospondin-1, the region between amino acids 131 and 141 that contains six basic amino acids is also important and apparently this region contains a secondary binding site for vitronectin. This was confirmed by using mutants that had substitutions for basic amino acids within the 131 to 141 region. The two mutants that were tested that contained these substitutions had significant attenuation of their ability to interact with vitronectin. This strongly suggests that this region is required for optimal IGFBP-5 binding to vitronectin. This property of vitronectin to bind to this region of IGFBP-5 differs significantly from these other three ECM proteins (13, 14). In this regard, the ability of vitronectin to bind to both of these regions of IGFBP-5 is similar to the serum protein, acid labile subunit (ALS). This protein is a component of the ternary complex that controls IGF-I bioavailability in plasma. ALS binds to both IGFBP-3 and IGFBP-5 to form stable high molecular weight complexes (27). It has been reported that IGFBP-5 binding to ALS is dependent upon basic amino acids within the region between amino acids 131–141 as well as the region between 201 and 218, which is similar to our findings for vitronectin binding (28).
The specific basic amino acids that are required for optimal binding of IGFBP-5 to vitronectin can only be definitively determined with mutants that have single substitutions. However, our results with combined substitution mutants strongly support that certain basic amino acids are important for vitronectin binding. Our data show that the charged amino acids in positions 134 and 136, 138, 139, 201, 202 and 206, 208 appear to be the most important for binding. The requirements for amino acids 201 and 202 for optimal binding is similar to the requirement for IGFBP-5 to bind to TSP-1 and differs from the requirements for IGFBP-5 binding to OPN and PAI-1. Amino acids 134, 136, 201, 202, and 206, 208 have also been shown to be important for IGFBP-5 binding to ECM (17). Although both the R134A R136A K211N and the R201A, K202N, R206A, R208A mutants had attenuated vitronectin binding and both showed a decreased ability to augment IGF-I actions compared with native IGFBP-5 there were some differences in cellular responsiveness. The R134A R136K K211N mutant was more effective in augmenting both the migration and DNA synthesis responses to IGF-I when cells were plated on plastic. The most likely explanation for this difference is that the R201A K202N R206A R208A mutant has marked attenuation in ECM binding (23). In contrast, the R134A R136A K211N mutant has much less attenuation in ECM binding relative to native IGFBP-5 unless a vitronectin-enriched ECM is used (data not shown). Therefore, when compared with native IGFBP-5, the R134A, R136A, K211N mutant shows less difference in its ability to enhance IGF-I actions unless the cells are plated on vitronectin.
Because glycosoaminoglycans have been shown to bind to both IGFBP-5 and to vitronectin, we determined if they would inhibit this interaction. Both heparin and heparan sulfate were potent inhibitors of IGFBP-5 binding. The glycosaminoglycan binding site within IGFBP-5 has been localized primarily to the region between amino acids 201 and 218, although the region between residues 131 and 141 contributes to binding (29). We determined if the combination of heparin plus peptide A had a greater effect than peptide A or heparin alone. The addition of heparin resulted in greater competing activity. This suggests that either heparin is competing for ability of both the peptide A and B regions of IGFBP-5 to bind to vitronectin or that heparin binding to binding sites within the C-terminal region of vitronectin (30) inhibits the ability of this region of vitronectin to bind to sites within IGFBP-5 other than those located in the peptide A region. The biologic significance of glycosaminoglycan binding to IGFBP-5 altering vitronectin binding is unknown; however, cell surface associated proteoglycans have been shown to bind IGFBP-5 and to alter the biologic activity of both IGFBP-5 and IGFBP-3 (31, 32). It is possible, therefore, that both proteoglycans and vitronectin can bind to IGFBP-5 simultaneously within ECM or on cell surfaces leading to further alteration in IGF/IGFBP-5 actions.
Other proteins in ECM and in extracellular fluids may also bind to both IGFBP-5 and to vitronectin. We showed in previous studies that vitronectin would compete with PAI-1 for binding to IGFBP-5 (13). Those results suggested that this was a direct competitive interaction for the same binding site on IGFBP-5 and that it was not due to a conformational change in PAI-1 that occurred following vitronectin association.
Another physiologically significant event that alters the abundance of IGFBP-5 within the ECM is proteolysis. Although IGFBP-5 is rapidly degraded in extracellular fluids by the complement component, C1s, (33) its anchorage to ECM appears to protect it from degradation (12). Because peptides derived from vitronectin have been shown to inhibit proteolysis of IGF binding proteins (34), it is possible that vitronectin is acting to directly inhibit IGFBP-5 proteolysis in ECM or that simply the physical anchoring of IGFBP-5 to ECM sequesters its proteolytic cleavage site.
An important result of ligand binding to Vß3 is the stimulation of
matrix metalloprotease (MMP) secretion. Vitronectin binding to Vß3
results in increased secretion of MMP2 and MMP9 by endothelial
cells (35). Some investigators have proposed that this is
required for cells to migrate in response to vitronectin (36, 37), and both of these forms of MMPs have been shown to degrade
IGFBP-5 (38). Therefore, both vitronectin and
glycosaminoglycan binding to IGFBP-5 may be an important mechanism
whereby IGF-I can be made available to receptors on migrating cells
even when activated MMPs are abundant in the microenvironment. As such,
vitronectin could have a dual role of protecting proteins, such as
IGFBP-5, from degradation even though it is simultaneously
stimulating the secretion of serine proteases or MMPs that are capable
of degrading them.
The in vivo significance of these observations is unclear;
however, it is remarkable that the proteins in ECM that have been shown
to bind to IGFBP-5; that is, thrombospondin-1, osteopontin,
vitronectin, and tenascin have all be shown to bind avidly to Vß3
(5, 9, 39). The only exception to this is plasminogen
activator inhibitor-1; however, it binds to vitronectin, and therefore
could indirectly modulate IGFBP-5 actions through this mechanism. The
findings suggest that the variables that regulate the synthesis and/or
bioavailability of these proteins in the pericellular space may be
important secondary modulators of IGF-I and IGFBP-5 actions both by
enhancing the amount of IGFBP-5 within the extracellular matrix and by
direct altering their interactions with Vß3. The ability of
multifunctional matricellular proteins to alter both sets of biologic
responses may be an important characteristic for their ability to
modulate cell growth and migration following injury and as well as
during normal development.
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Acknowledgments |
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Footnotes |
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Abbreviations: ALS, Acid labile subunit; ECM, extracellular matrix; IGFBP, IGF binding protein; IRS-1, insulin receptor substrate-1; MMP, matrix metalloprotease; SMC, smooth muscle cells.
Received March 5, 2001.
Accepted for publication September 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vß3
Intergrin associates with the activated insulin and PDGF receptor and
potentiates the biologic activity of PDGF. EMBO J 16:5600–5607[CrossRef][Medline]
Vß3 integrins in the activation
of vascular endothelial cell growth factor-2 receptor. EMBO J 18:882–892[CrossRef][Medline]
Vß3 integrin inhibits IGF-I signaling in vascular
smooth muscle cells. Proc Natl Acad Sci USA 95:11217–11222Vß3 integrin is necessary for smooth
muscle cell to migrate in response to insulin-like growth factor I.
Proc Natl Acad Sci USA 93:2482–2487Vß3 antagonists inhibit IGF-I
stimulated smooth muscle cell migration and replication. Endocrinology 140:4616–4621Vß3 in vascular biology. Thromb Haemost 80:726–234[Medline]
5ß1 integrin by means of its
Arg-Gly-Asp sequence. Proc Natl Acad Sci USA 90:10553–10557Vß3 integrin. Arterioscler Thromb Vasc Biol 20:1467–1478Vß3.
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.....
, P < 0.05 compared with the cultures
treated with IGF-I alone.