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Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599
Address all correspondence and requests for reprints to: David R. Clemmons, M.D., Division of Endocrinology, CB# 7170, 6111 Thurston-Bowles, Chapel Hill, North Carolina 27599-7170. E-mail: endo{at}med unc.edu.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsMaterials and MethodsResultsDiscussionReferences |
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ECM is an important component of atherosclerotic lesions. When lesions
are analyzed, both thrombonspondin-1 (TSP-1) and osteopontin (OPN) have
been shown to be preferentially synthesized by cells in lesions and to
be abundant within the ECM (8, 9, 10). Both proteins contain RGD sequences
and bind avidly to the
Vß3 integrin (11, 12).
SMC that migrate into neointima have been shown to possess abundant
Vß3 receptors, and
their migration and division are partly dependent upon
Vß3 expression
(13, 14, 15). OPN and TSP-1 have been shown to stimulate SMC migration and
division (14, 15), and it has been proposed that SMC remain in the
activated, synthetic state partly as a function of the enhanced
quantities of these specific matrix proteins within lesions (16, 17). During the course of studies to purify an IGFBP-5 protease from
pSMC-conditioned medium, IGFBP-5 affinity chromatography was used as a
purification step. A 140-kDa protein that was eluted from the affinity
column was sequenced. The first 29 residues of the amino-terminal
sequence were identical to TSP-1, suggesting that TSP-1 binds to
IGFBP-5 with relatively high affinity. Although OPN was not detected by
sequencing, it is also an abundant component of atherosclerotic lesion
ECM. As IGFBP-5 is also concentrated in lesions (18), and as it can act
to potentiate the effects of IGF-I on pSMC division (2), it was of
interest to determine whether TSP-1 or OPN would bind to IGFBP-5
and the functional consequences of IGFBP-5 binding on IGF-I
actions.
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Materials and Methods |
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Materials and Methods |
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Coimmunoprecipitation of human IGFBP-5 and TSP-1 or OPN
The ability of IGFBP-5 to bind to either OPN or TSP-1 was
analyzed by coimmunoprecipitation.
[125I]IGFBP-5 was prepared as previously
described (2). The specific activity was between 5–10 µCi/µg.
[125I]IGFBP-5 (50,000 cpm/ml) was incubated
with anti-TSP-1 (1:500 dilution) and TSP-1 (100 ng/ml) for 14 h at
4 C. The incubation buffer (250 µl) contained 30 mM
sodium phosphate (pH 7.4), 10 mM EDTA, 0.1% BSA, and 0.5%
Tween-80. The following day, normal mouse serum (0.5 µl) was added
with 3 mg protein G-Sepharose, and the mixture was centrifuged for 10
min at 12,000 x g. If OPN was to be
immunoprecipitated, the same incubation buffer was used, but OPN (200
ng/ml) was substituted for TSP-1. The anti-OPN antibody was used at a
1:250 dilution. No mouse serum was added, and protein A-Sepharose (3
mg) was substituted for protein G-Sepharose. The immune complexes were
then dissociated from either protein A- or protein G-Sepharose by
boiling the samples in 50 µl Laemmli sample buffer, and 40 µl were
removed and analyzed by SDS-PAGE, 12.5% gel. The gels were dried, and
the amount of IGFBP-5 bound was determined by autoradiography. The
amount of binding activity that could be detected using an excess of
unlabeled IGFBP-5 (500 ng/ml) was determined to assess the degree of
nonspecific binding. For quantification, the autoradiographs were
analyzed by PhosphorImager using ImageQuant SF software
(Molecular Dynamics, Inc., Sunnyvale, CA). The results are
expressed as arbitrary scanning units. In some experiments, the ability
of mutant forms of IGFBP-5 to bind to TSP-1 or OPN was determined. The
incubation conditions were identical to those previously described,
except that rather than adding unlabeled, nonmutated IGFBP-5, the
various mutants were added at a concentration of 1 µg/ml. The mutants
had had basic amino acids, between positions 201–218 within IGFBP-5,
converted to neutral residues. Some of the mutants contained amino acid
substitutions that had been shown to markedly alter their ability to
bind to ECM (2, 7). The mutants that were analyzed had received
substitutions for basic amino acids at positions K202N, R207A, and
R208A; K217A and R218A; K211N, R214A, K217A, and R218A; K207A and
K211N; R201A and K202N; K211N alone; or R214A alone. The preparation,
purification, and characterization of these mutants were previously
reported (2, 7). None had alterations in affinity for IGF-I.
In some experiments the capacity of glycosaminoglycans to compete for binding to IGFBP-5 with OPN or TSP-1 was analyzed. These compounds were added at 100 ng/ml, along with [125I]IGFBP-5 and unlabeled TSP-1 (100 ng/ml) or OPN (200 ng/ml). In other experiments, synthetic peptides that contained regions of the IGFBP-5 sequence with several charged residues were tested for the ability to compete with [125I]IGFBP-5 for binding to OPN or TSP-1. The peptides were added at a concentration of 1.0 µg/ml, then binding was determined as described above. The method of synthesis and purification as well as their amino acid sequences have been reported previously (7). The sequences are as follows: peptide A: RKGFYKRKQCKPSRGRKR; peptide B, AVKKDRRKKLT; peptide C, HALLHGRGVCLNEKS; and peptide D, RPKHTRISELKAE.
In some experiments, no [125I]IGFBP-5 was added, and the unlabeled IGFBP-1, -2, -3, or -4 (500 ng/ml) was coimmunoprecipitated with TSP-1 or OPN (1.0 µg/ml) as described above. Human IGFBP-1, -2, -3, and -4 were purified from conditioned medium from CHO cells that had been transfected with each complementary DNA as described previously (4). The method of purification and the purity of these proteins have been reported previously (1). After immunoprecipitation and separation by SDS-PAGE, the proteins were transferred to Immobilon membranes (Millipore Corp., Bedford, MA) and analyzed by Western ligand blotting using [125I]IGF-I as described previously (6, 22). [125I]IGF-I (150 µCi/µg) was added to the filters using 150,000 cpm/ml incubation buffer. The filters were washed before autoradiography as previously described (22). The results were quantified by scanning densitometry.
Measurement of binding affinity and biological actions
To determine the affinity of TSP-1 and OPN for IGFBP-5,
[125I]IGFBP-5 (40,000 cpm/tube),was incubated
with TSP-1 (100 ng/ml) or OPN (200 ng/ml). Increasing concentrations of
unlabeled IGFBP-5 (0.5–40 ng/tube) were added to duplicate tubes.
After overnight incubation, the bound
[125I]IGFBP-5 was precipitated as described
previously (6), and Scatchard analysis of the results was performed to
calculate the affinity constants. To determine whether TSP-1 or OPN
binding to IGFBP-5 would alter its affinity for IGF-I,
[125I]IGF-I (25,000 cpm/tube) was incubated
with IGFBP-5 (200 ng/ml) plus OPN (200 ng/ml) and/or TSP-1 (100 ng/ml)
and increasing concentrations of unlabeled IGF-I (0.5–100 ng/ml).
After overnight incubation, IGFBP-5 was precipitated with 6.25%
polyethylene glycol (Sigma), and the bound
[125I]IGF-I was determined as described
previously (23). The results were analyzed by the method of Scatchard.
To determine the ability of OPN and TSP-1 binding to alter the ability
of IGFBP-5 to mediate IGF-I actions, SMC were plated at 5000
cells/cm2 on 24-well plates in DMEM containing
2% FBS. Some of these plates had been precoated with TSP-1 or OPN
(2 µg/well). After 24 h to allow for attachment, the
medium was changed, and 0.5 ml fresh DMEM containing 0.2% human
platelet-poor plasma was added. After 24 h, 50 ng/ml IGF-I was
added to triplicate cultures. Additional wells also received IGFBP-5
(500 ng/ml). After 48 h, cell number was determined. Each point
represents the mean ± SD of triplicate determinations
from separate experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsMaterials and MethodsResultsDiscussionReferences |
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, [125I]IGFBP-5
bound specifically to both OPN and TSP-1, and binding could be competed
with unlabeled IGFBP-5. To determine whether the effect was specific
for IGFBP-5, unlabeled IGFBP-1 to -4 were incubated with TSP-1, and
IGFBP-1 to -3 were incubated with OPN. The amount of
[125I]IGF-I bound was determined by Western
ligand blotting. As shown in Fig. 2A, IGFBP-3 and -4 bound to TSP-1, but the nonspecific precipitation of
IGFBP-3 was high, and the signal intensity for IGFBP-4 was considerably
less than that for IGFBP-5. IGFBP-2 and -3 binding to OPN could be
detected, but, again, the signal intensities were substantially less
than that for IGFBP-5 (Fig. 2B). When the scanning densitometry results
were compared with those for IGFBP-5, the results showed that
substantially more IGFBP-5 bound to each protein compared with the
other forms of IGFBPs (Table 1). Because
of differences in the affinity of each binding protein for
[125I]IGF-I, absolute quantitative comparisons
cannot be made; however, the differences between the signal intensities
of the IGFBP-5 bands compared with those for IGFBP-2, -3, or -4
are so great (e.g. >20-fold) that they are unlikely to be
due solely to differences in affinity for IGF-I.
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). Chondroitin sulfate A and B had no
effect on binding, whereas dermatan sulfate was a weak inhibitor.
Unlike TSP-1, both heparin and heparan sulfate were potent competitive
inhibitors of IGFBP-5 binding to OPN, and chondroitin sulfate A, B, and
C had no effect (Fig. 3B).
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, the peptide termed peptide A
competitively inhibited binding to TSP-1 (the band intensity is reduced
59% compared with that in lane 2). In contrast, peptide B had no
effect on binding, and peptides C and D resulted in an increase.
Peptide A was also a potent competitive inhibitor of IGFBP-5 binding to
OPN (Fig. 4B), peptide B had a modest effect, peptide C had no effect,
and peptide D resulted in an increase. The results indicate clearly
that binding to TSP-1 and OPN is mediated primarily through the peptide
A region.
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. Mutants containing substitutions for
residues R201A, K202N or K211N, R214A, K217A, R218A, or R214A alone had
the greatest reduction in their ability to compete for binding. In
contrast, mutants containing substitutions for K202N, R206A, and R207A;
R207A and K211N; or K211N were strong competitors. When these results
were quantified by scanning densitometry, between 4–99% reductions in
binding were noted at the concentrations tested (Table 2). Therefore, residues R201 and R214
appeared to be the most important for binding to TSP-1. To determine
the amino acids that were important for binding to OPN, several mutants
were coincubated with [125I]IGFBP-5 and OPN,
and then [125I]IGFBP-5 was immunoprecipitated.
The K202N, R206A, R207A, and R201A, K202N mutants gave the greatest
reductions (Fig. 6 and Table 2). The
K211N mutant gave an intermediate result. The K217A and R218A; R214A;
and K211N, R214A, K217A, and R218A mutants showed no competition. The
R214A mutant had a major reduction in binding. This suggests that amino
acid R214 is the most important for binding to OPN and that K217 and
R218 are also important.
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To determine the effects of these proteins on the ability of IGFBP-5 to
modulate IGF-I action, the SMC growth response to IGF-I was measured in
the presence of IGFBP-5 alone or with IGFBP-5 plus either TSP-1 or OPN
added to ECM. As shown in Fig. 7, when
either OPN or TSP-1 was added with IGFBP-5, there was a significantly
enhanced cell growth response compared with that to OPN plus IGF-I,
TSP-1 plus IGF-I, or IGFBP-5 plus IGF-I. This suggests that both
proteins not only enhance IGF-I action, but also are interacting to
further enhance the cellular responses to IGF-I and IGFBP-5.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsMaterials and MethodsResultsDiscussionReferences |
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Vß3 integrin. This
binding occurs through their RGD sequences and can be blocked by
disintegrins. In addition, both TSP-1 and OPN have other cell-binding
domains and heparin-binding domains, which may be active in binding
heparan sulfate-containing proteoglycans, and both proteins have been
shown to bind to other proteins that are present in the ECM and on cell
surfaces (26, 27, 28, 29, 30). TSP-1 has the additional property of binding to
integrin-activating protein, a protein that binds
Vß3 and alters its
activity (31). Because both OPN and TSP-1 are abundant within ECM, and because the expression and ECM content of both are increased during the development of atherosclerotic lesions, we were interested in whether they would bind to IGFBP-5. Our interest was further stimulated by the fact that IGFBP-5 is the only form of IGFBP that binds preferentially to ECM (3), and the amount of IGFBP-5 within the ECM appears to be an important determinant of the ability of IGF-I to optimally stimulate SMC DNA synthesis (2, 3). In these studies we determined that both TSP-1 and OPN bind to IGFBP-5 with high affinity. However, unlike binding to ECM, the association of TSP-1 or OPN with IGFBP-5 did not significantly alter their affinities for IGF-I. This suggests that the decrease in the affinity of IGFBP-5 for IGF-I that is seen after IGFBP-5 association with ECM may be due to multivalent binding to several proteins, rather than to a single protein, or that a specific site in IGFBP-5 has to be occupied to alter its affinity for IGF-I and that that site is not involved in binding to TSP-1 or OPN. Alternatively, it could be due to the fact that adhesion to ECM results in an insoluble form compared with binding to OPN and TSP-1 in solution. This absence of a change in affinity was also noted when IGFBP-5 bound to PAI-1 in solution (6). Because of their abundance in ECM and their relatively high affinities for IGFBP-5, we conclude that these two proteins are important determinants of the amount of IGFBP-5 that localizes within the pSMC ECM.
The functional significance of binding was analyzed by determining the
ability of the combination of IGF-I, IGFBP-5, and TSP-1 or OPN to
stimulate cell growth. As shown in Fig. 7
, both TSP-1 and OPN
potentiated the cellular response to IGF-I. The effect of TSP-1 is not
surprising in view of our previously published findings that TSP-1,
when added with IGF-I, results in enhanced IGF-I receptor kinase
activity and increased phosphorylation of the principal signaling
element distal to the receptor, insulin receptor substrate-1 (32).
Furthermore, we have shown that blocking occupancy of the
Vß3 integrin by
ligands, such as vitronectin or TSP-1, with disintegrins, such as
echistatin, results in complete attenuation of IGF-I signaling (32).
Therefore, it is not surprising that both TSP-1 and OPN potentiated
IGF-I stimulation of cell growth. However, their interaction with
IGFBP-5 is another potential mechanism by which this process may be
controlled. In other studies we have shown that IGFBP-5 binding to ECM
also results in a potentiation of the cell growth response to IGF-I
(3). Our finding that the addition of either TSP-1 or OPN to ECM
results in further enhancement of the SMC growth response to IGF-I plus
IGFBP-5 suggests that these proteins bind IGFBP-5 within the ECM, thus
allowing an increased amount of IGF-I to be released to receptors over
an extended time period. This enhanced receptor stimulation could also
function with the additional TSP-1 and OPN to further stimulate
Vß3 and thus to
enhance IGF-I-stimulated actions (32). Thus, TSP-1 and OPN fulfill dual
roles of enhancing the amount of IGFBP-5 within ECM and binding to
Vß3, which facilitates
IGF-I-stimulated receptor activation. This conclusion is also further
supported by our observation that transfection of SMC with IGFBP-5
mutants that suppress the synthesis of endogenous wild-type IGFBP-5 and
are not bound to ECM results in no potentiation of IGF-I-stimulated DNA
synthesis in this cell type (2). This suggests that a basal amount of
IGFBP-5 needs to be associated with ECM to achieve optimum IGF-I
responsiveness and that the cellular response to IGF-I is proportional
to the amount of IGFBP-5 within the ECM. To definitively quantify the
amount of enhancement of IGF-I action that is due to binding of TSP-1
or OPN to Vß3 compared
with their abilities to increase the amount of IGFBP-5 within the ECM,
the effects of IGFBP-5 mutants that have low affinity for OPN or TSP-1
need to be compared with those of IGFBP-5 mutants that have normal
affinity for these proteins but low affinity for ECM.
The variables that determine the binding of OPN or TSP-1 to IGFBP-5 were analyzed. The binding to both proteins is perturbed by heparan sulfate. In contrast, the addition of heparin increased IGFBP-5 binding to TSP-1, but decreased binding to OPN. This suggests that sulfated glycosaminoglycans may have more complex effects on the TSP-1- IGFBP-5 interaction than they do on OPN binding to IGFBP-5, wherein it simply appears that the glycosaminoglycan-binding domain of OPN is directly involved in IGFBP-5 binding. Although TSP-1 has a heparin-binding domain, simple inhibition of IGFBP-5 binding by heparin could not be demonstrated, suggesting that after heparin binding, TSP-1 undergoes a conformational change that alters its interaction with IGFBP-5. In contrast, the ability of heparan sulfate to inhibit IGFBP-5 binding to both proteins suggests that heparan sulfate is binding to the glycosaminoglycan-binding domain of IGFBP-5 and inhibiting its ability to interact with either protein (5). This was further confirmed by using peptides containing specific sequence domains of IGFBP-5. Peptide A, which spans the glycosaminoglycan-binding region of IGFBP-5, was a potent inhibitor of interaction with either TSP-1 or OPN.
Further domain specificity was determined using a series of IGFBP-5 mutants that had had basic residues within the peptide A sequence altered. For TSP-1, residues R201 and R214 appeared to be the predominate residues that were required for optimum binding. In contrast, for OPN, residue R201 was not important. R214 was the most important, and residues K217 and R218 were also very important for optimum binding. Some experiments suggested that K211 was important, but this result was not consistent. These are somewhat different from the residues that are most important for binding to total ECM, which are predominantly R207 and R214, although R201, K217, and R218 have been shown to contribute to ECM binding. This suggests that specific sequences of basic residues within the peptide A domain are required for optimum binding to these proteins.
In atherosclerosis there is an accumulation of TSP-1 and OPN within
lesions. These proteins can bind to the SMC surface as well as the ECM
through both specific cell surface receptors, such as
Vß3, and through cell
surface proteins, such as integrin-activating protein. As IGFBP-5 can
also associate with cell surfaces, these interactions may provide a
molecular scaffold by which significant amounts of IGFBP-5 and IGF-I
may be concentrated focally near IGF-I surface receptors. Our in
vitro cell growth results suggest that both proteins are
potentially important modulators of IGF-I actions in vivo.
Further studies will be required to determine whether either protein
has a role in modulating the effect of IGF-I on lesion development and
the mechanisms by which they participate in this process.
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Acknowledgments |
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Footnotes |
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Received August 17, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsMaterials and MethodsResultsDiscussionReferences |
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Vß3
function through integrin associated protein. J Cell Biol 135:533–544Vß3 integrin inhibits IGF-I signaling in vascular smooth
muscle cells. Proc Natl Acad Sci USA 95:11217–11222This article has been cited by other articles:
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Q. Xu, B. Yan, S. Li, and C. Duan Fibronectin Binds Insulin-like Growth Factor-binding Protein 5 and Abolishes Its Ligand-dependent Action on Cell Migration J. Biol. Chem., February 6, 2004; 279(6): 4269 - 4277. [Abstract] [Full Text] [PDF]
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 L. A. Maile and D. R. Clemmons Integrin-Associated Protein Binding Domain of Thrombospondin-1 Enhances Insulin-Like Growth Factor-I Receptor Signaling in Vascular Smooth Muscle Cells Circ. Res., November 14, 2003; 93(10): 925 - 931. [Abstract] [Full Text] [PDF]
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L. A. Maile, J. Badley-Clarke, and D. R. Clemmons The Association between Integrin-associated Protein and SHPS-1 Regulates Insulin-like Growth Factor-I Receptor Signaling in Vascular Smooth Muscle Cells Mol. Biol. Cell, September 1, 2003; 14(9): 3519 - 3528. [Abstract] [Full Text] [PDF]
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 S. M. Firth and R. C. Baxter Cellular Actions of the Insulin-Like Growth Factor Binding Proteins Endocr. Rev., December 1, 2002; 23(6): 824 - 854. [Abstract] [Full Text] [PDF]
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 C. McCaig, C. M. Perks, and J. M. P. Holly Intrinsic actions of IGFBP-3 and IGFBP-5 on Hs578T breast cancer epithelial cells: inhibition or accentuation of attachment and survival is dependent upon the presence of fibronectin J. Cell Sci., November 15, 2002; 115(22): 4293 - 4303. [Abstract] [Full Text] [PDF]
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Y. G. Amaar, G. R. Thompson, T. A. Linkhart, S.-T. Chen, D. J. Baylink, and S. Mohan Insulin-like Growth Factor-binding Protein 5 (IGFBP-5) Interacts with a Four and a Half LIM Protein 2 (FHL2) J. Biol. Chem., March 29, 2002; 277(14): 12053 - 12060. [Abstract] [Full Text] [PDF]
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S. R. Rittling, Y. Chen, F. Feng, and Y. Wu Tumor-derived Osteopontin Is Soluble, Not Matrix Associated J. Biol. Chem., March 8, 2002; 277(11): 9175 - 9182. [Abstract] [Full Text] [PDF]
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 E. Tonner, M. C. Barber, G. J. Allan, J. Beattie, J. Webster, C. B. A. Whitelaw, and D. J. Flint Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice Development, January 10, 2002; 129(19): 4547 - 4557. [Abstract] [Full Text] [PDF]
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T. Nam, A. Moralez, and D. Clemmons Vitronectin Binding to IGF Binding Protein-5 (IGFBP-5) Alters IGFBP-5 Modulation of IGF-I Actions Endocrinology, January 1, 2002; 143(1): 30 - 36. [Abstract] [Full Text] [PDF]
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B. P. Eliceiri Integrin and Growth Factor Receptor Crosstalk Circ. Res., December 7, 2001; 89(12): 1104 - 1110. [Abstract] [Full Text] [PDF]
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D. R. Clemmons Use of Mutagenesis to Probe IGF-Binding Protein Structure/Function Relationships Endocr. Rev., December 1, 2001; 22(6): 800 - 817. [Abstract] [Full Text] [PDF]
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W. H. Busby Jr., T.-J. Nam, A. Moralez, C. Smith, M. Jennings, and D. R. Clemmons The Complement Component C1s Is the Protease That Accounts for Cleavage of Insulin-like Growth Factor-binding Protein-5 in Fibroblast Medium J. Biol. Chem., November 22, 2000; 275(48): 37638 - 37644. [Abstract] [Full Text] [PDF]
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