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Endocrinology, doi:10.1210/en.2002-221086
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Endocrinology Vol. 144, No. 7 2807-2815
Copyright © 2003 by The Endocrine Society

Structural and Functional Evidence for the Interaction of Insulin-Like Growth Factors (IGFs) and IGF Binding Proteins with Vitronectin

Jennifer A. Kricker, Chris L. Towne, Sue M. Firth, Adrian C. Herington and Zee Upton

Tissue BioRegeneration and Integration Program (J.A.K., C.L.T., A.C.H., Z.U.), School of Life Sciences, Queensland University of Technology, Brisbane, Queensland 4001, Australia; and Kolling Institute of Medical Research (S.M.F.), University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

Address all correspondence and requests for reprints to: Jennifer Kricker, Tissue BioRegeneration and Integration Program, School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia. E-mail: j.kricker{at}qut.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies demonstrated that IGF-II binds directly to vitronectin (VN), whereas IGF-I binds poorly. However, binding of VN to integrins has been demonstrated to be essential for a range of IGF-I-stimulated biological effects, including IGF binding protein (IGFBP)-5 production, IGF type-1 receptor autophosphorylation, and cell migration. Thus, we hypothesized that a link between IGF-I and VN must occur and may be mediated through IGFBPs. This was tested using competitive binding assays with VN and 125iodine-labeled IGFs in the absence and presence of IGFBPs. IGFBP-4, IGFBP-5, and nonglycosylated IGFBP-3 were shown to significantly enhance binding of IGF-I to VN, whereas IGFBP-2 and glycosylated IGFBP-3 had a smaller effect. Furthermore, binding studies with analogs indicate that glycosylation status and the heparin-binding domain of IGFBP-3 are important in this interaction. To examine the functional significance of IGFs binding to VN, cell migration in MCF7 cells was measured and found to be enhanced when VN was prebound to IGF-I in the presence of IGFBP-5. The effect required IGF:IGFBP:VN complex formation; this was demonstrated by use of a non-IGFBP-binding IGF-I analog. Together, these data indicate the importance of IGFBPs in modulating IGF-I binding to VN and that this binding has functional consequences in cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MITOGENIC EFFECTS of IGFs are modulated by members of the IGF binding protein (IGFBP) family. These proteins have been demonstrated to both inhibit and potentiate IGF action (1). In addition to the six IGFBPs, another group of proteins, termed IGFBP-related proteins, have also been shown to bind the IGFs, however with a much lower affinity. Upton et al. (2) have reported identification of another endogenous protein complex consisting of vitronectin (VN) and IGF-II. This is particularly interesting because VN is structurally unrelated to both the IGFBPs and IGFBP-related proteins.

VN, a multifunctional protein found in plasma and extracellular matrix, is a component of the urokinase system. A number of proteins bind to VN, including glycosaminoglycans (3, 4), which bind via a heparin-binding domain (HBD) in VN, and integrins, which bind via an RGD (Arg-Gly-Asp) sequence (5, 6). It is through the binding of various proteins to these motifs, as well as other domains within VN, that diverse physiological processes such as extracellular anchoring, cell spreading, and migration are mediated (7, 8, 9).

IGF-II has been shown to bind directly to VN, whereas only minimal binding of IGF-I to VN occurs (2). Nevertheless, it is intriguing that VN seems to be critical for a number of IGF-I-related effects, including cellular DNA synthesis, type-1 IGF receptor autophosphorylation, and cell migration (10, 11, 12). More specifically, Clemmons et al. (13) have shown that VN binding to the integrin {alpha}vß3 is critical for IGF-I-stimulated smooth muscle cell migration. In addition, inhibition of IGFBP-5 binding to porcine smooth muscle cell extracellular matrix also reduces cellular responses to IGF-I (14). Furthermore, the potentiating effects of IGFBPs on IGF action seem to require interaction with as-yet-unidentified cell-surface-associated proteins, which may include VN (15). For example, IGFBP-5 has been demonstrated to facilitate binding of IGF-I to bone independently of the IGF receptors (16), and IGFBP-3 has also been shown to potentiate IGF action markedly after binding to the cell surface (17).

Given the importance of IGFBPs and VN for regulation of IGF action, we hypothesized that IGFBPs may mediate direct binding of IGFs to VN and, in particular, that IGFBPs may be necessary for functional interaction of IGF-I with VN. We now provide substantial evidence to support this hypothesis, based on studies examining binding of labeled-IGF-I and -IGF-II to VN in the absence and presence of IGFBPs, and enhanced cell migration in the presence of these complexes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
IGF-I, IGF-II, Des(1–3)IGF-I, Des(1–6)IGF-II, [Leu27]IGF-II, and IGFBP-1, -2, and -4 were purchased from GroPep Pty. Ltd. (Adelaide, South Australia, Australia). IGFBP-5, glycosylated IGFBP-3, HBD mutant IGFBP-3, and mutant nonglycosylated IGFBP-3 were produced as described previously by Firth et al. (18), whereas glycosylated IGFBP-6 was kindly donated by Dr. Leon Bach (Department of Medicine, University of Melbourne, Victoria, Australia). Human breast carcinoma (MCF-7) cells were obtained from the American Type Culture Collection (Manassas, VA; no. HTB-22). Human VN was purchased from Promega Corp. (Madison, WI). RIA grade BSA, heparin, chloramine-T, sodium metabisulfite, and Sigmacote were purchased from Sigma (St. Louis, MO). Sodium 125iodine ([125I]), Sephadex G-50, and HiTrap heparin affinity columns were obtained from Amersham Pharmacia Biotech UK Ltd. (Buckinghamshire, UK). Other chromatography equipment for radiolabeling IGFs was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Hanks’ balanced salt solution, DMEM, DMEM-Ham’s F12 (DMEM-F12), trypsin, penicillin-streptomycin, and gentamycin were purchased from Invitrogen Australia Pty. Ltd. (Mt. Waverley, Victoria, Australia), whereas fetal calf serum was from Trace Scientific (Noble Park, Victoria, Australia). Removawell Immulon-4 HB wells were from Dynex Technologies Inc. (Chantilly, VA), whereas 80-cm2 culture flasks and 24-well plates were from Nagle Nunc International (Roskilde, Denmark). Transwells were purchased from Costar (New York, NY). Autoradiographic film was purchased from Eastman Kodak Co. (Rochester, NY), whereas low-molecular-weight protein markers were obtained from Bio-Rad Laboratories, Inc. All other reagents were of analytical grade. General plastic-ware used in experiments containing IGFBPs and VN was siliconized with Sigmacote and left to air-dry overnight.

Radiolabeling of proteins
IGF-I and IGF-II were iodinated according to the chloramine-T method, as described by GroPep Pty. Ltd. for IGFs, whereas IGFBP-3 (glycosylated and nonglycosylated) was iodinated as per Dr. Janet Martin (personal communication). The chloramine-T reactions were performed for 1 min for the IGFs (10 µg) and 15 sec for IGFBP-3 (5 µg). Labeled IGFs were purified using size exclusion on Sephadex G-50, with 50 mM sodium phosphate, 150 mM NaCl, 0.25% wt/vol BSA (pH 6.5) as the elution buffer, whereas labeled IGFBP-3 was purified using heparin affinity chromatography and 50 mM sodium phosphate, 0.1% wt/vol BSA (pH 6.5) as the equilibration buffer. The protein was eluted using elution buffers 1–3, consisting of the equilibration buffer containing: 1) 0.4 M NaCl; 2) 0.75 M NaCl; and 3) 1.0 M NaCl (all at pH 6.5), respectively. Confirmation that the [125I]-IGFBP-3 was the correct molecular size and was not fragmented during the labeling procedure was obtained by nonreducing SDS-PAGE. Ten thousand counts per minute of [125I]-IGFBP-3 fractions from peaks in the iodination elution profile were run on a 4% stacking/10% separation Tris-glycine gel, dried, and then exposed to autoradiographic film for 1–7 d.

Solid-plate binding assay
IGF:VN:IGFBP binding assays were performed in removable Immulon wells coated with or without 300 ng VN in 100 µl DMEM at 37 C, 5% CO2 for 2–4 h. Wells were rinsed twice with HEPES binding buffer (t: 0.1 M HEPES, 0.12 M NaCl, 5 mM KCl, 1.2 mM MgSO4, 8 mM glucose containing 0.5% wt/vol BSA, pH 7.6) to prevent nonspecific binding. [125I]-labeled protein (IGF-I, IGF-II, glycosylated IGFBP-3, or nonglycosylated IGFBP-3) (10,000 cpm) in HBB + 0.5% BSA in the absence or presence of increasing concentrations of unlabeled IGFs (0.1–100 ng), IGF analogs (0.1–100 ng), and/or IGFBPs (0.05–100 ng) were incubated overnight at 4 C in a final vol of 100 µl (19). Unbound radiolabeled protein was then removed by aspiration, and the wells were washed three times with Hanks’ balanced salt solution. Radioactivity remaining bound in each well was then determined using a {gamma}-counter. Each sample was measured in triplicate, and the experiment was repeated at least three times. Student’s paired t test was used to compare amounts of labeled protein of test wells with the control (absence of VN and presence of tracer). Differences were significant if the P value was less than 0.05.

Transwell cell migration assay
Human breast carcinoma (MCF-7) cells were grown in DMEM-F12 media supplemented with 10% fetal calf serum, penicillin (50 U/ml), streptomycin (0.1 µg/ml), and gentamycin (1 µg/ml). Cells were grown to 70–80% confluence at 37 C in a humidified environment with 5% CO2. Cell migration assays, using Transwells, were performed using cells from passages 24–34.

The lower chambers of 12-µm pore polycarbonate-tissue-culture-treated Transwells were precoated with 1 µg VN in serum-free DMEM-F12 and incubated at 37 C for 2 h. Medium containing unbound VN was then removed, and the lower chambers were washed twice with HBB containing 0.5% BSA. IGF-I or Des(1–3)IGF-I (1–100 ng) in DMEM-F12 + 0.05% BSA was added to the lower chamber, in the absence or presence of IGFBP-5 (1000 ng), and allowed to bind to the precoated VN, overnight at 4 C. The media containing unbound growth factors was removed, and the lower chambers were washed twice with DMEM-F12 + 0.05% BSA. MCF-7 cells that had been serum-starved for 4 h were trypsinized and seeded onto the microporous membrane in the upper chamber of the Transwell inserts (200,000 cells/well) and incubated at 37 C in 5% CO2 for 5 h. Cells that had migrated to the lower surface of the porous membrane were then fixed in 37% formaldehyde and stained with 0.01% crystal violet in 0.1 mM borate buffer (pH 9). The number of cells that had migrated to the lower side of the membrane was quantitated by extracting the crystal violet stain in 10% acetic acid and determining the optical density of these extracts at 595 nm. Treatments were expressed as a percentage of the response observed on VN alone. Data were pooled from duplicate samples from three experiments, and significant differences in responses were compared with VN or, between treatments, were determined by Tukey’s analysis of multiple means. Differences were significant if the P value was less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of IGFs and IGF-II analogs on binding of [125I]-IGF-II to VN
To demonstrate that the interaction of IGF-II with VN is specific, [125I]-IGF-II binding assays were conducted in the presence of IGF-II analogs with varying affinities for IGFBPs and/or IGF receptors. Des(1–6)IGF-II (which has a low affinity for IGFBPs) (20) and [Leu27]IGF-II (which has low affinity for the type-1 IGF receptor and IGFBP-3) (21) were equipotent with native IGF-II in their ability to displace [125I]-IGF-II bound to VN (data not shown). Half-maximal competitive effects were observed at approximately 1 ng. IGF-I, on the other hand, was much less effective at displacing [125I]-IGF-II, achieving approximately a 20% reduction at 0.2 ng, with no further reduction at higher doses up to 100 ng.

Effect of IGFBPs on modulating binding of [125I]-IGF-II to VN
The ability of IGFBPs to modulate IGF-II binding to VN was then investigated (Fig. 1Go). All six IGFBPs were examined (A–F), and each IGFBP was found to compete with radiolabeled IGF-II for binding to VN. However, IGFBP-5 was only effective at the highest dose tested (100 ng). On the other hand, IGFBP-1, -2, -3, -4, and -6 competed effectively, even at the lowest doses tested (0.05 ng and 0.2 ng), although IGFBP-2 and -4 had much less dramatic effects on binding of [125I]-IGF-II to VN, compared with IGFBP-1, -3, and -6.



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  		Figure 1. Effect of IGFBPs on modulating binding of [125I]-IGF-II to VN. A–F, Radiolabeled IGF-II binding to VN in the absence and presence of IGFBPs. Ten thousand counts per minute of IGF-II tracer were added to prebound VN with increasing amounts of IGFBPs. Data are expressed as percentage of control ([125I]-IGF-II and VN alone), where 100% is approximately 4500 cpm. Each data point is the mean ± SEM of triplicate wells from three experiments that have been corrected for nonspecific binding (400 cpm). Significant differences from VN-only value are indicated by: *, P < 0.05; **, P < 0.01.

 
Effect of IGFBPs on modulating binding of [125I]-IGF-I to VN
The effect of IGFBPs on modulating binding of [125I]-IGF-I to VN was also determined using the solid plate-binding assay (Fig. 2Go). IGF-I binding was very low (380 cpm), compared with that observed with IGF-II (4500 cpm), in the absence of IGFBPs. Addition of IGFBP-1 (A) had a significant inhibitory effect on the very small amount of [125I]-IGF-I binding directly to VN. IGFBP-6 was also inhibitory, but less so. In stark contrast, IGFBP-2, -3, -4, and -5 (B–E) share similar binding patterns, whereby they enhance binding of radiolabeled IGF-I to VN by 3-fold, 2-fold, 3.5-fold, and 8-fold, respectively, at their particular optimal concentrations. Maximum binding of labeled IGF-I to VN was observed with 0.5 ng IGFBP-3, whereas maximum binding of labeled IGF-I was found at 5 ng for IGFBP-2, -4, and -5.



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  		Figure 2. Effect of IGFBPs on modulating binding of [125I]-IGF-I to VN. A–F, Radiolabeled IGF-I binding to VN in the absence and presence of IGFBPs. Ten thousand counts per minute of IGF-I tracer were added to prebound VN with increasing amounts of IGFBPs. Data are expressed as percentage of control ([125I]-IGF-I and VN alone), where 100% is approximately 380 cpm. Each data point is the mean ± SEM of triplicate wells from three experiments that have been corrected for nonspecific binding (220 cpm). In the absence of VN, [125I]-IGF-I binding to IGFBP-5 was less than that of the nonspecific binding. Significant differences from VN-only value are indicated by: *, P < 0.05; **, P < 0.01.

 
Ability of IGF peptides to compete for binding of [125I]-IGF-I to VN in the presence of IGFBP-3 or IGFBP-5
To demonstrate that the enhanced binding of [125I]-IGF-I to VN in the presence of IGFBPs was specific and involved formation of IGF-I:IGFBP:VN complex, competitive binding studies were undertaken in the presence of IGFBP-3 or IGFBP-5 with unlabeled IGF-I or the IGF-I analog, Des(1–3)IGF-I, which has very low affinity for these IGFBPs (20). Des(1–3)IGF-I was much less effective than IGF-I at competing for binding of labeled IGF-I to VN in the presence of IGFBP-3, particularly at lower doses (Fig. 3AGo). Half-maximal competition for [125I]-IGF-I binding to VN occurred at much lower levels for IGF-I (<0.1 ng) than for Des(1–3)IGF-I (0.2 ng). Nevertheless, at the highest dose tested, both peptides were able to negate any enhancing effects of IGFBP-3, in terms of facilitating binding of IGF-I to VN. In the presence of IGFBP-5 (Fig. 3BGo), Des(1–3)IGF-I was ineffective in reducing binding of [125I]-IGF-I to VN. Half-maximal displacement for IGF-I occurred at 1 ng, whereas Des(1–3)IGF-I reduced binding by only 40% at the highest dose. These data strongly suggest that the binding of [125I]-IGF-I to VN requires the formation of IGF-I:IGFBP:VN complexes.



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  		Figure 3. Ability of IGF peptides to compete for binding of [125I]-IGF-I to VN in the presence of IGFBP-3 or IGFBP-5. A, Binding of IGF-I tracer to VN in the presence of 0.5 ng IGFBP-3 with increasing amounts of either ({diamondsuit}) IGF-I or ({square}) Des(1–3)IGF-I; B, The presence of 5.0 ng IGFBP-5. Data are represented as percentage of control (IGFBP in the presence of IGF-I tracer and VN), whereby additions of IGF-I or its analog reduce the additive effects of the complex. In the absence of VN, binding of [125I]-IGF-I to IGFBP-3 or -5 was less than the value for nonspecific binding (220 cpm). Values shown are the mean ± SEM of triplicate wells from three experiments. *, P < 0.05; **, P < 0.01.

 
Importance of IGFBP-3 HBD in IGFBP-3 in mediating [125I]-IGF-I binding to VN
HBDs are commonly important for many extracellular protein interactions (22, 23). IGFBP-3 contains such a domain. To determine the role of this domain in IGFBP-3 enhancement of IGF-I binding to VN, binding studies using glycosylated IGFBP-3 and HBD mutant IGFBP-3 were undertaken (Fig. 4Go). The HBD mutant has previously been demonstrated to bind IGF-I with similar affinity to that of the wild-type IGFBP-3 (24). As demonstrated earlier (Fig. 2CGo), the presence of IGFBP-3 increases the binding of labeled IGF-I to VN by 2-fold. However, when the HBD domain of IGFBP-3 is mutated, IGFBP-3-mediated binding of [125I]-IGF-I to VN is completely negated. Indeed, [125I]-IGF-I binding is inhibited, presumably because of the sequestration of the labeled IGF-I by the mutant IGFBP-3 because it does not bind to VN. These data again indicate the need for a complex involving IGFBPs to facilitate IGF-I binding to VN.



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  		Figure 4. Importance of IGFBP-3 HBD in IGFBP-3 mediation of [125I]-IGF-I binding to VN. IGF-I tracer was added with either ( ) glycosylated IGFBP-3 or ({blacksquare}) HBD mutant IGFBP-3 to wells prebound with VN. Data are expressed as percentage of control, which is IGF-I tracer and VN alone. Values shown are the mean ± SEM of triplicate wells from three experiments. **, P < 0.01.

 
Comparison of the effects of glycosylated and nonglycosylated IGFBP-3 on [125I]-IGF-I binding to VN
To examine whether glycosylation of IGFBP-3 was important for the enhancement of IGF-I binding observed in earlier assays, binding of labeled IGF-I to VN in the presence of glycosylated IGFBP-3 or nonglycosylated IGFBP-3 was also compared (Fig. 5Go). Nonglycosylated IGFBP-3 was approximately 15 times more effective in enhancing binding of labeled IGF-I to VN than glycosylated IGFBP-3 at 0.5 ng. However, this was not solely related to the ability of glycosylated or nonglycosylated IGFBP-3 to bind to VN. Experiments with radiolabeled glycosylated and nonglycosylated IGFBP-3 indicated that there was only a 2-fold-greater binding of nonglycosylated IGFBP-3 (1066 ± 217 cpm) to VN, compared with that observed with glycosylated IGFBP-3 (560 ± 98 cpm).



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  		Figure 5. Comparison of the effects of glycosylated and nonglycosylated IGFBP-3 on [125I]-IGF-I binding to VN. IGF-I tracer was added with either ( ) glycosylated IGFBP-3 or ({square}) nonglycosylated mutant IGFBP-3 to wells prebound with VN. Data are expressed as percentage of control, which is IGF-I tracer and VN alone. Values shown are the mean ± SEM of triplicate wells from three experiments. *, P < 0.05.

 
Effects of IGF-I and IGFBP-5 on cell migration in MCF-7 breast carcinoma cells
To determine whether the enhanced binding of IGF-I to VN in the presence of IGFBP-5 has functional consequences, the effects of the complexes on stimulating cells to migrate were examined using the MCF-7 cell line. Minimal cell migration was observed in the absence of VN, regardless of the presence or absence of IGF-I, IGFBP-5, or the combination of both (data not shown). In the presence of VN, but in the absence of IGFBP-5, 1 and 3 ng IGF-I exposed to VN resulted in nonsignificant decreases in cell migration on VN by 11 ± 4 and 4 ± 6%, respectively; whereas 10, 30, and 100 ng IGF-I exposed to VN resulted in increased migration of 21 ± 10, 35 ± 14, and 47 ± 11% (Fig. 6Go). Striking differences in responses were observed with the addition of 1 µg IGFBP-5, with 1, 3, 10, 30, and 100 ng IGF-I increasing cell migration on VN between 64–78 ± 17%. These increased responses, in the presence of IGFBP-5, were significant at 1, 3, and 10 ng IGF-I (P < 0.05). Moreover, the responses were not attributable to IGF-independent effects of IGFBP-5, because the presence of this binding protein alone resulted in a significant reduction in migration, compared with VN, of 14 ± 4% (P < 0.05).



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  		Figure 6. Migration of MCF-7 cells through Transwells in response to IGF-I prebound to VN in the absence or presence of IGFBP-5. MCF-7 cells were seeded onto Transwells that had been coated with VN ± IGF-I ± IGFBP-5 and allowed to migrate through the porous membrane for 5 h. The number of cells transversing the membrane in the presence of IGF-I were then expressed as a percentage of those that migrated on VN alone. The responses are shown as IGF-I exposed to VN in the absence of IGFBP-5 ({blacksquare}), the presence of IGFBP-5 ( ), and the response of IGFBP-5 alone ({square}). Values shown are the mean ± SEM of duplicate wells from three experiments. *, P < 0.05.

 
Comparison of the IGF-I peptides on MCF-7 cell migration in the absence and presence of IGFBP-5
To examine whether the increase in cell migration, after addition of IGF-I and IGFBP-5, involved the formation of a ternary IGF-I:IGFBP-5:VN complex, responses were compared between native IGF-I and the Des(1–3)IGF-I analog that has reduced affinity for IGFBPs while retaining its ability to activate the IGF-I receptor (20). In the absence of IGFBP-5, assays with either 10 ng native IGF-I or Des(1–3)IGF-I, in the presence of VN, resulted in increased migration on VN of 19 ± 5 and 27 ± 7%, respectively (Fig. 7Go). However, only the migration of cells in the Transwells with native IGF-I treatment was significantly increased by the addition of IGFBP-5 (an increase of 71 ± 10%, compared with VN). Responses observed with the Des(1–3)IGF-I in the presence of IGFBP-5 remained unchanged (an increase of 13 ± 9%, compared with VN).



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  		Figure 7. Migration of MCF-7 cells through Transwells in response to Des(1–3)IGF-I prebound to VN in the absence or presence of IGFBP-5. MCF-7 cells were seeded onto Transwells that had been coated with VN ± IGFBP-5 ± native or mutant IGF-I and allowed to migrate through the porous membrane for 5 h. The number of cells transversing the membrane in the presence of IGF-I were then expressed as a percentage of those that migrated on VN. The responses are shown as IGF-I exposed to VN in the absence of IGFBP-5 ({blacksquare}), the presence of IGFBP-5 ( ), and the response of IGFBP-5 alone ({square}). Values shown are the mean ± SEM of duplicate wells from three experiments. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The studies reported here extend previous observations in which VN was identified as a novel high-affinity IGF-II binding protein (2) that may be responsible for mediation of many effects of IGF-II in the extracellular environment. The same earlier studies (2) revealed that IGF-I did not bind directly to VN. This was somewhat surprising, given the increasing evidence suggesting a key role for VN in mediating a number of core cellular effects of IGF-I, such as cellular DNA synthesis, type-1 IGF receptor autophosphorylation, and cell migration (10, 11, 12, 25). To explain this, we proposed that IGFBPs may be specifically required to mediate binding of IGF-I to VN.

The present study provides evidence to support our hypothesis by demonstrating that IGF-I can only interact with VN via the intermediate involvement of IGFBPs. This investigation has shown for the first time that: 1) direct binding of IGF-II to VN does not require IGFBPs but is competitively inhibited by IGFBPs; 2) IGF-I binding to VN is significantly enhanced by all IGFBPs except for IGFBP-1 and -6; and 3) the role of IGFBPs is specific since: 1) Des(1–3)IGF-I is a poor competitor for binding of labeled IGF-I to VN in the presence of IGFBPs; and 2) IGFBP-3 enhancement of IGF-I binding to VN requires an intact IGFBP-3 HBD and is affected by the glycosylation state of IGFBP-3. In addition, we have shown that the IGF:VN interaction is functionally significant (cell migration) through the interaction of IGF-I indirectly (via IGFBP-5) with VN, whereas the direct interaction of IGF-II has been shown by others in our laboratory (Noble et al., unpublished data).

IGF-II binding to VN is independent of IGFBPs, this being demonstrated by two means; first, by the equivalent competitive inhibition of [125I]-IGF-II binding by wild-type IGF-II and by two analogs, Des(1–6)IGF-II and [Leu27]IGF-II, which have reduced affinity for IGFBPs and the type-1 IGF receptor, respectively (20, 21); second, by showing that all six IGFBPs inhibit IGF-II binding to VN, at least at the higher levels tested. The most effective IGFBPs were IGFBP-1, -3, and -6. Competition for binding of IGF-II to VN may occur either by IGFBPs directly competing with IGF-II for the IGF-II binding site on VN or by the IGFBPs binding and sequestering IGF-II in solution. Our studies, to date, cannot distinguish between these possibilities for IGFBP-3. For IGFBP-1 and -6, however, because these IGFBPs also inhibited IGF-I binding to VN, it is likely that these IGFBPs bind IGF-I and/or IGF-II in solution and hence primarily sequester the IGFs away from VN. It is likely that the very efficient inhibition of IGF-II:VN binding by IGFBP-6 reflects its high affinity for IGF-II (26). Previous biochemical data (2) have demonstrated, by 2-dimensional gel electrophoresis, that the purified VN used for IGF-II binding studies was devoid of any traces of contaminating IGFBPs. This is further substantiated by the inability of IGF-I to bind to VN.

The finding that IGF-I binding to VN is markedly enhanced in the presence of IGFBPs is of particular interest. All IGFBPs, except for IGFBP-1 and -6, enhanced binding of IGF-I to VN to varying degrees. The lack of effect observed with IGFBP-6 suggests that it does not bind to VN and/or may also be an indication of its low affinity for IGF-I (26). The inhibitory effect of IGFBP-1, on the other hand, suggests that the minor amount of binding of IGF-I directly to VN was blocked by IGFBP-1, presumably via IGFBP-1 sequestration of IGF-I in solution. These data, taken together with the presence of an RGD (Arg-Gly-Asp) integrin-binding motif in IGFBP-1 (27) and the finding that IGFBP-1 can bind directly to integrins to effect cell migration and proliferation (28, 29, 30), indicate that these IGFBP-1-stimulated cellular responses are unlikely to also involve VN.

The specificity and functional significance of the requirement for IGFBPs to facilitate IGF-I binding to VN was demonstrated in several ways. First, through competitive inhibition studies, it was shown that, whereas unlabeled IGF-I was effective in reducing the enhancing effects of both IGFBP-3 and -5 on binding of [125I]-IGF-I to VN, Des(1–3)IGF-I, which has a much reduced affinity for IGFBPs, especially IGFBP-3 (20), was a great deal less effective. These data reflect the necessity for IGFBP (-3 or -5) to mediate the binding of IGF-I to VN.

Second, we have demonstrated that the HBD motif in IGFBP-3 is a critical determinant of the ability of IGFBP-3 to enhance IGF-I binding to VN. IGFBP-3-mediated binding of IGF-I to VN was abolished when the IGFBP-3 HBD region was mutated, even though the affinity of IGF-I for this IGFBP-3 variant is similar to that of the wild-type IGFBP-3 (24). The heparin-binding site in proteins such as IGFBP-3 and VN has been previously implicated in cell association processes (7, 15, 31, 32, 33, 34). This observation mirrors other recent findings that the HBD of IGFBP-3 was required for binding to fibronectin, a protein with functions similar to those of VN (35).

Likewise, IGFBP-5 has been shown to bind, via its HBD, to VN with high affinity (36) and that functional effects of IGFBP-5 required trimeric complex formation with IGF-I. This complex was found to effect IGF-I-mediated functional responses through the {alpha}vß3 integrin (36). Interestingly, these IGF-I-stimulated responses were decreased in the presence of heparin, again highlighting the involvement of IGFBP basic amino acid residues in binding to VN. Indeed, the study by Nam et al. (36), where labeled IGFBP-5 was used to examine VN:IGFBP-5 complex formation, independently validates our own findings, which demonstrate IGF:IGFBP:VN complex formation using labeled IGF-I.

Third, we have shown that the ability of IGF-I to bind to VN is markedly influenced by the glycosylation state of IGFBP-3. In various IGF/IGFBP studies, the effect of the glycosylation state of the IGFBPs has received little attention. Indeed, glycosylation is reported to play little role in IGFBP-3-mediated IGF effects (37, 38, 39). In contrast, we demonstrate here that nonglycosylated IGFBP-3 markedly enhances binding of labeled IGF-I to VN, compared with glycosylated IGFBP-3. This was attributable, in part only, to a greater (2-fold) ability of nonglycosylated to bind to VN, demonstrating that factors other than glycosylation are important. Firth and Baxter (38) have previously demonstrated that deglycosylated IGFBP-3 has a higher affinity for the cell surface. In view of the present data, this difference may reflect preferential binding of deglycosylated IGFBP-3 to VN associated with the cell surface. Though nonglycosylated IGFBP-3 would not seem to be especially relevant in the physiological context, the observations in this study suggest that use of nonglycosylated IGFBP-3 in a trimeric protein complex with IGF-I and VN may well prove to be a potent way to facilitate delivery of IGF-I to the cell surface (a phenomenon which potentially could be used in therapeutic and industrial applications to manipulate cell processes).

The findings presented here, along with those by others (25, 36, 40), provide important new insights into the mechanism by which IGF-I mediates its effects via VN and VN-binding integrins. Although it has not been specifically addressed in this study, these findings also offer an explanation as to how IGF-II and IGF-I can exert different functions, because IGF-II seems to bind directly to VN, whereas IGF-I binds indirectly via select IGFBPs. Thus, despite their structural similarity, the IGFs have clearly evolved different regulatory mechanisms to provide the capacity for different cellular functional roles.

We propose that four of the IGFBPs, namely IGFBP-2, -3, -4, and -5, enhance IGF-I binding to VN by forming a heterotrimeric complex comprised of IGF:IGFBP:VN, and that this complex is required for cellular responses. We have shown here that the proposed heterotrimeric complex involving IGFBP-5 enhances MCF-7 breast carcinoma cell migration to a significantly greater extent than either VN:IGFBP or VN:IGF binary complexes. The functional requirement for IGFBPs in the complex also has been demonstrated by showing that Des(1–3)IGF-I, which binds poorly to IGFBP-3 or -5 (20), fails to stimulate MCF-7 cell migration in this system. Together, these data confirm our hypothesis that IGFBPs are directly involved in, and indeed required for, enhancing the cellular responsiveness of IGF-I in the presence of VN.

Previous studies by Clemmons et al. (13, 14) also indicated that there is a functional and specific connection between IGF-I and VN, because blocking of the VN receptor, {alpha}vß3, inhibited IGF-I-mediated cellular responses. Grulich-Henn et al. (25) have also recently demonstrated that transport of IGF-I across endothelial cell monolayers required IGF-I interacting with VN. These investigations also suggested that VN was not likely to be a primary binding site for IGF-I and that IGFBPs could be implicated. The results from the study reported here, in which IGF-I is linked to VN via IGFBPs, can potentially explain the observation that VN is critical in a number of IGF-I-stimulated cellular responses, such as those reported by Clemmons et al. (13) and Grulich-Henn et al. (25), despite there being only minimal direct binding of IGF-I to VN (2). Together, these findings give insights as to how IGF-I can mediate diverse effects such as cell migration and cellular DNA synthesis and, moreover, suggest that VN may have a critical role in linking effects requiring both activation of integrins and the type-1 IGF receptor, as demonstrated by Maile et al. (40). Thus, the IGF:IGFBP:VN complex seems to be important in normal growth and development, and further functional and structural investigation of this complex may provide mechanisms for maintaining these physiologies in altered diseased states.


   Acknowledgments
 
We acknowledge the technical advice of Dr. Janet Martin (Kolling Institute for Medical Research, Australia) in IGFBP-3 iodinations and the generosity of Dr. Leon Bach (University of Melbourne, Department of Medicine, Australia) in providing IGFBP-6 for these studies. We would also like to acknowledge Dr. D. R. Powell (Baylor College of Medicine, Houston, TX) for helpful discussion.


   Footnotes
 
This work was supported by the Queensland Cancer Fund and the Queensland University of Technology Small Grants Scheme.

Abbreviations: HBD, Heparin-binding domain; IGFBP, IGF binding protein; VN, vitronectin.

Received October 18, 2002.

Accepted for publication March 20, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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