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Endocrinology Vol. 147, No. 9 4400-4409
Copyright © 2006 by The Endocrine Society

Insulin-Like Growth Factor Binding Protein-3 in Extracellular Matrix Stimulates Adhesion of Breast Epithelial Cells and Activation of p44/42 Mitogen-Activated Protein Kinase

Janet L. Martin and Stan Jambazov

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

Address all correspondence and requests for reprints to: Janet L. Martin, Ph.D., Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: janetlm{at}med.usyd.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-binding protein-3 (IGFBP-3) is a multifunctional protein that regulates the potent mitogenic and antiapoptotic effects of IGF-I and IGF-II and exerts bioactivity independent of modulating IGF receptor activation. Previous studies have shown that in solution, IGFBP-3 binds constituent proteins of the extracellular matrix (ECM) such as fibronectin and collagen and is present in ECM deposited by fibroblasts in vitro; however, binding of IGFBP-3 to matrix has not been characterized, nor has its function in this environment been investigated. In this study, we show that IGFBP-3 binds to ECM deposited by human breast epithelial and cancer cells and neonatal human fibroblasts. IGF-I and heparin blocked binding of IGFBP-3 to matrix when added with the binding protein but were unable to displace IGFBP-3 already bound to the matrix. IGF-I bound to matrix-immobilized IGFBP-3 with approximately 25-fold reduced affinity compared with IGFBP-3 in solution. Mutation of the C-terminal basic domain of IGFBP-3 (228KGRKR->MDGEA) resulted in markedly reduced binding to matrix compared with wild-type IGFBP-3, whereas mutation of the adjacent consensus heparin-binding domain (220KKK->HSR) had relatively little effect. In the presence of matrix-bound IGFBP-3, adhesion of breast epithelial cells was increased by approximately 25%, and activation of the signaling pathway intermediate p44/42 MAPK was enhanced greater than 3-fold. These results indicate a previously unrecognized and potentially important role for IGFBP-3 in the extracellular matrix.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-BINDING PROTEIN-3 (IGFBP-3) is a member of the family of six IGFBPs named for their high-affinity binding to the mitogenic growth factors IGF-I and IGF-II and biologically significant for their regulation of this important growth system in a wide range of cell and tissue types (1, 2). Present at high concentrations in the circulation, IGFBP-3 moderates the potent insulin-like activity of the IGFs through their sequestration into binary complexes and, in the presence of the acid-labile subunit, biologically inert ternary complexes that have a long circulating half-life and impaired ability to move from the vasculature to target tissues (3). At the tissue level, IGFBP-3 modulates the interaction of IGFs with the signaling type I IGF receptor (IGFR1) and the IGF-II/mannose-6-phosphate receptor. Although IGFBP-3 is predominantly inhibitory to IGF action at the tissue level, there is also good evidence that in certain cell types and growth conditions, it may enhance the potent growth-promoting effects of IGF-I and IGF-II (4, 5, 6).

A number of studies have revealed that IGFBP-3 and other IGFBPs exert bioactivity independent of their ability to modulate interaction of IGFs with their receptors (7, 8, 9). IGFBP-3 may inhibit proliferation or promote apoptosis per se or enhance the apoptotic effects of other agents by modulating expression of pro- and antiapoptotic effectors such as Bad, Bax, and Bcl-2 (10) leading to activation of caspase 3 (11). IGFBP-3 is also reported to activate a phosphotyrosine phosphatase that down-regulates IGFR1 signaling pathways, thus impacting on IGF signaling via a mechanism unrelated to its direct binding of IGFs (12). The functional diversity of IGFBP-3 is further exemplified by studies showing that it may also promote cellular growth and proliferation, again apparently independently of modulating IGF signaling. This may be brought about through its enhancement of other signaling systems, such as the epidermal growth factor (EGF) receptor signaling system (13, 14), although the mechanisms involved remain unclear.

Interaction of IGFBP-3 with monolayers of cells in vitro has been demonstrated by a number of groups (5, 7, 8, 15), where it appears to correlate with some physiological parameters including apoptosis and growth inhibition (7, 8). It has been suggested that this reflects the existence of functional receptors for IGFBP-3 (7, 8), but signal transduction by any protein in response to direct binding of IGFBP-3 has not been demonstrated. Despite considerable effort to isolate, identify, and characterize the molecules involved in cell surface association of IGFBP-3 and define their role in IGFBP-3 action, this phenomenon remains poorly understood.

A second explanation for association of IGFBP-3 with cells in monolayer culture may lie in its potential for interaction with extracellular matrix (ECM). ECM is expressed and deposited by cells and plays a crucial role in the maintenance of cellular integrity and function by interacting with cell surface receptors to modulate cell communication, adhesion, migration, proliferation, and survival (16). A study of human fetal fibroblasts indicated the presence of small amounts of IGFBP-3 in ECM laid down by these cells (17), although the binding characteristics and function of matrix-bound IGFBP-3 were not studied further. ECM binding of other IGFBPs, most notably IGFBP-5 and IGFBP-2, has been demonstrated (17, 18, 19), and when matrix-bound, these proteins impact on cellular growth and proliferation. In this study, we have characterized the binding of IGFBP-3 to matrix laid down by breast epithelial and other cells in vitro and show that matrix-bound IGFBP-3 increases cell attachment and enhances MAPK signaling. This suggests an important, previously unrecognized role for IGFBP-3 in the ECM.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture reagents and plastic ware were purchased from Trace Biosciences (North Ryde, New South Wales, Australia) and Nunc (Roskilde, Denmark). BSA, bovine insulin, hydrocortisone, EGF, and heparin (sodium salt) were purchased from Sigma Chemical Co. (St. Louis, MO), and cholera enterotoxin was from ICN Biomedicals Australasia (Seven Hills, New South Wales, Australia). Recombinant human IGF-I and Leu24Ala31-IGF-I were the generous gifts of Genentech, Inc. (South San Francisco, CA). IGF-I analogs Long Arg3-IGF-I (LR3-IGF-I) and des(1–3)-IGF-I were purchased from GroPep (Adelaide, South Australia). Antibodies directed against phospho-Thr202/Tyr204 and total p44/42 MAPK were from Cell Signaling Technology (Beverley, MA), and {alpha}-tubulin antibody was from Sigma. Recombinant wild-type and mutant human IGFBP-3 and IGFBP-5 were expressed in human 911 retinoblastoma cells using an adenoviral expression system and purified by IGF-I-affinity chromatography and reverse-phase HPLC as previously described (20, 21). Electrophoresis and enhanced chemiluminescence (ECL) reagents were purchased from Bio-Rad (Hercules, CA), Amrad-Pharmacia (Ryde, New South Wales, Australia), and Pierce (Rockford, IL). IGF-I and protein A were radiolabeled with [125I]sodium iodide (ICN) using chloramine T and purified by size exclusion chromatography.

Preparation of radiolabeled IGFBP-3
Plasma-derived or recombinant human IGFBP-3 were radiolabeled with [125I]sodium iodide using chloramine T and then purified by heparin Sepharose chromatography. A 1-ml column of heparin Sepharose 6B was equilibrated in 50 mM sodium phosphate buffer containing 2.5 g/liter BSA and 0.15 M NaCl. The iodinated IGFBP-3 was applied to the column and run in with 200 µl buffer, and then the column was clamped for 1 h. The column was washed with buffer, and then the 30-kDa proteolyzed fragment of IGFBP-3 was eluted with buffer containing 0.4 M NaCl. Intact 43-kDa IGFBP-3 was eluted with buffer containing 0.75 M NaCl. Purity of the tracer preparations was confirmed by SDS-PAGE and autoradiography.

Cell culture
MCF-10A breast epithelial cells were maintained in DMEM/F12 containing 5% horse serum, 10 µg/ml insulin, 100 ng/ml cholera enterotoxin, 10 ng/ml EGF, and 0.5 µg/ml hydrocortisone, as previously described (22). Cells were passaged by trypsinization every 5–7 d. MCF-7 and Hs578T breast cancer cells obtained from American Type Culture Collection (Rockville, MD) and neonatal human fibroblast cultures established as previously described (15) were maintained in RPMI containing 5% fetal calf serum and 10 µg/ml insulin.

Preparation of matrix from cultured cells
Monolayer cultures of cells in flasks or multiwell plates were used to prepare extracellular matrix in situ. Confluent cells were changed from serum-containing medium to serum-free medium for 48 h before matrix preparation and then washed twice with PBS. Cells were lysed in 0.25 M NH4OH for 30 min at 37 C, and then lysates were aspirated from wells. Adhered matrix was washed five times with large volumes of sterile water and then once with 75% ethanol and air dried. Plates were stored at 4 C until use.

Matrix binding assays
Binding of IGFBP-3 to immobilized matrix was determined immunologically, as previously described for cells in monolayer culture (15). Briefly, matrix in 48-well plates was incubated with IGFBP-3, with or without additional peptides as indicated for individual experiments, overnight at 22 C in serum-free DMEM/F12 containing 1 g/liter BSA. Wells were washed with serum-free medium and incubated for another 2 h with anti-IGFBP-3 antiserum (R30, 1/2000 final dilution) in serum-free medium. Wells were again washed, and [125I]protein A (25,000 cpm/200 µl) was added for 2 h at room temperature. After washing, matrix was solubilized in 5 g/liter SDS overnight and then counted in a {gamma}-counter (Hewlett Packard).

An alternative protocol was carried out for competitive binding assays, using radiolabeled [125I]IGFBP-3. Matrix immobilized in 48-well plates was incubated overnight with [125I]IGFBP-3 (10,000 cpm/well), with or without unlabeled IGFBP-3, diluted in serum-free DMEM/F12 containing 2.5 g/liter BSA. Wells were washed in medium, and matrix was solubilized with 5 g/liter SDS overnight before counting.

Affinity labeling of IGFBP-3 to matrix
To characterize IGFBP-3 binding sites in matrix, MCF-10A matrix was prepared as described above in 75-cm2 flasks and incubated with serum-free medium containing 1 g/liter BSA with or without 100 ng/ml IGFBP-3 overnight at 37 C. Disuccinimidyl suberimidate was added to a final concentration of 0.25 mM, and flasks were incubated at 22 C for 30 min. Media were removed, and the matrix was washed briefly with PBS before addition of 1 ml SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, containing 20 g/liter SDS, 10% glycerol, and 0.1 g/liter bromphenol blue) to solubilize matrix. Samples were stored frozen until analysis by 7.5% SDS-PAGE and IGFBP-3 immunoblotting, as described below.

Cell attachment assays
Attachment of cells to immobilized matrix was carried out in six-well plates. To determine the effect of matrix-bound IGFBP-3, wells were preincubated with IGFBP-3 diluted in serum-free DMEM/F12 containing 1 g/liter BSA overnight at 37 C. Media were removed, and wells were washed with serum-free medium. Cells were prepared for attachment assays by trypsinization and diluted in serum-free DMEM/F12 to a concentration of 5 x 105 cells/ml. One milliliter of cells was added to each well, and plates were immediately returned to 37 C. At 5 and 15 min, unattached cells were removed and counted. Attached cells were solubilized in SDS-PAGE sample buffer containing 50 mM dithiothreitol at 4 C for 10 min, scraped into chilled Eppendorf tubes, and stored at –80 C before electrophoretic analysis.

SDS-PAGE and Western blotting
Cell lysates were resolved on 10 or 12% SDS-polyacrylamide gels and transferred to Hybond C nitrocellulose for Western analysis, as previously described (13). After transfer, filters were blocked in 50 g/liter skim milk powder in Tris-buffered saline with Tween 20 (TBS-T: 10 mM Tris, 150 mM NaCl, pH 7.4, containing 1 ml/liter Tween 20) and probed with anti-phospho-Thr202/Tyr204 p44/42 MAPK or total MAPK (1/1000 final dilution) at 4 C for 16 h. Filters were washed three times for 10 min each in cold TBS-T and then incubated with horseradish-peroxidase-labeled donkey antirabbit secondary antibody for 1–2 h at room temperature. Filters were washed and developed by ECL using Pierce reagents. Filters were then stripped using Restore stripping reagent (Pierce) and reprobed with {alpha}-tubulin antibody to confirm equal loading of total protein. For IGFBP-3 immunoblots, samples were resolved by 7.5 or 10% SDS-PAGE, transferred to nitrocellulose, blocked, and probed with anti-IGFBP-3 antibody R30 at 1/10,000 dilution in 10 g/liter BSA in TBS-T followed by secondary antibody (1/20,000 dilution) before ECL development.

Statistics
All experiments were performed at least twice, and individual experiments were carried out in triplicate or quadruplicate. Statistical analysis was performed using Statview for Macintosh (SAS Institute, Cary, NC) using ANOVA with Fisher’s protected least-significant differences. Differences were considered statistically significant where P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Binding of IGFBP-3 to matrix deposited by MCF-10A breast epithelial cells was initially determined immunologically as previously described for cell-associated IGFBP-3 in monolayer cultures (15). IGFBP-3 over the concentration range 0.3–100 nM (12–4000 ng/ml) was applied to matrix immobilized on 48-well plates and then detected by sequential addition of IGFBP-3 antiserum and [125I]protein A. As shown in Fig. 1AGo, a dose-dependent increase in IGFBP-3 binding to MCF-10A matrix was apparent over this concentration range, with half-maximal binding at approximately 5 nM IGFBP-3 and maximal binding at 10–20 nM IGFBP-3. To examine the IGFBP specificity of this binding, competitive binding curves were set up in which matrix was incubated with 2.5 nM IGFBP-3 in the presence of increasing concentrations of IGFBP-2 or IGFBP-5. As shown in Fig. 1BGo, IGFBP-5 showed some competition with IGFBP-3 for binding to matrix, but IGFBP-2 did not, even at 40-fold higher concentrations. Similarly, IGFBP-6 was unable to compete with IGFBP-3 for matrix binding (not shown). A time course of binding of radiolabeled IGFBP-3 to MCF-10A matrix revealed very rapid association with the matrix, such that 20% of maximal binding occurred within 5 min and 60% within 1 h; binding was maximal (~80% of added tracer) after 3 h incubation with matrix at 22 C (data not shown).


Figure 1
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  		FIG. 1. IGFBP-3 binds to MCF-10A matrix. A, MCF-10A matrix in 48-well plates was incubated with IGFBP-3 diluted to the indicated concentrations in serum-free medium overnight at 22 C. Bound protein was detected using anti-IGFBP-3 antibody (R30, 1/2000 final dilution) and 25,000 cpm [125I]protein A, as described in Materials and Methods. Results are shown as the percentage of total added [125I]protein A recovered in the solubilized matrix. Results shown are mean ± SE of quadruplicate determinations in one of five similar experiments. B, MCF-10A matrix in 48-well plates was incubated with 2.5 nM IGFBP-3 (100 ng/ml) in the presence of IGFBP-2 (bullet) or IGFBP-5 ({square}) in medium overnight at 22 C. Bound IGFBP-3 was detected as indicated for A. Results are shown as percentage of total added [125I]protein A recovered in the solubilized matrix and are mean ± SE of quadruplicate determinations in one of two similar experiments.

 
Partial characterization of the IGFBP-3 binding sites on ECM was achieved by cross-linking IGFBP-3 to matrix and then solubilizing the matrix and analyzing IGFBP-3 by Western blot. This revealed that in addition to a diffuse signal at approximately 43 kDa representative of non-cross-linked IGFBP-3, four bands of approximate molecular mass 80, 100, 140, and more than 200 kDa were detected (Fig. 2AGo). None of these bands were apparent in identical blots probed with nonimmune serum (not shown). To determine whether the IGFBP-3 component of these cross-linked species represented intact IGFBP-3 or a proteolyzed fragment of the protein, IGFBP-3 was incubated with matrix that was then solubilized without cross-linking and analyzed by SDS-PAGE and immunoblot for IGFBP-3. As shown in Fig. 2BGo, the IGFBP-3 recovered from the matrix was predominantly intact 40- to 43-kDa doublet IGFBP-3, although an additional minor band of approximately 30 kDa was also apparent that was not present in the preparation of IGFBP-3 added to the cells (Input). Analysis of the medium removed from the matrix after the incubation period revealed the presence of considerably more of this 30-kDa species, with an additional band of approximately 21 kDa also evident. These data suggest that proteolysis of IGFBP-3 was occurring during incubation with isolated matrix. The matrix metalloproteinase (MMP) ADAM-12, which is inhibitable by chelation of divalent cations, has been shown to proteolyze IGFBP-3 to fragments of approximately 30 and 21 kDa (23). However, incubation of matrix with IGFBP-3 in the presence of EDTA did not prevent the generation of the 30- and 21-kDa IGFBP-3 fragments (Fig. 2BGo), suggesting that ADAM-12, or other MMPs, are not responsible for IGFBP-3 cleavage by MCF-10A matrix.


Figure 2
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  		FIG. 2. IGFBP-3 binds to multiple sites in matrix and is proteolyzed by matrix. A, IGFBP-3 (100 ng/ml) was incubated with MCF-10A matrix in 75-cm2 flasks overnight at 37 C, and then cross-linking of proteins was carried out for 30 min at 22 C as described in Materials and Methods. Matrix was solubilized in Laemmli sample buffer, fractionated by 7.5% SDS-PAGE, and then immunoblotted with anti-IGFBP-3 antiserum R30 (1/10,000 final dilution) and ECL. The migration distances of molecular mass markers are shown on the left (kilodaltons). The positions of affinity-labeled bands are indicated by open arrowheads. B, IGFBP-3 in serum-free medium was incubated with matrix as described above in the absence or presence of 20 mM EDTA as indicated. Media were removed for analysis, and then matrix was solubilized in Laemmli buffer (without cross-linking). Solubilized matrix and matrix-exposed medium (medium post-matrix) were applied to 10% gels and then immunoblotted with IGFBP-3 antiserum. Input refers to medium containing IGFBP-3 incubated under identical conditions (37 C overnight) in the absence of matrix. The migration distances of molecular mass markers in kilodaltons are shown.

 
We examined whether IGFBP-3 could bind to matrix laid down by other cell types. Matrix derived from neonatal foreskin fibroblasts, the estrogen-receptor-negative breast cancer cell line Hs578T, and the estrogen-receptor-positive breast cancer cell line T47D was prepared in 48-well plates, and then IGFBP-3 binding was assessed. As shown in Fig. 3AGo, IGFBP-3 bound to matrix deposited by the breast cancer cells with similar potency as to MCF-10A epithelial cells, whereas binding to fibroblast-derived matrix was slightly lower. Competitive binding curves using 125I-labeled IGFBP-3 and unlabeled IGFBP-3 revealed further subtle differences between matrix derived from the different cells. Whereas cold IGFBP-3 competed with radiolabeled peptide for binding to MCF-10A, fibroblast and T47D matrix, binding of [125I]IGFBP-3 to Hs578T matrix was poorly competed by IGFBP-3 (Fig. 3BGo). In addition, a slight increase in tracer binding at low concentrations of competing IGFBP-3 was apparent for the MCF-10A, fibroblast, and T47D matrix but not Hs578T.


Figure 3
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  		FIG. 3. Binding of IGFBP-3 to matrix of other cell types. A, Matrix was prepared from MCF-10A cells (bullet), neonatal fibroblasts ({circ}), Hs578T breast cancer cells ({square}), or T47D breast cancer cells ({triangleup}). Binding of IGFBP-3 at the indicated concentrations was determined immunologically as described for Fig. 1Go. Results are shown as percentage of total added [125I]protein A recovered in the solubilized matrix and are the mean ± SE of quadruplicate determinations in one of three similar experiments. B, Matrices derived from the cells indicated in A were incubated with increasing concentrations of IGFBP-3 in the presence of 10,000 cpm [125I]IGFBP-3 overnight at 22 C and then washed and solubilized for counting as described in Materials and Methods. Results are shown as percent control, where the percentage of tracer bound in the absence of cold IGFBP-3 is expressed as 100% for each matrix type. Bound/total counts were 20–25% for each matrix. Symbols are the same as indicated for A. Data points shown are mean ± SE of quadruplicate determinations in one of three similar experiments.

 
We then determined whether IGF-I affected binding of exogenous IGFBP-3 to matrix. These experiments revealed that IGF-I was able to prevent binding of 2.5 nM IGFBP-3 to matrix (Fig. 4Go) with a maximal effect apparent at a 2-fold molar excess of IGF-I. An IGF analog with near normal affinity for IGFBP-3 but very weak binding to IGFR1, Leu24Ala31-IGF-I (24), was also able to prevent IGFBP-3 binding to matrix, although with slightly reduced potency compared with wild-type IGF-I. Surprisingly, LR3-IGF-I, an IGF analog that has relatively normal IGFR1 binding but markedly reduced affinity for IGFBPs (24), was also able to inhibit binding of IGFBP-3 to MCF-10A matrix but again with reduced potency compared with either wild-type IGF-I or Leu24Ala31-IGF-I. This was not a nonspecific effect of the growth factor because EGF at the same concentration had no effect on IGFBP-3 binding to matrix (Fig. 4Go).


Figure 4
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  		FIG. 4. IGFs block binding of IGFBP-3 to MCF-10A matrix. IGFBP-3 (2.5 nM) was bound to matrix in the presence of wild-type IGF-I (white bars), Leu24Ala31-IGF-I (diagonal stripes), LR3-IGF-I (shaded), or EGF (vertical bars) at the indicated concentrations overnight at 22 C, and then matrix-bound IGFBP-3 was determined by addition of antibody and [125I]protein A as described in Materials and Methods. Results are shown as percentage of total added [125I]protein A recovered in the solubilized matrix. Data points shown are mean ± SE of pooled data from three similar experiments each performed in quadruplicate. *, P < 0.0001 compared with control (no addition); ¶, P < 0.001 compared with wild-type IGF-I.

 
We have previously shown that cell-associated IGFBP-3 in monolayer cultures can be displaced by IGF-I (15) and examined whether matrix-bound IGFBP-3 could similarly be dissociated. We found, unexpectedly, that even when IGF-I was added at 10-fold molar excess to IGFBP-3 prebound to matrix, it was unable to displace IGFBP-3 from the matrix (data not shown). We investigated whether this was because the matrix-bound IGFBP-3 was sequestering the added IGF-I. IGFBP-3 was bound to matrix overnight, and then radiolabeled IGF-I was added for 4 h. After washing, matrix was solubilized and counted. As shown in Fig. 5AGo, IGF-I tracer bound to matrix-immobilized IGFBP-3, and this was fully competed by unlabeled IGF-I added with the tracer. Scatchard analysis of competitive binding curves generated by incubating matrix-immobilized IGFBP-3 with radiolabeled IGF-I in the presence of increasing concentrations of un-labeled peptide (Fig 5BGo) indicated an association constant of 1 x 109 liters/mol for IGF-I binding to matrix-bound IGFBP-3. Des(1–3)-IGF-I, which binds IGFBP-3 with approximately one third the potency of wild-type IGF-I in solution (25), showed approximately 100-fold reduced ability to compete with IGF-I for binding to matrix-bound IGFBP-3 (Fig. 5BGo). Similarly, heparin, which displaces cell-associated IGFBP-3 (15), prevented binding of IGFBP-3 when added with the binding protein but was unable to displace IGFBP-3 from matrix once it was bound (data not shown).


Figure 5
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  		FIG. 5. IGF-I binds to matrix-bound IGFBP-3. A, MCF-10A matrix was incubated overnight at 22 C with the indicated concentration of IGFBP-3. Wells were washed, and [125I]IGF-I (10,000 cpm) was added to each well in the absence (bullet) or presence ({circ}) of 200 ng unlabeled IGF-I. After 4 h at 22 C, wells were washed and matrix solubilized for counting. Results are expressed as percentage of total added [125I]IGF-I recovered in the solubilized matrix. Data shown are mean ± SE of triplicate wells from one of three similar experiments. B, MCF-10A matrix was incubated overnight at 22 C with 5 nM IGFBP-3. Wells were washed, and [125I]IGF-I (10,000 cpm) was added to each well in the presence of IGF-I (bullet) or des(1–3))-IGF-I ({circ}) at the indicated concentration. After 4 h at 22 C, wells were washed and matrix solubilized for counting. Bound/total counts in the absence of cold IGF-I are expressed as 100%, and displacement is expressed relative to this. Data shown are mean ± SE of triplicate wells from one of three similar experiments.

 
Mutation of 228KGRKR->MDGEA in the C-terminal region of IGFBP-3 has been shown to adversely affect its binding to cells in monolayer culture (26); therefore, we investigated whether this mutation also affected IGFBP-3 binding to matrix. As shown in Fig. 6Go, binding of this IGFBP-3 mutant to matrix was markedly reduced compared with wild-type IGFBP-3, although not completely abolished. By contrast, a second IGFBP-3 mutant in which the basic region that forms part of the consensus heparin-binding domain is substituted for the corresponding region of IGFBP-1 (220KKK->HSR) showed only slightly reduced binding to matrix compared with wild-type IGFBP-3 (Fig. 6Go).


Figure 6
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  		FIG. 6. Binding of IGFBP-3 mutants to matrix. MCF-10A matrix was incubated with wild-type IGFBP-3 ({circ}), C-terminal basic domain mutant IGFBP-3 (228KGRKR->MDGEA; bullet), or heparin-binding domain mutant IGFBP-3 (220KKK->HSR; {square}) at the indicated concentrations overnight at 22 C and then probed for bound IGFBP-3 using R30 antibody and [125I]protein A. Data are shown as percentage of added [125I]protein A bound and are mean ± SE of quadruplicate determinations of one of three identical experiments.

 
In light of the finding shown in Fig. 2Go in which there was some association of a 30-kDa form of IGFBP-3 with matrix, we characterized further the binding of proteolyzed IGFBP-3 to matrix. In these experiments, we used radiolabeled 30-kDa IGFBP-3 isolated from a plasma-derived IGFBP-3 preparation by heparin affinity chromatography as described in Materials and Methods. Comparison of binding of intact and proteolyzed IGFBP-3 to matrix revealed that 30-kDa IGFBP-3 exhibited approximately 10-fold decreased binding to MCF-10A matrix compared with intact IGFBP-3 (2.5% of total added tracer bound for 30 kDa compared with 24% of total tracer bound for intact IGFBP-3; data not shown). Competitive binding curves indicated that when B0 binding was expressed as 100% for each protein, binding of 30-kDa IGFBP-3 to matrix was competed with greater potency by intact IGFBP-3 than binding of 43-kDa IGFBP-3 (Fig. 7AGo), reflecting the higher affinity of intact IGFBP-3 for ECM compared with proteolyzed IGFBP-3 as described above. Consistent with its reported reduced ability to bind IGFs compared with intact IGFBP-3, binding of 30-kDa IGFBP-3 to matrix was not blocked by coincubation with IGF-I at concentrations that fully prevented binding of intact IGFBP-3 (Fig. 7BGo). Heparin (100 µg/ml) blocked binding of intact IGFBP-3 to matrix by 95% and binding of 30-kDa IGFBP-3 by 70% (Fig 7BGo), reflecting the different affinities of the intact and proteolyzed proteins for heparin revealed during heparin Sepharose purification (i.e. proteolyzed IGFBP-3 was eluted with 0.4 M NaCl, whereas intact required 0.75 M NaCl for elution).


Figure 7
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  		FIG. 7. Proteolyzed IGFBP-3 binds to matrix. A, [125I]-labeled intact ({square}) and proteolyzed (bullet) IGFBP-3 (10,000 cpm/well) was added to each well of MCF-10A matrix in the presence of the indicated concentration of unlabeled IGFBP-3. Binding was carried out overnight at 22 C, and then wells were washed and matrix solubilized for counting. Bound/total counts in the absence of unlabeled IGFBP-3 have been expressed as 100% for each. B0 binding for intact IGFBP-3 was approximately 25% of total and for proteolyzed IGFBP-3 approximately 2.5% of total. Data shown are mean ± SE of quadruplicate wells from one of three identical experiments. B, Binding of 10,000 cpm 125I-labeled intact (white bars) and proteolyzed (striped bars) IGFBP-3 to MCF-10A matrix was carried out in the absence or presence of IGF-I (100 ng/well) or heparin (50 µg/well), as indicated. Wells were washed and matrix solubilized and counted. Bound/total counts in the absence of IGF-I or heparin have been expressed as 100% for each. B0 binding for intact IGFBP-3 was approximately 25% of total and for proteolyzed IGFBP-3 approximately 2.5% of total. Data shown are mean ± SE of quadruplicate wells from one of three identical experiments.

 
We then investigated whether matrix-bound IGFBP-3 was functional by examining its effect on cell attachment, a process in which ECM proteins are known to play an important role. Matrix in six-well plates was preincubated with IGFBP-3, and then the attachment of MCF-10A cells was determined 5 and 15 min after dispensing 5 x 105 cells into each well. As shown in Table 1Go, MCF-10A cells adhered very rapidly to matrix, with more than half the cells attached within 5 min of addition to the plates. The number of cells attached at this time point was significantly increased in wells that had been preincubated with IGFBP-3 at either 10 or 100 ng/ml (Table 1Go), with 25–30% more cells attached in the presence of matrix-bound IGFBP-3 compared with its absence. At a later time point (15 min), 80% of cells were attached in control wells, and an effect of IGFBP-3 was no longer apparent.


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  		TABLE 1. Attachment of MCF-10A cells to immobilized matrix containing IGFBP-3

 
To further explore the bioactivity of matrix-bound IGFBP-3, we investigated the activation state of p44/42 MAPK, a signaling intermediate that has been shown to be linked to ECM and integrin modulation of cell adhesion and proliferation (16, 27). Cells attached to control or IGFBP-3-containing matrix at 5 min were lysed and analyzed for total and phospho-(Thr202/Tyr204) p44/42 MAPK by Western blotting (13). As shown by a representative blot in Fig. 8AGo, phosphorylation of p44/42 MAPK was evident in the control cells (i.e. in the absence of IGFBP-3) at this time point, indicating that adhesion of cells to matrix is coincident with increased phosphorylation of p44/42 MAPK. Where plating had occurred in the presence of matrix-bound IGFBP-3, phosphorylation of p44/42 MAPK was increased relative to control cells (Fig. 8AGo); analysis of total p44/42 MAPK in these samples confirmed that this was not increased in the presence of IGFBP-3 (data not shown). Densitometric analysis of data pooled from five similar experiments (Fig. 8BGo) indicated a greater than 3-fold increase in the level of p44/42 MAPK phosphorylation where cells had been plated onto matrix preincubated with 100 ng/ml IGFBP-3 (P < 0.001). The magnitude of the change at the highest dose of IGFBP-3 tested indicated that this was independent of an increase in total cell number (which increased ~25%, Table 1Go), and analysis of {alpha}-tubulin as loading control confirmed that differences in p44/42 MAPK phosphorylation were not a result of differences in loading on gels. Consistent with the effects on cell attachment, the increase in p44/42 MAPK phosphorylation was transient, and by 15 min, there was no evidence of phosphorylation of p44/42 MAPK in the presence or absence of IGFBP-3 (data not shown). To investigate whether p44/42 MAPK activation was important for cell attachment, attachment assays were carried out using cells that had been preincubated with an inhibitor of MAPK kinase activity, PD98059 (20 µM). Neither basal nor IGFBP-3-stimulated cell attachment was affected by blockade of MAPK kinase signaling (data not shown), indicating that the increase in p44/42 phosphorylation is coincident with cell attachment rather than integral to the process itself. Collectively, these findings indicate that matrix-bound IGFBP-3 is bioactive, affecting the attachment of cells to matrix and modulating activation of the p44/42 MAPK signaling pathway.


Figure 8
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  		FIG. 8. Matrix-bound IGFBP-3 enhances p44/42 MAPK phosphorylation during cell adhesion. A, MCF-10A matrix in six-well plates was incubated with the indicated concentration of IGFBP-3 overnight at 37 C. Media were removed, and wells were washed before addition of MCF-10A cells (5 x 105 cells per well). After 5 min at 37 C, nonadherent cells were removed, and attached cells were lysed in reducing SDS-PAGE sample buffer. Lysates were collected and analyzed for phospho-Thr202/Tyr204 p44/42 MAPK using a phosphospecific antibody. Filters were developed using ECL and then stripped and reprobed for {alpha}-tubulin as loading control. B, Densitometric analysis of data obtained from five experiments similar to that shown in A, each with duplicate samples. Data from each experiment were normalized and expressed as percent control (0 ng/ml IGFBP-3) and then pooled. Shown is mean ± SE of these pooled data; *, P < 0.05; ¶, P < 0.001 compared with control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ECM has a central role in cellular function through its integration of extracellular and intracellular signaling molecules and pathways (16). Therefore, molecules that interact with ECM and affect its function have the potential to impact significantly on the ability of a cell to survive and proliferate. The presence of IGFBP-3 in ECM deposited by fibroblasts in vitro was first documented over a decade ago (17), but its role in this environment has not been investigated, nor has the association between IGFBP-3 and this complex entity been analyzed. We have identified and characterized binding of IGFBP-3 to matrix deposited by MCF-10A human breast epithelial cells, breast cancer cells, and neonatal human fibroblasts. When IGFBP-3 was bound to matrix, both the rate of attachment of MCF-10A cells and the activation of p44/42 MAPK, a key molecule in the p44/42 MAPK (ERK1/2) signaling pathway implicated in numerous ECM- and integrin-driven cellular processes including adhesion, migration, growth, and survival (16, 28, 29) were transiently increased. Thus we have shown for the first time that when associated with the ECM, IGFBP-3 is able to modulate some aspects of cellular function.

The most well documented actions of IGFBPs relates to their high-affinity binding of IGFs and inhibitory or stimulatory effects on IGF bioactivity (reviewed in Ref. 2). In the present study, we showed that matrix-bound IGFBP-3 was able to bind IGFs, suggesting that in addition to directly affecting cellular adhesion and signal transduction, it also has the potential to function in this more passive role, i.e. providing a sink for IGFs, and in doing so may modulate IGF action. It has been proposed that in chondrocytes, enhancement of IGF-stimulated tissue repair by IGFBP-3 reflects the ability of the binding protein to localize at the cell surface or in the ECM and maintain a reservoir of IGFs proximal to the signaling IGFR1 (30). Similarly in fibroblasts, matrix-bound IGFBP-5 enhances the growth response to IGF-I (17). Further evaluation of the role of ECM-bound IGFBP-3, as distinct from soluble IGFBP-3, in the potentiation of IGF action (4, 5, 6) is clearly an important area for additional study.

Association of IGFBPs with ECM may alter their IGF-binding characteristics, as demonstrated for ECM-bound IGFBP-5, which has 7-fold reduced affinity for IGF-I (17). Similarly, we found that the affinity of IGF-I for IGFBP-3 is reduced when the binding protein is matrix bound, resulting in an association constant of 1 x 109 liters/mol. This is approximately 20- to 30-fold lower than the affinity of IGF-I for IGFBP-3 in solution (2 x 1010 to 3 x 1010 liters/mol) (31). Matrix-bound IGFBP-3 exhibited 100-fold lower potency for binding des(1–3)-IGF-I compared with wild-type IGF-I, even though in solution there is only a 3-fold difference (25). In this respect, matrix-bound IGFBP-3 is similar to IGFBP-1 and IGFBP-2, which show very poor binding to des(1–3)-IGF-I in solution (25). The structural basis of the difference between the IGFBPs for des(1–3)-IGF-I-binding is not known, so the mechanism underlying this reduced binding is unclear. It may reflect a conformational change in IGFBP-3 brought about by matrix binding or masking of residues involved in the interaction between des(1–3))-IGF-I and IGFBP-3 in solution.

Our observation that IGFs prevent IGFBP-3 binding to matrix, presumably because of sequestration, also suggests that reciprocal regulation of bioactivity exists between IGF-I and IGFBP-3; just as IGFBP-3 can modulate IGF bioactivity, IGFs can modulate the actions of IGFBP-3 in the matrix by blocking its localization therein. This is in marked contrast to binding of IGFBP-2 to ECM, which requires IGFs, presumably to induce a conformational change necessary for its interaction with the matrix (19, 32). Our studies indicate that no such conformational change is necessary for IGFBP-3 to interact with the matrix, which is consistent with observations that IGFBP-3 can bind to matrix proteins such as fibronectin in solution in the absence of IGFs (33).

Association of IGFBP-3 with cells in monolayer culture has been described in numerous cell systems (5, 7, 8, 15) and has been variously attributed to interaction with glycosaminoglycans (34), transferrin/transferrin receptor complexes, and caveolin at the cell surface (35) and specific cell surface receptors (36, 37). The present demonstration of IGFBP-3 binding to matrix laid down by cells suggests that at least a proportion of IGFBP-3 associated with cell monolayers represents binding to ECM components rather than to cells per se. Furthermore, our data showing that matrix-bound IGFBP-3 could not be displaced by IGFs might explain earlier observations that IGF-I is unable to completely displace IGFBP-3 from cell monolayers (15); it may be that ECM-associated IGFBP-3 in cell monolayers is binding IGFs rather than being displaced by them.

We have previously shown that there is cell-associated IGFBP-3 in MCF-10A cell monolayers (9), but it has been difficult to quantify IGFBP-3 in the matrix and cell-bound compartments to evaluate the relative contribution of the two to total cell-associated IGFBP-3. IGFBP-3 is stripped from matrix and rendered undetectable by RIA during ammonium hydroxide isolation of the matrix (our unpublished data), so we have been unable to measure the absolute amount of IGFBP-3 in matrix from any cell type. In relative terms, cell-associated IGFBP-3 (i.e. matrix- plus membrane-bound IGFBP-3) correlates roughly with the levels of secreted IGFBP-3 (15); the higher the level of expression of IGFBP-3, the greater the amount of IGFBP-3 associated with cell monolayers. Furthermore, transfection of IGFBP-3 cDNA into cells that don’t normally express the protein results in an increase in both secreted and cell-associated IGFBP-3 (26). In the present study, the concentrations of IGFBP-3 used to characterize its binding to matrix and bioactivity are of the same order of magnitude as those measured in the conditioned medium of MCF-10A cells (~1 nM) (9) and similar to or considerably less than those that have been shown to have biological effects in numerous other studies (5, 9, 14, 38, 39). Therefore, although we can’t specifically demonstrate that the concentrations of IGFBP-3 used in the present study are typical of endogenous matrix-bound IGFBP-3, we would argue that they are likely to be close to physiological levels.

Affinity labeling of IGFBP-3 to MCF-10A-derived matrix revealed that IGFBP-3 appears to associate with a number of moieties in the matrix, with four affinity-labeled bands, of approximately 80, 100, 120, and more than 200 kDa, resolved by 7.5% SDS-PAGE. Cross-linking of IGFBP-3 to cell monolayers in other studies has similarly indicated multiple binding sites for IGFBP-3, with proteins of 20, 26, and 50 kDa shown to interact with IGFBP-3 in monolayers of Hs578T cells (36), and proteins of 20–30 kDa indicated by affinity-labeling mink lung cell monolayers with radiolabeled IGFBP-3 (37). Neither study addressed whether these were matrix or cell-surface proteins, and their relationship to the proteins detected in the present study remains unclear.

IGFBP-3 bound poorly to matrix when its C-terminal basic domain residues 228KGRKR were mutated to the corresponding sequence of IGFBP-1 (MDGEA). This region has also been implicated in cell association of IGFBP-3 (26) and its nuclear transport mediated by importin ß (40). Mutation of an adjacent consensus heparin-binding domain, 220KKK to HSR, had relatively little effect on IGFBP-3’s matrix-binding activity, and IGFBP-6, which has a consensus heparin-binding domain in the same region, was unable to compete with IGFBP-3 for binding to matrix. Collectively, these findings suggest that it is unlikely that glycosaminoglycans play a predominant role in matrix binding of IGFBP-3. Our observation that heparin prevented matrix binding of IGFBP-3 probably reflects direct interaction between it and the binding protein rather than indicating specific disruption of binding of IGFBP-3 to glycosaminoglycans in ECM.

A 30-kDa fragment of IGFBP-3 isolated from a plasma-derived IGFBP-3 preparation bound to matrix, but in contrast with intact IGFBP-3, 30-kDa IGFBP-3 binding to matrix was not affected by simultaneous addition of IGF-I. This is consistent with data showing that most proteolyzed IGFBP-3 fragments have very low affinity for IGFs. Binding of 30-kDa IGFBP-3 was, however, blocked by heparin although with reduced potency compared with intact IGFBP-3. This may indicate the loss of the C-terminal heparin-binding domain (219YKKKQCRP226) responsible for high-affinity binding of IGFBP-3 to heparin and retention of a domain in the nonconserved midregion of IGFBP-3 (149KKGHA153) that allows binding to heparin with 4-fold lower affinity (41). The physiological relevance of 30-kDa IGFBP-3 binding to matrix is not clear. We have shown previously that IGFBP-3 in the medium conditioned by MCF-10A cells is intact (43 kDa) when cells are maintained under serum-free conditions (9). It is possible that IGFBP-3 fragments may be generated under physiological rather than experimental conditions, for example in the presence of the cells or growth factor-replete medium, or they may be available to matrix from paracrine or endocrine sources. We also found that incubation of intact IGFBP-3 with matrix led to the generation of significant amounts of 30-kDa IGFBP-3, some of which was recovered in solubilized matrix (as shown in Fig. 2BGo). This indicates the potential for proteolyzed IGFBP-3 not only to bind to matrix but also to be generated by exposure of intact IGFBP-3 to matrix. The failure of EDTA to prevent the formation of the 30-kDa IGFBP-3 suggests that MMPs in the ECM are not responsible for the proteolysis of IGFBP-3 in this instance; the identity of the protease(s) responsible is currently being investigated.

IGFBP-5, the binding protein most similar to IGFBP-3 in both structure and function, was able to compete with IGFBP-3 for matrix binding sites; however, binding of 2.5 nM IGFBP-3 could not be not fully competed by a 40-fold higher concentration of IGFBP-5, suggesting preferential binding of IGFBP-3 to the matrix of breast epithelial cells. This is in contrast to a study in fibroblasts, which showed markedly preferential binding of IGFBP-5 to matrix deposited by these cells compared with IGFBP-3 (17). Although the reason for this discrepancy is not clear, it may reflect differences in the composition of the ECM deposited by different cell types. IGFBP-3 and IGFBP-5 share a number of characteristics, including a common nuclear transport pathway in T47D breast cancer cells (42) and interaction with the retinoic acid receptor (39), and have some common binding partners (2), including matrix components such as glycosaminoglycans (34, 43), fibronectin (17, 33), and vitronectin (17, 44). However, important functional differences between IGFBP-3 and IGFBP-5 have been identified. For example, IGFBP-3 but not IGFBP-5 potentiates EGF action in breast epithelial cells (13), and in some cell models, the two proteins have opposite effects on cell survival and adhesion (38). Furthermore, despite similar affinity for IGFs, IGFBP-3 and IGFBP-5 may also have distinct roles in modulating IGF bioactivity in different tissues, with IGFBP-5 important in bone metabolism and IGFBP-3 in mammary gland biology (45, 46). Whether some of these differences can be attributed to different association with, and function in, the ECM compartment is clearly an intriguing area for future research.

Interaction of IGFBP-3 with isolated component proteins of ECM, including fibronectin (17, 33) and vitronectin (17, 44), and the effects of soluble IGFBP-3 on cell attachment to these proteins have been shown in previous studies (38, 44). McCaig and colleagues (38) showed that for some parameters, the response to IGFBP-3 was subject to modulation by individual matrix proteins. In particular, plating cells preexposed to IGFBP-3 onto purified fibronectin reversed the effects of IGFBP-3 on cell attachment (from inhibition to stimulation) and apoptosis (from proapoptotic to antiapoptotic) compared with plating on either laminin or type IV collagen (38). Although such studies can provide important insights into the functional interaction between IGFBP-3 and specific proteins, they fail to acknowledge the inherent complexity of cell-derived ECM, in which interaction of IGFBP-3 with the different component proteins may elicit quite specific, and perhaps opposite, effects. Investigating the actions of IGFBP-3 in the matrix deposited by cells, as in the present study, is arguably closer to revealing the functional significance of its interaction with the ECM in a more physiologically relevant context.

The mechanism underlying IGFBP-3’s effects when ECM bound are not clear. In the study of McCaig and colleagues (38), preincubation of cells with IGFBP-3 increased their attachment to fibronectin, but the authors argued that it was unlikely that the actions they described were a result of direct interaction between IGFBP-3 and the fibronectin or other ECM components. They suggested that, instead, IGFBP-3 may be interacting with other effectors of integrin signaling, such as focal adhesion kinase. However, direct interaction of IGFBP-3 with ECM could not be discounted, because IGFBP-3 was not removed from the cell suspension before plating. Identifying the matrix components with which IGFBP-3 interacts will be important for delineating the mechanism by which matrix-bound IGFBP-3 modulates cell function, and this is now underway.

We have previously shown that IGFBP-3 potentiates the growth-promoting effects of EGF in MCF-10A cells, enhancing EGF-stimulated DNA synthesis, cell proliferation, EGF receptor phosphorylation, and the activity of p44/42 and p38 MAPK (13). In experiments not shown in the present study, we sought to resolve whether matrix-bound IGFBP-3 might be involved in this phenomenon. We considered it important that these analyses were carried out in the absence of serum, which contains appreciable quantities of ECM proteins such as vitronectin, fibronectin, collagens, etc. that might affect the response to IGFBP-3 bound to matrix. However, although MCF-10A cells adhered readily to matrix in the absence of serum, they failed to proliferate in response to EGF, presumably because of a lack of other essential growth factors and hormones. Similarly, EGF alone did not stimulate cell attachment, and matrix-bound IGFBP-3 enhanced cell attachment to the same degree in the presence and absence of EGF (data not shown). Although these experiments do not provide clear data regarding a role for matrix-bound IGFBP-3 in the potentiation of EGF bioactivity, we nevertheless consider it unlikely that it does play a major role in modulating EGF bioactivity. In our earlier study (13), the 228KGRKR->MDGEA mutant IGFBP-3 had similar potency as wild-type IGFBP-3 in enhancing the effects of EGF on DNA synthesis, but as shown in the present study, this mutant binds very poorly to matrix.

In conclusion, we have shown that IGFBP-3 binds to ECM laid down by a variety of cell types and in this environment is able to affect cell attachment and activation of intracellular signaling pathways important in the growth and survival of normal and malignant cells. Elucidation of the mechanisms underlying these phenomena and the ECM components involved is clearly an important area for further investigation.


   Acknowledgments
 
We thank Dr. Sue Firth and Mr. Xiao-Lang Yan for providing the IGFBP-3 mutants and Professor Rob Baxter for helpful discussion.


   Footnotes
 
This work was supported by National Health and Medical Research Council Grants 107244 and 302153 (to J.L.M.) and the Cancer Institute NSW. J.L.M. is a Cancer Institute NSW Fellow.

Author declaration: J.L.M. and S.J. have nothing to declare.

First Published Online June 8, 2006

Abbreviations: ECM, Extracellular matrix; EGF, epidermal growth factor; IGFBP, IGF-binding protein; IGFR1, type I IGF receptor; MMP, matrix metalloproteinase; TBS-T, Tris-buffered saline with Tween 20.

Received January 25, 2006.

Accepted for publication May 30, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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