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Endocrinology, doi:10.1210/en.2003-0636
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Endocrinology Vol. 145, No. 2 620-626
Copyright © 2004 by The Endocrine Society

Regulation of Insulin-Like Growth Factor (IGF)-I Action by Matrix Metalloproteinase-3 Involves Selective Disruption of IGF-I/IGF-Binding Protein-3 Complexes

John L. Fowlkes, Delila M. Serra, R. Clay Bunn, Kathryn M. Thrailkill, Jan J. Enghild and Hideaki Nagase

Department of Pediatrics (J.L.F., R.C.B., K.M.T.), University of Arkansas for Medical Sciences and Arkansas Children’s Hospital Research Institute, Little Rock, Arkansas 72202; Department of Pediatrics (D.M.S.), Duke University Medical Center, Durham, North Carolina 27710; Department of Molecular and Structural Biology (J.J.E.), University of Aarhus, DK-8000 Aarhus C, Denmark; and Kennedy Institute of Rheumatology Division (H.N.), Faculty of Medicine, Imperial College London, London W6 8LH, United Kingdom

Address all correspondence and requests for reprints to: John L. Fowlkes, M.D., Professor and Chief, Division of Pediatric Endocrinology and Diabetes, University of Arkansas for Medical Sciences, Arkansas Children’s Hospital, Arkansas Children’s Hospital Research Institute, 800 Marshall Street, Slot 512-6, Little Rock, Arkansas 72202. E-mail: fowlkesjohnl{at}uams.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I and IGF-II play important roles in growth and development via interactions with cell-surface receptors; however, in nature, IGFs are sequestered by at least six soluble, high-affinity IGF-binding proteins (IGFBPs), namely IGFBPs 1–6. Herein, we demonstrate that the stromal cell-derived extracellular matrix-degrading metalloproteinase stromelysin 1 (matrix metalloproteinase 3) disrupts IGF/IGFBP-3 complexes and liberates free, intact IGFs, leading to phosphorylation of cell surface type 1 IGF receptors and cellular proliferation. Tissue inhibitor of metalloproteinases (TIMP-1) or an antibody to the type 1 IGF receptor mitigates IGF-mediated cellular proliferation. Thus, these studies suggest that matrix metalloproteinases, beyond their effects on extracellular matrix turnover, regulate cellular proliferation by modulating the bioavailability of IGFs, an event critical for such diverse phenomena as embryo development, morphogenesis, angiogenesis, and tumorigenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I AND IGF-II ARE PEPTIDE growth factors (~7.5 kDa) involved in cellular growth, differentiation, and metabolism (1, 2). Unlike their structurally homologous relative, insulin, almost all IGFs found in serum and other biological fluids are complexed with one of six highly homologous IGF-binding proteins (IGFBPs 1–6) (1, 2, 3). Through sequestration of IGFs, soluble IGFBPs inhibit IGF action by preventing their interaction with cell surface receptors (1, 2, 3). This is possible because all six IGFBPs have similar or higher affinities for IGFs than does the type 1 IGF receptor, the receptor that mediates metabolic and mitogenic effects of IGFs (4).

Because IGFs are essential for normal growth and development (1, 2, 3, 4, 5), mechanisms must exist by which complexed IGFs are released from IGFBPs to interact with type 1 IGF receptors. This may occur through several fundamental mechanisms, including: 1) binding of IGFBPs to extracellular matrix molecules, facilitating IGF sequestration in close proximity to type 1 IGF receptors; 2) phosphorylation of IGFBPs, which may alter the affinity of IGFBPs for IGFs; and 3) proteolysis of IGFBPs, which may reduce the affinities of IGFBPs for IGFs, thus favoring dissociation of IGFs from IGF/IGFBP complexes (1, 2, 3). Although each mechanism may play a pivotal role in regulating IGF action, IGFBP proteolysis has emerged as a major mechanism controlling IGF action; yet, in most instances, the proteinases involved have not been identified, nor their activities on IGF bioavailability clarified (6, 7).

Studies from our laboratories have identified members of the zinc-dependent metalloproteinase family, known as matrix metalloproteinases (MMPs), as IGFBP-degrading proteinases (8). MMPs, including collagenases, stromelysins, gelatinases, macrophage metalloelastase, and membrane-type MMPs, are considered to play a central role in degradation and remodeling of extracellular matrix and tissues during embryo development, morphogenesis, and tissue regeneration and in diseases such as arthritis, cancer, and metastatic disease (9, 10). The ability of MMPs to degrade IGFBP-3, the major carrier of IGFs in serum, both in vitro (8, 11, 12) and in vivo (13, 14), suggests that MMPs may participate in cellular growth and tissue morphogenesis not only through their capacity to alter the extracellular architecture but also by modulating the bioavailability of IGFs to cell surface receptors. Herein, we have investigated the physiologic consequences of IGFBP degradation by MMPs on IGF action.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Digestion of IGFBP-3 and IGF-I/IGFBP-3 complexes by MMP-3
Twenty micrograms of nonglycosylated recombinant human IGFBP-3 [(rh)IGFBP-3], produced in Escherichia coli (IGFBP-3Escherichia coli) (15), was incubated, with or without a 1:1 molar ratio of recombinant human IGF-I, for 2–4 h at room temperature. After complex formation was completed, IGFBP-3Escherichia coli alone or IGF-I/IGFBP-3Escherichia coli complexes were incubated with increasing concentrations of activated recombinant human MMP-3 (MMP-3:IGFBP-3 molar ratios of 1:100, 1:200, 1:400, and 1:1000), for 19 h at 37 C, under conditions similar to those described elsewhere (11, 16). Samples were separated by SDS-PAGE (15% acrylamide) under reducing conditions and electroblotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA) using a Bio-Rad (Hercules, CA) trans-blot unit. For immunoblotting, membranes were first blocked with 5% milk in Tris-buffered saline, then membranes were incubated with goat antihuman IGF-I antiserum (1:1,000 dilution, Sigma, St. Louis, MO), washed, and incubated with horseradish peroxidase-conjugated rabbit antigoat antiserum (1:10,000 dilution, Sigma). After washing, blots were imaged by enhanced chemiluminescence (Bio-Rad) and images were captured on a Versadoc 5000 (Bio-Rad). Band intensity was quantified using Quantity One software (Bio-Rad). For N-terminal sequencing, membranes were stained with 0.1% Coomassie blue, destained, and washed in dH2O. N-terminal amino acid sequencing was performed by first excising Coomassie-stained bands from the polyvinylidene difluoride membrane. Each band was then sequenced by automated Edman degradation in an Applied Biosystems (Foster City, CA) 477A Sequencer with on-line phenylthiohydantoin analysis using an Applied Biosystems 120A HPLC system as previously described (11).

Cross-linking of 125I-IGF-II to intact and fragmented IGFBP-3
IGFBP-3Escherichia coli (12 ng) and recombinant human 125I-IGF-II (~20,000 cpm) were incubated in the presence or absence of MMP-3 (300 ng) for 20 h at 37 C, brought up in 50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10 mM CaCl2, 0.02% NaN3, 0.05% Brij 35 (TNC buffer) in a total vol of 60 µl. To terminate the digestion by MMP-3, EDTA was added to the mix at a final concentration of 10 mM. Mixtures were then allowed to re-equilibrate by incubation at 4 C overnight to allow for 125I-IGF-II/IGFBP-3 fragment complex formation to occur. After the 4 C incubation, mixtures were then diluted into 2 vol (120 µl) of Eagles minimal essential media, containing 20 mM HEPES, and incubated at 22 C in the presence or absence of 12 ng glycosylated rhIGFBP-3 produced in Chinese Hamster ovary cells (IGFBP-3CHO) (15). Identical volumes were removed from each reaction at the indicated times, over a 6-h incubation period. 125I-IGF-II/IGFBP-3 interactions were assessed by chemical cross-linking with disuccinimidyl suberate (DSS; final concentration of 0.5 mM) for 20 min at 22 C. Termination of the cross-linking was performed by adding 1 M Tris-HCl (pH 7.5), to give a final concentration of 50 mM. Samples were then lyophilized, reconstituted in deionized H2O, and solubilized in reducing sample buffer. Proteins were separated by SDS-PAGE (15%), and then gels were dried under vacuum and exposed to x-ray film at -80 C for several days. Cross-linked complexes were detected by autoradiography.

Phosphorylation of the type 1 IGF receptor mediated by IGF-I
IGFBP-3Escherichia coli (60 µg) and IGF-I (8 µg) (a 2:1 molar ratio, respectively), were incubated with or without MMP-3 (200 ng) in a total vol of 60 µl TNC buffer and digested for 8 h at 37 C. The reaction was terminated with the addition of 5 µg recombinant TIMP-1 (tissue inhibitor of metalloproteinases) (17), and the IGF-I/IGFBP-3 complex or IGF-I/IGFBP-3-MMP-3 digest was stored at 4 C until used in the studies described below.

Murine 3T3 fibroblasts, transfected with the human type 1 IGF receptor (clone NWT C43, kindly provided by Dr. Derek LeRoith, National Institutes of Health (NIH), Bethesda, MD), were produced and grown as described in Ref.17 . NWT C43 cells were grown to near confluence in six-well culture dishes as described elsewhere (18), then quiesced in serum-free MEM overnight. Fibroblasts were treated with buffer alone, IGF-I (50 ng/ml), IGF-I/IGFBP-3 complexes (equivalent of 50 ng/ml IGF-I), or MMP-3-digested IGF-I/IGFBP-3 complexes (equivalent of 50 ng/ml IGF-I). At the indicated times, plates were flash frozen in liquid N2, then thawed into modified RIPA buffer [50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mm NaF]. Lysed samples were cleared by centrifugation, then separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Phosphorylation of the ß-subunit of the type I IGF receptor was analyzed by immunoblotting with an antiphosphotyrosine monoclonal antiserum (clone PT-66, Sigma). Detection of the PT-66 was performed according to the manufacturer’s instructions, using an HRP-labeled antimouse antibody and the chemiluminescent reagent provided in the enhanced chemiluminescence kit from Amersham (Piscataway, NJ).

Cell proliferation mediated by IGF-I
NWT C43 cells were seeded into 96-well plates at 3 x 103 cells/well. After 24 h, cells were changed to serum-free medium for an additional 24 h. The cells were then changed into medium containing buffer alone, IGF-I (50 ng/ml), IGF-I/IGFBP-3 complexes (50 ng/ml and 375 ng/ml, respectively), or IGFBP-3 alone (375 ng/ml), with or without MMP-3 (5 µg/ml), for 48 h at 37 C. At h 28 of the incubation XTT [sodium 3'-[1-(phenylaminocarbonyl0–3, 4-tetrazolium]bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate] and PMS (N-methyl dibenzopyrazine methyl sulfate) were added to each well according to the manufacturer’s instructions (Roche Molecular Biochemicals, Mannheim, Germany); and 20 h later, the plates were read at A490. In certain studies, cells were treated with IGF-I/IGFBP-3 complexes (50 ng/ml and 375 ng/ml, respectively) with increasing concentrations of MMP-3 (0–5 ug/ml). In other studies, cells were treated with IGF-I/IGFBP-3 complexes (50 ng/ml and 375 ng/ml, respectively), along with MMP-3 (5 ug/ml) in the absence or presence of TIMP-1 (5 ug/ml) or the antitype 1 IGF-receptor monoclonal antiserum {alpha}-IR3 (1:250 dilution; a kind gift of Steve Jacobs, Burroughs Wellcome Co., Research Triangle Park, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
For MMPs to regulate IGF action, they must first be capable of releasing functional IGFs from IGF/IGFBP complexes. Therefore, we tested stromelysin 1 (MMP-3), the most potent MMP shown to degrade IGFBP-3 (11), for its ability to digest IGFBP-3 alone and when complexed with IGF-I. Recombinant human MMP-3 digested nonglycosylated rhIGFBP-3 (IGFBP-3E. Coli) into six major fragments in a dose-dependent manner (Fig. 1AGo). MMP-3 also digested IGF-I/IGFBP-3 complexes, resulting in similarly sized IGFBP-3 fragments, as well as a unique band at approximately 7.5 kDa (Fig. 1BGo). N-terminal amino acid sequencing of the 7.5-kDa band revealed only the native N-terminal amino acid sequence of IGF-I (i.e. 1GPETLCGAELVD....). This, together with the fact that native intact IGF-I has a molecular mass of approximately 7.5 kDa and that the size and intensity of the approximately 7.5-kDa band did not change at any of the MMP-3 concentrations examined, suggests that MMP-3 does not significantly degrade IGF-I when complexed to IGFBP-3. Similar studies were also carried out using MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), or MMP-9 (gelatinase B) in place of MMP-3, and these experiments revealed both various degrees of proteolysis and different digestion patterns of IGFBP-3 (data not shown) (11). Nevertheless, the appearance of the approximately 7.5-kDa band and its N-terminal amino acid sequence in MMP-1 and MMP-2 digests was identical with that observed in MMP-3 digests. MMP-9 digests also revealed the identical approximately 7.5-kDa band, although amino acid sequencing of the band was not performed. Interestingly, when IGF-I was not precomplexed with IGFBP-3, and exposed to the highest concentration of MMP-3 used in these studies, almost all of the IGF-I was proteolyzed, resulting in a reduction of more than 90% of the intact protein (Fig. 2Go; P < 0.001). Taken together, these studies demonstrate that, during the proteolytic process, IGFBP-3 and IGFBP-3 fragments are capable of protecting IGF-I from degradation by MMP-3, as well as other MMPs, and MMP-mediated degradation of IGF-I/IGFBP-3 binary complexes results in dissolution of the binding protein, yet IGF-I is spared.



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  		FIG. 1. Digestion of IGFBP-3 and IGF-I/IGFBP-3 complexes by MMP-3. A, Nonglycosylated rhIGFBP-3 produced in Escherichia coli (IGFBP-3Escherichia coli) was digested with increasing concentrations of MMP-3 as described in Materials and Methods. The digestion resulted in the complete digestion of IGFBP-3E. Coli in a dose-dependent fashion with the production of six IGFBP-3 fragments, whose sequences have been reported elsewhere (11 ). B, IGF-I/IGFBP-3 complexes (1:1 molar ratio) were similarly digested with increasing concentrations of MMP-3 as described for A, resulting again in the complete digestion of IGFBP-3 in a dose-dependent fashion. N-terminal amino acid sequencing of the approximately 7.5-kDa band revealed only the natural N-terminal sequence of IGF-I (GPETLCGAELVDALQ.).

 


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  		FIG. 2. Digestion of IGF-I and IGF-I/IGFBP-3 complexes by MMP-3. IGF-I was incubated, with or without MMP-3 (320 ng), in the absence or presence of IGFBP-3, as described in Materials and Methods. Bars represent the mean percent ± SEM of intact IGF-I present at the end of the digest. Immunoreactive bands at approximately 7.5 kDa, representing intact IGF-I, were imaged and captured on a Versadoc 5000 (Bio-Rad). Band intensity was quantified using Quantity One software (Bio-Rad). *, P < 0.001.

 
To determine whether degradation of IGF-I/IGFBP-3 complexes by MMP-3 alters the dissociation of IGFs from IGF/IGFBP complexes, 125I-IGF-II/IGFBP-3 complexes were first digested by MMP-3. After proteolysis was terminated, complex formation was allowed to reoccur at 4 C. Complexes of 125I-IGF-II (Fig. 3Go) and MMP-3-generated IGFBP-3 fragments were then examined by covalent cross-linking in the absence or presence of intact glycosylated IGFBP-3 (IGFBP-3CHO; Mr 42–45,000), which binds IGFs with the same affinity as nonglycosylated IGFBP-3 used in the MMP-3 digests. 125I-IGF-II (Fig. 3Go) formed complexes with several MMP-3-generated IGFBP-3 fragments, and little or no dissociation of radiolabeled IGFs from IGFBP-3 fragments was observed over a 6-h period at ambient temperature in the absence of glycosylated IGFBP-3 (Fig. 3AGo). In contrast, the addition of glycosylated IGFBP-3 resulted in dissociation of all 125I-IGF-II from IGFBP-3 fragments, resulting in the recomplexing of 125I-IGF-II with intact glycosylated IGFBP-3 (Fig. 3BGo). Thus, degradation of IGF/IGFBP-3 complexes by MMP-3 results in the formation of several IGFBP-3 fragments that still bind functional IGF-II; yet, in the presence of intact IGFBP-3, IGF-II preferentially binds the high-affinity, full-length binding protein.



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  		FIG. 3. Redistribution of 125I-IGF-II among MMP-3-generated IGFBP-3 Escherichia coli fragments and intact glycosylated rhIGFBP-3CHO. A, IGFBP-3Escherichia coli and 125I-IGF-II were digested with MMP-3 for 20 h at 37 C as described in Materials and Methods. The digestion was then stopped, and complex formation was allowed to take place at 4 C. 125I-IGF-II/IGFBP-3Escherichia coli fragments interactions were observed at various time points, at ambient temperature, over a 6-h interval. B, The same study was carried out as in A, with the exception that IGFBP-3CHO (denoted by the arrow) was added to the 6-h incubation. Again, complexed 125I-IGF-II with IGFBP-3Escherichia coli fragments or intact IGFBP-3CHO was observed at various time points, at ambient temperature, over a 6-h interval.

 
Although redistribution of IGFs from IGFBP fragments to intact IGFBPs may account for relocalization or targeting of IGFs, IGF bioactivity is dependent on the interaction of IGF with the type 1 IGF receptor (1, 2, 3, 4, 5). To examine whether IGF-I/IGFBP-3 complex degradation by MMP-3 results in enhanced IGF/IGF receptor interaction, we measured type 1 IGF receptor ß-subunit tyrosine phosphorylation in 3T3 fibroblasts overexpressing the type 1 IGF receptor as an indicator of receptor-ligand interaction (4, 18). Because IGFBP-3 binds IGF-I with approximately 10- to 50-fold higher affinity than the type 1 IGF receptor (2, 6), little or no tyrosine phosphorylation of the receptor occurred when cells were exposed to a complex of IGF-I/IGFBP-3 at a 1:2 molar ratio (Fig. 4Go, A and B). However, when IGF was added alone, tyrosine phosphorylation of the type 1 IGF receptor increased dramatically at all time points examined. In contrast, when MMP-3-digested IGF-I/IGFBP-3 complexes were added to the cells, ß-subunit tyrosine phosphorylation occurred in a time-dependent manner, reaching a degree of phosphorylation similar to that of IGF-I alone by 30 min (Fig. 4Go, A and B). Thus, treatment of IGF-I/IGFBP-3 complexes with MMP-3 results in the production of IGFBP-3 fragments, which display affinities sufficiently low enough for IGF-I to dissociate from the MMP-3-generated IGFBP-3 fragments and to interact with cell surface type 1 IGF receptors in a time-dependent fashion. Furthermore, the IGF-I that is released is capable of activating its cognate receptor.



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  		FIG. 4. Effect of MMP-3 on IGF-I/IGFBP-3 complexes to phosphorylate type 1 IGF receptor. A, Murine 3T3 fibroblasts, which overexpress the human type 1 IGF receptor, were treated with buffer, IGF-I, or IGF-I/IGFBP-3 complexes that were or were not digested with MMP-3, as described in Materials and Methods. At the indicated times, cells were lysed, and tyrosine phosphorylation of the ß-subunit of the type 1 IGF receptor (denoted by the arrow) was analyzed by immunoblotting cell lysates with a monoclonal antibody that detects phosphotyrosine residues. Cells treated with buffer (lanes 1, 5, and 9) or undigested IGF-I/IGFBP-3 complexes (lanes 3, 7, and 11) showed little or no increase in tyrosine phosphorylation of the ß-subunit over a 30-min period. In contrast, IGF-I (lanes 4, 8, and 12) caused rapid tyrosine phosphorylation to occur as early as 5 min, and this was sustained throughout the entire time course. Cells treated with MMP-3-digested IGF-I/IGFBP-3 (lanes 2, 6, and 10) resulted in an incremental increase in tyrosine phosphorylation over the 30-min time period. B, Tyrosine phosphorylation of the ß-subunit of the type 1 IGF receptor in three separate experiments, as described in A, were quantitated by scanning densitometry and expressed as the mean ± SD, compared with control (i.e. buffer alone). Bars represent ß-subunit phosphorylation in cells treated with MMP-3-digested IGF-I/IGFBP-3 complexes (hatched bars), undigested IGF-I/IGFBP-3 complexes (solid bars), or IGF-I alone (dotted bars). *, P = 0.01; **, P < 0.001.

 
To determine the cellular response to disruption of IGF-I/IGFBP-3 complexes by MMP-3, 3T3 fibroblasts overexpressing the type 1 IGF receptor were treated with IGF-I, IGFBP-3, or IGF-I/IGFBP-3 complexes in the presence or absence of MMP-3. In the absence of MMP-3, only cells treated with IGF-I alone, but not with IGF-I/IGFBP-3 complexes, demonstrated significant proliferation (Fig. 5AGo). In contrast, when MMP-3 was added to the incubation, cells treated with either IGF-I alone or IGF-I/IGFBP-3 complexes demonstrated similar increases in proliferation (Fig. 5BGo). The addition of MMP-3 did not alter the cellular response to IGF-I or IGFBP-3 when used alone, suggesting that MMP-3 exerts its effects on IGF action exclusively through its ability to disrupt IGF-I/IGFBP-3 complexes. The effect of MMP-3 to enhance IGF-I interaction with the cell surface type 1 IGF receptor is dose-dependent (Fig. 5CGo), emphasizing that this mode of IGF release by MMPs can be finely regulated in the extracellular environment, dependent on the relative concentrations of active MMPs present in the pericellular milieu.



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  		FIG. 5. The effects of MMP-3 disruption of IGF-I/IGFBP-3 complexes on cellular proliferation. A and B, 3T3 fibroblasts overexpressing the human type 1 IGF receptor were incubated without (A) or with (B) MMP-3 in the presence of buffer alone (control, open bars), IGF-I (speckled bar), IGF-I/IGFBP-3 complexes (hatched bar), or IGFBP-3 (solid bar) alone for 48 h at 37 C. A, In the absence of MMP-3, only IGF-I significantly stimulated fibroblast proliferation, as measured by the XTT cellular proliferation assay. B, In the presence of MMP-3, IGF-I/IGFBP-3 complexes resulted in fibroblast proliferation similar to IGF-I alone. *, P < 0.001; **, P < 0.0001. C, Incubation of cells with IGF-I/IGFBP-3 complexes in the presence of increasing concentrations of MMP-3 resulted in a dose-dependent increase in fibroblast proliferation. D, The addition of TIMP-1 (open bar) or {alpha}-IR3 (solid bar) to incubations containing IGF-I/IGFBP-3 complexes and MMP-3 resulted in a significant decrease in fibroblast proliferation, when compared with IGF-I/IGFBP-3 complexes and MMP-3 alone (hatched bar). Values were normalized to data obtained from cells treated similarly but in the absence of MMP-3. *, P < 0.01. All values are expressed as the mean ± SD.

 
In keeping with this idea, TIMP-1, a naturally occurring specific inhibitor of all known MMPs (19), inhibited significantly (64%, P < 0.01) the ability of MMP-3 to enhance IGF-I action on cellular proliferation (Fig. 5DGo). The specificity of MMP-3 on IGF-I bioavailability is confirmed by the finding that a monoclonal antibody specific for the human type 1 IGF receptor ({alpha}-IR3) (20) inhibits the effects of MMP-3 on cellular proliferation by 76% (P < 0.01) (Fig. 5DGo). Together, these data illustrate the precision by which MMP-3 can regulate IGF bioavailability at the cell surface and how IGF is modulated by inhibitors of IGFs (i.e. IGFBPs) as well as MMPs (i.e. TIMPs).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The release of growth factors by proteinases, both in physiologic and pathologic conditions, has been hypothesized (21, 22), and only recently has there been evidence to support that such release is a physiologically relevant phenomena. For example, proteases can release basic fibroblast growth factor and TGF-ß1 from extracellular matrix to interact with their respective receptors (22, 23). The demonstration that MMPs selectively degrade IGFBP-3, as well as other IGFBPs (8, 11, 12, 13, 14, 24), thereby releasing bioactive IGFs, provides a mechanism by which this family of proteinases can indirectly influence cellular growth and differentiation. Our findings emphasize an ever-growing new concept regarding MMPs: that these enzymes are not only involved in degradation of extracellular matrix molecules, the originally identified target molecules ascribed to MMPs, but that MMP activity is also involved in cellular growth and differentiation that is critical for normal development, morphogenesis, tissue remodeling, and tumorigenesis. This is illustrated in virgin mice that develop premature breast growth associated with precocious breast duct development and ß-casein expression when MMP-3 is overexpressed exclusively in breast tissue (25). Furthermore, some of these mice go on to demonstrate frank breast tumors (26), emphasizing the significant growth-promoting effects that MMP-3 displays in this model. Because no direct effect of MMP-3 on proliferation has been demonstrated, it is possible that the overexpression of MMP-3 in these mice may lead to increased IGF concentrations within breast tissue, resulting in the tissue responses observed. In keeping with the idea of MMPs as growth-promoting agents, several studies have now demonstrated that synthetic inhibitors of MMPs inhibit tumor growth in a variety of malignancies in vivo (27, 28, 29, 30, 31, 32). Indeed, recently we have reported that overexpression of TIMP-1 in an SV40-mediated hepatocellular tumor model prevents hepatocellular proliferation and subsequent tumorigenesis (14). Using a variety of approaches, we demonstrated that TIMP-1 overexpression in hepatic tissues inhibits IGFBP-3 proteolysis and thereby decreases bioavailable IGF-II, resulting in enhanced cell proliferation and tumorigenesis.

The physiologic consequences of IGFBP proteolysis may be particularly important in pathologic conditions in which increased IGFBP proteolysis has been reported, such as cancer, type I and type II diabetes, burn injury, and arthritis (7). In many of these conditions, alterations in MMP production have also been reported (9, 10) and therefore may provide a mechanism by which IGF bioavailability is altered in states of MMP dysregulation. Further studies are needed to assess the overall contribution that MMPs may play in regulating the in vivo effects of IGFs; however, the data presented herein supports the notion that MMPs are capable of accentuating IGFs effects through selective degradation of the sequestering binding protein, and subsequent release of an intact and bioactive IGF molecule. Because IGFBP-degrading proteinases have emerged as probable regulators of IGF action at the cell surface, it will be important to determine how each identified IGFBP-degrading proteinase affects IGF activity. For instance, proteinases such as MMPs may enhance IGF action though degrading IGFBP-IGF complexes near the cell surface, releasing functional IGFs to bind to their cell-surface receptors, thus promoting IGF-mediated events. MMPs may also serve an additional role through degrading so-called free IGFs, not bound to IGFBPs, thereby eliminating IGF-signaling. This mechanism of eliminating IGFs from the pericellular milieu is in contrast to that used by other IGFBP-degrading proteinases, such as cathepsins, which are capable of cleaving both IGFBPs and IGFs when complexed, thereby allowing for the inactivation and clearance of binary complexes from the pericellular environment (6, 7, 33). In addition to their effects on IGF-mediated events, IGFBP-degrading proteinases, such as MMPs, by cleaving IGFBPs, may produce IGFBP fragments that might have novel and IGF-independent effects, as has been described for IGFBP fragments generated by other proteinases such as plasmin (7, 34). Taken together, our findings suggest that, because IGFs and MMPs have been independently implicated in a variety of processes, including tumor cell growth and invasion, morphogenesis, trophoblast growth and invasion, cartilage and bone repair and turnover, and wound healing and angiogenesis (9, 10, 35), the interaction of these two systems may play a crucial role in the delicate balance among biologic and pathologic growth, remodeling, and tissue destruction.


   Acknowledgments
 
We thank C. Maack for providing rhIGFBP-3Escherichia coli and rhIGFBP-3CHO. We also acknowledge the gifts of recombinant human IGF-I and recombinant human IGF-II from Genentech Inc. and Lilly Research Laboratories, respectively. Ko Suzuki is thanked for his technical assistance.

This work was presented, in part, at the New York Academy of Sciences Colloquium entitled: Inhibition of Matrix Metalloproteinases: Therapeutic Applications; Tampa, FL, October 21–25, 1998.


   Footnotes
 
This work was partially supported by Grant R01 DK055653 from NIH (to J.L.F.).

Abbreviations: rhIGFBP-3, Recombinant human IGFBP-3; IGFBP, IGF-binding protein; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases.

Received May 23, 2003.

Accepted for publication October 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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