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[Cancer Research 65, 7363-7369, August 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Insulin-Like Growth Factor Binding Protein-5 Is a Target of Matrix Metalloproteinase-7: Implications for Epithelial-Mesenchymal Signaling

Elaine Hemers, Cedric Duval, Catherine McCaig, Mark Handley, Graham J. Dockray and Andrea Varro

Department of Physiology, University of Liverpool, Liverpool, United Kingdom

Requests for reprints: Andrea Varro, Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom. Phone: 44-0-151-794-5331; Fax: 44-0-151-794-5315; E-mail: avarro{at}liverpool.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Matrix metalloproteinase-7 (MMP-7) is localized to epithelial cells and is up-regulated in many cancers and in inflammation. We now report that MMP-7 targets a key mesenchymal cell type, the myofibroblast. Recombinant MMP-7 stimulated the proliferation and migration of human colonic myofibroblasts. These responses were partly attributable to activation of other MMPs, notably MMP-3 and MMP-8, and to stimulation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways. Using a proteomic approach, we identified insulin-like growth factor binding protein-5 (IGFBP-5) as a previously unsuspected target of MMP-7 produced by colonic myofibroblasts. We present evidence that the MMP-7 cleavage of IGFBP-5 liberates IGF-II that functions as an autocrine myofibroblast growth factor. Thus, MMP-7 may act as a signal from epithelial cells for local recruitment of myofibroblasts and stimulation of their proliferation. Similar effects of MMP-7 produced in epithelial tumors might account for the expansion of stroma through activation of myofibroblasts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumors of epithelial origin typically exhibit an expansion of subepithelial or stromal cells implying disordered signaling between the two cell populations. Subepithelial cells (e.g., myofibroblasts) produce a variety of key growth factors thought to play a role both in the maintenance of normal epithelial cell proliferation and to drive tumor growth (1). Moreover, these cells synthesize extracellular matrix proteins and are likely to be important in the remodeling events that restore normal mucosal organization following epithelial cell damage, as well as in stroma deposition in cancer (2). In contrast, the factors derived from normal or neoplastic epithelial cells that might regulate the proliferation and migration of myofibroblasts remain relatively poorly understood.

The matrix metalloproteinases (MMP) are a growing group of proteolytic enzymes involved in remodeling of the extracellular matrix. Members of the group are frequently increased in cancer and in inflammatory conditions (3, 4). Conversion of precursor molecules to their proteolytically active form can be mediated by other MMPs giving rise to the idea that they can act in cascades (5, 6). Most MMPs are produced in subepithelial cells, but an exception is MMP-7 (matrilysin or PUMP) that is predominantly expressed in epithelial cells including those of the gastrointestinal tract, airways, mammary gland, and urogenital tract (7–9). There is increased MMP-7 expression in many tumors, in bacterial infections of normal epithelia, in inflammation, and in injury (9–13). In addition to digestion of extracellular matrix proteins, it is now clear that MMP-7 like other MMPs, may act on a variety of substrates to liberate growth factors and other signaling molecules, thereby potentially producing a diverse range of cellular responses (3, 8, 12, 14, 15). In view of the increased expression of MMP-7 in epithelial cells in a range of different conditions associated with fibrosis or deposition of stroma, it seems plausible to suppose that MMP-7 might play a role in dysfunctional interactions between epithelial and mesenchymal cells.

In the present study, we hypothesized that MMP-7 acts as a mediator of epithelial-mesenchymal signaling and in particular targets myofibroblasts. We report here that MMP-7 stimulates myofibroblast proliferation and migration through a mechanism dependent on activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. Using a proteomic approach, we identify insulin-like growth factor binding protein-5 (IGFBP-5) as a previously unidentified substrate of MMP-7 and provide evidence that the effects of MMP-7 are a consequence of liberation of IGF-II which then acts as an autocrine growth factor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and drugs. Human primary colonic myofibroblasts (CCD18co; ref. 16) were obtained from the American Type Culture Collection (ATCC, Manassas, VA), cultured in DMEM (ATTC) supplemented with 10% fetal bovine serum (Perbio, Cramlington, United Kingdom) and 1% penicillin/streptomycin and used up to 10 passages. Before studies, expression of the myofibroblast cell markers {alpha}-smooth muscle actin and vimentin was verified by immunohistochemistry. Human recombinant MMP-7, recombinant MMP-8, mouse purified epidermal growth factor (EGF), GM6001, LY-294002, Akt inhibitor, AG1024, MMP-3 inhibitor IV, MMP-2/9 inhibitor I, and MMP-8 inhibitor I were purchased from Calbiochem (Nottingham, United Kingdom). Recombinant MMP-3 was bought from Oncogene (Boston, MA) and U0126 from Cell Signaling Technology (Beverly, MA). Human purified platelet-derived growth factor (PDGF), recombinant IGFBP-5, IGF-I, and IGF-II were obtained from R&D Systems (Abingdon, Oxon, United Kingdom). All other chemicals were obtained from Sigma (Poole, Dorset, United Kingdom).

Immunoprecipitation and Western blots. Media from colonic myofibroblasts were collected and concentrated (see below). Protein extracts were prepared in lysis buffer [20 mmol/L Tris (pH 7.8), 150 mmol/L NaCl, 2 mmol/L EDTA, 0.5% NP40, 10 mmol/L NaF, 10 mmol/L sodium orthovanadate, and 2 mmol/L phenylmethylsulfonyl fluoride)] containing protease inhibitor cocktail set III (10 µL mL–1) and phosphatase inhibitor cocktail set II (10 µL mL–1; Calbiochem). Protein concentrations were estimated by Bio-Rad detergent-compatible protein assay (Bio-Rad, Hercules, CA). Both media and cell extracts were resolved by SDS-PAGE electrophoresis, transferred to nitrocellulose (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), and incubated with antibodies to MMP-1, MMP-2, IGF-1R (Santa Cruz Biotechnology, Santa Cruz, CA), MMP-3 (R&D Systems), MMP-8 (Oncogene), MMP-9 (Chemicon, Chandlers Ford, United Kingdom), urokinase-type plasminogen activator (uPA, American Diagnostics, Stamford, CT), phospho-p42/44 MAPK (New England BioLabs, Inc., Hertford, United Kingdom), phospho-Akt (Ser473; Cell Signaling Technology), IGFBPs 1-6, and IGF-I and IGF-II (R&D Systems) followed by horseradish peroxidase–conjugated secondary antibody and detection by incubation with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and HyperFilm (Amersham, Chalfont St. Giles, United Kingdom) as previously described (17). Samples from cellular extracts were reprobed for total MAPK using anti-ERK1 (BD Transduction Laboratories, Bedford, MA) or total Akt anti-Akt antibodies (BD Transduction Laboratories) as appropriate. Cell extracts were immunoprecipitated with mouse or rabbit antihuman IGF-IR{alpha} antibody (Santa Cruz Biotechnology) using protein agarose A or G bead slurry (Upstate, Lake Placid, NY). Western blot analysis was done on control and small interfering RNA (siRNA)–treated myofibroblasts (see later) using a mouse or rabbit anti IGF-IR{alpha} antibody and HeLa cell extracts as positive control (Santa Cruz Biotechnology).

Fluorogenic substrate assays. Media from myofibroblasts treated with MMP-7 were incubated with specific MMP-3 and MMP-8 fluorogenic substrates (Calbiochem) and fluorescence was measured using excitation at 280 nm and emission at 360 nm. For collagen IV breakdown assays, FITC-labeled bovine type IV collagen (Chemicon) was used; in this case, substrate and MMP-7 were incubated with MMP-3 and MMP-8 inhibitors according to the manufacturer's instruction.

Migration assays. Migration of human colonic myofibroblasts in response to MMP-7 was studied in 8-µm-pore Boyden chambers as previously described (17). Cells (25,000-30,000) were added to wells in serum-free medium; MMP-7 or drugs were added to the lower chamber. Cells migrating through the membrane were detected using DifQuick (Dade Behring, Inc., Newark, DE). The total cells in five fields per well were counted, and mean of three wells per experiment taken.

Proliferation assays. The incorporation of [methyl-3H]-thymidine (Amersham) into human colonic myofibroblasts was studied using methods previously described (18). Cells (25,000-30,000) were cultured in 6-well plates in serum-free medium for 48 hours, stimulated for 18 hours with MMP-7 and other compounds, and [3H]-thymidine (2 µCi mL–1) added for the last 2 hours; cells were processed as described (18).

Proteomic identification of proteins in media. Colonic myofibroblasts were plated into 10-cm Petri dishes and grown to 80% confluence. Cells were serum starved for 24 hours before stimulation with MMP-7 (4.0 µg mL–1), MMP-8 (0.1 µg mL–1), or MMP-3 (0.4 µg mL–1). Medium was then collected on ice, concentrated (final volume of 500 µL) using an Ultrafree-15 Biomax Membrane 5 kDa (Millipore, Bedford, MA) filter unit at 2,000 x g (20 minutes, 4°C). Following concentration, an equal volume of 20% trichloroacetic acid was added to the supernatant, which was then incubated on ice (120 minutes) and centrifuged (14,000 rpm, 5 minutes, 4°C). The pellet was washed twice with H2O-saturated diethyl ether and resuspended in 100 µL of 9 mol/L urea containing 2 mol/L thio-urea, 4% CHAPS, 40 mmol/L Tris base, and 10 µg mL–1 DTT; centrifuged (45,000 rpm, 30 minutes, 4°C) to remove DNA; and the supernatant stored at –80°C.

Two-dimensional gel electrophoresis and mass spectrometry. Protein extracts were diluted in 350 µL of 9 mol/L urea containing 2% CHAPS and a trace of bromophenol blue. Just before use, immobilized pH gradient (IPG: 4-7 or 3-10) buffer (Amersham) and DTT were added to final concentrations of 2% and 0.28%, respectively. Isoelectric focusing strips (18 cm; Amersham Biosciences) were rehydrated overnight at room temperature in a rehydration chamber (Amersham Biosciences). Isoelectric focusing of samples was done in a Multiphor II System (Amersham Biosciences) with an initial linear gradient of 0 to 500 V over 1 minute and a linear gradient from 500 to 3,500 V over 90 minutes, with a final step at 3,500 V for 340 minutes. Strips were then stored frozen at –80°C, before second dimension separation in which strips were incubated twice (15 minutes) in equilibration buffers [1.5 mol/L Tris-HCl (pH 8.8), 6 mol/L urea, 34.5% glycerol, 2%SDS, and a trace of bromophenol blue], with shaking at room temperature. The first buffer contained 1% DTT and the second buffer contained 2.5% iodoacetamide. After one rinse in electrophoresis buffer (25 mmol/L Tris base, 192 mmol/L glycine, and 0.1% w/v SDS), strips were applied to a 12.5% precast Ettan DALT II polyacrylamide gel (Amersham Biosciences) and sealed with melted agarose. Electrophoresis was done using the Ettan Dalt II Separation Unit and Buffer Kit (Amersham Biosciences) at 25°C, with an initial 2.5 W per gel (30 minutes) followed by a 20 W per gel until the dye front had run ~18 cm. Gels were then immersed in fixative (40% methanol, 7% acetic acid) overnight; stained for 4 hours in 80% methanol and a 20% solution of 0.1% Coomassie brilliant blue-G250 dye, 2% orthophosphoric acid, and 10% ammonium sulfate; and destained for 1 minute in 10% acetic acid, 25% methanol with destaining continued in 25% methanol. Finally, gels were stored in 25% methanol at 4°C. Gel images were acquired using a Bio-Rad GS-710 scanner at 300 dpi resolution and data captured using PDQuest software (Bio-Rad). Differentially abundant spots in control, MMP-7-, MMP-3-, or MMP-8-treated samples were picked from the gel and stored in 25% methanol. Identification of spots was based on tryptic digestion coupled to peptide sequencing using a QTOF mass spectrometer (Functional Genomics and Proteomics Service, University of Birmingham, Birmingham, United Kingdom). The peptide masses obtained were used for database protein identifications using Mascot (Matrix Science Ltd., London, United Kingdom). Individual ion scores of >40 indicate identity or extensive homology at a significance level of P < 0.05. Protein scores are derived from ion scores and assignments were only made above a significance threshold of P < 0.05.

Small interference RNA of insulin-like growth factor-1 receptor. Transfection of siRNA SMARTpool (four pooled SMART-selected siRNA duplexes) for IGF-1R (Dharmacon, Inc., Chicago, IL) was done on cells in suspension using Amaxa Nucleofection Apparatus (Amaxa GmbH, Koln, Germany), solution R, program G16, according the manufacturer's instruction. Treated cells were extracted for detection of IGF-1R by Western blot (see above). In addition, to determine receptor binding after siRNA treatment, we did assays on cells (10–5 cells plated in 24-well plates for 72 hours) incubated for either 4 hours at 22°C or 16 hours at 4°C using recombinant human [125I]-IGF-I (receptor grade, DSL, Inc., Webster, TX) according a previously published protocol (18, 19). Nonspecific binding was determined by addition of 1.0 µg mL–1 IGF-II.

Statistics. Results are presented as means ± SE; comparisons were made using ANOVA or Student's t tests where appropriate and were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased proliferation and migration of colonic myofibroblasts in response to matrix metalloproteinase-7. In initial studies, we found increased proliferation and migration of colonic myofibroblasts exposed to MMP-7 (1-4 µg mL–1) using [3H]-thymidine incorporation and Boyden chamber migration assays, respectively. Both responses were inhibited by the broad specificity MMP inhibitor GM6001 (12.9 µmol/L), indicating that the enzymatic activity of MMP-7 was responsible for these effects (Supplementary Fig. 1A and B). The colonic myofibroblasts we used are known to express MMP-2 (1) and we have also found expression of MMP-1, MMP-3, MMP-8, and MMP-9 (but not MMP-7), as well as uPA (data not shown). Because MMPs are frequently involved in activating each other (20), we examined the proliferative and migratory responses to MMP-7 in the presence of specific inhibitors of some other MMPs. Thus, inhibitors of MMP-3 (4.8 µmol/L) and MMP-8 (27 nmol/L) each significantly reduced both responses (Fig. 1A and B). In control experiments, the specificity of these inhibitors was verified by showing no significant effect in a FITC-conjugated collagen IV breakdown assay using MMP-7 (MMP-7 alone, 100 ± 2.3%; MMP-7 + MMP-3 inhibitor, 91.7 ± 3.7%; MMP-7 + MMP-8 inhibitor, 90 ± 3.9%). Consistent with these observations, media from colonic myofibroblasts treated with MMP-7 exhibited increased abundance of proMMP-3 and MMP-3 and of proMMP-8 and MMP-8 detected by Western blot (Fig. 1C), but there was no increase in abundance in the cell extracts (data not shown). Moreover, there was increased MMP-3 and MMP-8 enzyme activity in specific fluorogenic substrate assays of media from MMP-7-treated myofibroblasts (Fig. 1D). MMP-3 and MMP-8 also stimulated proliferation in a concentration-dependent manner, but the maximal response in both cases was less than that of MMP-7 (Supplementary Fig. 2). In contrast to the MMP-3 and MMP-8 inhibitors, an inhibitor of MMP-2/9 (1.3 µmol/L) had no significant effect on the responses to MMP-7 (MMP-7-stimulated [3H]-thymidine incorporation, 100 ± 3.9%, compared with 110.3 ± 13.8% for MMP-7 + MMP-2/9 inhibitor; MMP-7-stimulated migration, 100 ± 17.6%, compared with 95.4 ± 12.3% for MMP-7 + MMP-2/9 inhibitor).



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  		Figure 1. MMP-7 stimulates colonic myofibroblast proliferation and migration partly via activation of MMP-3 and MMP-8. A, colonic myofibroblast proliferation is stimulated by MMP-7 (2 µg mL–1) and this is significantly reduced by specific MMP-3 (4.8 µmol/L) and MMP-8 (27 nmol/L) inhibitors (n = 3). The incorporation of [3H]-thymidine in control cells was 2,724 ± 460 cpm per 106 cells. B, colonic myofibroblast migration is stimulated by MMP-7 (2 µg mL–1) and is reduced by the same inhibitors (n = 3). The migration of control cells was 6.7 ± 1.0 per field. There is no effect of the inhibitors on the proliferation and migration of control cells. C, Western blot of myofibroblast medium after treatment of cells with MMP-7 for 24 hours showing increased abundance of proMMP-3 and proMMP-8 and of MMP-3 and MMP-8. D, in assays using specific fluorogenic substrates (see Materials and Methods), MMP-3 and MMP-8 enzyme activities were each increased in medium from MMP-7-treated colonic myofibroblasts (n = 3). *, P < 0.05.

 
Matrix metalloproteinase-7 stimulates colonic myofibroblast proliferation and migration via mitogen-activated protein kinase and phosphatidylinositol 3-kinase. To examine possible cellular mechanisms mediating the effects of MMP-7 on cell proliferation and migration, we then considered the involvement of the MAPK and PI3K pathways. Western blots indicated transiently increased abundance of phosphorylated p42/44 extracellular signal-regulated kinase (ERK) in myofibroblasts treated with MMP-7 after 10 minutes. Similarly, there was increased phosphorylation of the PI3K target Akt, although the time course was distinct from that of p42/44 ERK phosphorylation with a progressive increase in phosphorylation up to 240 minutes (Fig. 2A and B). Compatible with these observations, an inhibitor of the p42/44 kinase kinase, MAPK kinase (i.e., U0126, 10 µmol/L) reversed the effect of MMP-7 on both proliferation and migration and so too did the PI3K inhibitor LY294002 (50 µmol/L) and an inhibitor (3.4 µmol/L) of Akt (Fig. 2C and D).



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  		Figure 2. MMP-7 activates p42/44Erk and PI3K in colonic myofibroblasts. Western blots show (A) increased abundance of phospho-p42/44Erk 10 minutes after addition of MMP-7 (2 µg mL–1) and (B) increased abundance of phospho-Akt with a maximum at 120 minutes after addition to cells of MMP-7. C, stimulation of [3H]-thymidine incorporation by MMP-7 (2 µg mL–1) in colonic myofibroblasts was virtually abolished by the MAPK kinase inhibitor U0126 (10 µmol/L), the PI3K inhibitor LY294002 (50 µmol/L) and significantly reduced by an Akt inhibitor (3.4 µmol/L). In control cells, U0126 reduced [3H]-thymidine incorporation by 34.7 ± 2.7% and this was taken into account in presenting the data; LY294002 and the Akt inhibitor had no effect in control cells. D, migration of myofibroblasts in a Boyden chamber in response to MMP-7 (2 µg mL–1) was also greatly reduced by U0126, LY294002, and the Akt inhibitor. In control cells, there were small but not significant effects of U0126, LY294002, and the Akt inhibitor.

 
Identification of putative matrix metalloproteinase-7 substrates in the medium of colonic myofibroblasts. To identify putative substrates of MMP-7, we separated medium from colonic myofibroblasts by two-dimensional gel electrophoresis with mass spectrometric identification of peptides exhibiting differential abundance (Fig. 3). In media from MMP-7-treated cells there were seven relatively abundant spots corresponding to proteins of 25 to 50 kDa that were absent in control media. These included collagen {alpha}1 types I, III, and VI, and fibronectin. Strikingly, however, two relatively abundant proteins yielded tryptic peptides corresponding to fragments of IGFBP-5 (Fig. 3). Essentially similar identifications were made using two different isoelectric focusing conditions, pH 3-10 and pH 4-7 (Table 1). The assignment of IGFBP-5 as a putative MMP-7 substrate was based on identification of four of seven tryptic peptides in the sequence 34 to 135 (69% coverage). Because our studies had identified MMP-3 and MMP-8 as implicated in the response to MMP-7, we also sought proteins that were differentially abundant in media from colonic myofibroblasts treated with MMP-3 (0.4 µg mL–1) and MMP-8 (0.1 µg mL–1). In both cases, there was increased abundance of spots corresponding to IGFBP-5 (Supplementary Fig. 3; Table 1), although these were relatively less acidic than those generated by MMP-7 treatment. The spots generated by MMP-3 and MMP-8 were identified as including three of the tryptic peptides also found in MMP-7-treated samples, as well as a fourth tryptic peptide corresponding to the sequence 50-73. Moreover, in the case of MMP-8 an additional tryptic peptide covering the sequence 144-153 was also identified suggesting that MMP-8 cleaved IGFBP-5 at a site COOH-terminal to the MMP-7 cleavage site (Supplementary Fig. 4).



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  		Figure 3. Identification of putative MMP-7 substrates in the medium of colonic myofibroblasts. Two-dimensional gel electrophoresis images of media from control (A) and MMP-7 treated (B) myofibroblasts (24 hours) showing several spots exhibiting differential abundance. C, identification of tryptic peptides (bold) in the primary sequence of IGFBP-5 in differentially abundant proteins in the medium of colonic myofibroblasts treated with MMP-7. One spot (a, see B) in MMP-7-treated samples yielded peptides corresponding to IGFBP-5, 60-73 and 98-104; a second spot (b) yielded peptides corresponding to IGFBP-5, 34-49, 60-73, 84-104, and 118-135.

 

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  		Table 1. Identification of IGFBP-5, collagens, and other proteins in the medium of colonic myofibroblasts treated with MMP-7, MMP-3, and MMP-8

 
Cleavage of insulin-like growth factor binding protein-5 by matrix metalloproteinase-7 and release of insulin-like growth factor-II. To independently verify whether MMP-7 acted on IGFBP-5, we then showed by Western blot that (a) MMP-7 increased the abundance of a 20-kDa fragment of IGFBP-5 in the media of colonic myofibroblasts within 1 hour, (b) MMP-7 was capable of cleaving rhIGFBP-5 (Fig. 4A), and (c) with the exception of small amounts of IGFBP-4, other members of this family (i.e., IGFBP-1, IGFBP-2-, IGFBP-3, and IGFBP-6 were undetectable; data not shown). With prolonged exposure of colonic myofibroblasts to MMP-7 up to 24 hours, there was a progressive increase in the abundance of the 20-kDa IGFBP-5 fragment and the appearance of proteins of 18 and 16 kDa. In control media incubated for 24 hours, intact IGFBP-5 (38 kDa) and a partial degradation product of ~25 kDa were detected but no proteins of 16 to 20 kDa (Fig. 4A). Based on the results presented above, we then hypothesized that the proliferative and migratory responses of myofibroblasts treated with MMP-7 reflected stimulation by IGF released after cleavage of IGFBP-5. In keeping with the hypothesis, Western blot analysis showed an increase of IGF-II in the media of colonic myofibroblasts after 1 hour of MMP-7 stimulation and a decrease of IGFBP-5 and IGF-II in treated cells (Fig. 4B-C). In contrast, IGF-I was not detected in the media of colonic myofibroblasts either in control conditions, confirming previous observations (21) or after MMP-7 treatment (Fig. 4B). IGF-II increased phosphorylation of p42/44 ERK and Akt (data not shown), and stimulated proliferation, which although not unexpected, was a necessary requirement to sustain the hypothesis.



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  		Figure 4. Time-dependent cleavage of IGFBP-5 by MMP-7 and release of IGF-II. A, Western blots of medium from colonic myofibroblasts incubated with MMP-7 (2 µg mL–1) up to 24 hours show the appearance of a fragment of 20 kDa and a progressive increase with time of products of 18 and 16 kDa (arrows). Note the presence of a larger cleavage product in the unstimulated medium after 24 hours. Right, effect of recombinant MMP-7 on recombinant IGFBP-5. B, Western blots of medium from colonic myofibroblasts incubated with MMP-7 show increased IGF-II abundance at 60 minutes but not IGF-I. C, abundance of IGFBP-5 (left), IGF-II (right), and GAPDH in extracts of cells treated with MMP-7 for 1 hour.

 
Functional significance of increased insulin-like growth factor-II on myofibroblast function. To directly establish whether enhanced IGF-II signaling might be responsible for the proliferative and migratory responses to MMP-7, we then showed that a specific IGF-1R tyrosine kinase inhibitor, AG1024 (3.2 µmol/L), significantly reduced both responses. Moreover, a neutralizing antibody to IGF-II (1.25 µg mL–1) also reduced MMP-7-stimulated proliferation, whereas neutralizing antibody to IGF-I (1.25 µg mL–1) had no effect (Fig. 5A). Finally, siRNA knockdown of IGF-1R (200 nmol/L), which reduced IGF-1R abundance and IGF-1R binding to 34.3 ± 7.5% and 31.2 ± 7.2%, respectively, significantly inhibited the proliferative responses to both MMP-7 (2 µg mL–1) and IGF-I (50 ng mL–1) but had no effect on responses to PDGF (10 ng mL–1) or EGF (50 ng mL–1; Fig. 5B).



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  		Figure 5. MMP-7 increases proliferation and migration of colonic myofibroblasts via IGF-1R. A, an IGF-1R tyrosine kinase inhibitor AG1024 (3.2 µmol/L) and a neutralizing IGF-II antibody (1.25 µg mL–1) significantly inhibited MMP-7 (2 µg mL–1) stimulated [3H]-thymidine incorporation and migration (n = 3-6). *, P < 0.05. B, pretreatment with siRNA (200 nmol/L) to down-regulate IGF-1R also significantly reduced both MMP-7- and IGF-I-stimulated [3H]-thymidine incorporation, while EGF (50 ng mL–1) and PDGF (10 ng mL–1) stimulated [3H]-thymidine incorporation were not affected (n = 3-6).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results show that MMP-7 stimulates colonic myofibroblast proliferation and migration, that IGFBP-5 produced by myofibroblasts is a substrate for MMP-7, and that myofibroblast responses to MMP-7 are mediated at least partly by IGF-II, activation of IGF-1R and stimulation of the MAPK and PI3K pathways. The data are compatible with the view that IGF-II functions as an autocrine growth factor for myofibroblasts (21), but they extend this model by showing that the action of IGF-II is limited by autologous production of IGFBP-5 in these cells, and that MMP-7 modulates the activity of the IGF-signaling system by partial proteolysis of IGFBP-5. Expression of MMP-7 is a feature of epithelial cells; thus, release of MMP-7 acts, in effect, as a signal from the epithelium that determines local recruitment of myofibroblasts and their proliferation. One implication of these findings is that increased MMP-7 expression by epithelial tumor cells might trigger the expansion of stroma by activation of myofibroblasts.

The MMPs are generally found in subepithelial cells but MMP-7 is relatively unusual in that its expression is predominantly epithelial. In keeping with this, we have found MMP-1, MMP-2-, MMP-3, MMP-8, and MMP-9, but not MMP-7, in colonic myofibroblasts. There is a substantial body of evidence to indicate that increased MMP-7 expression occurs in many different epithelial tumors, including breast, pancreas, colon, and stomach (22–24). In addition, increased MMP-7 expression occurs in a wide variety of epithelial cells including those of the airways, urogenital, and gastrointestinal tracts in response to bacterial infection and chronic inflammation (9, 13, 25). It has previously been reported that MMP-7 is increased in association with IGF-II and IGF-I in colorectal cancer (26), but the functional links between MMP-7 and IGF-signaling have not attracted attention until recently.

The MMPs have long been recognized to play a role in tumor cell invasion by digestion of the extracellular matrix required for tumor cell spread. In the case of MMP-7, there is also a growing body of evidence that there are other important substrates including growth factors such as heparin-binding EGF, Fas-ligand, syndecan-1 (which tethers the CXC chemokine KC), and prodefensins (12, 27). Together, these actions may lead either to gain or loss of function and are potentially important in host defense and in the regulation of cell proliferation, apoptosis, and migration (3, 8). In addition, observations in MMP-7 null mice suggest a role in the development of fibrosis in a model of chronic inflammation (28), although the relevant cellular mechanisms are poorly understood. The present findings identify IGFBP-5 as a novel target for MMP-7 implicated in proliferative and migratory responses of myofibroblasts.

The IGF-signaling system consists of IGF-I and IGF-II, the receptor IGF-1R and six different binding proteins, IGFBP1-6 (29, 30). In solution, the latter bind IGFs to prevent IGF-1R activation; however, IGFBPs may also associate with extracellular matrix or the cell surface to promote receptor stimulation. Furthermore, recent evidence points to biological actions of IGFBPs that are independent of IGF binding (30). The IGF signaling system is important in normal growth and development, in tissue remodeling, and in the progression to cancer. In particular, increased expression of IGF-I, IGF-II, IGF-1R, and IGFBP-3 and IGFBP-5 have all been reported in tumors of the gastrointestinal tract (26, 31–33). Limited proteolysis of IGFBPs and the consequent liberation of IGFs has been extensively studied (30, 34). Within IGFBP-5, there are IGF binding sites in both the NH2- and COOH-terminal domains (35), and cleavage reduces the affinity for IGF-II. A number of proteases have previously been identified as cleaving IGFBP-5 including complement C1s, ADAM12s, ADAM-9, and PAPPA-2 (36–40). A role for MMP-1 and MMP-2, together with an unidentified 97-kDa proteinase, in degradation of IGFBP-5 in murine osteoblasts has also been described (41, 42). Moreover, MMP-1, MMP-2, MMP-3, and MMP-7 have all been shown to act on IGFBP-3 in vitro inhibiting its capacity to bind IGF-I and leading to increased proliferation of the colon cancer cell lines, CoLo201 and HT29, or 3T3 fibroblasts (43–46). The present study indicates that MMP-7 acts on IGFBP-5 to liberate a NH2-terminal fragment of ~20 kDa. Our data do not suggest that other IGFBP-s (with the possible exception of IGFBP-4 that is expressed at a low level) are produced by myofibroblasts. Neither do they suggest IGF-1 is present in this system. Thus, at least as far as autocrine control of colon myofibroblast proliferation and migration is concerned, the action of MMP-7 to modulate IGFBP-5 binding of IGF-II is likely to be functionally important.

It is well established that MMPs are generally produced as inactive precursors that are activated by cleavage after secretion (20). Many interactions have been described indicating that these and other proteases may function in cascades. We found that human colon myofibroblasts express a number of MMPs including MMP-3 and MMP-8 and that in the presence of MMP-7 there are increases in the abundance of both proMMP-3 and proMMP-8 indicating enhanced release of precursors, as well as increased abundance of the active enzymes suggesting enhanced conversion. We also found evidence that MMP-3 and MMP-8 might contribute to the action of MMP-7 in stimulating myofibroblast proliferation and migration. Furthermore, with prolonged incubation of unstimulated myofibroblasts there was limited spontaneous cleavage of IGFBP-5 generating a fragment of ~25 kDa that could be attributed to the activity of MMP-3 and MMP-8. There have been previous reports of MMP-3 expression by myofibroblasts (47) but MMP-8 expression seems not to have been described before; clearly, further work is now needed to systematically define the role of different MMPs produced in these cells.

Taken as a whole the present data identify MMP-7 as a previously unsuspected regulator of myofibroblast cell function. Although the present studies were based on colonic myofibroblasts, we have also observed MMP-7 stimulation of proliferation and migration of gastric myofibroblasts via cleavage of IGFBP-5. Myofibroblasts are a key stromal cell type thought to produce growth factors for epithelial cells; the data therefore suggest a novel mechanism by which epithelial cells might regulate stromal cell number and location. Myofibroblasts are also important for deposition of extracellular matrix, and in addition to IGBFP-5, we identified collagen {alpha}1 types I, II, and VI as substrates for MMP-7. Further work is now needed to determine whether the MMP-7-regulated mechanism described here also leads to increased collagen production compatible with stimulation of myofibroblast cell function. Given the role of myofibroblasts in the control of the stromal reaction in cancer (2), the identification of MMP-7 as a novel mediator of epithelial-mesenchymal signaling therefore raises new possibilities for manipulating tumor progression.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the Medical Research Council, Wellcome Trust, and the North West Cancer Research Fund.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 1/17/05. Revised 4/22/05. Accepted 6/ 7/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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