Originally published In Press as doi:10.1074/jbc.M308708200 on December 16, 2003
J. Biol. Chem., Vol. 279, Issue 10, 8592-8601, March 5, 2004
Differential Inhibition of Membrane Type 3 (MT3)-Matrix Metalloproteinase (MMP) and MT1-MMP by Tissue Inhibitor of Metalloproteinase (TIMP)-2 and TIMP-3 Regulates Pro-MMP-2 Activation*
Huiren Zhao
,
M. Margarida Bernardo,
Pamela Osenkowski,
Anjum Sohail,
Duanqing Pei
,
Hideaki Nagase¶,
Masahide Kashiwagi¶,
Paul D. Soloway||,
Yves A. DeClerck**, and
Rafael Fridman
From the
Department of Pathology, School of Medicine, Wayne State University, Detroit, Michigan 48201, the Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, the ¶Kennedy Institute of Rheumatology Division, Imperial College London, London W6 8LH, United Kingdom, the ||Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, and the **Division of Hematology-Oncology and Department of Pediatrics, Children's Hospital Los Angeles, University of Southern California, Los Angeles, California 90027
Received for publication, August 6, 2003
, and in revised form, December 1, 2003.
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ABSTRACT
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The membrane type (MT)-matrix metalloproteinases (MMPs) constitute a subgroup of membrane-anchored MMPs that are major mediators of pericellular proteolysis and physiological activators of pro-MMP-2. The MT-MMPs also exhibit differential inhibition by members of the tissue inhibitor of metalloproteinase (TIMP) family. Here we investigated the processing, catalytic activity, and TIMP inhibition of MT3-MMP (MMP-16). Inhibitor profile and mutant enzyme studies indicated that MT3-MMP is regulated on the cell surface by autocatalytic processing and ectodomain shedding. Inhibition kinetic studies showed that TIMP-3 is a high affinity inhibitor of MT3-MMP when compared with MT1-MMP (Ki = 0.008 nM for MT3-MMP versus Ki = 0.16 nM for MT1-MMP). In contrast, TIMP-2 is a better inhibitor of MT1-MMP. MT3-MMP requires TIMP-2 to accomplish full pro-MMP-2 activation and this process is enhanced in marimastatpretreated cells, consistent with regulation of active enzyme turnover by synthetic MMP inhibitors. TIMP-3 also enhances the activation of pro-MMP-2 by MT3-MMP but not by MT1-MMP. TIMP-4, in contrast, cannot support pro-MMP-2 activation with either enzyme. Affinity chromatography experiments demonstrated that pro-MMP-2 can assemble trimolecular complexes with a catalytic domain of MT3-MMP and TIMP-2 or TIMP-3 suggesting that pro-MMP-2 activation by MT3-MMP involves ternary complex formation on the cell surface. These results demonstrate that TIMP-3 is a major regulator of MT3-MMP activity and further underscores the unique interactions of TIMPs with MT-MMPs in the control of pericellular proteolysis.
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INTRODUCTION
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The matrix metalloproteinases (MMPs),1 a multidomain family of zinc-dependent endopeptidases, degrade all structural components of the extracellular matrix (ECM) and many bioactive molecules, thereby playing essential roles in many physiological and pathological processes (1–4). Based on structural organization and subcellular localization, the MMP family is divided into secreted and membrane-anchored enzymes (1, 5). The membrane type-MMPs (MT-MMPs) comprise six members of plasma membrane-tethered MMPs, which include four type I transmembrane enzymes: MT1-, MT2-, MT3-, and MT5-MMP, and two glycosylphosphatidylinositol-anchored enzymes: MT4- and MT6-MMP (4, 6–8). Although there is some tissue-specific expression of MT-MMPs, there is also significant overlap both in expression and in function raising the question of how cells command the repertoire of MT-MMPs at their disposition during proteolytic events. A major mechanism for controlling MT-MMP activity in the pericellular space is mediated by the action of tissue inhibitors of metalloproteinases (TIMPs). However, as opposed to soluble MMPs, which are almost equally inhibited by all TIMPs (9, 10), the MT-MMPs are highly selective when it comes to TIMP interactions. For example, the type I transmembrane MT-MMPs are poorly inhibited by TIMP-1 but are relatively well inhibited by TIMP-2, TIMP-3, and TIMP-4. In contrast, the glycosylphosphatidylinositol-anchored MT-MMPs are inhibited by both TIMP-1 and TIMP-2 (11). The differential sensitivity to TIMP inhibition among members of the MT-MMP family may facilitate the control of MT-MMP activity in the pericellular space.
In addition to being potent ECM-degrading enzymes, the type I transmembrane MT-MMPs can also initiate a cascade of zymogen activation on the cell surface. MT-MMPs activate the zymogenic form of MMP-2 (pro-MMP-2 or pro-gelatinase A) (6, 8), which in turn can activate pro-MMP-9 (pro-gelatinase B) (12). MT1-MMP also promotes the activation of pro-collagenase 3 (MMP-13) (13), a potent collagenolytic protease. Because the gelatinases are efficient gelatinolytic enzymes, the MT-MMP/MMP-13/gelatinase axis represents a well adapted proteolytic system designed to promote coordinated and complete collagen degradation in the pericellular space. Although the type I transmembrane MT-MMPs have been shown to activate pro-MMP-2 (6), the specific mechanism(s) by which each member of this subgroup accomplishes pro-MMP-2 activation and the accessory molecules involved in this process are not completely understood. In the case of MT1-MMP, pro-MMP-2 activation requires the participation of TIMP-2, which acts as a molecular link between an active MT1-MMP on the cell surface and pro-MMP-2 (14, 15). The N-terminal region of TIMP-2 binds to the active site of MT1-MMP, whereas the C-terminal region of the inhibitor binds to the hemopexin-like domain of pro-MMP-2 forming a so-called "ternary complex" (16, 17). The propeptide of the bound pro-MMP-2 is then cleaved at the Asn37-Leu38 peptide bond by a neighboring TIMP-2-free active MT1-MMP molecule. This is followed by a second cleavage event in which the intermediate MMP-2 form is cleaved at the Asn80-Tyr81 peptide bond by a fully active MMP-2 in an autocatalytic intermolecular manner resulting in full activation (13). TIMP-2-dependent pro-MMP-2 activation occurs only at low TIMP-2 concentrations relative to MT1-MMP (9, 15, 18). This permits availability of enough TIMP-2-free active MT1-MMP to hydrolyze the prodomain of pro-MMP-2 bound in the ternary complex. Thus, under limited conditions, TIMP-2 promotes zymogen activation, whereas high levels of TIMP-2 relative to MT1-MMP inhibit activation by blocking all free MT1-MMP molecules (14, 18–20). Besides its role in regulation of pro-MMP-2 activation, TIMP-2 also controls the turnover of active MT1-MMP on the cell surface by inhibiting its autocatalytic processing, which in turn can positively influence MT1-MMP activity (18). Paradoxically, reversible synthetic MMP inhibitors, which also inhibit the autocatalytic turnover of MT1-MMP, can enhance pro-MMP-2 activation by MT1-MMP in the presence of TIMP-2 (21).
The role of TIMPs in pro-MMP-2 activation varies depending on the MT-MMP. Whereas TIMP-2 is required for the efficient activation of pro-MMP-2 by MT1-MMP (18) via ternary complex formation (14, 22), it is not required for MT2-MMP (23). However, how pro-MMP-2 interacts with MT2-MMP in the absence of TIMPs is unknown. Although TIMP-4 is capable of binding pro-MMP-2 (24) and is an effective inhibitor of MT1-MMP and MT2-MMP (22, 23, 25), it cannot support pro-MMP-2 activation (22, 26). Consistently, TIMP-4 has been shown to be unable to generate ternary complexes with these MT-MMPs (22). Likewise, TIMP-3, which binds pro-MMP-2 and inhibits MT-MMPs (27–29), does not support pro-MMP-2 activation by MT1-MMP (28). However, the role of TIMP-3 in pro-MMP-2 activation by other MT-MMPs is unknown. Together, this accumulating evidence also indicates that TIMPs differentially regulate the ability of MT-MMPs to initiate cascades of zymogen activation at the cell surface.
MT3-MMP (MMP-16), which was originally cloned from human melanoma tissue and human placenta (30), is expressed in a variety of normal (30–33) and tumor (7, 33–35) tissues. Studies with a purified active MT3-MMP lacking the transmembrane domain have shown that the truncated enzyme cleaves a variety of ECM components and is inhibited by TIMP-2 and TIMP-3 but not by TIMP-1 (29). However, the kinetics of MT3-MMP inhibition by TIMPs was not determined. Functionally, expression of MT3-MMP in human WM1341D melanoma cells (36), but not in monkey kidney COS-1 cells (37), facilitated in vitro collagen I invasion. Also, expression of MT3-MMP in hamster CHO-K1 and canine Madin-Darby canine kidney cells induced the expression of a fibrin invasive phenotype (38). Expression of MT3-MMP in various cell lines (30, 37, 39–41) promoted pro-MMP-2 activation and this activity was inhibited by addition of exogenous TIMP-2 (30, 42) or synthetic MMP inhibitors (40, 42). However, the role of TIMPs in MT3-MMP-mediated pro-MMP-2 activation has not been determined. To gain insight into the regulation of MT3-MMP, we expressed it in mammalian cells and investigated its processing, inhibition by TIMPs, and its ability to activate pro-MMP-2 in the presence of TIMPs.
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EXPERIMENTAL PROCEDURES
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Cell Culture—Immortalized heterozygous (+/–) and homozygous (–/–) TIMP-2-deficient mouse fibroblasts were isolated as previously described (18, 43) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics. Monkey kidney immortalized BS-C-1 (CCL-26), and human HeLa S3 cells were cultured as described (21). Monkey kidney immortalized COS-1 (CRL-1650) and CV-1 (CCL-70) cells and human fibrosarcoma HT-1080 (CCL-121) cells were all obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM containing 10% fetal bovine serum and antibiotics. All other cell culture reagents were obtained from Invitrogen.
Recombinant Proteins, Protease Inhibitors, and Antibodies—Human recombinant pro-MMP-2, TIMP-2, and TIMP-1 were expressed in HeLa S3 cells and purified to homogeneity, as previously described (44). Human TIMP-4 and TIMP-3 were purchased from R&D Systems Inc. (Minneapolis, MN) and their concentrations were determined by activesite titration using active MMP-2 with known concentration, as previously described (45). The catalytic domains of MT1-MMP (MT1-MMPcat) and MT3-MMP (MT3-MMPcat) were purchased from Calbiochem (San Diego, CA). N-TIMP-2, an N-terminal inhibitory domain of human TIMP-2 ending at Cys128 was expressed in mammalian cells and purified as previously described (46). N-TIMP-3 comprising the N-terminal region of mature human TIMP-3, residues Cys1 to Asn121, with a His tag attached to the C terminus was expressed in Escherichia coli and purified as described (47). The hydroxamate-based MMP inhibitors batimastat (BB94) and marimastat (BB2516) were obtained from British Biotech (Oxford, United Kingdom). A rabbit polyclonal antibody against human MT3-MMP was purchased from Calbiochem. The monoclonal antibody to TIMP-2 (CA-101) has been previously described (48).
Recombinant Vaccinia Viruses—The generation of the recombinant vaccinia virus expressing bacteriophage T7 RNA polymerase (vTF7-3) has been described by Fuerst et al. (49). Recombinant vaccinia viruses expressing MT1-MMP (vT7-MT1), pro-MMP-2 (vT7-GelA), or TIMP-2 (vSC59-T2) were generated by homologous recombination as previously described (18, 48, 49). To generate a recombinant vaccinia virus expressing the wild-type human MT3-MMP enzyme, the full-length human MT3-MMP cDNA was amplified by PCR using specific primers containing the appropriate restriction sites, and the resulting fragment was cloned into the NcoI and BamHI sites of the pTF7EMCV-1 expression vector, under the control of the T7 promoter (49). After sequence verification of the insert in both directions, the resulting pTF7-MT3 plasmid was used to generate a recombinant vaccinia virus (vT7-MT3) by homologous recombination with wild-type vaccinia virus, as previously described (48, 49).
Generation of MT3-MMP Mutants—To generate a catalytically inactive MT3-MMP (E/A-MT3-MMP), Glu247 was substituted for Ala using the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the wild-type MT3-MMP cDNA in pTF7EMCV-1 as a template. A cytosolic tail (CT) deletion mutant (
CT-MT3-MMP) was constructed by introducing a termination codon at Gln586. Likewise, a transmembrane domain and cytosolic tail deleted MT3-MMP (TM/CT-MT3-MMP) was constructed by introducing a termination codon at Ala564. The fidelity of the inserts was verified by DNA sequencing in both directions. Recombinant viruses expressing these MT3-MMP mutants were generated by homologous recombination with wild-type vaccinia virus, as previously described (48, 49).
Expression of MT3-MMP and TIMP-2 by Co-infection—To express MT3-MMP, cells (various types) in six-well plates were co-infected with 10 plaque-forming units (pfu)/cell each of vTF7-3 and vT7-MT3 viruses (encoding for either wild-type or mutant enzymes) for 45 min in infection media (DMEM supplemented with 2.5% fetal bovine serum and antibiotics) at 37 °C. As a control, the cells were infected only with the vTF7-3 virus. To co-express MT3-MMP with TIMP-2, and to modulate the level of inhibitor expression, BS-C-1 cells were co-infected with 5 pfu/cell each of vTF7-3 and vT7-MT3 viruses and increasing amounts (0–20 pfu/cell) of the TIMP-2-expressing vaccinia virus vSC59-T2 as described (18). The infected cells were then used for pro-MMP-2 activation and MT3-MMP processing studies by gelatin zymography and immunoblot analysis, respectively, as described (50). In some experiments, the media of cells co-expressing MT3-MMP and increasing amounts of TIMP-2 were collected and analyzed for TIMP-2 concentration by enzyme-linked immunosorbent assay (catalog number QIA40, Oncogene Research Products, San Diego, CA) as described by the manufacturer.
Pro-MMP-2 Activation by Membrane-bound and Soluble MT3-MMP—Cells (various types) in six-well plates were co-infected to express MT3-MMP as described above. After these procedures, the media were aspirated and cells were rinsed once with serum-free DMEM, followed by a 4-h incubation with serum-free DMEM. The media were then aspirated and replaced with serum-free DMEM (1 ml/well) supplemented with pro-MMP-2 without or with various amounts of TIMP-2, N-TIMP-2, TIMP-3, TIMP-4, or marimastat. At varying times, the media were collected for gelatin zymography and the cells were lysed on ice with cold lysis buffer (25 mM Tris-HCl, pH 7.5, 1% IGEPAL CA 630 (Sigma), also known as Nonidet P-40, 100 mM NaCl, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 2 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride). Media and lysates were analyzed by gelatin zymography for pro-MMP-2 activation and by immunoblot analysis for MT3-MMP forms, as previously described (50). To assess the ability of soluble MT3-MMP to process pro-MMP-2, BS-C-1 and TIMP-2–/– cells were grown in a 100-mm tissue culture dish each and co-infected (vTF7-3 and vT7-MT3 viruses) to express MT3-MMP or infected with vTF7-3 virus alone, as described above. After infection, the cells were washed and incubated overnight at 37 °C in serum-free media (6 ml/dish). Then, the media were collected, clarified by centrifugation, and concentrated 
30 times with Centricon Plus-20. The concentrated media were then subjected to ultracentrifugation at 100,000 x g for 1 h at 4 °C. The supernatants were collected and 20 µl of this fraction were incubated (24 h, 37 °C) with various concentrations of pro-MMP-2 (25, 100, and 500 nM) in collagenase buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.02% Brij-35) in a final reaction volume of 50 µl. Pro-MMP-2 processing was monitored by gelatin zymography.
Enzyme Inhibition Studies—The enzymatic activity of MT1-MMP and MT3-MMP catalytic domains (MT1-MMPcat and MT3-MMPcat) was monitored using the synthetic fluorogenic peptide (7-methoxycoumarin-4-yl)acetyl-L-prolyl-L-glycyl-leucyl-(N3-(2,4-dinitophenol)-L-2,3-diaminopropionyl)-L-alanyl-L-arginine amide (MOCAcPLGLA2pr(Dnp)AR-NH2 from Peptides International, Louisville, KY), as described (51), on a Photon Technology International spectrofluorometer, at excitation and emission wavelengths of 328 and 393 nm, respectively. The concentration of MT1-MMPcat and MT3-MMPcat was determined using a solution of recombinant TIMP-2 with known concentration. All of the kinetic assays were carried out in a buffer consisting of 50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM CaCl2, 0.01% Brij-35, and 1% Me2SO (1% v/v) (buffer R), at 25 °C, in a thermostatted cuvette holder. Less than 10% hydrolysis of the fluorogenic substrate was monitored (51). The kinetic parameters kcat and Km for the reaction of MT3-MMPcat with the fluorogenic substrate were determined from fitting the substrate concentration dependence of the initial rates of substrate hydrolysis to the Michaelis-Menten equation, using the program Scientist (MicroMath, Salt Lake City, UT). MT1-MMPcat and MT3-MMPcat inhibition by TIMPs was carried out essentially as described (52). The concentrations of functional full-length TIMPs and N-TIMP-3 were determined by active site titration using active MMP-2 with known concentration, as previously described (45). MT1-MMPcat or MT3-MMPcat (0.5 nM) was added to a mixture of synthetic substrate (7 µM) and various inhibitor concentrations (up to 16 nM), in buffer R, in acrylic cuvettes with stirring, and hydrolysis of the substrate was followed for 30 min. Analysis of these progress curves yielded the association and dissociation rate constants (kon and koff) and the inhibition constant values (Ki = koff/kon), as described (52). For TIMP-3, progress curves for enzyme inhibition were done with N-TIMP-3 because of adsorption of the wild-type inhibitor to the acrylic cuvettes used, which precluded kinetic analysis of the data. Of note, TIMP-3 titration with MMP-2, an end point experiment, was still feasible, as described above. The koff values were independently determined from the enzyme activity recovered after dilution of a solution of pre-formed enzyme-inhibitor complex, obtained by incubating enzyme (25 and 50 nM) with inhibitor (50 and 100 nM, respectively) for 1 h, at room temperature. The complex was diluted (400-fold) into 2 ml of buffer R, containing 10 µM of the fluorogenic peptide substrate. Complex dissociation was monitored for up to 1 h. The dissociation curves were analyzed as described previously (52) to yield the koff values. To monitor MT3-MMPcat inhibition by marimastat and batimastat, initial rates were obtained by adding the enzyme (0.5 nM) to a solution containing the fluorogenic substrate (7 µM), and various concentrations of the inhibitor (up to 40 nM), in buffer R, in semi-microquartz cuvettes, and monitoring substrate hydrolysis for 10 min. The initial velocities were determined by linear regression analysis of the fluorescence versus time traces using FeliXTM software. Analysis of the initial rate dependence on the inhibitor concentration, according to a competitive model of inhibition, yielded the Ki values, as previously described (52).
Cell Surface Biotinylation and Plasma Membrane Isolation—To detect MT3-MMP forms on the cell surface, CV-1 cells were infected/transfected to express either wild-type or CT-MT3-MMP followed by surface biotinylation, as described (18). Briefly, cells in six-well plates were infected with 10 pfu/cell of vTF7-3 virus in infection media for 45 min followed by transfection of plasmids containing either wild-type or CT-MT3-MMP cDNA, using Effectene (Qiagen, Valencia, CA) as described by the manufacturer. Control cells were infected with vTF7-3 virus but received no plasmid DNA. Eighteen hours later, the cells were rinsed with cold phosphate-buffered saline (PBS) containing 0.1 mM CaCl2 and 1 mM MgCl2 and half of the dishes were incubated with 0.5 mg/ml sulfo-NHS-biotin as described (53), whereas the other half of the dishes received no biotin. The biotinylated and non-biotinylated cells were lysed with lysis buffer, followed by addition of streptavidin beads. After 12 h at 4 °C, the beads were washed four times with harvest buffer (0.5% SDS in 60 mM Tris-HCl, pH 7.5, 2 mM EDTA) supplemented with 2.5% Triton X-100 (final concentration) followed by one wash with Tris-buffered saline (50 mM Tris-HCl, pH 7.5, 150 mM NaCl). The biotinylated proteins bound to the streptavidin beads were eluted with Laemmli SDS sample buffer, boiled, and resolved by reducing 10% SDS-PAGE followed by transfer to a nitrocellulose membrane. The biotinylated MT3-MMP forms were detected with anti-MT3-MMP as described (54). Plasma membrane fractions were isolated from BS-C-1 cells infected to express MT3-MMP or from human fibrosarcoma HT1080 cells, which express natural MT3-MMP. Briefly, HT1080 cells were grown to confluence in complete media. BS-C-1 cells were coinfected to express MT3-MMP as described above. Infected BS-C-1 cells and HT1080 cells (three 150-mm dishes each) were washed with cold PBS, scraped, and centrifuged at 1,000 x g for 5 min. The cells were re-suspended in 25 mM Tris-HCl (pH 7.4) containing 8.5% sucrose, 50 mM NaCl, and protease inhibitor mixture (Roche Diagnostics) followed by homogenization in a Dounce homogenizer. The homogenates were centrifuged at 3,000 x g for 10 min in a refrigerated centrifuge. The resulting supernatants were centrifuged at 100,000 x g for 2 h at 4 °C and the pellets were re-suspended in a 25 mM Tris-HCl (pH 7.4), 50 mM NaCl buffer containing protease inhibitors. The fractions were separated further on a discontinuous sucrose gradient (20, 30, 50, and 60% sucrose), and centrifuged at 100,000 x g for 2 h at 4 °C. The plasma membrane band (30/50% sucrose interface) was collected, pelleted at 100,000 x g for 2 h, and stored at –80 °C. The protein concentration was determined using the BCA assay (Pierce). Aliquots of the plasma membrane fractions were subjected to immunoblot analysis for detection of MT3-MMP and MT1-MMP using the appropriate antibodies as described (50).
Affinity Chromatography—Generation of a trimolecular complex between MT3-MMP, TIMP-2/TIMP-3, and pro-MMP-2 was investigated using the method developed by Bigg et al. (22). Briefly, human recombinant pro-MMP-2 (750 nM) was incubated (2 h, 4 °C) with 750 nM each of either human recombinant TIMP-2 or TIMP-3 in PBS. MT3-MMPcat (375 nM) was then added to the mixture (35 µl final volume, molar ratio of pro-MMP-2:TIMP-2/3/MT3-MMPcat = 1:1:0.5) for an additional 2-h incubation at 4 °C. As a control, some samples contained one or two components (in various combinations) of the reaction proteins in the same amounts as described above. The samples were then loaded onto a column packed with gelatin-agarose beads (column volume of 100 µl) that was pre-equilibrated with PBS (for TIMP-2) or PBS supplemented with 0.025% (v/v) Brij-35 (for TIMP-3). The loaded samples were then incubated for 1 h at 4 °C followed by a brief spin and the flow-through was collected (unbound fraction). The column was then washed three times with 500 µl each of PBS with 0.025% (v/v) Brij-35 and the bound proteins were dissociated from the beads with 50 µlof4x Laemmli SDS sample buffer. The columns were then briefly centrifuged and the eluates were subjected to 12% SDS-PAGE under reducing conditions. The proteins were visualized by staining the gels with the Gelcode SilverSNAP Stain Kit II (Pierce).
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RESULTS
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Expression and Processing of MT3-MMP—Human MT3-MMP was expressed in TIMP-2–/– fibroblasts (Fig. 1A, lane 2), BS-C-1 cells (Fig. 1A, lane 3), and COS-1 cells (Fig. 1A, lane 4) and was detected as a major form of 63 kDa and two minor species of 61 and 59 kDa. In addition, a 35-kDa form, which specifically reacted with the polyclonal antibody to MT3-MMP, was clearly detected in the TIMP-2–/– fibroblasts (Fig. 1A, lane 2). BS-C-1 and COS-1 cells showed lower levels of the low molecular weight species, which was usually detected as a doublet of 35,000–37,000 (Fig. 1A, lanes 3 and 4). Although the polyclonal antibody to MT3-MMP exhibits significant cross-reactivity with proteins in the range of 50–40 kDa, proper controls indicated the specificity of the species described. The profile of the MT3-MMP species in the cell lysates and on the cell surface (shown in Fig. 1C) was undistinguishable even when cells were infected with very low virus levels (data not shown) indicating that the level of MT3-MMP expression had no significant effect on cellular distribution. The appearance of the 35-kDa form of MT3-MMP in the lysate of TIMP-2–/– cells prompted us to examine the cell culture medium for the presence of shed MT3-MMP forms. A 32-kDa soluble protein reactive with the polyclonal antibody to MT3-MMP was specifically identified in the media of TIMP-2–/– fibroblasts expressing MT3-MMP (Fig. 1B, lane 2), which was absent in the control cells (Fig. 1B, lane 1). Surface-biotinylated CV-1 cells expressing MT3-MMP showed that the 63-, 59-, and 35-kDa species were present on the cell surface, albeit at different levels (Fig. 1C). CV-1 cells expressing CT-MT3-MMP exhibited on the cell surface forms of 62, 58, and 33 kDa, consistent with a truncation at the C-terminal end. However, at similar levels of expression, CT-MT3-MMP showed much greater levels of the low molecular weight form when compared with the wild-type enzyme (Fig. 1C, lane 2). Purified plasma membrane fractions isolated from BS-C-1 cells co-infected to express MT3-MMP (Fig. 1D, lane 3) or from HT1080 cells (Fig. 1D, lane 2) contained mostly the 63-kDa species of MT3-MMP. The 59-kDa species of MT3-MMP was also detected in the plasma membranes of BS-C-1 cells (Fig. 1D, lane 3). The 35–37-kDa form was barely detected, suggesting that it is not retained after plasma membrane isolation. As expected, the plasma membranes of HT1080 cells contained the 57-kDa form of MT1-MMP (Fig. 1D, lane 1). However, higher amounts of membrane protein were required to detect MT3-MMP (10 µg) when compared with MT1-MMP (5 µg), indicating the low level of MT3-MMP expression in HT1080 cells.
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FIG. 1.
Expression of MT3-MMP. A, TIMP-2–/– (lanes 1 and 2), BS-C-1 (lane 3), and COS-1 (lane 4) cells in six-well plates were infected with 10 pfu/cell of vTF7-3 (lane 1) or co-infected with 10 pfu/cell each of vTF7-3 and vT7-MT3 (lanes 2–4). After infection, the cells were incubated (24 h, 37 °C) with serum-free DMEM. The cell lysates were subjected to reducing 10% SDS-PAGE followed by immunoblot analysis with anti-MT3-MMP polyclonal antibody. B, shedding of MT3-MMP. TIMP-2–/– cells were infected with control virus alone (lane 1) or co-infected to express MT3-MMP (lane 2) as described in A. After 24 h, the media were collected, concentrated, and subjected to immunoblot analysis as described in A. C, cell surface forms of MT3-MMP. CV-1 cells in six-well plates were infected/transfected to express wild-type MT3-MMP (lane 1) or CT-MT3-MMP (lane 2). Control cells were infected with vTF7-3 virus but received no plasmid DNA (lane 3). Eighteen hours after transfection, the cells were surface-biotinylated (Biotin) as previously described (50). Parallel plates of cells were similarly infected/transfected but the cells were not biotinylated (No biotin). The cell lysates were then precipitated with streptavidin beads and the bound proteins were resolved by reducing 10% SDS-PAGE, transferred to a nitrocellulose membrane, and detected with the polyclonal antibody against MT3-MMP. D, plasma membranes isolated from HT1080 cells (lanes 1 and 2) or from BS-C-1 cells co-infected to express MT3-MMP (lane 3) were subjected to reducing 7.5% SDS-PAGE followed by immunoblot analyses with anti-MT1-MMP (lane 1) and anti-MT3-MMP (lanes 2 and 3) antibodies. Lane 1, 5 µg/lane; lane 2, 10 µg/lane; and lane 3, 1 µg/lane. The asterisks in A, B, and C show nonspecific bands. Mark 12TM unstained molecular weight standards (Invitrogen) were used as a reference for relative molecular mass (kDa).
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We expressed various MT3-MMP mutants including a catalytic inactive mutant (E/A), a cytosolic tail deletion mutant (CT), and a transmembrane domain and cytosolic tail-deleted mutant (TM/CT) (Fig. 2A). In general, all of these mutants exhibited enzyme forms consistent with the presence of the 63-, 61-, and 59-kDa species. However, the relative electrophoretic migration and amount of these forms varied accordingly with the type of mutation/truncation. The catalytic inactive MT3-MMP (E/A-MT3-MMP) did not produce the 35–37-kDa species indicating that this form is the product of autocatalytic degradation. In contrast, cells expressing CT-MT3-MMP showed greater levels of the low molecular weight form (Fig. 2A). The TM/CT-MT3-MMP showed lower levels of the 35–37-kDa form when compared with the wild-type enzyme suggesting that MT3-MMP autolysis requires membrane insertion. In TIMP-2–/– fibroblasts expressing wild-type MT3-MMP, processing to the 35-kDa species was dose dependently inhibited by marimastat (Fig. 2B). Marimastat also caused a slight, but readily detectable accumulation of the 61- and 59-kDa species (Fig. 2B). However, quantitatively, disappearance of the 35–37 kDa did not correlate with an equivalent accumulation of the 59-kDa species, probably because of a differential reactivity of these species to the antibody or to another unknown effect.
Role of TIMP-2 in MT3-MMP Processing and Pro-MMP-2 Activation—MT3-MMP and TIMP-2 were co-expressed in BS-C-1 cells by co-infection using the appropriate vaccinia viruses. The level of MT3-MMP expression was kept constant, whereas the level of TIMP-2 was varied by increasing the amounts of TIMP-2 virus (vSC59-T2). An enzyme-linked immunosorbent assay showed that the concentration of TIMP-2 in the media increased from 3.5 nM (1 pfu/cell; Fig. 3, lane 3) to 40 nM at the highest pfu/cell of the TIMP-2 virus (20 pfu/cell; Fig. 3, lane 9). At 3.5 nM TIMP-2 (Fig. 3A, lane 3), significant activation of pro-MMP-2 was observed. Above this concentration of TIMP-2, pro-MMP-2 activation remained almost constant up to the highest level (40 nM; Fig. 3A, lane 9) of inhibitor in which a slight inhibition of activation was observed. Thus, TIMP-2 enhances the activation of pro-MMP-2 by MT3-MMP. Without addition of exogenous TIMP-2, mostly the intermediate form of MMP-2 was detected (Fig. 3A, lane 2) suggesting that the first cleavage site in the pro-domain of pro-MMP-2 is specifically mediated by MT3-MMP and occurs independently of TIMP-2 expression. Immunoblot analysis showed the characteristic 63-, 61-, 59-, and 35–37-kDa species of MT3-MMP (Fig. 3B). However, the level of the 35–37-kDa forms decreased as a function of TIMP-2 expression. In contrast, high levels of TIMP-2 correlated with a slight but noticeable accumulation of the 59-kDa species of MT3-MMP (Fig. 3B, lanes 5–9). Thus, MT3-MMP undergoes an autocatalytic degradation process that is regulated by TIMP-2 and synthetic MMP inhibitors.
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FIG. 3.
Pro-MMP-2 activation by membrane-bound and soluble MT3-MMP and role of TIMP-2 in processing and activation. BS-C-1 cells in six-well plates were co-infected to express MT3-MMP alone or MT3-MMP with increasing amounts of TIMP-2 using the appropriate viruses, as described under "Experimental Procedures." Four h post-infection, the cells were incubated (24 h, 37 °C) with serum-free DMEM (1 ml/well) supplemented with 10 nM pro-MMP-2. The media were collected and subjected to gelatin zymography (A). The cell lysates were analyzed for MT3-MMP forms (B) and TIMP-2 (C) expression by immunoblot analysis. The media of each sample were subjected to a TIMP-2 enzyme-linked immunosorbent assay and the following amounts of TIMP-2 were measured: lane 1, 1.3 nM; lane 2, 0.36 nM; lane 3, 3.5 nM; lane 4, 15.4 nM; lane 5, 24.2 nM; lane 6, 23.3 nM; lane 7, 27.5 nM; lane 8, 28 nM; and lane 9, 40 nM. The asterisk in B shows a nonspecific band. D, pro-MMP-2 (lanes 1 and 2, 25 nM; lanes 3 and 4, 100 nM, and lanes 5–8, 500 nM) was incubated (24 h, 37 °C) with an aliquot of ultracentrifuged (supernatant fraction) serum-free media collected from BS-C-1 (lanes 1–6) and TIMP-2–/– (lanes 7 and 8) cells infected with vTF7-3 virus alone (lanes 1, 3, 5, and 7) or co-infected with vTF7-3 and vT7-MT3 (lanes 2, 4, 6, and 8). Processing of pro-MMP-2 was monitored by gelatin zymography. L, latent; I, intermediate; A, active MMP-2.
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Shed MT3-MMP Can Process Pro-MMP-2—We next examined whether the soluble 32-kDa fragment of MT3-MMP (shown in Fig. 1B, lane 2) is catalytically active, as described under "Experimental Procedures." These studies demonstrated that pro-MMP-2 was specifically processed when exposed to the conditioned media of cells (BS-C-1 and TIMP-2–/– cells) expressing MT3-MMP (Fig. 3D). In contrast, media from cells infected with vTF7-3 virus alone, as a control, had no effect (Fig. 3D, lanes 1, 3, 5, and 7). Processing of pro-MMP-2 by the MT3-MMP-containing media was dependent on zymogen concentration. At doses of 25 and 100 nM pro-MMP-2 (Fig. 3D, lanes 2 and 4, respectively), the intermediate form of MMP-2 was detected most. However, full activation was detected at pro-MMP-2 concentrations of 500 nM (Fig. 3D, lanes 6 and 8), consistent with the effect of the pro-MMP-2 concentration on zymogen activation via the second MMP-2 autocatalytic step, as previously reported (55). The 32-kDa species is a true soluble form because its activity was completely recovered in the supernatant fraction after ultracentrifugation. Thus, MT3-MMP sheds a 32-kDa soluble form that is catalytically active.
TIMP-2 Is Required for Full Pro-MMP-2 Activation by MT3-MMP—Because BS-C-1 cells express small amounts of endogenous TIMP-2 (21), we examined pro-MMP-2 activation by MT3-MMP in mouse fibroblasts devoid of TIMP-2. As shown in Fig. 4A, significant pro-MMP-2 activation was observed after addition of exogenous TIMP-2 in the homozygous and heterozygous TIMP-2-deficient cells. However, in the absence of exogenous TIMP-2, active MMP-2 was only observed in the heterozygous TIMP-2+/– cells, which, as expected, express endogenous TIMP-2 (21). Dose dependent studies in the homozygous TIMP-2–/– cells showed maximal pro-MMP-2 activation with 5–10 nM TIMP-2 (Fig. 4B). At high concentrations (50–200 nM), TIMP-2 was inhibitory consistent with a biphasic effect of TIMP-2 on pro-MMP-2 activation, as reported for MT1-MMP (14, 15, 18, 19). Although full pro-MMP-2 activation was only achieved in the presence of TIMP-2, formation of the intermediate form by MT3-MMP did not require exogenous TIMP-2, as previously reported with MT1-MMP (22, 23). Activation of pro-MMP-2 in the presence of TIMP-2 (10 nM) and increasing concentrations of N-TIMP-2 (up to 200 nM) caused a dose-dependent inhibition in the generation of the fully active form of MMP-2 but had no effect on the formation of the intermediate form (Fig. 4C). These results suggest that a ternary complex may be involved in the activation of pro-MMP-2 by MT3-MMP. Consistently, administration of N-TIMP-2 alone (1–200 nM), which cannot bind to the hemopexin-like domain of pro-MMP-2, did not support pro-MMP-2 activation by MT3-MMP but inhibited formation of the intermediate form, consistent with inhibition of MT3-MMP (Fig. 4D).
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FIG. 4.
Activation of pro-MMP-2 by MT3-MMP in TIMP-2-deficient cells. A, TIMP-2–/– and TIMP-2+/– cells in six-well plates were co-infected to express MT3-MMP. Four h post-infection, the cells were incubated (24 h, 37 °C) with serum-free DMEM (1 ml/well) supplemented with 10 nM pro-MMP-2, with (+) or without (–) 10 nM TIMP-2. B, TIMP-2 dose-dependent activation of pro-MMP-2 in TIMP-2–/– cells expressing MT3-MMP. TIMP-2–/– cells in six-well plates were co-infected to express MT3-MMP. Four h post-infection, the cells were incubated (24 h, 37 °C) with serum-free DMEM (1 ml/well) supplemented with 10 nM pro-MMP-2 and increasing amounts of TIMP-2 (1–200 nM). C and D, effect of N-TIMP-2 on pro-MMP-2 activation by MT3-MMP in the presence (C) or absence of wt TIMP-2 (D). TIMP-2–/– cells in six-well plates were co-infected to express MT3-MMP. Four h postinfection, the cells were incubated (24 h, 37 °C) with serum-free DMEM (1 ml/well) supplemented with 10 nM each of pro-MMP-2 (C and D) and wt TIMP-2 (C) and increasing amounts (1–200 nM) of N-TIMP-2 (C and D). The media of the experiments in A–D were collected and analyzed by gelatin zymography. The control (Ctrl.) shows the media of TIMP-2–/– cells infected only with vTF7-3 virus and supplemented with pro-MMP-2. L, latent; I, intermediate; A, active MMP-2.
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Marimastat and TIMP-2 Enhance Pro-MMP-2 Activation by MT3-MMP—We previously reported that pretreatment of cells expressing MT1-MMP with reversible hydroxamic-based synthetic MMP inhibitors resulted in enhanced pro-MMP-2 activation after exposure to TIMP-2 (21). Here we report that this effect is also evident in cells expressing MT3-MMP. As shown in Fig. 5, exposure of TIMP-2–/– cells to various concentrations of marimastat (1 nM to 1 µM), followed by washing of the cells to remove the excess synthetic MMP inhibitor and incubation with TIMP-2 (10 nM), results in a dose-dependent activation of pro-MMP-2, as determined by the presence of active MMP-2 in both the medium and cell lysates. Thus, as reported for MT1-MMP (21), reversible synthetic MMP inhibitors (at low doses) can enhance the TIMP-2-dependent pro-MMP-2 activation by MT3-MMP.
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FIG. 5.
Effect of marimastat and TIMP-2 on pro-MMP-2 activation by MT3-MMP. TIMP-2–/– cells in six-well plates were co-infected to express MT3-MMP. After infection, the cells were incubated (16 h, 37 °C) with serum-free DMEM (1 ml/well) supplemented with increasing concentrations of marimastat (1–1000 nM), followed by washing of the cells to remove excess inhibitor. The cells were then incubated (24 h, 37 °C) with serum-free DMEM (1 ml/well) supplemented with 10 nM each of pro-MMP-2 and TIMP-2. The media and cell lysates were then subjected to gelatin zymography. The control (Ctrl.) shows the media of TIMP-2–/– cells infected only with vTF7-3 virus and supplemented with pro-MMP-2. L, latent; I, intermediate; A, active MMP-2.
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Pro-MMP-2 Activation by MT1-MMP and MT3-MMP and TIMP-2 Inhibition—We compared the activation of pro-MMP-2 by MT1-MMP and MT3-MMP expressed in TIMP-2–/– cells in the presence of exogenous TIMP-2. As shown in Fig. 6, TIMP-2–/– cells expressing MT1-MMP activated pro-MMP-2 more rapidly and efficiently than cells expressing MT3-MMP. Because full pro-MMP-2 activation is TIMP-2-dependent, we investigated the kinetics of MT1-MMPcat and MT3-MMPcat inhibition by TIMP-2 using the synthetic fluorogenic peptide substrate MOCAcPLGLA2pr(Dnp)AR-NH2. Preliminary experiments showed that both enzyme species reacted with practically identical kinetic efficiencies with the peptide substrate (kcat and Km values of 0.67 ± 0.03 s–1 and 6.9 ± 0.6 µM for MT1-MMPcat (21) and 0.74 ± 0.04 s–1 and 6.8 ± 0.9 µM for MT3-MMPcat). The TIMP inhibition data were analyzed according to a slow binding model of inhibition, as described under "Experimental Procedures." Table I shows that TIMP-2 binds MT3-MMPcat with a 2–5-fold lower affinity than MT1-MMPcat due to a faster dissociation (koff) rate because the association rates are comparable (kon = 1.78 x 106 and 2.74 x 106 M–1 s–1, respectively). It should be mentioned that, although not clearly evident in the values shown in Table I, the difference in dissociation (koff) rates between the MT3-MMPcat·TIMP-2 and MT1-MMPcat·TIMP-2 complexes may be even more pronounced, because, under the experimental conditions used, the direct koff determination for the MT1-MMPcat-TIMP-2 interaction was not feasible, and the value listed is an estimate based on a 10-fold difference between the slopes of the linear portions of the dissociation curves for the complexes of MT1-MMPcat with N-TIMP-2 (steady-state rate) and full-length TIMP-2, as previously described (21). In contrast, dissociation of the MT3-MMPcat·TIMP-2 complex was readily observed yielding a koff value between 3 and 6 x 10–4 s–1. Thus, the differential affinity of TIMP-2 toward MT1-MMP and MT3-MMP may account, at least in part, for the observed differences in pro-MMP-2 activation.
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FIG. 6.
Time course of pro-MMP-2 activation by MT1- and MT3-MMP. TIMP-2–/– cells in six-well plates were co-infected to express MT1- or MT3-MMP, as described under "Experimental Procedures." Sixteen h post-infection, the cells were incubated with serum-free DMEM (1 ml/well) supplemented with 10 nM each of pro-MMP-2 and TIMP-2. At the indicated times, the media were collected and subjected to gelatin zymography. L, latent; I, intermediate; A, active MMP-2.
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TABLE I
Association, dissociation, and inhibition constants for MT1-MMPcat and MT3-MMPcat interactions with natural and synthetic MMP inhibitors
The enzymes (0.5 nM) were incubated with increasing concentrations of the inhibitors, at 25 °C, in buffer R. The kinetic parameters were determined as described under "Experimental Procedures."
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TIMP-3 but Not TIMP-4 Supports Pro-MMP-2 Activation by MT3-MMP—TIMP-4 (24) and TIMP-3 (28), like TIMP-2, are known to form a non-covalent complex with pro-MMP-2. Therefore, we examined whether TIMP-4 and TIMP-3 could promote pro-MMP-2 activation by MT3-MMP in TIMP-2–/– cells. As shown in Fig. 7A, TIMP-3 promoted pro-MMP-2 activation. However, as opposed to TIMP-2, the active enzyme produced in the presence of TIMP-3 was mostly detected in the cell lysates, whereas the culture medium exhibited the latent and intermediate forms of MMP-2 (Fig. 7A). TIMP-3 enhancement of pro-MMP-2 activation was also dose dependent and at a concentration of 100 nM, pro-MMP-2 processing was inhibited in both the media and the cells (Fig. 7B). At the highest levels (40–100 nM) of TIMP-3, more pro-MMP-2 was also associated with the cells. As shown with TIMP-2 (Fig. 4), formation of the intermediate form of MMP-2 was also TIMP-3 independent (Fig. 7B). N-TIMP-3 (1–30 nM) had no enhancing effect on pro-MMP-2 activation by MT3-MMP but inhibited formation of the intermediate form (data not shown). Furthermore, TIMP-3 had no effect on the activation of pro-MMP-2 by MT1-MMP (data not shown), in agreement with a previous study (28). TIMP-4 did not support the activation of pro-MMP-2 by MT3-MMP and, in fact, inhibited the formation of the intermediate form of MMP-2 at the highest concentrations (Fig. 7C).
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FIG. 7.
TIMP-3 but not TIMP-4 promotes pro-MMP-2 activation by MT3-MMP. A, effect of TIMP-3 and TIMP-2 on pro-MMP-2 activation. TIMP-2–/– cells in six-well plates were co-infected to express MT3-MMP. Four h post-infection, the cells were incubated (24 h, 37 °C) with serum-free DMEM (1 ml/well) supplemented with 10 nM pro-MMP-2 and the indicated amounts of either TIMP-3 or TIMP-2. B, TIMP-3 dose-dependent activation of pro-MMP-2. Four h post-infection, the MT3-MMP-expressing TIMP-2–/– cells were incubated (24 h, 37 °C) with serum-free DMEM (1 ml/well) supplemented with 10 nM pro-MMP-2 and increasing amounts (1–100 nM) of TIMP-3. C, effect of TIMP-4 on pro-MMP-2 activation. Four h post-infection, the MT3-MMP-expressing TIMP-2–/– cells were incubated (24 h, 37 °C) with serum-free DMEM (1 ml/well) supplemented with 10 nM pro-MMP-2 and increasing amounts (1–50 nM) of TIMP-4. The media and lysates of the experiments in A–C were collected and analyzed by gelatin zymography. The control (Ctrl.) shows the media and lysates of TIMP-2–/– cells infected only with vTF7-3 virus and supplemented with pro-MMP-2. L, latent; I, intermediate; A, active MMP-2.
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We studied the kinetics of inhibition of MT1-MMPcat and MT3-MMPcat by TIMPs and synthetic MMP inhibitors. N-TIMP-3 was used in these studies because of nonspecific adsorption of wild-type TIMP-3 to the acrylic cuvettes. As shown in Table I, N-TIMP-3 exhibits the typical kinetic characteristics of a tight, slow-binding, reversible inhibitor of both enzymes. However, N-TIMP-3 inhibited MT3-MMPcat with a significantly higher affinity (20-fold) than that for MT1-MMPcat (Ki values of 0.008 and 0.16 nM, respectively), due to a lower dissociation rate (koff = 2.9 x 10–5 and 1.7 x 10–4 s–1, respectively) because the kon values were practically indistinguishable. TIMP-4 exhibited a comparable lower affinity for both MT3-MMPcat and MT1-MMPcat (Ki = 0.3 nM) (Table I). The affinity of TIMP-4 for MT1-MMPcat is in good agreement to that previously reported by Troeberg et al. (25). TIMP-1 interaction with MT1-MMPcat has been shown to be practically negligible (22, 55) and, at concentrations up to 9 nM, showed no inhibition of MT3-MMPcat (data not shown). In contrast to the TIMPs, the synthetic hydroxamate inhibitors were unable to discriminate between the two MT-MMPs and both marimastat and batimastat inhibited the MT-MMPs competitively, with similar but considerably lower affinity (Ki values in the low nanomolar range) (Table I).
TIMP-3 and TIMP-2 each Form a Trimolecular Complex with Pro-MMP-2 and MT3-MMPcat—Because both TIMP-3 and TIMP-2 promoted pro-MMP-2 activation by MT3-MMP, we examined the ability of these proteins to form a trimolecular complex using gelatin affinity chromatography as previously described (22). Pro-MMP-2 was incubated with TIMP-3 or TIMP-2 to form zymogen-inhibitor complexes, which then were incubated with MT3-MMPcat as described under "Experimental Procedures." The mixtures were subjected to gelatin affinity chromatography and the bound (Fig. 8, A and B) and unbound (Fig. 8, C and D) fractions were analyzed by silver-stained SDS-PAGE. As shown in Fig. 8, most of the MT3-MMPcat was specifically recovered in the bound fraction with TIMP-3 (Fig. 8A, lane 4) or with TIMP-2 (Fig. 8B, lane 4) only in the samples containing pro-MMP-2 consistent with the formation of trimolecular complexes. In contrast, when MT3-MMPcat, TIMP-3, and TIMP-2 were incubated, alone or in combination, with gelatin beads in the absence of pro-MMP-2, the proteins were recovered in the unbound fraction (Fig. 8, C and D). Some processing of pro-MMP-2 to the intermediate form occurred when MT3-MMPcat was incubated with pro-MMP-2 in the absence of TIMPs, which was recovered in the bound fraction, as expected (Fig. 8, A and B, lanes 5). However, MT3-MMPcat did not bind to the beads under these conditions and was recovered in the unbound fraction, even in the presence of TIMP-3 or TIMP-2 (Fig. 8, C and D, lanes 3 and 6). TIMP-3 and TIMP-2 were also recovered in the bound fraction when complexed with pro-MMP-2 (Fig. 8, A and B, lanes 7), as expected.
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FIG. 8.
Affinity chromatography of TIMP-3/TIMP-2 and MT3-MMPcat with pro-MMP-2. Pro-MMP-2·TIMP-3 (A and C) and pro-MMP-2·TIMP-2 (B and D) complexes were generated and incubated with MT3-MMPcat as described under "Experimental Procedures." As a control, some samples contained one or two components (in various combinations) of the reaction proteins (indicated by + or –). The samples were subjected to gelatin affinity chromatography and the bound (A and B) and unbound (C and D) fractions were subjected to reducing 12% SDS-PAGE. The proteins were detected by silver staining.
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DISCUSSION
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Accumulating evidence indicates that type I transmembrane MT-MMPs, as opposed to the secreted MMPs, are uniquely regulated by TIMPs (1, 6, 56). The TIMPs not only act in inhibition of catalysis but also have profound effects on MT-MMP turnover (processing and internalization), which eventually determines the level of mature enzyme on the cell surface (6, 56–58). In the case of TIMP-2 and MT1-MMP, their interaction can also promote pericellular proteolysis by supporting the activation of pro-MMP-2 via formation of ternary complexes (14, 22). The present study shows that these properties can also be applied to the interactions of MT3-MMP with TIMP-2 and with TIMP-3. TIMP-3 is a member of the TIMP family of metalloproteinase inhibitors and is mostly bound to the ECM (9, 10, 59–62). Although only a few quantitative studies on the kinetics of TIMP-3 inhibition have been published and the number of MMPs that were examined is rather small, the consensus is that TIMP-3 is an efficient MMP inhibitor (9). In regards to MT-MMPs, TIMP-3 inhibition of MT1- and MT2-MMP is similar to that of TIMP-2 (27, 55). In addition, TIMP-3 is a potent inhibitor of several members of the closely related family of metalloproteinases, the ADAMs (a disintegrin and a metalloproteinase domain) (47, 55, 63–65), further demonstrating the unique properties of TIMP-3. Our inhibition kinetic studies revealed that N-TIMP-3 binds MT3-MMPcat with significant higher affinity than MT1-MMPcat (Ki = 0.008 and 0.057/0.16 nM, respectively), mainly because of a lower rate of dissociation (2.9 x 10–5 and 1.7 x 10–4 s–1, respectively). Moreover, N-TIMP-3 inhibits MT3-MMPcat considerably more strongly than TIMP-1 (Ki > 9 nM), TIMP-2 (Ki = 0.17/0.31 nM), and TIMP-4 (Ki = 0.34 nM). Thus, it seems reasonable to propose that, among the members of the TIMP family of inhibitors, TIMP-3 is the most likely candidate to act as an MT3-MMP inhibitor, under physiological conditions.
Although MT3-MMP is an activator of pro-MMP-2 (29, 30, 40, 42), the role of TIMPs in this process has not been established. Here we showed for the first time that both TIMP-2 and TIMP-3 enhance the activation of pro-MMP-2 by MT3-MMP. However, there were significant differences between TIMP-3- and TIMP-2-mediated activation of pro-MMP-2 by MT3-MMP. First, TIMP-3-mediated pro-MMP-2 activation was much less efficient than that elicited in the presence of TIMP-2 and second, active MMP-2 generated by MT3-MMP in the presence of TIMP-3 was mostly detected in the cell lysate, whereas with TIMP-2, MMP-2 was readily detected in both the cell lysate and in the medium (20). At present the mechanism(s) regulating the release of active MMP-2 from the cell surface is unknown but it is likely to be a consequence of the interactions and affinities among the components of the activation complex (MT3-MMP, TIMP-3, and MMP-2). It is also possible that active MMP-2 remains cell associated via TIMP-3 bound to polyanionic molecules on the cell surface (28, 66) or to the pericellular ECM (60, 62). When compared with MT1-MMP, the activation of pro-MMP-2 by MT3-MMP in the presence of TIMP-2 was significantly less efficient. Although several reasons may account for this difference, TIMP-2 exhibits a slightly reduced affinity (2–5-fold) for MT3-MMP when compared with MT1-MMP, which is because of a faster dissociation rate. A recent study using chimeric MT-MMPs demonstrated that the catalytic domains of MT1- and MT3-MMP, which are responsible for TIMP-2 binding, are major determinants for the lower efficiency of MT3-MMP in promoting pro-MMP-2 activation (67). Together, these observations suggest that under conditions of MT1-MMP and MT3-MMP co-expression in cells (35, 68), activation of pro-MMP-2 is likely to proceed preferentially via the MT1-MMP/TIMP-2 axis.
The role of TIMP-2 in pro-MMP-2 activation by MT1-MMP is to facilitate the assembly of a ternary complex on the cell surface (14, 22). Our data suggest that a similar mechanism may apply for the activation of pro-MMP-2 by MT3-MMP in the presence of TIMP-2 and TIMP-3. First, the effect of TIMP-2 on activation was dose dependent and consistent with a biphasic effect because of titration of free active MT3-MMP on the cell surface, analogous to the process reported with MT1-MMP and TIMP-2 (14, 15, 18, 19). Second, N-TIMP-2, which cannot bind to the hemopexin-like domain of pro-MMP-2, did not support activation and inhibited this process in the presence of TIMP-2. Third, TIMP-3, like TIMP-2, which binds pro-MMP-2 (28) and inhibits MT3-MMP with high affinity (shown here), supported the activation of pro-MMP-2 by MT3-MMP. In contrast, N-TIMP-3 did not support activation (data not shown). And fourth, pre-formed pro-MMP-2·TIMP-3 and pro-MMP-2·TIMP-2 complexes bound to the catalytic domain of MT3-MMP in solution yielding complexes that were specifically recovered in the bound fraction after gelatin affinity chromatography. Together, these results strongly suggest that activation of pro-MMP-2 by MT3-MMP in the presence of TIMP-2 or TIMP-3 proceeds via formation of ternary complexes on the cell surface, as reported for the activation of pro-MMP-2 by MT1-MMP in the presence of TIMP-2 (14, 15, 22). Structurally, the ability of TIMP-2 to support pro-MMP-2 activation via a ternary complex has been attributed at least in part on the anionic character of its C-terminal tail, which may account for the high affinity interaction with the positively charged hemopexin-like domain of pro-MMP-2 (28, 69–71). The binding affinity and conformation of the pro-MMP-2·TIMP-2 complex allows the N-terminal region of TIMP-2 to dock into the active site of MT1-MMP yielding trimolecular complexes either in solution (22) or on the cell surface (14). Because the C-terminal tail of TIMP-3 has a net charge of zero, based on amino acid sequence (70), it has been suggested that the interaction of TIMP-3 with the positively charged hemopexin-like domain of pro-MMP-2 may be further weakened (70). In addition, studies using gelatin affinity chromatography suggested a lower affinity of pro-MMP-2 for TIMP-3 when compared with TIMP-2 (28). Therefore, the ability of TIMP-3 to support pro-MMP-2 activation via a ternary complex with MT-MMPs has been questioned (70). Although the affinities between TIMP-3/TIMP-2 and pro-MMP-2 in the presence and absence of MT3-MMP need to be measured directly in future studies, the results presented here could not have been predicted solely from the amino acid sequence and charge distribution within the C-terminal tail of TIMP-3 and the hemopexin-like domain of pro-MMP-2, which under physiological conditions may very well be fully solvated. Solvation of charged amino acids on the surfaces of proteins may at times be so effective that their involvement in interactions with other structural partners may be minimized. As such, it is entirely conceivable that other structural elements such as strong hydrogen bonding or hydrophobic interactions may also contribute to the binding affinities and assembly of ternary complexes. On the other hand, the reported weaker interaction of pro-MMP-2 with TIMP-3 when compared with TIMP-2 (28) may partly explain why TIMP-3 was less efficient than TIMP-2 in promoting pro-MMP-2 activation by MT3-MMP. Considering that TIMP-3 can bind tightly to the pericellular ECM, it is possible that zymogen activation may also ensue as a consequence of docking of pro-MMP-2 via its hemopexin-like domain to ECM-bound TIMP-3, providing the interaction of pro-MMP-2 with MT3-MMP on the cell surface is not compromised. However, under the experimental conditions used in this study, this is not a likely scenario because TIMP-3 did not support pro-MMP-2 activation by MT1-MMP (our data and Ref. 28), further demonstrating the unique relationship between MT3-MMP and TIMP-3.
Our data also showed that TIMP-4 is a potent inhibitor of MT3-MMP as it is of MT1-MMP (22, 25, 71) and MT2-MMP (23). However, despite its ability to bind pro-MMP-2 (24), albeit with lower affinity than TIMP-2 (25), TIMP-4 was unable to support pro-MMP-2 activation by MT3- (shown here), MT1- (22, 26), or MT2-MMP (23). A pro-MMP-2·TIMP-4 complex does not bind MT1-MMP (22), an inability that has been attributed to specific structural constrains in the C-terminal region of TIMP-4 (24, 70, 71). Although not examined in this study, the relative low affinity and the structural orientation of the pro-MMP-2·TIMP-4 complex may preclude formation of a ternary complex with MT3-MMP and therefore activation does not ensue. Thus, TIMP-4, as opposed to TIMP-1, is emerging as a general and potent inhibitor of both soluble and membrane-anchored MMPs.
Expression of MT3-MMP in a variety of cells yielded a complex pattern of enzyme forms including four cell-associated forms of 63, 61, 59, and 35–37 kDa and a soluble form of 32 kDa. Although determination of the true nature of these species awaits isolation and sequencing, based on molecular mass, the 63- and 59-kDa forms may represent the zymogen and active forms, respectively. The 63-kDa form of MT3-MMP was consistently detected on the cell surface and in the plasma membrane fraction of HT1080 cells and in infected cells. If this species is the zymogen form, this observation would imply that pro-MT3-MMP may also traffic to the cell surface, as has been reported with pro-MT1-MMP (18, 72, 73). Zymogen activation may then be accomplished on the cell surface by furin, which was also reported to be capable of trafficking to the extracellular milieu (74). The 59-kDa species, on the other hand, may represent the active enzyme because its relative amount increased in cells exposed to TIMP-2 or marimastat, consistent with stabilization of the mature enzyme by metalloproteinase inhibitors, as reported for the mature form of MT1-MMP (18, 72, 75). The 35–37-kDa form is an autocatalytic degradation product because of its sensitivity to TIMP-2 and synthetic MMP inhibitors and its absence in cells expressing the inactive E/A-MT3-MMP mutant. Thus, the level of MT3-MMP on the cell surface is regulated by autolysis of the active enzyme, as reported in previous studies (41, 76). Here we showed that autolysis of MT3-MMP to the 35–37-kDa species was significantly enhanced after deletion of the cytosolic tail. In MT1-MMP, the cytosolic tail is required for endocytosis via clathrincoated vesicles, a process that is regulated by dynamin (77, 78). Unpublished evidence from our laboratory using a dominant-negative mutant of dynamin 1 (dynamin K44A) show that co-expression of dynamin K44A with MT3-MMP results in enhanced pro-MMP-2 activation in the presence of TIMP-2. This result suggests that, analogous to MT1-MMP, the cytosolic tail of MT3-MMP regulates enzyme internalization and, indirectly, the rate of autocatalytic processing. The ability of MT1- and MT3-MMP to undergo autocatalytic processing may be usurped by reversible synthetic MMP inhibitors to produce unwarranted proteolytic effects because in the presence of TIMP-2, exposure to low doses of marimastat resulted in enhanced pro-MMP-2 activation. This process has been attributed to inhibition of autocatalytic turnover and/or to displacement of reversible synthetic MMP inhibitors such as marimastat from the active site by TIMP-2, which may promote formation of ternary complexes resulting in enhanced pro-MMP-2 activation (6, 21). Further studies are required to establish the precise mechanism behind this paradoxical effect of reversible synthetic MMP inhibitors on MT-MMP regulation of catalytic activity. Finally, the soluble 32-kDa species found in the media of cells expressing MT3-MMP is a functional protease because it promoted the processing of pro-MMP-2 in solution. Thus, ectodomain shedding is also a property of MT3-MMP, which may regulate pericellular proteolysis at the cell surface and in the extracellular space. In summary, our data contribute to the emerging amount of evidence demonstrating the unique properties of the members of the MT-MMP subfamily and their differential regulation by TIMPs, which may endow cells with the ability to elicit the most effective proteolytic program at the pericellular microenvironment in both physiological and pathological conditions.
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FOOTNOTES
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* This work was supported by National Institutes of Health Grants NCI-CA61986 and NCI-CA100475 (to R. F.), and AR39198 (to H. N.), and Wellcome Trust Grant 057508 (to H. N.). 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. 
To whom correspondence should be addressed: Dept. of Pathology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1218, Fax: 313-577-8180; E-mail: rfridman{at}med.wayne.edu.
1 The abbreviations used are: MMP, matrix metalloproteinase; MT-MMP, membrane type-matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; ECM, extracellular matrix; pfu, plaqueforming unit; DMEM, Dulbecco's modified Eagle's medium; MOCAcPLGLA2pr(Dnp)AR-NH2, (7-methoxycoumarin-4-yl)acetyl-Lprolyl-L-glycyl-L-leucyl-(N3-(2,4-dinitophenol)-L-2,3-diaminopropionyl)-L-alanyl-L-arginine amide; CT, cytosolic tail; PBS, phosphate-buffered saline.
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ACKNOWLEDGMENTS
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We thank Steven Singson for excellent technical assistance. We also thank Dr. S. Mobashery (University of Notre Dame) for comments and suggestions.
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REFERENCES
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- Visse, R., and Nagase, H. (2003) Circ. Res. 92, 827–839[Abstract/Free Full Text]
- Overall, C. M. (2002) Mol. Biotechnol. 22, 51–86[CrossRef][Medline]
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