HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 25, 17391-17405, June 20, 2008

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 283/25/17391    most recent M708943200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cho, J.-A. Articles by Fridman, R. Search for Related Content PubMed PubMed Citation Articles by Cho, J.-A. Articles by Fridman, R. Social Bookmarking                      What's this?

The Inactive 44-kDa Processed Form of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Enhances Proteolytic Activity via Regulation of Endocytosis of Active MT1-MMP^*

From the Department of Pathology and Proteases and Cancer Program, Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201

Received for publication, October 31, 2007 , and in revised form, April 7, 2008.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Membrane type 1 (MT1) matrix metalloproteinase (MMP-14) is a membrane-tethered MMP considered to be a major mediator of pericellular proteolysis. MT1-MMP is regulated by a complex array of mechanisms, including processing and endocytosis that determine the pool of active proteases on the plasma membrane. Autocatalytic processing of active MT1-MMP generates an inactive membrane-tethered 44-kDa product (44-MT1) lacking the catalytic domain. This form preserves all other enzyme domains and is retained at the cell surface. Paradoxically, accumulation of the 44-kDa form has been associated with increased enzymatic activity. Here we report that expression of a recombinant 44-MT1 (Gly²⁸⁵–Val⁵⁸²) in HT1080 fibrosarcoma cells results in enhanced pro-MMP-2 activation, proliferation within a three-dimensional collagen I matrix, and tumor growth and lung metastasis in mice. Stimulation of pro-MMP-2 activation and growth in collagen I was also observed in other cell systems. Expression of 44-MT1 in HT1080 cells is associated with a delay in the rate of active MT1-MMP endocytosis resulting in higher levels of active enzyme at the cell surface. Consistently, deletion of the cytosolic domain obliterates the stimulatory effects of 44-MT1 on MT1-MMP activity. In contrast, deletion of the hinge turns the 44-MT1 form into a negative regulator of enzyme function in vitro and in vivo, suggesting a key role for the hinge region in the functional relationship between active and processed MT1-MMP. Together, these results suggest a novel role for the 44-kDa form of MT1-MMP generated during autocatalytic processing in maintaining the pool of active enzyme at the cell surface.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Membrane type 1 matrix metalloproteinase (MT1-MMP,² MMP-14) is a type I-transmembrane protease and a major mediator of pericellular proteolysis. MT1-MMP is responsible for the proteolytic cleavage of multiple pericellular and membrane-associated substrates, including collagens and other extracellular matrix proteins, growth factors, growth factor receptors, cell adhesion proteins and their receptors, cytokines, protease inhibitors, and proteases, just to mention a few (1–5). MT1-MMP is also the major physiological activator of pro-MMP-2 (pro-gelatinase A) on the cell surface (6, 7), a process that further contributes to pericellular proteolysis. As a multifunctional protease, MT1-MMP elicits profound effects on cell behavior and has been implicated in the pathogenesis of various human diseases, including cancer (8–10), diabetes (11, 12), vascular (13, 14), and connective tissue diseases (2).

The importance of MT1-MMP for pericellular proteolysis demands a tight control of its catalytic activity at the cell surface. This is partly achieved by the action of endogenous protease inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), which bind to the active site inhibiting catalysis (15). In addition, by virtue of being a membrane-anchored protease, MT1-MMP developed a unique set of regulatory mechanisms that control enzymatic activity independently of TIMPs. These processes include the targeting of active enzyme to specific plasma membrane locations, endocytosis, and autocatalytic processing (1, 16–19). Together, these distinct processes determine the level of active enzyme on the cell surface. However, how these processes are integrated to control the pool of active MT1-MMP is not understood.

The processing of MT1-MMP is a cell surface event in which the active enzyme is usually autocatalytically cleaved in trans to generate a major membrane-anchored product of 44 kDa (also referred to as the 43- or 45-kDa species in some studies) and a soluble 18-kDa inactive fragment of the catalytic domain (6, 20–25). The 44-kDa product of MT1-MMP is detected in cultured cells expressing natural MT1-MMP (20, 26–32) and has been found in platelets (33), human tumors extracts (34–36), and extracts of arthritic synovial tissues (37). MT1-MMP processing is stimulated by a variety of factors known to stimulate MT1-MMP expression, trafficking, and/or endocytosis, including phorbol ester (21, 24, 38, 39), concanavalin A (conA) (24, 40–45), bafilomycin A₁ (46, 47), cytochalasin D (40, 44), transforming growth factor-β1 (26), extracellular calcium (48), and extracellular matrix components (30, 43, 49–51). Expression of constitutively active Rac1 in HT1080 cells also promotes MT1-MMP processing (30). In addition, high levels of enzyme expression (36, 48, 52–54) and low levels of TIMP-2 relative to MT1-MMP (6, 21) are associated with enhanced MT1-MMP processing. Finally, agents such as conA and cytochalasin D, which are known to inhibit clathrin-dependent endocytosis (55, 56), promote MT1-MMP processing, which indicates that the rate of MT1-MMP internalization influences processing. Collectively, these findings suggest a relationship between processing and the level and activity of MT1-MMP at the cell surface.

Although MT1-MMP processing is thought to terminate activity on the cell surface, the structural characteristics of the remnant 44-kDa product suggest that it may play a more complex role in enzyme regulation. The 44-kDa species is composed of the hinge region, the hemopexin-like domain, and the complete anchoring apparatus with its cytosolic tail. Furthermore, the 44-kDa fragment is retained on the cell surface, and like active MT1-MMP it is also cleared from the cell surface by endocytosis (28, 48). Previous studies showed that expression of recombinant species of MT1-MMP lacking the catalytic domain, analogous to the 44-kDa fragment, inhibited MT1-MMP-dependent pro-MMP-2 activation (57–59), collagen degradation (60), in vitro tumor cell migration and invasion (61, 62), and in vivo tumor growth (62). These studies suggested that the 44-kDa species of MT1-MMP behaves as a dominant negative regulator of enzyme function. Paradoxically, and contrary to these observations, a plethora of studies have shown a positive correlation between enhanced processing of MT1-MMP and enzymatic activity (21, 29, 30, 36, 37, 46, 52–54, 63–65). Given these conflicting observations, we set forth to investigate the functional relationship between active MT1-MMP and its processed 44-kDa form in various cellular settings and its effect on enzyme function in vitro and in vivo. We present evidence that the 44-kDa form of MT1-MMP positively influences the level and activity of surface MT1-MMP by regulating enzyme internalization. This positive effect of the processed form depends on the presence of the hinge region. We propose a model in which the 44-kDa form of MT1-MMP regulates the endocytosis of the active protease to preserve a viable level of MT1-MMP on the cell surface.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—Human fibrosarcoma HT1080 cells, human breast carcinoma MDA-MB-231 cells, human breast carcinoma T47D cells, and monkey kidney epithelial BS-C-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). HT1080, MDA-MB-231, and BS-C-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. T47D cells were cultured in RPMI 1640 medium supplemented with 10% FBS, insulin, and antibiotics. Human glioma U87 cells were a gift from Dr. Bonnie Sloane (Wayne State University, Detroit, MI) and were cultured in minimum essential media supplemented with 10% FBS and antibiotics. Human breast carcinoma BT549 cells, a kind gift from Dr. Hyeong-Reh Choi Kim (Wayne State University, Detroit, MI), were cultured in DMEM/F-12 supplemented with 10% FBS and antibiotics. HeLa cells (CCL-2.2) were obtained from the ATCC and were cultured in Spinner medium (Quality Biologicals Inc., Gaithersburg, MD) supplemented with 5% horse serum and antibiotics.

Recombinant Vaccinia Viruses—The production of the recombinant vaccinia virus (vTF7-3) expressing bacteriophage T7 RNA polymerase has been described (66). Recombinant vaccinia viruses expressing human pro-MMP-2, TIMP-2, or TIMP-1 were obtained by homologous recombination, as described previously (66).

Recombinant Proteins and Antibodies—Human recombinant pro-MMP-2, TIMP-2, and TIMP-1 were expressed in HeLa cells infected with the appropriate recombinant vaccinia viruses and purified to homogeneity, as described previously (67). The rabbit polyclonal antibody (pAb) 437, raised against residues 437–454 of the hemopexin-like domain of human MT1-MMP was described previously (6). The rabbit pAb 198 raised against a synthetic peptide including residues 160–174 of the catalytic domain of human MT1-MMP was a generous gift from Dr. Q. X. Amy Sang (Florida State University) (68). The LEM2/15 monoclonal antibody (mAb), a generous gift from Dr. A. Arroyo (Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain), was raised against a synthetic peptide comprising residues 218–233 of the catalytic domain of human MT1-MMP (69). The rabbit pAb to the cytosolic domain of MT1-MMP (RP2-MMP-14) was purchased from Triple Point Biologics, Inc. (Forest Grove, OR). The rabbit pAb 815 against the hinge domain of MT1-MMP was purchased from Chemicon (Temecula, CA). The mAb to the human transferrin receptor was purchased from Zymed Laboratories Inc. The mAb to β-actin was purchased from Sigma.

MT1-MMP cDNA Constructs—To express the 44-kDa form of MT1-MMP (referred to as 44-MT1 in Fig. 1A), the entire catalytic domain of MT1-MMP (Tyr¹¹² to Gly²⁸⁴) was deleted by mutagenic PCR from pro-MT1-MMP using the QuikChange^TM site-directed mutagenesis kit (Stratagene, La Jolla, CA), specific primers, and wild-type pro-MT1-MMP cDNA as the template. This procedure generated a cDNA fragment in which the furin-recognition motif ending at Arg¹¹¹ was positioned immediately upstream of Gly²⁸⁵ at the beginning of the hinge region. This cDNA fragment was cloned into the pcDNA3.1 (Invitrogen) expression vector for stable transfection, the pTF7-EMCV-1 vector for transient expression in the vaccinia expression system (66), and the pIND vector for inducible expression in response to ponasterone A (Invitrogen). In addition, we generated a series of cDNA constructs to express 44-MT1 lacking the cytosolic tail (ending at Arg⁵⁶³ and referred to as 44CT; Fig. 1A) and 44-MT1 lacking the hinge region (residues 285–315 and referred to as 44H; Fig. 1A). These cDNA constructs were inserted into the pcDNA3.1 expression vector. The correct sequence of all the cDNA constructs was verified by DNA sequencing of both strands.

Stable Transfections—Cells (HT1080, MDA-MB-231, U87, and T47D) were stably transfected with empty pcDNA3.1 vector (referred to as EV cells) or pcDNA3.1/44-MT1 vector (referred to as 44-MT1 cells) using the Effectene transfection reagent (Qiagen, Valencia, CA) as described by the manufacturer. T47D breast carcinoma cells were also transfected with wild-type MT1-MMP in pcDNA3.1 vector. Geneticin® (G418 sulfate, Invitrogen)-resistant pooled populations were selected using conventional approaches. HT1080 and U87 cells were also transfected with 44CT and 44H in pcDNA3.1, and both clones and pooled populations were obtained by Geneticin® selection. For most studies, both pooled populations and representative single clones were used. For in vivo studies, two representative clones with each construct, which varied in detected amounts of recombinant protein expression, were pooled (pooled clones).

Inducible Expression System—BT549 breast carcinoma cells were double-transfected with the pVgRXR vector (Invitrogen), which expresses the heterodimeric ecdysone receptor and the retinoid X receptor, and the pIND-inducible expression vector that expresses a modified ecdysone-response element upstream of a minimal heat shock promoter and a multiple cloning site. In the presence of ponasterone A, heterodimeric receptors binding to a modified ecdysone-response element induce expression of the 44-MT1 cDNA that was previously inserted into the pIND-inducible expression plasmid (Invitrogen).

Infection-Transfection—BS-C-1 cells seeded in 6-well plates were infected with 1 plaque-forming unit/cell of vTF7-3 vaccinia virus for 45 min in DMEM containing 2.5% FBS. The media were removed, and the infected cells were co-transfected with pTF7-EMCV-1 vector expressing wild-type MT1-MMP alone or 44-MT1 at 1:1 and 1:10 molar ratios (MT1-MMP:44-MT1) or with empty vector alone, up to a total of 3 µg of plasmid DNA/well using Effectene. The next day, the cells were incubated with 10 nM recombinant pro-MMP-2 for 30 min at 37 °C in serum-free media. The lysates and media were collected and subjected to immunoblot analysis and gelatin zymography, respectively, as described below.

Immunoblot Analyses—Cells were lysed with lysis buffer (25 mM Tris/HCl, pH 7.5, 1% Nonidet P-40, and 100 mM NaCl) supplemented with EDTA-free protease inhibitor mixture (Roche Applied Science), on ice for 1 h. The lysates were then clarified by centrifugation (13,000 rpm, 30 min). The supernatants were collected, and protein concentration was determined using the BCA procedure (Pierce) as described by the manufacturer. Equal amounts of protein were mixed with reducing Laemmli SDS sample buffer, boiled, and resolved by SDS-PAGE, followed by transfer to a nitrocellulose membrane. Detection of antigen was carried out with the appropriate antibody followed by enhanced chemiluminescence according to the manufacturer's instructions (Pierce). β-Actin was used as a loading control.

Gelatin Zymography—Serum-free conditioned media of stable or transiently transfected cells were collected after 16 h of incubation, clarified by centrifugation, and mixed with Laemmli sample buffer without reducing agents and heating. In some experiments, the stably transfected cells were treated with 10 µg/ml of concanavalin A (conA, Sigma) overnight in serum-free media. In other experiments, HT1080 transfectants suspended in complete media were seeded in 12-well plates, which were uncoated or previously coated with 1.5 mg/well of rat tail collagen I (Trevigen, Gaithersburg, MD). Six h later, the media were aspirated and replaced with 0.3 ml/well serum-free media followed by an overnight incubation. Pro-MMP-2 activation was monitored in the media by gelatin zymography, as described previously (70).

Cell Surface Biotinylation—Cells in 6-well plates were cooled down for 5 min, rinsed with cold phosphate-buffered saline (PBS), pH 7.4, containing 0.1 mM CaCl₂ and 1 mM MgCl₂ (PBS-CM), and then biotinylated with 0.5 mg/ml EZ-link sulfo-NHS-biotin (Pierce) for 30 min at 4 °C, as described previously (24). As a control, a parallel plate of cells received PBS without biotin. Biotin was quenched with 50 mM NH₄Cl in PBS-CM for 10 min at 4 °C. The cells were lysed with 0.2 ml/well of lysis buffer and centrifuged (13,000 rpm, 20 min), and the supernatant was incubated with 50 µl of streptavidin beads (Pierce) overnight at 4 °C. The beads were washed four times with harvest buffer (0.5% SDS, 60 mM Tris/HCl, pH 7.5, 2 mM EDTA) supplemented with 2.5% Triton X-100 (final concentration). The bound biotinylated proteins were eluted with reducing Laemmli SDS sample buffer, boiled, and resolved by 10% SDS-PAGE, followed by transfer to a nitrocellulose membrane. The biotinylated MT1-MMP forms were detected with the pAb 437, pAb 815, or mAbLEM2/15 MT1-MMP antibody. The blot was reprobed with transferrin receptor antibodies as a control for loading.

Semiquantitative RT-PCR—Total RNA of HT1080 cells was extracted with RNeasy® mini kit (Qiagen, Valencia, CA). RT-PCR was performed with 1 µg of each total RNA sample using SuperScript^TM III reverse transcriptase (Invitrogen) and subsequently HotStar Taq® master mix kit (Qiagen) following the manufacturer's instructions. The sequences of the specific primers (IDT, Coralville, IA) for human TIMP-2 used are as follows: forward, 5'-GAA ACG ACA TTT ATG GCA ACC-3'; reverse, 5'-GCT GGA CCA GTC GAA ACC-3'. Thirty cycles of PCR were performed. The housekeeping gene GAPDH was also amplified and used as an internal control. The sequences of the human GAPDH primers (IDT) are as follows: forward, 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3'; reverse, 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3'. The amplified fragments were resolved by 1% agarose gels and detected by ethidium bromide staining.

TIMP-2 ELISA—Cells in 6-well plates were incubated at 37 °C with serum-free media. Forty eight hours later, the media were collected, and the cells were rinsed twice with PBS, solubilized with cold lysis buffer, and centrifuged. The conditioned media were collected and clarified by centrifugation and concentrated with a Microcon YM-10 centrifugal filter device. Concentrated media and the lysate samples were analyzed for TIMP-2 protein by ELISA (QIA40, Oncogene Research Products, San Diego) as described by the manufacturer's instructions.

Growth of Cells within a Three-dimensional Collagen I Matrix—Cells suspended in 50 µl of serum free-media were added to a solution of 3.0 mg/ml rat tail collagen I, which was previously neutralized with 0.1 N NaOH. The mixture of cells and collagen was poured into the wells (0.5 ml/well, 10⁴ cells/well for HT1080 and MDA-MB-231 cells, and 2 x 10⁴cells/well for U87 cells) of a 12-well tissue culture plate in the absence or presence of recombinant 12.5 µg/ml TIMP-1 or 5 µg/ml TIMP-2, and the plates were incubated at 37 °C for 1 h to allow polymerization. Then each well received 3 ml of complete media (DMEM + 10% FBS + antibiotics and Geneticin®). After 11 days incubation for HT1080 cells and 14 days for MDA-MB-231 and U87 cells, the media were removed, and the collagen matrix was washed twice with PBS. Then 10 units of bacterial collagenase (Sigma) per 1 mg of collagen were added to the wells and incubated for 15 min at 37 °C. After the collagen gel was completely dissociated, complete media were added to stop collagenase activity, and the solution was centrifuged (1,000 rpm) for 5 min and washed twice with PBS. The cells were counted in the presence of trypan blue. Triplicate wells were used for each of the stable transfectant cell lines. Each experiment was performed in triplicate, and quantification values represent the means of three independent experiments ± S.D. Statistical significance was determined using the Student's t test.

RNA Interference—Gene silencing of endogenous MT1-MMP in pooled populations of HT1080 cells transfected with 44-MT1 was performed using sequence-specific small interfering RNA (siRNA), which were pre-designed by Ambion (Austin, TX). siRNAs used here to specifically target the human sequence of catalytic domain of MT1-MMP were as follows: forward, 5'-CGG AGA AUU UUG UGC UGC CTT-3'; reverse, 5'-GGC AGC ACA AAA UUC UCC GTG-3'. As a negative control, scramble and GAPDH siRNAs (Ambion) were used. Briefly, HT1080 cells were seeded in 12-well plates (8 x 10⁴ cells/well) in complete media. The next day, the cells were transiently transfected with several concentrations of the MT1-MMP and control siRNAs using Effectene. Twenty four h post-transfection, the media were diluted 2-fold with fresh serum-free media, and the cells were incubated an additional 24 h before analyses.

Indirect Immunofluorescence Microscopy—Cells grown on 22-mm² glass coverslips were rinsed with PBS and acid-washed (5 min on ice) with a buffer composed of 50 mM glycine-HCl, pH 3.0, and 150 mM NaCl to dissociate TIMPs bound to the cell surface. The cells were then washed several times with ice-cold PBS-CM, and nonspecific sites were blocked with 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) in PBS-CM buffer. Following several washes with ice-cold PBS-CM, the cells were incubated (1–2 h, 4 °C) with 73 µg/ml pAb 198 against the catalytic domain of MT1-MMP. After washing with PBS-CM, the cells were incubated (1 h, 4 °C) with a 1:50 dilution of FITC-conjugated donkey anti-rabbit IgG antibody (Jackson ImmunoResearch) in PBS supplemented with 1% bovine serum albumin. Following repeated washes with ice-cold PBS-CM, the cells were fixed with ice-cold 4% paraformaldehyde in PBS, pH 7.4, washed with PBS supplemented with 100 mM glycine and incubated (10 min, room temperature) with DAPI (Molecular Probes, Eugene, OR) for nuclei staining. Finally, the coverslips were mounted with anti-fade reagent (Molecular Probes).

To detect total MT1-MMP (intracellular and surface), the cells were acid-washed as described above and then fixed with ice-cold 4% paraformaldehyde in PBS followed by permeabilization with 0.5% Triton X-100in PBS at room temperature. The cells were then incubated with primary and secondary antibodies as described above with the exception that all of the incubations were performed at room temperature. The nuclei were stained with DAPI, and the coverslips were mounted with 50% glycerol in PBS. Negative controls included cells incubated only with secondary antibody or cells incubated with normal rabbit IgG as a primary antibody. These conditions produced no specific signals (data not shown).

The samples were examined and photographed using a 60x oil-immersion objective with a Zeiss LSM-510 META laser scanning confocal microscope at the Microscopy and Imaging Resources Laboratory at Wayne State University School of Medicine. DAPI was excited with a 745 nm multiphoton laser, and FITC was excited with a 488 nm argon laser. To compare the intensity of the fluorescence signal on the cell surface among the various transfectants, all the parameters for brightness and contrast were held consistent during the experiment. Average intensity of fluorescence signal per cell (as identified by DAPI staining) was measured and analyzed using the Metamorph^TM software, version 6.3, revision 2 (Molecular Devices, Sunnyvale, CA). Each experiment was performed in triplicate, and quantification values represent the means of three independent experiments ± S.D. Statistical significance was determined using the Student's t test.

Quantitative Fluorescence-based MT1-MMP Antibody Uptake Assay—This assay was performed as described (54, 71). Briefly, HT1080 transfectants (EV, 44-MT1, 44CT, and 44H) were seeded (7 x 10⁵ cells per well) on 22-mm² glass coverslips placed on 6-well plates. Cells were cooled down for 5 min on ice and acid-washed with 50 mM glycine-HCl, 0.1 M NaCl, pH 3, for 5 min on ice followed by several washes with ice-cold PBS-CM, as described above. The cells were then incubated with 73 µg/ml pAb 198 to MT1-MMP in ice-cold PBS-CM for 2 h at 4 °C. Negative controls included cells incubated with equal amount of normal rabbit IgG as a primary antibody or only with secondary antibody. These conditions produced no specific signals (data not shown). Endocytosis was then allowed to proceed by shifting the cells to 37 °C for 10 min in warm PBS-CM. Endocytosis was stopped by the addition of 0.01% NaN₃, pH 7.2, in PBS-CM until the end of experiment. Cells were washed several times with ice-cold PBS-CM containing 0.01% NaN₃, pH 7.2, and then incubated with normal donkey serum (5% in PBS-CM) for 1 h at 4 °C. Following several washes with ice-cold PBS-CM at 4 °C, the cells were incubated with FITC-conjugated anti-rabbit IgG antibody (1:50 dilution in ice-cold PBS-CM) for 1 h at 4 °C. After washing with ice-cold PBS-CM, the cells were fixed with ice-cold 4% paraformaldehyde for 20 min at 4 °C and washed with PBS supplemented with 100 mM glycine. The nuclei were stained with DAPI, and the coverslips were mounted with anti-fade reagent (Molecular Probes). In some experiments, the cells were seeded on collagen I-coated plates (50 µg collagen I/well) before the antibody uptake assay. Samples were examined and photographed with a Zeiss LSM-510 META laser scanning confocal microscope using a 60x oil-immersion objective. The intensity of the fluorescence signal on the cell surface was analyzed as described above. Each experiment was performed in triplicate, and quantification values represent the means of three independent experiments ± S.D. Statistical significance was determined using the Student's t test.

Reversible Biotinylation Assay—We followed the procedure of Wu et al. (72). To this end, we used HT1080 transfectants (EV and 44-MT1) and parental HT1080 cells seeded in 6-well plates. In the case of HT1080 parental cells, the cells were treated overnight at 37 °C with conA (20 µg/ml) in the presence or absence of 10 µM GM6001 (Chemicon) in serum-free media. The next day, the cells were rinsed and cooled down for 5 min with cold PBS-CM and then biotinylated with 0.5 mg/ml disulfide-cleavable EZ-link sulfo-NHS-SS-biotin (Pierce) for 30 min. Excess biotinylating reagent was quenched with 50 mM NH₄Cl in PBS-CM for 10 min at 4 °C followed by two washes of the cells with cold PBS-CM. To initiate internalization, the wells received pre-warmed serum-free media (3 ml/well), and the plates were immediately incubated at 37 °C for various times (0–120 min). After each time point, the plates were cooled down in ice to halt internalization, and the media were aspirated and the cells washed with cold PBS-CM. Cell surface-bound biotinylating reagent was stripped by incubating the cells (20 min on ice, twice) in 2 ml/well of reducing solution (150 mM NaCl, 1 mM EDTA, 1% bovine serum albumin, 20 mM Tris, pH 8.6, supplemented with 40 mM glutathione). In each experiment, a plate of biotinylated cells was maintained at 4 °C for 0–120 min, as a control of total surface MT1-MMP. These plates labeled "Total" did not receive reducing solution; instead they received PBS-CM. After addition of the reducing solution (endocytosed MT1-MMP) or PBS-CM (total MT1-MMP), the cells were washed twice with cold PBS-CM and lysed with 0.2 ml/well of Nonidet P-40 lysis buffer. The lysates were centrifuged (13,000 rpm, 20 min), and the supernatants were collected. Protein concentration was then determined in each supernatant using the BCA method. An equal amount of protein from each lysate was then incubated (overnight at 4 °C) with 50 µl of immobilized neutravidin protein beads (Pierce) to pull down biotinylated proteins. The beads were washed four times with harvest buffer (0.5% SDS, 60 mM Tris/HCl, pH 7.5, 2 mM EDTA) supplemented with 2.5% Triton X-100 (final concentration). The bound biotinylated proteins were eluted with reducing Laemmli SDS sample buffer, boiled, and resolved by 10%, reducing SDS-PAGE followed by transfer to a nitrocellulose membrane. The total and endocytosed MT1-MMP forms were detected with pAb 815 to the hinge region of MT1-MMP. The blots were then reprobed with a mAb to human transferrin receptor. Densitometric analysis of the bands of internalized active MT1-MMP in the same blot was performed with the US-SCAN-IT program (Silk Scientific Inc., Orem, UT). The density (area per square unit) was plotted against time and expressed in arbitrary units.

Tumorigenicity and Experimental Metastasis Assays—Tumorigenicity assays were performed on 4–6-week-old male homozygous CB-17 SCID/SCID mice and female NCr nude mice (Taconic Farms, Germantown, NY) according to the Animal Welfare Regulations at Wayne State University. HT1080 cell transfectants (pool populations and pooled clones) were harvested and resuspended in serum-free DMEM. Each mouse was inoculated subcutaneously with 5 x 10⁵ cells in 100 µl of serum-free DMEM. The volume of the tumor was measured twice a week with a caliper, using the formula V = (LW²) x 0.4 (where V is the volume (mm³); L is the longest diameter (mm), and W is the shortest diameter (mm)). Data were analyzed for statistical significance by unpaired t test with Welch correction using the GraphPad InStat® version 3.0 (GraphPad Software, San Diego). The differences were considered to be statistically significant at p < 0.05. Tumor incidence represents the percentage of mice bearing a tumor larger than 100 mm³ at day 28. The mice were euthanized 4 weeks after tumor cell inoculation, and the tumors were harvested and frozen at -80 °C until use. Experimental metastasis assays were conducted in CB-17 SCID/SCID mice. Briefly, each mouse was inoculated in the tail vein with 1 x 10⁶ HT1080-EV or HT1080-44-MT1 cells (pooled populations) suspended in 100 µl of serum-free DMEM. Twelve days later the mice were sacrificed, and the lungs were harvested for assay of metastatic burden by real time PCR and counting of lung colonies in hematoxylin and eosin sections as described below.

Determination of Lung Metastatic Burden—Twelve days after tail vein cell inoculation, the mice were sacrificed and the lungs were harvested. Equal portions from each lung were snapfrozen in liquid nitrogen or formalin-fixed. Frozen lung tissues were used for quantitative analyses of human alu sequences by quantitative real time PCR, as described (73). Briefly, genomic DNA was extracted from harvested mouse lung tissues using the Puregene DNA purification kit (Genta Systems, Minneapolis, MN). The primers specific for the human alu sequences were as follows: sense, 5'-ACG CCT GTA ATC CCA GCA CTT-3', and antisense, 5'-TCG CCC AGG CTG GAG TGC A-3'. Real time PCR was performed using the SYBR® Green QRT-PCR master mix kit according to the manufacturer's protocol (Stratagene). PCR conditions included polymerase activation at 95 °C for 10 min followed by 40 cycles at 95 °C for 30 s, 63 °C for 30 s, and 72 °C for 30 s. Each assay included a negative control (diethyl pyrocarbonate water, Ambion), a positive control (human genomic DNA), a no-template control, and the experimental samples in duplicate. A quantitative measurement of mouse DNA was obtained through amplification of the mouse GAPDH genomic DNA sequence with mouse GAPDH primers (sense, 5'-AAC GAC CCC TTC ATT GAC-3', and antisense, 5'-TCC ACG ACA TAC TCA GCA C-3') using the same PCR conditions described for alu. The threshold cycle (Ct) of each sample was recorded as a quantitative measure of the amount of PCR product in the sample using a Stratagene Mx 4000^TM PCR machine. The alu signal was normalized against the relative quantity of mouse GAPDH and expressed as Ct = (Ct_alu - Ct_GAPDH). The changes in alu signal relative to the total amount of mouse genomic DNA were expressed as Ct =Ct_treatment - Ct_control. Relative changes in metastasis were then calculated as 2^-Ct. Data processing and statistical analyses were performed using Microsoft Excel (Microsoft Corp., Redmond, WA).

Formalin-fixed lung tissues were sectioned and processed for hematoxylin and eosin staining using conventional procedures. Lung colonies were visualized and counted in serial sections under a light microscope. At least three different slides from each tumor and five different tumors from each cell line were evaluated for the number of metastatic colonies.

View larger version (50K): [in this window] [in a new window]   FIGURE 1. Recombinant 44-MT1 expression. A, schematic diagram of 44-kDa MT1-MMP constructs and their domain organization. Wt MT1-MMP, wild-type MT1-MMP; 44-MT1, catalytic domain-deleted MT1-MMP (Gly285–Val582); 44CT, catalytic and cytosolic domain-deleted MT1-MMP (Gly285–Arg563); 44H, catalytic domain and hinge-deleted MT1-MMP (Pro316–Val582); SS, signal sequence; Pro, pro-domain; Cat, catalytic domain; HPX, hemopexin domain; TM, transmembrane domain; CT, cytosolic domain; S, disulfide bond; RRKR, furin-recognition site in the prodomain. B–D, immunoblot analyses of active (57-kDa) MT1-MMP and 44-kDa MT1-MMP forms in lysates of HT1080 transfectants (B, panel i, pooled population; panels ii and iii, clones, indicated by numbers after name), BT-549 cells treated with or without ponasterone A (Pon A) (C), and infected-transfected BS-C-1 cells with various ratios of wild-type MT1-MMP and 44-MT1 (D), as described under "Experimental Procedures." Lysates were resolved by 10% reducing SDS-PAGE followed by immunoblot analysis with antihinge pAb 815 (B, panel i, and C and D), anti-hemopexin-like domain pAb 437 (B, panel ii), or anti-cytosolic domain pAb (B, panel iii) followed by enhanced chemiluminescence. β-Actin is shown as a loading control. 57 kDa refers to active MT1-MMP. Arrow shows 44-MT1 (44 kDa); black arrowhead, 44CT (40 kDa), and open arrowhead, 44H (38 kDa). The asterisk in D indicates a nonspecific band.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of 44-MT1 Forms on Pro-MMP-2 Activation—We first examined the effects of 44-MT1 forms on pro-MMP-2 activation, a major function of MT1-MMP (75). Unstimulated parental HT1080 and HT1080-EV cells showed no pro-MMP-2 activation (Fig. 2, A and B, respectively). In contrast, HT1080-44-MT1 and, to a much lesser extent, HT1080-44CT cells exhibited constitutive full pro-MMP-2 activation in the absence of conA (Fig. 2B). ConA treatment induced a robust and almost complete activation of pro-MMP-2 in parental HT1080 and HT1080-EV cells, as reported (45) (Fig. 2, A and B). However, expression of 44-MT1 had no effect on conA-stimulated pro-MMP-2 activation when compared with untreated HT1080-44-MT1 cells (Fig. 2B). Although expression of 44CT in HT1080 cells resulted in unstimulated activation of pro-MMP-2 (Fig. 2B), 44CT expression was associated with a reduced activation of pro-MMP-2 when compared with that observed in conA-stimulated HT1080-EV cells (Fig. 2B, 44CT versus EV) suggesting a role for the cytosolic tail in this activity of 44-MT1.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Recombinant 44-MT1 expression correlates with enhanced pro-MMP-2 activation. Analyses of pro-MMP-2 activation by gelatin zymography. HT1080 (A, parental; B, pooled populations and clones), MDA-MB-231 (F), and U87 (G) cells grown in 6-well plates were treated (16 h, 37 °C) without (-) or with (+) 10 µg/ml conA in serum-free media. C, HT1080 cells were seeded in 6-well plates uncoated (PL) or coated (COL) with collagen I (1.5 mg/well), and the media were collected. D, BT549 cells transfected with the inducible 44-MT1 expression vector were treated (48 h, 37 °C) without (-) or with (+) 10 µM ponasterone A (PonA) in serum-free media. E, BS-C-1 cells were infected-transfected to express wild-type MT1-MMP alone or with 44-MT1 at 1:1 or 1:10 (wt MT1:44-MT1) molar ratio. Sixteen h later, the infected-transfected cells received 10 nM of recombinant pro-MMP-2 for a 30-min incubation at 37 °C. H, T47D cell transfectants were incubated (16 h, 37 °C) with 10 nM of recombinant pro-MMP-2 in the presence of 10 nM TIMP-2 in serum-free media. The media were collected, and equal amounts (normalized by lysate protein concentration) were subjected to gelatin zymography. L, latent; I, intermediate; A, active MMP-2 forms. I, HT1080-44-MT1 cells (pooled population) were transiently transfected with either MT1-MMP (siMT1) or scramble (Scr.) siRNA, as described under "Experimental Procedures." Forty eight h later, the conditioned media were collected and analyzed by gelatin zymography (I, upper panel). The lysates were resolved by reducing 10% SDS-PAGE followed by immunoblot analyses with anti-MT1-MMP hinge pAb 815 (I, middle panel). β-Actin is shown as a loading control (I, lower panel). The results are representative of at least four independent experiments. 57 kDa indicates the endogenous active MT1-MMP.

Previous studies demonstrated that expression of an N-terminally tagged 44-kDa form inhibits MT1-MMP-mediated pro-MMP-2 activation (57, 59, 76). These species, as opposed to 44-MT1, lack significant parts of the hinge region, which may affect the experimental outcome (see "Discussion"). To address this possibility, we deleted the entire hinge (Gly²⁸⁵–Gly³¹⁵) of 44-MT1 to generate 44H (Fig. 1A) and expressed this form in HT1080 cells (Fig. 1B). As shown in Fig. 2B, two clones expressing 44H did not exhibit constitutive (unstimulated) activation of pro-MMP-2. In fact, 44H expression partially inhibited conA-induced pro-MMP-2 activation, and only the intermediate form of MMP-2 was detected, when compared with HT1080-EV cells (Fig. 2B). Thus, removal of the hinge region is associated with a negative effect of 44-MT1 on conA-stimulated pro-MMP-2 activation. Together, these results show that 44-MT1 expression (extending from Gly²⁸⁵ to Val⁵⁸²) can promote unstimulated pro-MMP-2 activation in HT1080 cells, and this effect is reduced by the absence of the hinge region.

To determine whether the constitutive stimulation of pro-MMP-2 activation observed in HT1080-44-MT1 cells is MT1-MMP-dependent, we used RNA interference to specifically knock down endogenous MT1-MMP expression without affecting the expression of 44-MT1. As shown in Fig. 2I, MT1-MMP siRNA significantly inhibited expression of MT1-MMP (57 kDa) in HT1080-44-MT1 cells, whereas expression of 44-MT1 expression was barely affected. Zymography of the media revealed that the MT1-MMP siRNA-transfected HT1080-44-MT1 cells lacked activation of pro-MMP-2 (Fig. 2I, upper panel). In contrast, constitutive pro-MMP-2 activation was evident in scrambled siRNA-transfected HT1080-44-MT1 cells. These results suggest that the enhanced activation of pro-MMP-2 by 44-MT1 is mediated by interactions between active and processed MT1-MMP. Consistent with this assertion, expression of 44-MT1 in T47D cells, which do not express endogenous MT1-MMP, did not result in pro-MMP-2 activation (Fig. 2H). However, active MMP-2 was readily detected in T47D cells transfected to express wild-type MT1-MMP, as expected (Fig. 2H).

Because activation of pro-MMP-2 depends on the level of TIMP-2 (6), we measured TIMP-2 mRNA and protein in the HT1080 transfectants. All of the transfectants exhibited similar levels of TIMP-2 mRNA (supplemental Fig. 1D). However, ELISA data showed a differential distribution of TIMP-2. In HT1080-44-MT1 and HT1080-44CT cells, TIMP-2 was mostly associated with the cell lysate, whereas in HT1080-EV and HT1080-44H cells most of the TIMP-2 was present in the conditioned media (supplemental Fig. 1C). The differential distribution of TIMP-2 observed in these transfectants correlated with their pattern of pro-MMP-2 activation (Fig. 2B). Because the cell association of TIMP-2 is mainly mediated by MT1-MMP, these results suggest that different levels of surface active MT1-MMP among the transfectants may explain pro-MMP-2 activation results, as is shown below.

We next examined the effects of 44-MT1 on pro-MMP-2 activation using a ponasterone-inducible system in BT549 cells, which express endogenous MT1-MMP and pro-MMP-2, and in BS-C-1 cells infected-transfected to express MT1-MMP with or without 44-MT1. These expression systems allow functional evaluation of the gene of interest without issues of selection. Exposure of BT549 cells to ponasterone induced expression of 44-MT1 (Fig. 1C), which resulted in pro-MMP-2 activation (Fig. 2D). Likewise, co-expression of wild-type MT1-MMP with 44-MT1 in BS-C-1 cells also induced activation of pro-MMP-2, which was more evident at higher levels of 44-MT1 protein (Fig. 1D and Fig. 2E).

Treatment of MDA-MB-231-EV and U87-EV cells with conA induced pro-MMP-2 activation, as expected (Fig. 2, F and G). However, expression of 44-MT1 in these cells had no effect on activation of pro-MMP-2 regardless of conA treatment (Fig. 2, F and G). Furthermore, expression of 44CT or 44HinU87 cells had no effect on conA-stimulated pro-MMP-2 activation when compared with U87-EV cells (Fig. 2G).

44-MT1 Effect on Cell Growth within Three-dimensional Collagen I—MT1-MMP was described previously as conferring tumor cells with the ability to grow within a three-dimensional collagen I matrix (77). Therefore, we examined the effects of 44-MT1 expression on the proliferation of HT1080 and U87 cells within three-dimensional collagen I. As shown in Fig. 3, both HT108-44-MT1 and U87-44-MT1 cells exhibited enhanced growth within three-dimensional collagen I when compared with control cells. In contrast, 44-MT1 expression had no detectable effect on the rate of growth of MDA-MB-231 cells in three-dimensional collagen I (Fig. 3). Expression of 44CT or 44H in HT1080 or U87 cells had no significant effect on cell growth when compared with EV cells (Fig. 3), further demonstrating that both the cytosolic domain and the hinge region mediate the biological effect of 44-MT1. Next, we treated HT1080-44-MT1 cells growing within three-dimensional collagen I with either TIMP-2 or TIMP-1. As shown in Fig. 3, TIMP-2 but not TIMP-1 was associated with inhibited growth of HT1080-44-MT1 cells, consistent with this growth effect mediated by MT1-MMP. Thus, 44-MT1 expression promotes growth of two cell lines within a three-dimensional collagen I matrix. No differences in cell growth were detected when the HT1080 or U87 cell transfectants were cultured on uncoated dishes or on a two-dimensional collagen I matrix (data not shown).

View larger version (20K): [in this window] [in a new window]   FIGURE 3. 44-MT1 expression is associated with enhanced growth within three-dimensional collagen I in HT1080 and U87 cells. Cell suspensions of HT1080 (pooled populations), U87 (clones), and MDA-MB-231 (pooled populations) transfectants were mixed with a solution of 3 mg/ml of neutralized rat tail collagen I and plated (HT1080 and MDA-MB-231, 1 x 104 cells/well; U87, 2 x 104 cells/well) in 12-well plates, as described under "Experimental Procedures." Some wells of HT1080 cell transfectants were also supplemented with either recombinant TIMP-1 (12.5 µg/ml) or TIMP-2 (5 µg/ml). The cells were cultured for 11–14 days in complete media supplemented with Geneticin®. The collagen gels were then treated with bacterial collagenase, and viable cells were counted in the presence of trypan blue. The results shown are mean ± S.D. of triplicate wells; **, p < 0.01; Student's t test. NS, not statistically significant relative to EV cells. This experiment was repeated at least three times with similar results.

View larger version (71K): [in this window] [in a new window]   FIGURE 4. Enhanced surface expression of active MT1-MMP in HT1080-44-MT1 cells. A, HT1080 transfectants (clones) were immunostained at 4 °C with pAb 198 to the catalytic domain of MT1-MMP followed by an incubation with FITC-conjugated donkey anti-rabbit IgG antibody and fixed to detected surface MT1-MMP (Surface). For visualization of total (surface plus intracellular) MT1-MMP (Total), the cells were fixed, permeabilized, and then immunostained with pAb 198 followed by incubation with the secondary FITC-labeled antibody, as described under "Experimental Procedures." The nuclei were stained with DAPI. Cells were examined by confocal microscopy and photographed under a 60x oil immersion objective. Insets show representative images of magnified single cells, bar = 10 µm. Pictures represent the result of at least three independent experiments. B, intensity of fluorescence signal per cell was quantified using the MetamorphTM software in at least three different fields for each transfectants. The results shown are mean ± S.D. of at least three fields; *, p < 0.05; Student's t test; NS, not statistically significant relative to HT1080-EV cells. Similar results were obtained in three independent experiments.

Because collagen I was shown to regulate MT1-MMP internalization (54, 61), the antibody uptake assay was also conducted in HT1080 cell transfectants seeded on collagen I-coated coverslips, as described under "Experimental Procedures." These data also showed a delayed internalization of active MT1-MMP in HT1080-44-MT1 cells when compared with control EV cells or cells expressing 44CT or 44H (supplemental Fig. 2). These results indicate that the process of MT1-MMP regulation by endocytosis is not influenced by the presence of collagen I.

We also followed the internalization of MT1-MMP using a reversible biotinylation assay (72). This assay was performed using a cleavable, membrane-permeable biotin. After incubations at 37 °C, residual surface biotin was stripped with glutathione, a membrane-impermeable reducing agent, and internalized biotinylated MT1-MMP was captured by avidin beads and detected by immunoblot analyses. Fig. 6A shows a representative blot depicting the endocytosed and total biotinylated active endogenous MT1-MMP and 44-MT1 form in the HT1080 transfectants. The data of Fig. 6B depict the densitometric analysis of internalized active MT1-MMP as a function of time. Using this approach, we observed more endocytosed active MT1-MMP (glutathione-resistant) in HT1080-EV cells when compared with HT1080-44-MT1 cells under the same experimental conditions. These data further demonstrated a delayed internalization of active MT1-MMP as a function of 44-MT1 expression, in agreement with the antibody uptake assay.

View larger version (68K): [in this window] [in a new window]   FIGURE 5. Delayed endocytosis of active MT1-MMP in HT1080-44-MT1 cells. A–C, HT1080 cell transfectants (clones) were incubated on ice with pAb 198 against the catalytic domain of MT1-MMP. After the indicated times at 37 °C, the cells were incubated with FITC-labeled secondary antibodies and fixed, as described under "Experimental Procedures." The nuclei were then stained with DAPI. Cells were analyzed by confocal microscopy and photographed. A, photographs show representative images for each transfectants using a 60x oil immersion objective. Images at the indicated times within the same cell transfectant were photographed under same brightness and contrast conditions. Bar = 10 µm. B, representative images of HT1080-EV and HT1080-44-MT1 cells at 0 min were photographed under same brightness and contrast conditions to demonstrate the relative fluorescence intensity between these transfectants. C, remaining surface active MT1-MMP per cell was quantified using the MetamorphTM software in at least three different fields for each transfectant. Bars indicate S.D., Student's t test; **, p < 0.01; *, p < 0.05; NS, not statistically significant relative to 0 min. Similar results were obtained in three independent experiments.

Effect of 44-MT1 Forms on Tumor Growth and Metastasis—The HT1080 cell transfectants were inoculated subcutaneously in athymic mice, and tumor growth was monitored for 28 days. Initially we used pooled populations and SCID mice (Fig. 7, A and B). These studies showed that HT1080-44-MT1 cells formed significantly larger tumors than HT1080-EV cells (Fig. 7, A and B). In contrast, expression of 44CT had no significant effect on tumor growth. Surprisingly, we found that 7/8 mice inoculated with HT1080-44H cells showed no evidence of tumor formation (Fig. 7, A and B). We repeated this experiment using a mixture of two clones of each transfectant, which were inoculated in NCr mice to account for potential differences because of clonal variation and mouse strain. These studies essentially reproduced the previous results with the exception that the HT1080-44CT clones grew faster than control clones (Fig. 7, C and D). Taken together, these results demonstrate that although 44-MT1 expression is associated with enhanced growth of HT1080 cells in mice, expression of 44H is strongly correlated with decreased tumorigenic potential. These results suggest that the presence of the hinge regulates the activity of the processed form of MT1-MMP in vivo. We next examined the HT1080 transfectants for differences in their ability to form lung colonies after intravenous inoculation in SCID mice. Mice were sacrificed 12 days after cell inoculation and lungs harvested, and tumor burden was determined by a quantitative real time PCR of genomic DNA using human alu sequences and mouse GAPDH as an internal control (73). As shown in Fig. 8A, the results of alu PCR indicated that mice inoculated with HT1080-44-MT1 cells contained significantly greater amounts of human genomic DNA in their lungs when compared with lungs of mice injected with HT1080-EV cells. As observed with the subcutaneous tumor assay, expression of 44H also inhibited lung colonization after intravenous inoculation, as determined by alu PCR (Fig. 8A). These results were confirmed by counting tumor colonies in the lungs of mice (Fig. 8B). Thus, 44-MT1 expression in HT1080 cells is associated with enhanced tumor growth and lung colonization.

View larger version (35K): [in this window] [in a new window]   FIGURE 6. Endocytosis of active MT1-MMP in the presence of recombinant and natural 44-kDa species by reversible biotinylation assay. A, surface proteins in HT1080-EV and HT1080-44-MT1 cells were labeled at 4 °C using sulfo-NHS-SS-biotin and allowed to internalize for 0–60 min at 37 °C. At the indicated times, the cells were treated with (+) or without (-) glutathione (40 mM) at 4 °C and lysed. Biotinylated proteins were recovered with streptavidin-agarose beads and endocytosed (glutathione-resistant). MT1-MMP was detected by immunoblotting with pAb 815 to the hinge region (upper panels). 57 kDa indicates the endogenous active MT1-MMP. Arrow shows 44-MT1 (44 kDa). Blots were reprobed with an antibody to transferrin receptor (TrfR, 90 kDa, lower panel). B, biotinylated bands of internalized active MT1-MMP (+Glutath.) in HT1080-EV and HT1080-44-MT1 cells as shown in A were quantified by densitometry as described under "Experimental Procedures." C, parental HT1080 cells were treated (16 h, 37 °C) with conA (20 µg/ml) in the presence or absence of 10 µM GM6001. Time-dependent endocytosis of MT1-MMP was then examined as described in A. D, biotinylated bands of internalized active MT1-MMP (+Glutath.) as shown in C (left panel) were quantified by densitometry, as described under "Experimental Procedures." Data in A–D are representative results of at least three independent experiments with similar results.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Not all MT1-MMP-mediated activities were equally affected by expression of 44-MT1, and its effects were dependent on the structural organization of the 44-kDa form, the type of activity assayed, and the cell type, just to mention a few. At present, the reasons why different cell types respond differently to 44-MT1 expression in selected activities are unclear. Regardless, in none of the cellular systems used here did we observe a dominant negative effect of 44-MT1 on MT1-MMP function, including in vivo systems. Contrary to our findings, previous studies showed that expression of a recombinant 44-kDa form inhibited MT1-MMP activity (57, 59, 60). This inhibitory function was ascribed to a competitive interference of the hemopexin and cytosolic domains of the 44-kDa form with the tendency of MT1-MMP to undergo homophilic interactions (1, 57, 59, 62) and to cleave collagen I (60, 76). According to these studies, the 44-kDa species acts in a manner consistent with being a dominant negative regulator of MT1-MMP activity. The results presented here, however, do not support the universality of this model because pro-MMP-2 activation, growth within collagen I, in vivo tumor growth, and lung metastasis were not disrupted by 44-MT1 expression. Consequently, our observations do not support the notion that homophilic interactions of active MT1-MMP are critical for enzymatic activity, if indeed a function of the 44-kDa form is to disrupt these interactions via its hemopexin-like domain and/or the cytosolic tail, as proposed earlier (57–59, 62, 76). In fact, our data show that the processed form of MT1-MMP, which only lacks the catalytic domain, does not disrupt enzyme activity. Interestingly, Hotary et al. (80) showed that MT1-MMP devoid of the hemopexin-like domain confers cells with the ability to degrade basement membranes suggesting that this domain is dispensable for this activity of MT1-MMP. Nevertheless, even if 44-MT1 forms were to disrupt homodimeric-oligomeric complexes of active MT1-MMP, the evidence presented here would suggest a lesser importance of these complexes for enzyme function than previously considered (1). Possibly, dimeric and oligomeric forms of MT1-MMP only represent a minor fraction of the pool of active enzyme on the cell surface (59), and thus their presence may not be essential for proper enzyme function.

View larger version (40K): [in this window] [in a new window]   FIGURE 7. Effect of 44-kDa recombinant forms on tumor growth of HT1080 cells. A–D, HT1080 cell transfectants were inoculated subcutaneously into SCID (A and B) or NCr (C and D) athymic mice on day 0 as described under "Experimental Procedures." The animals were monitored for tumor formation every 3–4 days until day 28. Tumors were measured every 3–4 days, and tumor volume was calculated. Points represent mean of tumor volume for each group, and bars indicate S.E. Unpaired t test; *, p < 0.05. Similar results were obtained in three independent experiments. B and D, tumor incidence and volume of tumors at day 28 from data shown in A and C.*, p values relative to HT1080-EV tumors. NS, not statistically significant relative to HT1080-EV tumors.

View larger version (23K): [in this window] [in a new window]   FIGURE 8. Effect of 44-kDa recombinant forms on experimental metastasis of HT1080 cells. A, SCID mice (n = 8) were inoculated with HT1080 transfectants (EV, 44-MT1, and 44H) into the tail vein. After 12 days, the mice were sacrificed, and the lungs were harvested, and total lung DNA was analyzed by real time alu PCR, as described under "Experimental Procedures." Relative metastasis represents the quantitative detection of human alu sequences normalized against mouse GAPDH relative to alu values from lungs of mice inoculated with HT1080-EV cells. Bars indicate S.D., Student's t test; *, p < 0.05. B, number of metastatic colonies in the lungs of SCID mice inoculated with HT1080 transfectants (EV, 44-MT1, and 44H) as described in A. Values represent the number of colonies per field in hematoxylin and eosin-stained tissue sections. Data shown are the average of at least three different slides from each tumor and five different tumors from each cell lines. Bars indicate S.D. Student's t test; *, p < 0.05.

Our data indicate that expression of 44-MT1 in HT1080 cells correlates with higher levels of surface MT1-MMP because of a slower rate of MT1-MMP endocytosis, as determined in two distinct assays. The relatively higher levels of surface-active MT1-MMP correlated with higher levels of TIMP-2 associated with the cell lysates of HT1080-44-MT1 cells (supplemental Fig. 1C). Together, these findings suggest that generation of the 44-kDa form by autocatalytic turnover serves to preserve a basal level of functional protease at the cell surface by regulating the rate of endocytosis of active MT1-MMP. Given that 44-MT1 maintains the cytosolic tail and thus possesses the same endocytic signal of the full-length protease, this process may be due to competitive interactions between the cytosolic tails of the MT1-MMP forms for components of the endocytic machinery (82). Indeed, expression of 44CT had no effect on the rate of active MT1-MMP internalization. This suggests the possibility that components of the endocytic pathway, including those mediated by di-leucine motifs, as present in the cytosolic domain of MT1-MMP, are saturable and thus prone to competition within the same type of receptors, as established in previous studies with several transmembrane receptors (82–84). Competition for the endocytotic machinery between MT1-MMP species may be due to the accumulation of the 44-kDa form as a consequence of autocatalytic processing. Alternatively, preferential internalization of the processed form cannot be ruled out. At present, the data presented here do not allow us to discriminate between these possibilities. This will require determining the rate of internalization of each species alone and in combination. However, this study is complicated by the need to express comparable levels of each species in cells without endogenous MT1-MMP and to prevent processing of the wild-type enzyme, which further complicates this type of analyses. Nevertheless, our data strongly suggest that there is a direct relationship between processing and endocytosis which serves to maintain a viable level of protease at the cell surface.

Based on the results presented here, we propose a model for the regulation of active MT1-MMP by its processed form. Conditions that increase the level of active MT1-MMP at the cell surface augment the probability of autocatalytic processing (78). This leads to the formation of a membrane-anchored 44-kDa degradation product (Gly²⁸⁵–Val⁵⁸²) that lacks the entire catalytic domain. Processing decreases the amount of active protease, and the overall activity of MT1-MMP at the cell surface is reduced. The net amount of active MT1-MMP can be recovered by de novo synthesis, enhanced trafficking, and/or reduced turnover and endocytosis. MT1-MMP processing also leads to accumulation of the 44-kDa product. Consequently, the 44-kDa form may saturate the local endocytic pathway resulting in delayed internalization of active MT1-MMP, which would eventually lead to recovery and preservation of enzymatic activity. Although the precise mechanism(s) by which the 44-kDa species delays the internalization of active MT1-MMP needs to be elucidated, this model provides a plausible explanation to the positive association between high levels of processed MT1-MMP (43–45-kDa forms) and increased enzymatic activity reported in many studies. We also propose that the regulation of active MT1-MMP by its processed 44-kDa form is also analogous to the stabilization model of active MT1-MMP by TIMPs and synthetic MMP inhibitors, which posits that under certain conditions these can also promote MT1-MMP activity by inhibiting autocatalytic processing (17, 85). The findings of this study should facilitate the re-evaluation of the contribution of MT1-MMP autocatalytic processing and its degradation product to enzyme function.

	FOOTNOTES

 
^* This work was supported, in whole or in part, by National Institutes of Health Grant NCI-CA61986 (to R. F.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ 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.

² The abbreviations used are: MT1-MMP, membrane type 1 matrix metalloproteinase; FITC, fluorescein isothiocyanate; pAb, polyclonal antibody; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; siRNA, small interfering RNA; DAPI, 4',6-diamidino-2-phenylindole; EV, empty vector; conA, concanavalin A; TIMP, tissue inhibitor of metalloproteinase.

	ACKNOWLEDGMENTS

 
The Microscopy and Imaging Resources Laboratory is supported in part by National Institutes of Health Center Grants P30ES06639 and P30CA22453. We thank Dr. Lorena Perrone (Dept. of Anatomy and Cell Biology, Wayne State University) for useful comments and suggestions. We also thank the assistance of Dr. Moin (Dept. of Pharmacology, Wayne State University) with the confocal microscopy analyses.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Itoh, Y., and Seiki, M. (2006) J. Cell. Physiol. 206, 1-8[CrossRef][Medline] [Order article via Infotrieve]
2. Holmbeck, K., Bianco, P., Yamada, S., and Birkedal-Hansen, H. (2004) J. Cell. Physiol. 200, 11-19[CrossRef][Medline] [Order article via Infotrieve]
3. Butler, G. S., and Overall, C. M. (2007) Curr. Pharm. Des. 13, 263-270[CrossRef][Medline] [Order article via Infotrieve]
4. Tam, E. M., Morrison, C. J., Wu, Y. I., Stack, M. S., and Overall, C. M. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 6917-6922[Abstract/Free Full Text]
5. Barbolina, M. V., and Stack, M. S. (2008) Semin. Cell Dev. Biol. 19, 24-33[CrossRef][Medline] [Order article via Infotrieve]
6. Hernandez-Barrantes, S., Toth, M., Bernardo, M. M., Yurkova, M., Gervasi, D. C., Raz, Y., Sang, Q. A., and Fridman, R. (2000) J. Biol. Chem. 275, 12080-12089[Abstract/Free Full Text]
7. Strongin, A. Y., Collier, I., Bannikov, G., Marmer, B. L., Grant, G. A., and Goldberg, G. I. (1995) J. Biol. Chem. 270, 5331-5338[Abstract/Free Full Text]
8. Seiki, M. (2003) Cancer Lett. 194, 1-11[CrossRef][Medline] [Order article via Infotrieve]
9. Deryugina, E. I., and Quigley, J. P. (2006) Cancer Metastasis Rev. 25, 9-34[CrossRef][Medline] [Order article via Infotrieve]
10. Sato, H., Takino, T., and Miyamori, H. (2005) Cancer Sci. 96, 212-217[CrossRef][Medline] [Order article via Infotrieve]
11. Savinov, A. Y., and Strongin, A. Y. (2007) IUBMB Life 59, 6-13[CrossRef][Medline] [Order article via Infotrieve]
12. Savinov, A. Y., Rozanov, D. V., and Strongin, A. Y. (2006) FASEB J. 20, 1793-1801[Abstract/Free Full Text]
13. Filippov, S., Koenig, G. C., Chun, T. H., Hotary, K. B., Ota, I., Bugge, T. H., Roberts, J. D., Fay, W. P., Birkedal-Hansen, H., Holmbeck, K., Sabeh, F., Allen, E. D., and Weiss, S. J. (2005) J. Exp. Med. 202, 663-671[Abstract/Free Full Text]
14. Arroyo, A. G., Genis, L., Gonzalo, P., Matias-Roman, S., Pollan, A., and Galvez, B. G. (2007) Curr. Pharm. Des. 13, 1787-1802[CrossRef][Medline] [Order article via Infotrieve]
15. Nagase, H., Visse, R., and Murphy, G. (2006) Cardiovasc. Res. 69, 562-573[Abstract/Free Full Text]
16. Genis, L., Galvez, B. G., Gonzalo, P., and Arroyo, A. G. (2006) Cancer Metastasis Rev. 25, 77-86[CrossRef][Medline] [Order article via Infotrieve]
17. Hernandez-Barrantes, S., Bernardo, M., Toth, M., and Fridman, R. (2002) Semin. Cancer Biol. 12, 131-138[CrossRef][Medline] [Order article via Infotrieve]
18. Itoh, Y., and Seiki, M. (2004) Trends Biochem. Sci. 29, 285-289[CrossRef][Medline] [Order article via Infotrieve]
19. Zucker, S., Pei, D., Cao, J., and Lopez-Otin, C. (2003) Curr. Top. Dev. Biol. 54, 1-74[Medline] [Order article via Infotrieve]
20. Ellerbroek, S. M., Wu, Y. I., Overall, C. M., and Stack, M. S. (2001) J. Biol. Chem. 276, 24833-24842[Abstract/Free Full Text]
21. Lehti, K., Lohi, J., Valtanen, H., and Keski-Oja, J. (1998) Biochem. J. 334, 345-353[Medline] [Order article via Infotrieve]
22. Lehti, K., Valtanen, H., Wickstrom, S., Lohi, J., and Keski-Oja, J. (2000) J. Biol. Chem. 275, 15006-15013[Abstract/Free Full Text]
23. Maquoi, E., Frankenne, F., Noel, A., Krell, H. W., Grams, F., and Foidart, J. M. (2000) Exp. Cell Res. 261, 348-359[CrossRef][Medline] [Order article via Infotrieve]
24. Toth, M., Hernandez-Barrantes, S., Osenkowski, P., Bernardo, M. M., Gervasi, D. C., Shimura, Y., Meroueh, O., Kotra, L. P., Galvez, B. G., Arroyo, A. G., Mobashery, S., and Fridman, R. (2002) J. Biol. Chem. 277, 26340-26350[Abstract/Free Full Text]
25. Wu, Y. I., Munshi, H. G., Sen, R., Snipas, S. J., Salvesen, G. S., Fridman, R., and Stack, M. S. (2004) J. Biol. Chem. 279, 8278-8289[Abstract/Free Full Text]
26. Munshi, H. G., Wu, Y. I., Mukhopadhyay, S., Ottaviano, A. J., Sassano, A., Koblinski, J. E., Platanias, L. C., and Stack, M. S. (2004) J. Biol. Chem. 279, 39042-39050[Abstract/Free Full Text]
27. Preaux, A. M., Mallat, A., Nhieu, J. T., D'Ortho, M. P., Hembry, R. M., and Mavier, P. (1999) Hepatology 30, 944-950[CrossRef][Medline] [Order article via Infotrieve]
28. Remacle, A., Murphy, G., and Roghi, C. (2003) J. Cell Sci. 116, 3905-3916[Abstract/Free Full Text]
29. Cowell, S., Knauper, V., Stewart, M. L., D'Ortho, M. P., Stanton, H., Hembry, R. M., Lopez-Otin, C., Reynolds, J. J., and Murphy, G. (1998) Biochem. J. 331, 453-458[Medline] [Order article via Infotrieve]
30. Zhuge, Y., and Xu, J. (2001) J. Biol. Chem. 276, 16248-16256[Abstract/Free Full Text]
31. Koshikawa, N., Giannelli, G., Cirulli, V., Miyazaki, K., and Quaranta, V. (2000) J. Cell Biol. 148, 615-624[Abstract/Free Full Text]
32. Haseneen, N. A., Vaday, G. G., Zucker, S., and Foda, H. D. (2003) Am. J. Physiol. 284, L541-L547
33. Kazes, I., Elalamy, I., Sraer, J. D., Hatmi, M., and Nguyen, G. (2000) Blood 96, 3064-3069[Abstract/Free Full Text]
34. Golubkov, V. S., Boyd, S., Savinov, A. Y., Chekanov, A. V., Osterman, A. L., Remacle, A., Rozanov, D. V., Doxsey, S. J., and Strongin, A. Y. (2005) J. Biol. Chem. 280, 25079-25086[Abstract/Free Full Text]
35. Guo, P., Imanishi, Y., Cackowski, F. C., Jarzynka, M. J., Tao, H. Q., Nishikawa, R., Hirose, T., Hu, B., and Cheng, S. Y. (2005) Am. J. Pathol. 166, 877-890[Abstract/Free Full Text]
36. Remacle, A. G., Chekanov, A. V., Golubkov, V. S., Savinov, A. Y., Rozanov, D. V., and Strongin, A. Y. (2006) J. Biol. Chem. 281, 16897-16905[Abstract/Free Full Text]
37. Konttinen, Y. T., Ceponis, A., Takagi, M., Ainola, M., Sorsa, T., Sutinen, M., Salo, T., Ma, J., Santavirta, S., and Seiki, M. (1998) Matrix Biol. 17, 585-601[CrossRef][Medline] [Order article via Infotrieve]
38. Aznavoorian, S., Moore, B. A., Alexander-Lister, L. D., Hallit, S. L., Windsor, L. J., and Engler, J. A. (2001) Cancer Res. 61, 6264-6275[Abstract/Free Full Text]
39. Lohi, J., Lehti, K., Westermarck, J., Kahari, V. M., and Keski-Oja, J. (1996) Eur. J. Biochem. 239, 239-247[Medline] [Order article via Infotrieve]
40. Gingras, D., Page, M., Annabi, B., and Beliveau, R. (2000) Biochim. Biophys. Acta 1497, 341-350[Medline] [Order article via Infotrieve]
41. Annabi, B., Pilorget, A., Bousquet-Gagnon, N., Gingras, D., and Beliveau, R. (2001) Biochem. J. 359, 325-333[CrossRef][Medline] [Order article via Infotrieve]
42. Theret, N., Lehti, K., Musso, O., and Clement, B. (1999) Hepatology 30, 462-468[CrossRef][Medline] [Order article via Infotrieve]
43. Lee, H., Overall, C. M., McCulloch, C. A., and Sodek, J. (2006) Mol. Biol. Cell 17, 4812-4826[Abstract/Free Full Text]
44. Zucker, S., Hymowitz, M., Conner, C. E., DiYanni, E. A., and Cao, J. (2002) Lab. Investig. 82, 1673-1684[Medline] [Order article via Infotrieve]
45. Gervasi, D. C., Raz, A., Dehem, M., Yang, M., Kurkinen, M., and Fridman, R. (1996) Biochem. Biophys. Res. Commun. 228, 530-538[CrossRef][Medline] [Order article via Infotrieve]
46. Maquoi, E., Peyrollier, K., Noel, A., Foidart, J. M., and Frankenne, F. (2003) Biochem. J. 373, 19-24[CrossRef][Medline] [Order article via Infotrieve]
47. Zucker, S., Hymowitz, M., Conner, C., DeClerck, Y., and Cao, J. (2004) Exp. Cell Res. 293, 164-174[CrossRef][Medline] [Order article via Infotrieve]
48. Munshi, H. G., Wu, Y. I., Ariztia, E. V., and Stack, M. S. (2002) J. Biol. Chem. 277, 41480-41488[Abstract/Free Full Text]
49. Maquoi, E., Noel, A., Frankenne, F., Angliker, H., Murphy, G., and Foidart, J. M. (1998) FEBS Lett. 424, 262-266[CrossRef][Medline] [Order article via Infotrieve]
50. Stanton, H., Gavrilovic, J., Atkinson, S. J., d'Ortho, M. P., Yamada, K. M., Zardi, L., and Murphy, G. (1998) J. Cell Sci. 111, 2789-2798[Abstract]
51. Barbolina, M. V., Adley, B. P., Ariztia, E. V., Liu, Y., and Stack, M. S. (2007) J. Biol. Chem. 282, 4924-4931[Abstract/Free Full Text]
52. Ratnikov, B. I., Rozanov, D. V., Postnova, T. I., Baciu, P. G., Zhang, H., DiScipio, R. G., Chestukhina, G. G., Smith, J. W., Deryugina, E. I., and Strongin, A. Y. (2002) J. Biol. Chem. 277, 7377-7385[Abstract/Free Full Text]
53. Rozanov, D. V., Hahn-Dantona, E., Strickland, D. K., and Strongin, A. Y. (2004) J. Biol. Chem. 279, 4260-4268[Abstract/Free Full Text]
54. Lafleur, M. A., Mercuri, F. A., Ruangpanit, N., Seiki, M., Sato, H., and Thompson, E. W. (2006) J. Biol. Chem. 281, 6826-6840[Abstract/Free Full Text]
55. Budd, D. C., Rae, A., and Tobin, A. B. (1999) J. Biol. Chem. 274, 12355-12360[Abstract/Free Full Text]
56. Gottlieb, T. A., Ivanov, I. E., Adesnik, M., and Sabatini, D. D. (1993) J. Cell Biol. 120, 695-710[Abstract/Free Full Text]
57. Itoh, Y., Takamura, A., Ito, N., Maru, Y., Sato, H., Suenaga, N., Aoki, T., and Seiki, M. (2001) EMBO J. 20, 4782-4793[CrossRef][Medline] [Order article via Infotrieve]
58. Inaki, N., Tsunezuka, Y., Kawakami, K., Sato, H., Takino, T., Oda, M., and Watanabe, G. (2004) J. Heart Lung Transplant. 23, 218-227[CrossRef][Medline] [Order article via Infotrieve]
59. Lehti, K., Lohi, J., Juntunen, M. M., Pei, D., and Keski-Oja, J. (2002) J. Biol. Chem. 277, 8440-8448[Abstract/Free Full Text]
60. Tam, E. M., Wu, Y. I., Butler, G. S., Stack, M. S., and Overall, C. M. (2002) J. Biol. Chem. 277, 39005-39014[Abstract/Free Full Text]
61. Galvez, B. G., Matias-Roman, S., Yanez-Mo, M., Sanchez-Madrid, F., and Arroyo, A. G. (2002) J. Cell Biol. 159, 509-521[Abstract/Free Full Text]
62. Nonaka, T., Nishibashi, K., Itoh, Y., Yana, I., and Seiki, M. (2005) Mol. Cancer Ther. 4, 1157-1166[Abstract/Free Full Text]
63. Annabi, B., Lachambre, M. P., Bousquet-Gagnon, N., Page, M., Gingras, D., and Beliveau, R. (2002) Biochim. Biophys. Acta 1542, 209-220[Medline] [Order article via Infotrieve]
64. Annabi, B., Lachambre, M., Bousquet-Gagnon, N., Page, M., Gingras, D., and Beliveau, R. (2001) Biochem. J. 353, 547-553[CrossRef][Medline] [Order article via Infotrieve]
65. Ellerbroek, S. M., Fishman, D. A., Kearns, A. S., Bafetti, L. M., and Stack, M. S. (1999) Cancer Res. 59, 1635-1641[Abstract/Free Full Text]
66. Fuerst, T. R., Earl, P. L., and Moss, B. (1987) Mol. Cell. Biol. 7, 2538-2544[Abstract/Free Full Text]
67. Olson, M. W., Gervasi, D. C., Mobashery, S., and Fridman, R. (1997) J. Biol. Chem. 272, 29975-29983[Abstract/Free Full Text]
68. Li, H., Bauzon, D. E., Xu, X., Tschesche, H., Cao, J., and Sang, Q. A. (1998) Mol. Carcinog. 22, 84-94[CrossRef][Medline] [Order article via Infotrieve]
69. Galvez, B. G., Matias-Roman, S., Albar, J. P., Sanchez-Madrid, F., and Arroyo, A. G. (2001) J. Biol. Chem. 276, 37491-37500[Abstract/Free Full Text]
70. Toth, M., Gervasi, D. C., and Fridman, R. (1997) Cancer Res. 57, 3159-3167[Abstract/Free Full Text]
71. Wang, X., Ma, D., Keski-Oja, J., and Pei, D. (2004) J. Biol. Chem. 279, 9331-9336[Abstract/Free Full Text]
72. Wu, X., Gan, B., Yoo, Y., and Guan, J. L. (2005) Dev. Cell 9, 185-196[CrossRef][Medline] [Order article via Infotrieve]
73. Zijlstra, A., Mellor, R., Panzarella, G., Aimes, R. T., Hooper, J. D., Marchenko, N. D., and Quigley, J. P. (2002) Cancer Res. 62, 7083-7092[Abstract/Free Full Text]
74. Fridman, R., Fuerst, T. R., Bird, R. E., Hoyhtya, M., Oelkuct, M., Kraus, S., Komarek, D., Liotta, L. A., Berman, M. L., and Stetler-Stevenson, W. G. (1992) J. Biol. Chem. 267, 15398-15405[Abstract/Free Full Text]
75. Belkaid, A., Fortier, S., Cao, J., and Annabi, B. (2007) Neoplasia (Bratisl.) 9, 332-340[CrossRef]
76. Itoh, Y., Ito, N., Nagase, H., Evans, R. D., Bird, S. A., and Seiki, M. (2006) Mol. Biol. Cell 17, 5390-5399[Abstract/Free Full Text]
77. Hotary, K. B., Allen, E. D., Brooks, P. C., Datta, N. S., Long, M. W., and Weiss, S. J. (2003) Cell 114, 33-45[CrossRef][Medline] [Order article via Infotrieve]
78. Osenkowski, P., Toth, M., and Fridman, R. (2004) J. Cell. Physiol. 200, 2-10[CrossRef][Medline] [Order article via Infotrieve]
79. D'Alessio, S., Ferrari, G., Cinnante, K., Scheerer, W., Galloway, A. C., Roses, D. F., Rozanov, D. V., Remacle, A. G., Oh, E. S., Shiryaev, S. A., Strongin, A. Y., Pintucci, G., and Mignatti, P. (2008) J. Biol. Chem. 283, 87-99[Abstract/Free Full Text]
80. Hotary, K., Li, X. Y., Allen, E., Stevens, S. L., and Weiss, S. J. (2006) Genes Dev. 20, 2673-2686[Abstract/Free Full Text]
81. Osenkowski, P., Meroueh, S. O., Pavel, D., Mobashery, S., and Fridman, R. (2005) J. Biol. Chem. 280, 26160-26168[Abstract/Free Full Text]
82. Marks, M. S., Woodruff, L., Ohno, H., and Bonifacino, J. S. (1996) J. Cell Biol. 135, 341-354[Abstract/Free Full Text]
83. Warren, R. A., Green, F. A., and Enns, C. A. (1997) J. Biol. Chem. 272, 2116-2121[Abstract/Free Full Text]
84. Wiley, H. S. (1988) J. Cell Biol. 107, 801-810[Abstract/Free Full Text]
85. Toth, M., Bernardo, M. M., Gervasi, D. C., Soloway, P. D., Wang, Z., Bigg, H. F., Overall, C. M., DeClerck, Y. A., Tschesche, H., Cher, M. L., Brown, S., Mobashery, S., and Fridman, R. (2000) J. Biol. Chem. 275, 41415-41423[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				H. Zhao, A. Sohail, Q. Sun, Q. Shi, S. Kim, S. Mobashery, and R. Fridman Identification and Role of the Homodimerization Interface of the Glycosylphosphatidylinositol-anchored Membrane Type 6 Matrix Metalloproteinase (MMP25) J. Biol. Chem., December 12, 2008; 283(50): 35023 - 35032. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 283/25/17391    most recent M708943200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cho, J.-A. Articles by Fridman, R. Search for Related Content PubMed PubMed Citation Articles by Cho, J.-A. Articles by Fridman, R. Social Bookmarking                      What's this?