From the Department of Pathology and the Karmanos
Cancer Institute, Wayne State University, Detroit, Michigan 48201, the
¶ Division of Immunology Shigei Medical Research Institute 2117 Yamada, Okayama 701-02, Japan, and the
Department of Molecular
Biology and Biochemistry, Okayama University Medical School,
Shikata-cho, Okayama 700, Japan
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ABSTRACT |
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Association of matrix metalloproteinases (MMPs)
with the cell surface and with areas of cell-matrix contacts is
critical for extracellular matrix degradation. Previously, we showed
the surface association of pro-MMP-9 in human breast epithelial MCF10A
cells. Here, we have characterized the binding parameters of pro-MMP-9 and show that the enzyme binds with high affinity
(Kd ~22 nM) to MCF10A cells and other
cell lines. Binding of pro-MMP-9 to MCF10A cells does not result in
zymogen activation and is not followed by ligand internalization, even
after complex formation with tissue inhibitor of metalloproteinase-1
(TIMP-1). A 190-kDa cell surface protein was identified by ligand blot
analysis and affinity purification with immobilized pro-MMP-9.
Microsequencing and immunoblot analysis revealed that the 190-kDa
protein is the 2(IV) chain of collagen IV. Specific pro-MMP-9
surface binding was competed with purified
2(IV) and was
significantly reduced after treatment of the cells with active MMP-9
before the binding assay since
2(IV) is hydrolyzed by MMP-9. A
pro-MMP-9·TIMP-1 complex and MMP-9 bind to
2(IV), suggesting that
neither the C-terminal nor the N-terminal domain of the enzyme is
directly involved in
2(IV) binding. The closely related pro-MMP-2
exhibits a weaker affinity for
2(IV) compared with that of
pro-MMP-9, suggesting that sites other than the gelatin-binding domain
may be involved in the binding of
2(IV) to pro-MMP-9. Although
pro-MMP-9 forms a complex with
2(IV), the proenzyme does not bind to
triple-helical collagen IV. These studies suggest a unique interaction
between pro-MMP-9 and
2(IV) that may play a role in targeting the
zymogen to cell-matrix contacts and in the degradation of the collagen IV network.
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INTRODUCTION |
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The degradation of ECM1
components is partly achieved by proteolytic enzymes closely associated
with discrete areas of cell-matrix contacts. These include the
plasminogen/plasmin system (1), cathepsins (2), and MMPs (3-6). The
gelatinases A (MMP-2) and B (MMP-9) are two members of the MMP family
that have been shown to play a central role in many normal and
pathological conditions involving ECM degradation, including wound
healing, angiogenesis, embryogenesis, arthritis, and tumor metastasis
(3-6). Like other members of the MMP family, the gelatinases are
produced in a latent form
(pro-MMP)2 that requires
activation to become proteolytically active. Thus, activation is a
critical step in the regulation of MMP-dependent proteolytic activity. Considerable evidence has associated the gelatinases with the ability of tumor cells to metastasize due to their
ability to degrade basement membrane collagen IV and to their elevated
expression in malignant tumors (5, 6). Previous studies have shown that
tumor cells contain gelatinase activity in the plasma membranes
consistent with cell surface association (7-10). We have shown a
surface localization of both gelatinases in carcinoma cells of breast
tumors (11, 12) and epithelial cells of fibrocystic breast disease
(12), and others have shown the localization of pro-MMP-9 in the tumor
basement membrane zone of skin tumors (13) colocalizing with collagen IV.3 In recent years, much
information has been gained on the cell surface association of
pro-MMP-2. It has been shown that pro-MMP-2 binds to the cell surface
via membrane type 1-MMP (MT1-MMP), a subclass of MMPs bound to plasma
membranes (14, 15). Interestingly, the binding of pro-MMP-2 to MT1-MMP
requires the participation of tissue inhibitor of metalloproteinase-2
(TIMP-2) (14). The trimer complex allows for pro-MMP-2 activation on
the cell surface, possibly by another MT1-MMP molecule. A later study
suggested that the cell surface association of pro-MMP-2 can also occur through the binding of the C-terminal domain of the enzyme to integrin
v
3 (16). Thus, several mechanisms may
play a role in the surface localization of pro-MMP-2.
Pro-MMP-9 is structurally similar to pro-MMP-2, with both enzymes
containing three tandem copies of a 58-amino acid residue fibronectin
type II-like module (17, 18), known as the gelatin-binding domain,
which plays a role in binding to extracellular matrix components
(19-21). Furthermore, both latent enzymes can bind a TIMP molecule in
the C-terminal domain of the zymogen with pro-MMP-9 binding to TIMP-1
(17) and pro-MMP-2 to TIMP-2 (4, 5). Pro-MMP-9 also contains a 54-amino
acid proline-rich extension of unknown function that is similar to the
2(V) chain of collagen V and in addition is a glycosylated enzyme
(17). In contrast to pro-MMP-2, little is known about the interactions
of pro-MMP-9 with the cell surface, yet pro-MMP-9 has been found to be
present on the cell surface. Recent studies by Partridge et
al. (22) showed that pro-MMP-9 is present in the plasma membrane
and focal contacts of cultured endothelial cells. Pro-MMP-9 was also
detected in the plasma membranes of human fibrosarcoma HT1080 cells
(23), and the enzyme could be activated by a
plasmin-dependent mechanism (24). We have recently shown
that, upon induction with
12-O-tetradecanoylphorbol-13-acetate, pro-MMP-9 binds to the
surface of human breast epithelial MCF10A cells (25). In this report,
we further examined the binding of pro-MMP-9 to a variety of cell lines
and demonstrated the existence of a single high affinity
(Kd ~22 nM) binding site. Using immobilized pro-MMP-9, ligand blot analysis and co-immunoprecipitation experiments with surface biotinylated cells, we have identified the
2(IV) chain of collagen IV as the major pro-MMP-9-binding protein.
These studies provide novel evidence on the interactions of pro-MMP-9
with
2(IV) that may play a role in the localization of zymogen at
cell-matrix contacts and degradation of basement membranes.
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EXPERIMENTAL PROCEDURES |
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Expression and Purification of Recombinant Gelatinases and TIMP-1-- Human recombinant pro-MMP-9, pro-MMP-2, and TIMP-1 were all expressed in HeLa cells using a recombinant vaccinia virus expression system and purified to homogeneity as described previously (26, 27). To obtain 35S-pro-MMP-9 and 35S-pro-MMP-2, expression of recombinant enzymes in infected HeLa cells was carried out in the presence of 15 µCi/ml [35S]methionine. The specific activities of 35S-pro-MMP-2 and 35S-pro-MMP-9 were 0.0159 µCi/pmol and 0.0106 µCi/pmol, respectively.
Antibodies--
The monoclonal antibodies to human pro-MMP-9
(CA-209) and to pro-MMP-2 (CA-801 and CA-805) have been described
previously (11, 12, 25, 28). An anti-pro-MMP-9 rabbit polyclonal antibody (pAB109) raised against a synthetic peptide
(APRQRQSTLVLTPGDLRT) from the N-terminal domain of human pro-MMP-9 was
a generous gift from Dr. Stetler-Stevenson (National Cancer Institute,
National Institutes of Health, Bethesda, MD) (25). A polyclonal
antibody against human TIMP-1 was raised in rabbits and reacts with
TIMP-1 but not with TIMP-2 (25). Anti-pro-MMP-2 polyclonal antibodies were raised in rabbits against human recombinant pro-MMP-2 (29). Mouse
monoclonal antibodies (mAbs) against human TIMP-1 (IM 322) were
purchased from Calbiochem (San Diego, CA). Chain-specific rat mAbs to
human collagen IV were prepared as described previously (30). A mAb to
the 2(IV) chain of human collagen IV (mAb 1910) was purchased
from Chemicon (Temecula, CA).
Iodination of Pro-MMP-9 and TIMP-1-- Pro-MMP-9 or TIMP-1 (50-100 µg) in 100 µl of collagenase buffer (50 mM Tris-HCl (pH 7.5), 5 mM CaCl2, 150 mM NaCl, and 0.02% Brij-35) were placed in a vial coated with IODOGEN (Pierce) as described by the manufacturer and allowed to incubate for 1 min at 25 °C. Na125I (500 µCi) was added to each vial, and the iodination reaction was allowed to continue for 3 min at 25 °C. The reaction was quenched by the addition of 200 µg (100 µl) of bovine serum albumin (BSA) and 2 mM NaI. Unincorporated Na125I was removed by a 1-ml Sephadex G-25 (fine) column equilibrated with collagenase buffer. The specific activity of 125I-pro-MMP-9 and 125I-TIMP-1 was determined after trichloroacetic acid precipitation and quantitation by densitometry of the proteins in Coomassie Blue-stained SDS-polyacrylamide gels relative to a standard curve of unlabeled purified proteins. Typically, the specific activities of 125I-TIMP-1 and 125I-pro-MMP-9 were 0.049 µCi/pmol and 0.322 µCi/pmol, respectively. No detectable autocatalytic/degradation forms of MMP-9 were observed in the iodinated enzyme as determined by both gelatin-zymography and autoradiography.
Cells-- Human immortalized breast epithelial MCF10A cells (31) were grown as described previously (25). MDA-MB-231 breast cancer cells and PC3 prostate cancer cells were provided by Dr. Fred Miller (Karmanos Cancer Institute, Detroit, MI) and grown in DMEM supplemented with 10% fetal bovine serum (FBS) and RPMI 1640 medium supplemented with 10% FBS, respectively. Human HT1080 fibrosarcoma (CCL-121) cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in DMEM supplemented with 10% FBS. Rat vascular endothelial (RVE) cells derived from brain capillaries (32) and mouse lung microvascular endothelial (CD3) cells were provided by Dr. Diglio (Department of Pathology, Wayne State University) and grown in DMEM supplemented with 10% FBS.
Binding Assays--
Cells (MCF10A, MDA-MB-231, PC3) grown to
80% confluence (3-5 days after seeding) in 12-well (22-mm) plates
were rinsed with PBS and incubated (15 min at 4 °C) with cold
binding medium (25 mM Hepes (pH 7.5) with 0.5% BSA in
DMEM). The medium was aspirated, and various concentrations (1-40
nM) of 125I-pro-MMP-9 diluted in binding medium
were added to each well (300 µl) in the presence or absence of
80-fold excess unlabeled pro-MMP-9. After a 45-min incubation at
4 °C, the medium was aspirated and the cells were washed three times
with cold PBS containing 0.1% BSA. The cells were lysed with 0.5 ml/well of 0.5 M NaOH for determination of radioactive
counts in a counter (Packard model 5650), and the results were
expressed as the mean of the values obtained from triplicate samples.
The number of cells in each well was determined in quadruplicate wells.
Time-course experiments were similarly done, except that the
concentration of 125I-pro-MMP-9 for each well was kept
constant at 18 nM and the cells were harvested after
various times at 4 °C. The nonspecific binding of
125I-pro-MMP-9 was determined in the presence of 80-fold
excess unlabeled pro-MMP-9. Typically, specific binding represented an
average of approximately 20-30% of total bound
125I-pro-MMP-9. The association rate constant
(kon) of pro-MMP-9 was determined from the
time-course experiment following logarithmic transformation of the
amount of specifically bound 125I-pro-MMP-9
versus time. Binding of 125I-pro-MMP-9 to the
cell surface follows a second-order binding isotherm. Thus, the
kon is determined from the slope of a line plotted as ln([LReq]/([LReq]
[LR])) versus time as described previously
(33), where LReq is the quantity of
125I-pro-MMP-9 bound at 120 min and LR is the
quantity of 125I-pro-MMP-9 bound at the times 0, 2, 15, and
30 min. The slope of this line was determined by linear regression
analysis using Microsoft ExcelTM, and the error represents the standard
deviation of the slope. The equilibrium binding constant
(Kd) and the number of binding sites per cell were
determined by nonlinear curve-fitting analysis using the GraphPad
PrismTM software version 2.0 and by Scatchard analysis. The slope and
the intercept from the Scatchard analysis were determined by linear
regression analysis using Microsoft ExcelTM. For the latter, the error
represents the standard deviation of the slope and intercept. For
HT1080 and RVE cells, binding was performed with
125I-pro-MMP-9 (3.6 nM) in the presence and
absence of 80-fold excess unlabeled pro-MMP-9 for 45 min at 4 °C and
the amount of specific ligand bound (fmol/cells) was determined.
Competition of 125I-pro-MMP-9 binding with pro-MMP-2 was
carried out in a ligand binding assay as described above, except that
80-fold excess unlabeled pro-MMP-2 was used instead of unlabeled
pro-MMP-9.
Cellular Distribution of Bound
125I-Pro-MMP-9--
MCF10A cells were incubated with cold
binding medium and then incubated with 18 nM/well of
125I-pro-MMP-9 in triplicate wells for 45 min at 4 °C.
The medium was aspirated, and the cells were washed four times with
cold binding medium. Each well then received 0.5 ml of prewarmed
(37 °C) binding medium, and the plates were incubated at 37 °C
for various times. At each time point, the medium was recovered and the
cells were washed with PBS, followed by the addition of 0.5 ml/well of
0.25% Pronase E (Sigma) in PBS. The cells were incubated at 4 °C
for 30 min, and the monolayer was dislodged by gentle pipetting and
transferred to a microcentrifuge tube. The samples were centrifuged for
5 min at 2000 × g, and the supernatant (cell
surface-bound fraction) was transferred to a new tube. The pellet
(internalized fraction) was washed once with PBS and then resuspended
in 0.5 ml of PBS. The radioactivity of the three fractions, in
triplicate, was measured in a counter. Internalization studies in
the presence of TIMP-1 were performed similarly, except that the cells
were incubated with a 125I-pro-MMP-9·TIMP-1 complex that
was previously formed by incubating 125I-pro-MMP-9 with
TIMP-1 for 30 min at 22 °C.
Competition of 125I-Pro-MMP-9 Binding with Purified
2(IV)--
125I-Pro-MMP-9 (3.6 nM) was
incubated for 1.5 h at 4 °C with ~2-fold molar excess of
affinity-purified
2(IV) at a final concentration of 7 nM
in the presence of 5 mM EDTA. Binding assays with MCF10A cells were carried out as described above.
Binding of 125I-Pro-MMP-9 to Cells Treated with MMP-9-- MCF10A cells were incubated for 45 min at 37 °C in binding medium with 1.2 pmol/well (final concentration of 4 nM) of either purified 82-kDa active species of MMP-9 or MMP-9 that was previously incubated with TIMP-1 (2.4 pmol) for 30 min at 22 °C. After treatment, the cells were washed twice with cold binding medium and incubated for 15 min in binding medium at 4 °C. Binding assays were carried out as described above.
Coupling of Pro-MMP-9 to Affi-Gel 10-- One milligram of recombinant pro-MMP-9 in 50 mM Hepes (pH 7.5), 5 mM CaCl2, 150 mM NaCl, and 0.02% Brij-35 was allowed to bind to 1 ml of Affi-Gel 10 (Bio-Rad) for 5 h at 4 °C with rotation in the presence of 60 mM CaCl2. After coupling, the matrix (Affi-Gel 10-pro-MMP-9) received a 150-µl volume of 1 M ethanolamine (pH 8) and incubated with rotation for 1 h at 4 °C. The matrix was allowed to settle, and the supernatant was subjected to SDS-PAGE to determine the amount of uncoupled pro-MMP-9. The Affi-Gel 10-pro-MMP-9 matrix was washed four times with collagenase buffer. The immobilized pro-MMP-9 maintained its capability to bind TIMP-1, as determined by binding of 125I-TIMP-1 compared with soluble enzyme.
Affinity Purification of the 190-kDa Protein-- MCF10A cells were lysed with (0.8 ml/150-mm plate) ice-cold lysis buffer (25 mM Tris (pH 7.5), 100 mM NaCl, 1% Nonidet P-40, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 5 mM benzamidine, and 1 mM PMSF). After a centrifugation (20 min, 14,000 rpm), the supernatant was collected and incubated (4 °C) with Affi-Gel 10-pro-MMP-9 batchwise overnight, poured into a column (PolyprepTM, Bio-Rad), and the flow-through fraction collected. The matrix was washed with 20 ml of 25 mM Tris (pH 7.5), 500 mM NaCl, 0.1% Nonidet P-40, 2 mM PMSF, 5 mM benzamidine, 10 µg/ml leupeptin, and 10 µg/ml aprotinin (wash 1), followed by 10 ml of the same buffer as described above but containing 150 mM NaCl (wash 2). The 190-kDa protein was eluted from the column with 4.5 ml of 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM CaCl2, 20% Me2SO, 2 mM PMSF, 5 mM benzamidine, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Three 1.5-ml fractions of eluate were collected. Forty microliters of the load, flow-through, wash 1, wash 2, and eluate fractions were analyzed by silver-stained SDS-PAGE (34) and ligand blot as described below. The protein concentrations of each column fraction were determined by the BCA protein assay (Pierce) reagent. The same procedure was used with lysates of surface biotinylated cells, except that 0.5-1 ml of lysates were incubated with 50 µl of Affi-Gel 10-pro-MMP-9 or uncoupled Affi-Gel 10 matrix. After binding, the matrix was washed with 1 ml of cold HNTG buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Nonidet P-40, 10% glycerol) followed by one wash in the same buffer containing 500 mM NaCl and three washes in HNTG buffer. After a brief centrifugation, the bound proteins were eluted with 40 µl of collagenase buffer with 10% Me2SO and subjected to SDS-PAGE, blotting, and detection by streptavidin-HRP or ligand blot as described below.
Microsequencing-- The affinity-purified 190-kDa protein was subjected to 7% SDS-PAGE under reducing conditions, followed by staining with 0.25% Coomassie Brilliant Blue. The band containing the 190-kDa protein was excised from the gel, and the pieces were washed three times (15 min each) with Millipore water. The gel slices were washed three times (5 min each) in 50% acetonitrile (Aldrich, HPLC grade), frozen in dry ice, and then sent to Dr. William Lane at the Harvard Microchemistry sequencing facility (Cambridge, MA).
Stokes Radius Determination--
Affinity-purified 2(IV) (250 ng) was chromatographed on a Superose 6 (10/30) column (Amersham
Pharmacia Biotech) equilibrated with 50 mM Tris (pH 7.5),
150 mM NaCl, 5 mM EDTA, and 0.02% Brij-35 using an FPLC system at a flow rate of 0.15 ml/min and 0.3 ml fractions
were collected. Samples (200 µl) from each fraction were
trichloroacetic acid-precipitated (10% v/v) and analyzed for the
presence of
2(IV) by SDS-PAGE under reducing conditions followed by
ligand blot analysis. The column was calibrated with thyroglobulin,
ferritin, catalase, aldolase, bovine serum albumin, ovalbumin, and
chymotrypsin (Amersham Pharmacia Biotech).
Glycerol Gradient Sedimentation--
Purified 2(IV) (500 ng)
was layered onto two 15-30% glycerol gradients in 50 mM
Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.02% Brij-35, 2 mM PMSF, and 4 mM benzamidine in
polyallomer tubes. The tubes were then centrifuged (35,000 rpm for
54 h at 4 °C) in an SW 41 rotor. The fractions (0.2 ml) were
collected and precipitated with trichloroacetic acid (10% v/v). The
precipitates were subjected to SDS-PAGE under reducing conditions
followed by ligand blot analysis as described below.
Cell Surface Biotinylation-- Surface proteins were biotinylated with sulfo-NHS-biotin (Pierce) as described previously (25). The biotinylated cells were lysed with 2 ml/dish of ice-cold lysis buffer, the lysates incubated for 1 h on ice, and the supernatant collected after a 15-min centrifugation (13,000 × g) at 4 °C. The supernatants were analyzed immediately for the presence of pro-MMP-9-binding proteins by pro-MMP-9-affinity chromatography or co-immunoprecipitation with pro-MMP-9 and detection with streptavidin-HRP as described below.
Preparation of Conditioned Medium-- Confluent cultures of MCF10A cells were incubated with serum-free DMEM/F-12 (15 ml/150-mm dish) for 24 h at 37 °C. The medium was collected, centrifuged, and concentrated (6-fold) with a Centriprep-10 (Amicon). The medium was analyzed by immunoblot analysis as described above. In some experiments, cells were grown in the presence of daily additions of ascorbate (75 µg/ml) and the conditioned medium was obtained as described.
Co-immunoprecipitation of 2(IV) with Pro-MMP-9--
Lysates
of surface-biotinylated or non-biotinylated cells in lysis buffer or
samples of concentrated serum-free conditioned medium were incubated
with 0.5-1 µg/ml purified pro-MMP-9 for 1 h at 4 °C. In some
experiments, the lysates received active MMP-9, pro-MMP-2, active
MMP-2, or TIMP-1 in the presence or absence of 10 mM EDTA.
Five micrograms of the appropriate antibody or control IgG were added
for another 16-h incubation at 4 °C. Each sample received 25 µl of
protein G-Sepharose 4 Fast Flow matrix (Amersham Pharmacia Biotech) and
incubated for 3 h at 4 °C with continuous rocking. The matrix
was washed with ice-cold HNTG buffer, followed by one wash in the same
buffer supplemented with 500 mM NaCl and three additional
washes with HNTG buffer. The samples were boiled in the presence of 20 µl of 2× Laemmli sample buffer (35) with reducing agent, and the
supernatants were subjected to SDS-PAGE and the proteins transferred to
a nitrocellulose membrane for immunoblot analysis or ligand blot
analysis as described below.
Immunoblot and Ligand Blot Analysis-- Samples resolved by SDS-PAGE were transferred to a BA-S 85 nitrocellulose membrane (Schleicher & Schuell). The membrane was blocked (12 h at 4 °C) with Blotto (3% BSA and 3% nonfat dry milk in 100 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.02% NaN3) and washed twice with T-TBS (20 mM Tris (pH 7.5), 137 mM NaCl, and 0.1% Tween 20). For immunoblot analysis, the membranes were incubated with the appropriate primary antibody diluted in T-TBS containing 0.5% nonfat dry milk, washed three times with T-TBS and incubated with the HRP-conjugated secondary antibody. The immunodetection of the antigen was performed using ECL (Amersham Pharmacia Biotech) according to the manufacturer's instructions. For biotinylated samples, the membranes were incubated with streptavidin-HRP (Amersham Pharmacia Biotech) and developed by ECL. For ligand blot analysis, the membranes were incubated (1 h at 25 °C) with 1 µg/ml pro-MMP-9 in T-TBS containing 0.5% nonfat dry milk and 1% Nonidet P-40, followed by three washes with T-TBS. After washing, the membranes were incubated (1 h at 25 °C) with anti-MMP-9 antibodies diluted in T-TBS containing 0.5% nonfat dry milk. After three washes in T-TBS, the membranes were incubated (1 h at 25 °C) with the HRP-conjugated secondary antibody in T-TBS, followed by three washes with T-TBS. The proteins were detected as described above.
Binding of Mouse-EHS Collagen IV to Immobilized Pro-MMP-9-- EHS native collagen IV (a generous gift from Dr. Hynda Kleinman, National Institutes of Health, Bethesda, MD) was diluted (10 µg/ml) in lysis buffer (0.5 ml) and incubated (12 h at 4 °C) with 30 µl of Affi-Gel 10-pro-MMP-9 matrix. After incubation, the beads were centrifuged (13,000 × g, 1 min at 4 °C) and the supernatant was collected (unbound fraction). The beads were washed with ice-cold HNTG buffer, twice with HNTG buffer containing 500 mM NaCl and a final wash in HNTG buffer. The beads were resuspended in 50 µl of 10% Me2SO in collagenase buffer and mixed for 30 min at 4 °C, followed by a brief centrifugation to obtain the supernatant (bound fraction). The bound and unbound fractions were mixed with sample buffer under reducing conditions and subjected to SDS-PAGE, followed by ligand blot analysis as described above.
Determination of the Affinity of 2(IV) for Pro-MMP-2 and
Pro-MMP-9--
Affinity-purified
2(IV) (9.5 nM) was
allowed to complex with varying concentrations of either
35S-pro-MMP-2 (20-750 nM) or
35S-pro-MMP-9 (2.5-250 nM) for 1 h at
4 °C. The samples were then subjected to gel filtration using a
Superose 6 column (10/30) (Amersham Pharmacia Biotech) equilibrated
with 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, and 0.02% Brij-35 at a flow rate of 0.2 ml/min,
and 70 fractions (0.3 ml) were collected. Aliquots (0.2 ml) of each
fraction were placed in 5 ml of scintillation fluid and counted to
determine the amount (pmol) of enzyme bound. Complex formation was
evaluated based on the relative inclusion volume of pro-MMP-2,
pro-MMP-9, or
2(IV) alone or in complex. The dissociation constant
(Kd) was determined from the binding profiles
(picomoles of pro-MMP bound versus pro-MMP concentration) by
extrapolating the point where 50% maximal binding was obtained.
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RESULTS |
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Characteristics of 125I-Pro-MMP-9 Binding to MCF10A
Cells--
Previously, we showed that exposure of MCF10A cells to
12-O-tetradecanoylphorbol-13-acetate results in the
secretion of pro-MMP-9 that associates with the cell surface (25). To
measure the binding of pro-MMP-9, we carried out binding assays of
radioiodinated enzyme to untreated MCF10A cells to avoid interference
with the endogenously produced pro-MMP-9 (25). Fig.
1A shows
time-dependent binding of 125I-pro-MMP-9 to
MCF10A cells at 4 °C. Logarithmic transformation of the data
revealed a kon value of 6.94 ± 0.67 × 104 M1 s
1 (Fig.
1A, inset). Incubation of the cells with
increasing concentrations (1-40 nM) of
125I-pro-MMP-9 in the presence of 80-fold excess of
unlabeled enzyme demonstrated a specific saturable binding (Fig.
1B). Scatchard analysis revealed a Kd
value of 2.16 ± 0.16 × 10
8 M and
1.3 × 105 sites/cell (Fig. 1B,
inset). Similar values were obtained after nonlinear
curve-fitting analysis of the data (Kd = 2.49 ± 0.27 × 10
8 M and 1.4 × 105 sites/cell). We found no evidence of cell surface
activation of 125I-pro-MMP-9, as determined by SDS-PAGE
followed by autoradiography or by gelatin zymography (data not
shown).
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Cellular Distribution of 125I-Pro-MMP-9 and a
125I-Pro-MMP-9·TIMP-1 Complex after Binding to MCF10A
Cells--
We investigated the fate of pro-MMP-9 after binding using
ligand internalization studies as described under "Experimental Procedures." Fig. 2A shows
that the amount of 125I-pro-MMP-9 associated with the cell
pellet (internalized ligand) reached a maximum of 21% of the total
ligand bound after 10 min of incubation at 37 °C. Later time points
showed little or no change (<5%) in the radioactivity of the pellet
fraction. Concurrently, after a 10-min incubation, ~80% of the total
cell-associated radioactivity was recovered by Pronase E digestion
(representing cell surface-bound enzyme). This fraction exhibited a
decline in radioactivity consistent with dissociation of cell
surface-bound ligand and its appearance in the supernatant fraction
(Fig. 2A). SDS-PAGE analysis demonstrated that the
radioiodinated enzyme in the supernatant fraction remained in the
latent form at all times examined. In contrast, the internalized enzyme
was degraded to low (20 kDa) molecular mass fragments (data not
shown). Thus, binding of pro-MMP-9 to MCF10A cells is not followed by
significant ligand internalization.
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Identification of a 190-kDa Cell Surface Pro-MMP-9-binding Protein-- The above binding data were consistent with the existence of a single pro-MMP-9-binding component on the cell surface. To identify the putative pro-MMP-9-binding protein, we carried out an affinity purification using immobilized pro-MMP-9 (Affi-Gel 10-pro-MMP-9). Lysates of surface-biotinylated MCF10A cells were incubated with Affi-Gel 10-pro-MMP-9 matrix or uncoupled Affi-Gel 10 and the bound proteins were eluted with 10% Me2SO. The eluted proteins were then detected by either streptavidin-HRP or ligand blot analysis as described under "Experimental Procedures." Fig. 3 shows that MCF10A cells express a major 190-kDa protein that specifically and consistently binds to the Affi-Gel 10-pro-MMP-9 matrix (Fig. 3, A, lane 2; and B, lanes 2, 4, and 5) but not to the uncoupled matrix (Fig. 3, A and B, lanes 1 and 3). Several minor biotinylated proteins (~70-90 kDa) were found to bind, albeit inconsistently, to the pro-MMP-9 affinity matrix (Fig. 3A, lane 2). The 190-kDa protein was detected by streptavidin-HRP (Fig. 3A, lane 2) consistent with biotinylation and consequently, cell surface localization. The streptavidin detection was specific since blots of samples derived from lysates of non-biotinylated cells and incubated with Affi-Gel 10-pro-MMP-9 matrix were negative (Fig. 3A, lane 4). The presence of the 190-kDa protein in the non-biotinylated cells was confirmed by ligand blot analysis (Fig. 3B, lane 4). Electrophoretic migration of the 190-kDa protein was similar under nonreducing (Fig. 3B, lane 5) or reducing conditions (Fig. 3, A, lane 2, and B, lanes 2 and 4). In contrast, under nonreducing conditions, pro-MMP-9 exhibited the presence of monomer and dimer forms (Fig. 3B, lane 6), as expected (37).
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Affinity Purification of the 190-kDa Protein and Hydrodynamic Studies-- The 190-kDa protein was purified from lysates of MCF10A cells using the Affi-Gel 10-pro-MMP-9 matrix as described under "Experimental Procedures." Fig. 4 shows the analysis of the column fractions in a silver-stained SDS-PAGE (Fig. 4A) and by ligand blot analysis (Fig. 4B). The 190-kDa protein was detected in the 20% Me2SO eluate (Fig. 4, A and B, lane 5). Analysis of the protein content in each fraction indicated a ~3300-fold purification. The affinity-purified 190-kDa protein was subjected to hydrodynamic studies for determination of the sedimentation coefficient and Stokes radius to determine the native molecular mass. As shown in Fig. 5A, the 190-kDa protein was detected in six fractions derived from the gradient as shown by ligand blot analysis (Fig. 5A, inset). A sedimentation coefficient of 7.9 S was calculated from two determinations using known protein standards. The Stokes radius of the 190-kDa protein was determined by gel filtration using protein standards (Fig. 5B) and was calculated to be 52.6 Å. By combining the sedimentation and Stokes radius (38), a native molecular mass of 192,000 was calculated. These data and the electrophoretic mobility of the 190-kDa protein on SDS-PAGE (Fig. 3) indicate that this protein is monomeric.
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The 190-kDa Pro-MMP-9-binding Protein Is the 2(IV) Chain of
Collagen IV--
The affinity-purified 190-kDa protein was submitted
for microsequencing as described under "Experimental Procedures."
Analyses of three HPLC-purified peptides obtained after tryptic
digestion revealed the following amino acid sequences:
GVSGFPGADGIPGHPGQGGP, DGYQGPDGPRG, and KIAIQPGTVGPQG, which correspond
to residues 109-128, 325-335, and 1396-1408, respectively, of the
human
2(IV) chain of collagen IV (39). Immunoblot analysis of the
190-kDa protein demonstrated reactivity with three different mAbs (H22,
H25, and H21) against the human
2(IV) chain (Fig.
6A). Furthermore,
co-immunoprecipitation of the 190-kDa protein from an MCF10A cell
lysate with exogenous pro-MMP-9 using an anti-MMP-9 antibody followed
by immunoblot analysis with H22 mAb further demonstrated that the
coprecipitated 190-kDa protein is the
2(IV) chain (Fig.
6B).
|
Specificity of the Binding of 125I-Pro-MMP-9 to
2(IV)--
To evaluate the role of
2(IV) in the surface binding
of pro-MMP-9, a binding assay was carried out with
125I-pro-MMP-9 that was preincubated with affinity-purified
2(IV), as described under "Experimental Procedures." The results
showed a 78% reduction in specific binding, from 4.5 to 1.0 fmol of
pro-MMP-9 bound/1.3 × 105 cells, when
125I-pro-MMP-9 (3.6 nM) was incubated with
~2-fold molar excess (~7 nM) of
2(IV) before the
binding assay. Since
2(IV) is a substrate of MMP-9 (shown below),
MCF10A cells were incubated (45 min, 37 °C) with MMP-9 (1.2 pmol/well) to remove surface-associated
2(IV). This treatment
resulted in an 80% reduction in 125I-pro-MMP-9-specific
binding (from 6.4 to 1.4 fmol/1.3 × 105 cells). In
contrast, when the cells were treated with MMP-9 in the presence of
TIMP-1, a significant recovery of 125I-pro-MMP-9-specific
binding (from 1.4 to 4.4 fmol/1.3 × 105 cells) was
observed, suggesting that TIMP-1 prevented the degradation of the
surface-associated
2(IV). Collectively, these experiments suggest
that the cell surface binding of pro-MMP-9 is mediated by
2(IV).
Expression of 2(IV) in Cultured Cells and Binding to
Pro-MMP-9--
We examined the expression of
2(IV) in cell extracts
and on the surface of MCF10A, MDA-MB-231, HT1080, RVE, and CD3 cells by
surface biotinylation, immunoblots and ligand blot analyses. Fig.
7 shows that all the cells examined
express
2(IV) that specifically bound to the pro-MMP-9-affinity
matrix (Fig. 7, A-D) or co-precipitated with pro-MMP-9
(Fig. 7E). Surface biotinylation followed by Affi-Gel 10-pro-MMP-9 purification demonstrated that
2(IV) is readily detected on the surface of MCF10A (Fig. 7B, lane
1), HT1080 (Fig. 7B, lane 3) and RVE (Fig.
7D) cells but could not be detected on the surface of
MDA-MB-231 cells (Fig. 7B, lane 2). However, in
the latter,
2(IV) was identified in the cell lysate (Fig. 7A, lane 2) consistent with the ligand binding
data demonstrating a reduced number of pro-MMP-9 binding sites/cell in
the MDA-MB-231 cells. Expression of
2(IV) was also detected in mouse
endothelial CD3 cells by co-immunoprecipitation with pro-MMP-9 and
detection by ligand blot analysis (Fig. 7E, panel
5). Thus, these studies demonstrate that monomeric
2(IV) is
expressed by a variety of cells and can be detected on the cell
surface.
|
Binding of Pro-MMP-9 to Collagen IV--
The identification of
2(IV) and its binding to pro-MMP-9 raised the question about
interactions of this enzyme with triple-helical collagen IV. Since the
most abundant collagen IV is a heterotrimeric protein composed of two
1(IV) and one
2(IV) chains (40), we examined whether MCF10A cells
produce trimeric collagen IV and whether it binds to pro-MMP-9.
Concentrated serum-free conditioned medium of MCF10A cells was
subjected to SDS-PAGE under nonreducing conditions, followed by
immunoblot analysis using various antibodies to collagen IV. As shown
in Fig. 8, mAbs to either the
2(IV) (Fig. 8, lane 1, mAb 1910) or
1(IV) (Fig. 8, lane
2, H11) chains recognized a protein of >450 kDa, likely to
represent trimeric collagen IV. In contrast, H22 antibodies against
2(IV) did not react with trimeric collagen IV (Fig. 8, lane
3) suggesting that its epitope is not exposed under nonreducing
conditions. A protein of ~160 kDa was also detected, although to a
lower extent, with both H11 and H22 antibodies. The nature of this
protein is unknown. It should be noted that, under these conditions,
monomeric
2(IV) chain could not be detected in the media of MCF10A
cells by immunoblot and ligand blot analysis. Furthermore, growth of
MCF10A cells in the presence of daily added ascorbate (75 µg/ml) did
not alter the localization and amounts of the
2(IV) chain (data not
shown).
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|
Domain Analysis of Pro-MMP-9-2(IV) Interactions--
To define
the pro-MMP-9 domains that interact with
2(IV), we examined the
binding of a pro-MMP-9·TIMP-1 complex since TIMP-1 is known to bind
to the C-terminal domain of pro-MMP-9 (4, 37, 41). In addition, we
questioned whether the active form of MMP-9, lacking the N-terminal
domain, binds
2(IV). A lysate of MCF10A cells was incubated with
either a pro-MMP-9·TIMP-1 complex or with MMP-9. The proteins were
then immunoprecipitated with either anti-MMP-9 or anti-TIMP-1
antibodies and the immunoprecipitates were resolved by ligand blot
analysis. As shown in Fig. 10,
2(IV) coprecipitated with pro-MMP-9, as expected (Fig. 10A,
lane 1). In contrast, TIMP-1 alone did not bind to
2(IV)
(Fig. 10A, lane 2). In the presence of a
pro-MMP-9·TIMP-1 complex,
2(IV) was co-immunoprecipitated by
either anti-TIMP-1 (Fig. 10A, lane 4) or
anti-MMP-9 antibodies (Fig. 10A, lane 3). Thus,
the pro-MMP-9·TIMP-1 complex interacts with
2(IV). Development of
the same blot with anti-TIMP-1 antibodies (Fig. 10B,
lanes 5-8) showed TIMP-1 in the samples containing
inhibitor (Fig. 10B, lanes 6-8). When MMP-9 was
added to MCF10A lysates,
2(IV) could not be detected in the immunoprecipitate (Fig. 10C, lane 9) or in the
supernatant fraction of the immunoprecipitates suggesting that it was
probably degraded by MMP-9. However,
2(IV) coprecipitated with the
active enzyme (Fig. 10C, lane 10) when the
experiment was carried out in the presence of 10 mM EDTA.
Thus, neither the N-terminal nor the C-terminal domain (at least the
region of TIMP-1 interaction) of pro-MMP-9 appears to be critical for
binding to
2(IV). However, we have consistently found that a
pro-MMP-9·
2(IV) complex failed to co-precipitate with
gelatin-agarose matrix (data not shown), suggesting a role for the
gelatin-binding domain in the interaction of pro-MMP-9 with
2(IV).
|
Affinity of Pro-MMP-9 and Pro-MMP-2 for 2(IV)--
Since
pro-MMP-9 bears a high degree of sequence similarity with pro-MMP-2 and
both possess a gelatin-binding domain (41), we determined the ability
of pro-MMP-2 to compete for the binding of
2(IV) to pro-MMP-9. The
2(IV) was co-imunoprecipitated with pro-MMP-9 and anti-MMP-9
antibodies in the absence or presence of pro-MMP-2 (equimolar or 5-fold
molar excess, relative to pro-MMP-9) and in the presence of EDTA to
prevent degradation of
2(IV) by any trace of MMP-2. The
immunoprecipitates were resolved by ligand blot analysis. As shown in
Fig. 11,
2(IV) co-precipitates only in the presence of pro-MMP-9 (Fig. 11A, lanes
2-4). No significant differences in the amounts of
2(IV)
co-precipitating with pro-MMP-9 were detected in the presence of equal
molar amounts (Fig. 11A, lane 3) or excess
(5-fold) molar amounts of pro-MMP-2 (Fig. 11A, lane
4), suggesting that under these conditions only pro-MMP-9 bound to
2(IV). In agreement with these results,
2(IV) incubated with
pro-MMP-2 alone did not form a co-precipitable complex as determined
using anti-MMP-2 antibodies and ligand blot analysis (Fig.
11A, lane 6). Consistently, pro-MMP-2 was
detected in the immunoprecipitates when the same blot was developed
with anti-MMP-2 antibodies (Fig. 11B, lane
8).
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DISCUSSION |
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Previously, we have shown the surface association of pro-MMP-9 in 12-O-tetradecanoylphorbol-13-acetate-treated MCF10A cells (25). Other studies have described the presence of pro-MMP-9 on plasma membranes of HT1080 cells (23) and in focal contacts of endothelial cells (22). Here, we have characterized the binding parameters of pro-MMP-9 and demonstrated that the proenzyme binds with high affinity (Kd ~20-30 nM) to the surface of a variety of cell types. In MCF10A cells, internalization studies demonstrated that binding of pro-MMP-9, free or in complex with TIMP-1, is not followed by ligand internalization. Furthermore, under the experimental conditions, surface binding of pro-MMP-9 does not result in zymogen activation, possibly due to the lack of activators in the cell culture system. However, recent studies demonstrated that surface-associated pro-MMP-9 can be activated by a plasmin-dependent mechanism (23, 24). Taken together, these findings suggest that the surface association of pro-MMP-9 is not directly associated with activation and/or internalization but plays a role in confinement of proenzyme in areas of cell-matrix contacts, where it could become the target for potential pro-MMP-9-activating proteinases.
We identified a 190-kDa cell surface protein in various cell lines that
specifically bound to a pro-MMP-9-affinity matrix and formed a
co-precipitable complex with pro-MMP-9. Microsequencing of the
affinity-purified protein revealed a complete sequence homology to the
2(IV) chain of human collagen IV (39). Additionally, immunoblot
analysis with chain-specific antibodies (30) corroborated that the
190-kDa protein is
2(IV). Several observations indicate that the
cell surface association of pro-MMP-9 is mediated by
2(IV). (i)
Preincubation of pro-MMP-9 with affinity-purified
2(IV)
significantly inhibited ligand binding. (ii) Pretreatment of MCF10A
cells with MMP-9 reduced by 80% the binding of proenzyme, likely due
to the degradation of surface-associated
2(IV) as observed in
experiments with soluble
2(IV) and MMP-9. Accordingly, treatment of
cells with MMP-9 and TIMP-1 resulted in the recovery of the majority of
ligand binding; (iii) Binding of pro-MMP-9 to the cells exhibited an
affinity (Kd ~22 nM) that was in close
agreement with the affinity (Kd ~45
nM) of the enzyme for purified
2(IV). In addition, there
was a good correlation between the number of pro-MMP-9 binding sites
determined in the binding assays and the level of expression of
surface-associated
2(IV). For example, the breast cancer MDA-MB-231
cells that exhibited 10-fold less binding sites than MCF10A cells
showed undetectable levels of surface-associated
2(IV). The reason
for the low levels of
2(IV) on the surface of these malignant cells
is unknown but may be related to their inability to retain
2(IV) on
the cell surface since
2(IV) was detected intracellularly. Thus,
in vitro, the surface association of pro-MMP-9 would depend
on the ability of each cell type to express and retain
2(IV) on the
cell surface. Whether pro-MMP-9 and pro-MMP-2 bind also to monomeric
1(IV) is yet unknown and remains to be determined. However, the fact that only a single polypeptide,
2(IV), was specifically and
consistently co-precipitated with pro-MMP-9 or bound to immobilized
proenzyme suggests a unique and preferential interaction between
pro-MMP-9 and
2(IV).
Co-immunoprecipitation experiments using a pro-MMP-9·TIMP-1 complex
or MMP-9 demonstrated that neither the C-terminal nor the N-terminal
domains of pro-MMP-9 appeared to be critical for interactions with
2(IV). However, we could not precipitate the
2(IV)/pro-MMP-9
complex with gelatin-agarose beads, suggesting a role for the
gelatin-binding domain. In this regard, it was interesting to observe
that pro-MMP-2, when compared with pro-MMP-9, exhibited a lower
affinity for
2(IV) as determined by the co-immunoprecipitation and
gel filtration experiments. Since pro-MMP-2 contains a similar gelatin
binding domain (41), this suggests that the interactions of
2(IV)
with pro-MMP-9 are also influenced by sites/domains other than the
gelatin binding domain and/or by the three-dimensional conformation of
pro-MMP-9. It would be of interest to determine whether the 54-amino
acid proline-rich
2(V)-like extension that is uniquely present in
pro-MMP-9 (17, 41), plays any role in the binding of the enzyme to
2(IV). In the cell binding assays, pro-MMP-2 slightly competed with
pro-MMP-9 binding, suggesting that surface-associated
2(IV) is also
available for pro-MMP-2 binding. However, given the lower affinity of
pro-MMP-2 for
2(IV) and the existence of alternate high affinity
surface binding sites for pro-MMP-2 (14), this enzyme would be expected
to bind to the cell surface via a different mechanism (14, 16).
The binding of gelatinases to collagen IV has been examined in previous
studies (21, 42); however, the sites of interaction were not defined.
The results presented here suggest that a major high affinity binding
site for pro-MMP-9 in collagen IV must reside within the 2(IV)
chain. However, our data also indicate that neither triple-helical
collagen IV secreted by MCF10A cells nor EHS collagen IV bound to
pro-MMP-9 under conditions of
2(IV) binding. In agreement with these
results, we have recently observed a weak affinity
(Kd = 2.15 µM) of pro-MMP-9 for EHS
collagen IV as determined by surface plasmon
resonance4. Thus, whereas
monomeric
2(IV) forms a tight complex with pro-MMP-9 (Kd nM), trimeric collagen IV binds
pro-MMP-9 with very low affinity (Kd
µM). This is also consistent with the studies of
Steffensen et al. (21), who showed a weaker affinity of a
recombinant gelatin-binding domain of pro-MMP-2 for trimeric collagen
IV compared with that for denatured collagen IV. This has led to the
suggestion that binding sites for the gelatin-binding domain in
denatured collagen IV may be masked in triple-helical collagen IV (21).
Therefore, it is conceivable that the binding of pro-MMP-9 to
2(IV)
is mediated by sites that are cryptic in collagen IV and that are
exposed after partial denaturation and/or degradation of the collagen
IV molecule. If so, these sites would have to be conserved after
partial proteolysis to allow binding of pro-MMP-9. Such a scenario
would probably involve a partial degradation of the collagen IV network
by a protease(s) other than the gelatinases as the catalytic efficiency
of MMP-2 and MMP-9 against native collagen IV has been reported to be
limited (43-45). After collagen IV degradation, secretion of pro-MMP-9 by pro-MMP-9 producing-cells in close association with the basement membrane would then facilitate binding of pro-MMP-9 to
2(IV). After
activation of the
2(IV)-bound pro-MMP-9, the enzyme would then
contribute to the complete degradation of the collagen IV network
consistent with its ability to degrade denatured collagens (41).
Another possible scenario for the results observed here may involve
binding of pro-MMP-9 to a surface-associated 2(IV). Although the
origin and binding mechanism of
2(IV) chains to the cell surface
remain to be elucidated, our in vitro studies with a variety of cell lines (epithelial, endothelial and fibrosarcoma) clearly show
that, although some
2(IV) chains are assembled with
1(IV) into
trimeric collagen IV molecules, a portion of single
2(IV) chains are
secreted and deposited on the cell surface. The surface-associated
2(IV) chains appear to be stable as determined by surface
biotinylation and pulse-chase analysis. Indeed, we have found that
2(IV) chains can be detected in the cellular compartment of both
MCF10A and HT1080 cells and in the supernatant of the latter even after
a 10-h chase period without any evidence of processing and/or
degradation.5 These findings
are in contrast with the general notion that
(IV) chains that fail
to form trimeric collagen molecules are unstable and/or rapidly
hydrolyzed. This is generally true if active proteinases are present in
the experimental system, as they can hydrolyze non-triple-helical
collagen chains as shown here with
2(IV) and MMP-9. Thus, the
integrity and eventually the detection of
(IV) chains would depend
on the presence of active proteinases and/or inhibitors in each
particular culture system and tissue. Detection of monomeric
(IV)
chains may also depend on the extraction procedures and/or on the
antibodies used since some antibodies may only recognize
(IV) chains
in triple-helical conformation. Here we have used immobilized pro-MMP-9
and chain-specific mAbs raised against synthetic peptides, which
independently allowed for the detection of native and stable
2(IV)
chains on the cell surface. Although the processing, localization and
stability of monomeric
2(IV) warrants further in vitro
and in vivo studies, it is tempting to speculate that surface-associated
2(IV) may anchor pro-MMP-9 on the cell surface after autocrine or paracrine secretion. After activation by
pro-MMP-9-activating enzymes, MMP-9 would then play a role in localized
surface proteolysis. Since the binding of pro-MMP-9 to the
2(IV)
chain is of high affinity and involves the zymogen form, it will be
important in future studies to address the activation of pro-MMP-9 and
its interactions with TIMP-1 in the context of pro-MMP-9 bound to
2(IV).
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FOOTNOTES |
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* This work was supported by Grant CA-61986 from the National Institutes of Health and Grant DAMD17-94-J-4356 from the Department of Defense (both to R. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
** To whom all correspondence should be addressed: Dept. of Pathology, Wayne State University, 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: ECM, extracellular matrix; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; MT1-MMP, membrane type 1-MMP; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; FBS, fetal bovine serum; PMSF, phenylmethylsulfonyl fluoride; HRP, horseradish peroxidase; EHS, Engelbreth-Holm-Swarm; ECL, enhanced chemiluminescence; mAb, monoclonal antibody; RVE, rat vascular endothelial.
2 Latent enzyme will be referred to as "pro-MMP" and active enzyme as "MMP."
3 L. Coussens, personal communication.
4 M. Olson and R. Fridman, unpublished results.
5 M. Toth and R. Fridman, unpublished results.
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REFERENCES |
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