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INTRODUCTION |
Degradation of connective tissue extracellular matrix
(ECM)1 and dissolution of
epithelial and endothelial basement membrane are remodeling processes
that occur during tumor invasion and metastasis. A family of
proteolytic enzymes that have been functionally linked to these
remodeling processes are the matrix metalloproteinases (MMPs) (1, 2).
The members of this family are multidomain, zinc-containing, neutral
endopeptidases and include the collagenases, stromelysins, gelatinases,
and membrane-type metalloproteases (3, 4). Different MMPs can be
induced in a variety of embryonic, and adult cell types and distinct
subsets are often found to be elevated in tumor tissue and malignant
cells in culture (5-7). Each MMP has a preferred substrate specificity
toward individual matrix proteins, but there is overlapping specificity
within the whole family (3, 4), and thus a few MMPs acting in tandem have the potential to catalyze the complete degradation of the proteinaceous components of the basement membrane and ECM. However, the
catalytic manifestations of MMP enzymes are highly regulated. First,
the MMPs are expressed as inactive zymogens and require distinct
activation processes to convert them to active enzymes (8, 9), and
second, a family of proteins, the tissue inhibitors of
metalloproteinases (TIMPs), are correspondingly widespread in tissue
distribution and function as highly effective MMP inhibitors (Ki ~10
10 M) (3). How
the invading tumor cells that utilize the MMP's degradative capacity
circumvent these negative regulatory controls is not well understood.
Within the cytokine- and oncoprotein-enriched tumor tissue environment,
MMP expression more than likely will be up-regulated, and thus the
limiting reaction in MMP-mediated tumor invasion may be zymogen
activation. Activation of MMP zymogens involves disruption of a
coordination bond formed between a highly conserved unpaired cysteine
in the amino-terminal propeptide of the pro-MMP molecule and the zinc
ion at the active center (8). Disruption of this bond can be mediated
by chemical and/or proteolytic mechanisms, often leading to an
autoproteolytic event, resulting in the removal of the
cysteine-containing propeptide domain, generating a lower molecular
weight, catalytically active form of the MMP. If an active MMP is
involved in the initial proteolytic step or if an autolytic processing
event is part of the final step, then the presence of TIMPs in the
surrounding milieu will prevent zymogen activation. MMP activation can
occur through intracellular, extracellular, and cell surface-mediated
proteolytic mechanisms. Intracellular activation of stromelysin 3 (MMP-11) occurs in the Golgi network and is mediated by the
intracellular serine protease furin (10). Activation of interstitial
collagenase (MMP-1) and stromelysin (MMP-3) occurs extracellularly and
can be mediated by the serine protease, plasmin (11, 12). Cell surface
activation of an MMP can occur when gelatinase A (MMP-2) is brought
into contact with a membrane-associated MMP, MT-1 MMP (13, 14).
Interestingly, gelatinase B (MMP-9), a close structural homologue of
MMP-2 does not appear to be activated by the same mechanism, as it
remains a zymogen under identical cellular conditions where a majority of co-expressed pro-MMP-2 is activated via the cell surface mechanism (15, 16).
MMP-9 is produced by mesenchymal, epithelial, and hematopoietic cells
and also by distinct tumor cell types (17-20). While MMP-2 appears to
be constitutively expressed by many cell types in culture, MMP-9
expression is induced by cytokines (21, 22), growth factors (23), and
cell/stroma interactions (19, 20, 24). MMP-9 expression has been
correlated with a number of physiological and pathological processes,
including trophoblast implantation (25), bone resorption (26),
inflammation (18, 19), and arthritis (27). Tumor cell invasion in a
number of instances also has been linked with MMP-9 activity (5-7,
20). Furthermore, Muschel and colleagues (28, 29) have shown by both
transfection and ribozyme-based approaches that MMP-9 is directly
involved in tumor metastasis, and more recently MMP-9 activity has been linked with the process of tumor cell intravasation (30). Thus in these
malignant cell systems, the regulatory controls that maintain MMPs as
inactive zymogens were circumvented, since conversion of pro-MMP-9 to
active enzyme had clearly occurred. The exact mechanism of MMP-9
activation in malignant tissue, however, has not been defined. A number
of purified proteases, including trypsin (31, 32), chymase (33), MMP-2
(34), tissue kallikrein (35), trypsin-2 (36), plasmin (37, 38), MMP-7
(39), MMP-13 (40), and MMP-3 (31-33, 37, 38, 41, 42) have been reported to activate pro-MMP-9 in vitro. MMP-3, based on
in vitro kinetic and catalytic parameters, appears to be the
most efficient activator of pro-MMP-9 (32) and may be a natural
activator in vivo. It is not clear, however, if MMP-3 will
always be present in the tumor tissues where pro-MMP-9 is expressed.
Furthermore, even if MMP-3 is available in the tissues, it is produced
as a zymogen and also requires activation. In addition, the inhibitory potential of the TIMPs must be circumvented in the tumor tissue to
allow for catalytic manifestations of both the activating MMP-3 and the
activated MMP-9.
To demonstrate a possible tumor-associated mechanism of MMP-9
activation and the circumvention of TIMP-mediated control, we have
examined cultures of a breast carcinoma cell line, MDA-MB 231, which
expresses both MMP-9 and TIMP-1 and TIMP-2. MDA-MB-231 cultures do not
appear to express MMP-2, also a potent gelatinase. Since MMP-9
activation is monitored by the generation of gelatinase activity, the
absence of any interfering MMP-2 gelatinase activity in the cultures
was critical for quantitating zymogen activation. Our results indicate
that MMP-9 produced by the breast tumor cells is a stable zymogen even
when exceptionally high levels of MMP-9 are induced and when active
plasmin is generated in the cultures. However, when pro-MMP-3 is
introduced into the system at concentrations that stoichiometrically
exceed the endogenous TIMP levels, plasmin activates the MMP-3 which in
turn efficiently processes and activates the pro-MMP-9. The activated
MMP-9 contributes significantly to tumor cell-mediated ECM degradation
and basement membrane invasion, and the specificity of these mechanisms
is demonstrated through the use of neutralizing anti-MMP-9 monoclonal antibodies.
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MATERIALS AND METHODS |
Cell Culture--
MDA-MB-231 cells, HT1080 cells, and MDA-MMP-9
cells were cultured at 37 °C in 8% CO2 in DMEM
containing 10% heat inactivated fetal bovine serum (FBS) supplemented
with 0.1 mM nonessential amino acids, 2 mM
glutamine, 1 mM sodium pyruvate, and 100 units/ml penicillin with 100 µg/ml streptomycin. For harvesting serum-free conditioned medium, the cell cultures were washed with DMEM and incubated for 24 h with serum-free DMEM that included the above mentioned supplements.
MMP-9-expressing Cell Line--
Cultures of MDA-MB-231 (5 × 105 cells/10-cm culture dish) were transfected with a
pRc/RSV/MMP-9 cDNA construct (20 µg) in calcium phosphate buffer
(125 mM CaCl2, 140 mM NaCl, 25 mM HEPES, 0.75 mM
Na2HPO4, pH 7.1). The MMP-9 cDNA construct
was provided by Dr. Ruth Muschel, University of Pennsylvania,
Philadelphia, PA (28). After 48 h, the transfected cells were
replated (5 × 105 cells/10-cm dish) in supplemented
DMEM, 10% FBS containing 600 µg/ml Geneticin (Life Technologies,
Inc.). After 2 weeks in culture, the Geneticin-resistant cells were
cloned by limiting dilution in 96-well culture plates. The supernatants
from the cloned cultures were analyzed by enzyme-linked immunosorbent
assay for secreted pro-MMP-9. A number of clones expressed increased
levels of pro-MMP-9 over that of the nontransfected, parental cultures.
One stable cell line, MDA-MMP-9, was propagated under standard culture
conditions and analyzed in detail.
MMP-9 Enzyme-linked Immunosorbent Assay--
Microtiter 96-well
plates were precoated with an anti-MMP-9 monoclonal antibody (6-6B
IgG2b) (1 µg/ml, 50 µl/well) in PBS for 1 h at
room temperature and blocked with 1% bovine serum albumin/PBS (100 µl/well) for 1 h at room temperature. Conditioned medium (50 µl/well) was added for 1 h at 37 °C followed by washing and the addition of a secondary anti-MMP-9 monoclonal antibody, 7-11C IgG1, (1 µg/ml, 50 µl/well) in 1% bovine serum
albumin/PBS for 1 h at 37 °C. The cells were washed with PBS,
0.5% Tween, incubated with alkaline phosphatase-conjugated goat
anti-mouse IgG (1:1000 in 1% bovine serum albumin/PBS), followed by
washing and the addition of p-nitrophenyl phosphate
substrate and the development of color according to manufacturer
(Kirkegaard and Perry, Gaithersburg, MD) instructions.
Western Blot Analysis--
Samples of conditioned medium were
first separated by SDS-polyacrylamide gel electrophoresis and then
electrophoresed onto nitrocellulose membranes (Millipore, Burlington,
MA). The membranes were blocked in 5% non-fat milk in Tris-buffered
saline, 0.5% Tween, washed, and incubated overnight with purified
monoclonal antibodies (1-5 µg IgG/ml). The blots were then washed
and incubated for 2-4 h with secondary antibody, horseradish
peroxidase-conjugated goat anti-mouse IgG (Kirkegaard and Perry) at
1:1000 dilution. The blots were developed using the ECL
chemiluminescence detection system (Amersham Pharmacia Biotech).
Substrate and Reverse Zymography--
Gelatin substrate
zymography was performed in SDS-polyacrylamide (10%) gels
copolymerized with gelatin, as described previously (21). Reverse
zymography, used to detect and quantitate TIMP levels, was performed in
SDS-polyacrylamide (15%) gels copolymerized with gelatin (60 µg/ml)
and conditioned medium (1.0 ml) from a cell line expressing MMP-2 (43).
After electrophoresis, the gels were washed for 2 h in Triton
X-100 (2.5%) and incubated overnight in buffer (50 nM
Tris, pH 7.25, 200 mM NaCl, 10 nM
CaCl2, 0.05% Brij-35, 0.02% NaN3). The gels
were stained with Coomassie Brilliant Blue, and dark zones marked the
TIMP-mediated inhibition of gelatin degradation.
Activation of Pro-MMP-9 in Culture--
The MDA-MB-231 cells and
MDA-MMP-9 cells were plated at 1 × 105
cells/cm2 in DMEM, 10% FBS. After 24 h the cells were
rinsed once with 2 mM 6-amino-n-caproic acid in
serum-free DMEM to remove any cell surface plasminogen. The cells were
rinsed twice with serum-free DMEM and incubated in the same medium in
the presence or absence of the following components: plasminogen (25 nM), pro-MMP-3 (2-16 nM), active MMP-3 (5 nM), aprotonin (40 µg/ml), TIMP-1 (20 nM), anti-MMP-9 IgG (200 nM), and normal mouse IgG (200 nM) added singularly or in the indicated combinations. The
cell cultures were incubated for 24-48 h and the conditioned medium
collected and stored at
70 °C before analysis. Pro-MMP-3 was
expressed as a recombinant protein in Chinese hamster ovary K-1 cells
and purified from conditioned medium as described previously (44).
Active MMP-3 (MMP-3
C) was a C-terminal truncated derivative of MMP-3
that spontaneously activated and yielded a specific activity equivalent
to native MMP-3. It was expressed in Escherichia coli and
purified as described previously (45). Anti-MMP-9 monoclonal antibody
7-11C blocks activation of pro-MMP-9 and was purified from hybridoma
conditioned medium as described previously (46).
Preparation of Cell Extracts--
MDA-MMP-9 cells were plated
and grown to confluence in 100-mm dishes (1.5-2 × 107 cells/plate in DMEM, 10% FBS. Each plate was washed
twice with serum-free DMEM with aprotinin 40 µg/ml) and once with
serum-free DMEM alone. The cells were incubated in 5 ml of serum-free
DMEM either alone, with 2 µg/ml plasminogen, with 16 nM
pro-MMP-3, or in combination for 20 h at 37 °C. At the end of
this time, the conditioned media were collected, and 40 µg/ml
aprotinin was added. The cells were washed twice with
phosphate-buffered saline and extracted at 4 °C with constant
shaking in 1 ml of lysis buffer (0.1 M Tris, pH 8.0, 0.5%
Triton X-100) with aprotinin (40 µg/ml). The extracts were
centrifuged at 12,000 rpm for 10 min at 4 °C. The resulting
supernatants were each incubated for 1 h at 4 °C in an
end-over-end mixer with 100 µl of gelatin-Sepharose that had been
equilibrated with 50 nM Tris, pH 7.25, 200 nM
NaCl, 10 nM CaCl2, 0.05% Brij-35. The beads
were washed five times in 1 ml of equilibration buffer, and the total
cellular MMP-9 was eluted from the beads by resuspending the slurry
twice with 75 µl equilibrium buffer containing 10%
Me2SO, centrifuging twice, and recovering and combining the
supernatants (150 µl).
Enzyme Activity in Solution--
MMP-9 gelatinase activity was
measured in solution using heat denatured 3H-acetylated
type I collagen (gelatin) purified from rat tails (47). Conditioned
medium (75-150 µl) was incubated with the labeled gelatin (20 µg/ml, 2000 cpm/µg) in buffer (50 mM Tris, pH 7.5, 200 nM NaCl, 10 mM CaCl2, 0.05%
Brij-35, 0.02% NaN3) for 4-48 h followed by
trichloroacetic acid precipitation, as described previously (48). MMP-3
activity was measured using a fluorogenic peptide,
Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(DNP)-NH2. The peptide, designated NFF-3, is selectively hydrolyzed by MMP-3 and
exhibits little reactivity with MMP-1, MMP-2, and MMP-9 (49). The assay
was performed by incubating 10-100 µl of conditioned medium with 1 µM peptide at 37 °C in 200 µl of solution.
Fluorescence was read continuously at
ex = 325 nm and
em = 393 nm as described previously (49). Plasmin
activity was determined by measuring hydrolysis of the specific peptide
Spectrozyme PL according to manufacturer's instructions (American
Diagnostica, Greenwich, CT). Human uPA activity was measured in a
plasminogen-dependent coupled assay as described previously
(50).
ECM Degradation Assays--
[3H]Proline-labeled
rat smooth muscle cell ECM was prepared as described by Jones et
al. (51). Degradation experiments were carried out on ECM
predigested with trypsin (5 µg/ml for 2 h at 37 °C in PBS).
Following digestion, trypsin activity was inhibited with soybean
trypsin inhibitor (10 µg/ml), and the matrices were washed three
times with DMEM (500 µl). For the matrix degradation assays, the
cells were seeded on radiolabeled ECM-coated 48-well plates at 1 × 105 cells/cm2 in 250 µl of DMEM. After
24 h the cells were rinsed once with serum-free DMEM and incubated
in the same medium (250 µl) in the presence or absence of the
following components: plasminogen (25 nM), pro-MMP-3 (16 nM), aprotonin (40 µg/ml), 7-11C anti-MMP-9 IgG (200 nM), control IgG (200 nM), and TIMP-1 (20 nM). To follow the progressive degradation of the ECM,
aliquots (50 µl) of the culture supernatants were harvested after 24, 48, and 72 h, and the soluble radioactivity quantitated in a
scintillation counter.
Basement Membrane Invasion Assay Using
Matrigel--
Matrigel-coated filter inserts (8 µm pore size) that
fit into 24-well invasion chambers were obtained from Becton Dickinson (Bedford, MA). The MDA-MMP-9 cells to be tested for invasion were detached from tissue culture plates with a nonenzymatic cell
dissociation solution (Sigma), washed, and resuspended in DMEM (5 × 104 cells/200 µl) and added to the upper compartment
of the invasion chamber in the presence or absence of the indicated
components. Culture medium (500 µl) was added to the lower
compartment of the invasion chamber. The Matrigel invasion chambers
were incubated at 37 °C for 48 h in 8% CO2. After
incubation the filter inserts were removed from the wells, and the
cells on the upper side of the filter were removed using cotton swabs.
The filters were fixed, mounted, and stained according to the
manufacturer's instructions (Becton Dickinson). The cells that invaded
through the Matrigel and were located on the under side of the filter
were counted. Three to five invasion chambers were used per condition.
The values obtained were calculated by averaging the total number of
cells from three filters.
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RESULTS |
Enhanced Expression of MMP-9 from Human Tumor Cells That Do Not
Express MMP-2--
To examine the activation of pro-MMP-9 in a
cellular setting that is devoid of MMP-2, cultures of the human breast
carcinoma cell, MDA-MB-231, were treated with cytokines known to
stimulate the production of pro-MMP-9. Gelatin substrate zymography, a
method that can detect subnanogram amounts of gelatinase, was used to monitor levels of MMP-9 in the conditioned medium harvested from the
cultures. A parallel culture of HT1080, a human fibrosarcoma cell line
that produces both MMP-2 and MMP-9 was used as control. Treatment of
MDA-MB-231 cells with PMA and interleukin-1 resulted in increased
levels of MMP-9 expression while platelet-derived growth factor
treatment yielded no change in MMP-9 levels (Fig. 1). There was no evidence of MMP-2
expression in the treated cultures. Although PMA stimulation resulted
in a substantial increase in MMP-9 expression, an 82-kDa form was not
observed, indicating that the induced enzyme was not activated but
remained in the 92-kDa zymogen form.

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Fig. 1.
Zymographic analysis of MMP-9 and MMP-2
expression in stimulated and unstimulated cultures of MDA-MB-231
cells. Cultures of MDA-MB-231 cells (5 × 104
cells/cm2) were treated with either PMA (100 ng/ml),
interleukin-1 (10 ng/ml), platelet-derived growth factor (10 ng/ml) or
left untreated (unstim.). Serum-free conditioned medium was
collected after 24 h from the cultures, and a 40-µl aliquot was
subjected to gelatin substrate zymography. The conditioned medium from
a parallel culture of PMA-treated HT 1080 cells (5 × 104 cells/cm2) was analyzed as a positive
control for MMP-9 and MMP-2 expression. The electrophoretic positions
of the 92-kDa pro-MMP-9 zymogen, the 72-kDa pro-MMP-2 zymogen, and the
82- and 62-kDa activated forms of MMP-9 and MMP-2, respectively, are
indicated.
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Generation of Active Stromelysin (MMP-3) in MDA-MB-231 Cultures
Fails to Activate Pro-MMP-9--
To determine if a previously proposed
MMP-3-dependent mechanism of pro-MMP-9 activation (41)
could occur in a complex cell culture system, a proteolytic cascade
that would yield active MMP-3 was initiated in the MDA-MB-231 cultures.
These cultures produce uPA at levels sufficient to catalyze the
conversion of plasminogen to plasmin. Plasmin is a known activator of
pro-MMP-3 (12), and active MMP-3 had been shown to activate pro-MMP-9 under in vitro conditions using purified preparations of
MMP-3 and pro-MMP-9 (32, 38). The proteolytic cascade was initiated by
supplementation of the PMA-treated MDA-MB-231 cultures with pro-MMP-3
(16 nM) and plasminogen (25 nM). The activation
of pro-MMP-3 was monitored by immunoblot analysis and activation of
pro-MMP-9 was measured by zymography (Fig.
2). In the absence of plasminogen, the
addition of pro-MMP-3 had no effect on the status of the pro-MMP-9 as
it remained in the 92-kDa zymogen form (Fig. 2, zymograph, lane
2). The pro-MMP-3 also was maintained as the 55-kDa zymograph (Fig. 2, immunoblot, lane 2). Addition of plasminogen plus
pro-MMP-3 caused a conversion of the 55-kDa pro-MMP-3 to the 45-kDa
active MMP-3 (immunoblot, lane 3) but, unexpectedly, little
or no activation of pro-MMP-9 occurred; only trace levels of an 82-kDa
form of MMP-9 appeared in the zymograph (lane 3), and no
gelatinase activity was detectable. Plasmin was generated in the
culture system since plasmin activity as measured by a specific peptide
hydrolysis assay was detected only in the plasminogen-containing
cultures (data not shown). The plasmin that was generated in this
system was responsible for the conversion of pro-MMP-3 because the
addition of aprotinin, a specific inhibitor of plasmin, blocked the
conversion of pro-MMP-3 from the 55-kDa form to the 45-kDa form (Fig.
2, immunoblot, lane 4).

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Fig. 2.
Zymographic and immunoblot analyses of
pro-MMP-9 and pro-MMP-3 activation in MDA-MB-231 cultures.
Cultures of MDA-MB-231 (1 × 105
cells/cm2) in serum-free DMEM were treated with PMA (100 mg/ml) to enhance production of pro-MMP-9. The treated cultures
(MDA·PMA) were incubated in the absence or presence of human
pro-MMP-3 (16 nM), human plasminogen (25 nM),
and the plasmin inhibitor aprotinin (40 µg/ml), as indicated. After
24 h, the conditioned medium was collected from the cultures and
analyzed by gelatin substrate zymography and immunoblotting using an
anti MMP-3 monoclonal antibody (1 µg/ml) that recognizes both the
55-kDa pro-MMP-3 and the processed 45-kDa active form of MMP-3.
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The absence of conversion of pro-MMP-9 to active MMP-9 in the presence
of a functioning proteolytic cascade, which clearly had generated
active MMP-3, was further investigated. The relative concentrations of
pro-MMP-9, TIMP-1, and TIMP-2 in the cultures were determined (Table
I). Interestingly, the levels of
pro-MMP-9 in the PMA-stimulated cultures were increased 8-fold over the levels in the unstimulated culture (0.25 nM
versus 0.03 nM), but the endogenous TIMP
concentrations still remained 10-20-fold higher than the MMP-9
concentration in these cultures (3-4 nM versus 0.25 nM). These data suggested that TIMP levels were
controlling the progression to active MMP-9 and that activation might
only occur when the total MMP concentration exceeded the TIMP
concentration in the immediate environment. Within the MDA-MB-231 cell
culture system, the molar concentration of pro-MMP-9 would have to be increased at least 20-fold to exceed the endogenous TIMP levels.
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Table I
Levels of pro-MMP-9, TIMP-1, and TIMP-2 in cultures of parental,
PMA-treated, and transfected MDA-MB-231 cells
The indicated cultures were incubated at 1 × 105
cells/cm2 in serum-free medium for 48 h. The conditioned
medium was harvested and analyzed for pro-MMP-9, TIMP-1, and TIMP-2 by
enzyme-linked immunosorbent assay. The values represent the mean ± S.D. of three separate determinations. The TIMP-1 and TIMP-2 values
were added together to represent total TIMP concentration.
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An MDA-MB-231-transfected Cell Line Expressing Increased Levels of
MMP-9--
To achieve increased levels of MMP-9 expression, MDA-MB-231
cells were transfected with an MMP-9 cDNA construct (28), selected, cloned, and analyzed for MMP-9 expression by zymography and immunoblot analysis. A stable cell line that secreted substantially increased levels of MMP-9 was isolated and designated MDA-MMP-9 (Fig. 3, A and C). Like the
parent MDA-MB-231 cells, this cell line did not express MMP-2 (Fig. 3,
B and C), MMP-3 (Fig. 3D),
or MMP-1 (Fig. 3E) and did express significant levels of uPA
(Fig. 3H). A direct enzyme assay indicated that the uPA
concentration in the cell culture supernatant was 0.1-0.2 µg/ml,
which could very effectively activate microgram quantities of
plasminogen. Although this cell line expressed increased levels of
MMP-9, the TIMP levels were the same as the parent cell line and
slightly less than the TIMP levels expressed by the PMA-treated
MDA-MB-231 cells (Fig. 3, F and G). Quantitation
of MMP-9 levels expressed by the MDA-MMP-9 cell line showed that the
MMP-9 concentration now exceeded the concentration of both TIMP-1 and
TIMP-2 (8 nM versus ~3 nM) (Table
I, third column). However, the MMP-9 in the transfected cultures
remained in the 92-kDa zymogen form despite the significant increase in
the MMP-9:TIMP ratio (Fig. 3, A and C), and no
gelatinase activity was manifested by the transfected cultures (data
not shown). Furthermore, when the transfected cultures were incubated
in the presence of plasminogen, which was rapidly converted to plasmin,
the 92-kDa zymogen form was maintained (see Fig.
4, inset, lane
1).

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Fig. 3.
Production of MMPs, TIMPs, and uPA by
parental, PMA-stimulated, and transfected MDA-MB-231 cells.
Cultures of parental MDA-MB-231 cells (MDA), MDA-MB-231
cells treated with 100 µg/ml PMA (MDA·PMA), and
MDA-MB-231 cells stably transfected with an MMP-9 cDNA construct
(MDA-MMP-9) were incubated (1 × 105
cells/cm2) in serum-free DMEM for 24 h. The
conditioned medium was collected, subjected to SDS-polyacrylamide gel
electrophoresis, and analyzed by immunoblotting, gelatin substrate
zymography, and reverse zymography. The immunoblots were probed with
the indicated antibodies at 1-2 µg/ml and developed using
chemiluminescence. Known standards were added to the first lanes in
each gel and included 2-10 ng of purified MMP-9 (A), MMP-2
(B), TIMP-1 (F), uPA (H), 25 µl of
conditioned medium from HT 1080 cultures (C), and 40 µl of
conditioned medium from interleukin-1-treated human dermal fibroblasts
(D and E). The MDA-MMP-9 cultures expressed high
levels of pro-MMP-9; moderate levels of uPA, TIMP-1 and TIMP-2; and no
detectable levels of MMP-2, MMP-3, and MMP-1.
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Fig. 4.
Activation of endogenous pro-MMP-9 in
MDA-MMP-9 cultures supplemented with plasminogen and pro-MMP-3.
Cultures of MDA-MMP-9 cells (1 × 105
cells/cm2) were incubated in serum-free DMEM containing
human plasminogen (2.5 µg/ml) and purified human pro-MMP-3 (0-16
nM), as indicated. After 48 h the conditioned medium
was collected and analyzed for: pro-MMP-3 conversion to active MMP-3 by
measuring specific fluorogenic peptidolytic activity expressed in
fluorescent (F) units (bar graph), MMP-9
activation by measuring the degradation of 3H-labeled
gelatin expressed in counts/min hydrolyzed (line graph), and
pro-MMP-9 processing by gelatin substrate zymography (zymograph
inset).
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MMP-3-dependant Activation of the Pro-MMP-9 Secreted by the
MDA-MMP-9 Cells--
The activation of pro-MMP-9 in the tumor cell
culture system was re-analyzed using the transfected MDA-MMP-9 cells.
By adding plasminogen (2.5 µg/ml) and pro-MMP-3 at increasing
concentrations to the cell culture system, the activation of MMP-9
occurred in a dose-dependent manner (Fig. 4). The
appearance of a gelatinolytic zone with an apparent molecular mass of
82 kDa was observed upon the addition of 8-16 nM pro-MMP-3
(Fig. 4, zymograph inset), and this change in molecular mass
was accompanied by a corresponding increase in solution phase
gelatinolytic activity (Fig. 4, line graph). In the presence
of 16 nM pro-MMP-3, more than half of the 92-kDa form of
the enzyme had been processed, and significant gelatinolytic activity
was observed.
The presence in the cell culture system of active MMP-3 generated from
the added pro-MMP-3 was measured using a fluorogenic peptide assay that
is specific for MMP-3 (49). Enzyme activity above background was
observed only in cultures containing pro-MMP-3 at concentrations
8
nM (Fig. 4, bar graph), which corresponded with
the appearance of both the 82-kDa form of MMP-9 and gelatinase activity. At concentrations less than 8 nM pro-MMP-3, MMP-3
activity as well as conversion of MMP-9 and gelatinase activity were at background levels. These data suggested that the effective generation of gelatinolytic activity was dependent on concentrations of MMP-3 and
MMP-9 that exceeded the endogenous TIMP concentrations.
To determine the pro-MMP-3 concentration necessary to activate MMP-9 in
the absence and presence of stoichiometric levels of TIMP-1, the
plasmin-induced proteolytic cascade was reconstituted in
vitro using purified components (Fig.
5). Purified pro-MMP-9 was added at 8 nM, the concentration present in the MDA-MMP-9 cell culture
system (Table I). Plasmin was added at 10 nM, the concentration determined to be generated from 25 nM
plasminogen in a uPA-containing culture, and pro-MMP-3 was added at
increasing concentrations (0-16 nM). Plasmin alone failed
to convert pro-MMP-9 (Fig. 5A, lane 1). The
conversion of pro-MMP-9 to the 82-kDa form of the enzyme was observed
with as little as 2 nM of pro-MMP-3 in the absence of TIMP
(Fig. 5A, lanes 2-5). However, the addition of
purified TIMP-1 at a concentration of 4 nM prevented the
conversion of pro-MMP-9 until 8 nM pro-MMP-3 was added and
then the 82-kDa form is observed (Fig. 5B). Almost complete
conversion of the enzyme occurred at a pro-MMP-3 concentration of 16 nM, which yields an effective concentration of 6-8
nM active MMP-3 (40-50% conversion). This activation
pattern of MMP-9 closely resembled the pattern observed in the crude
cell culture system (Fig. 4) and provided additional evidence that the
activation of MMP-9 was regulated by the levels of TIMP in the
system.

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Fig. 5.
Activation of pro-MMP-9 by plasmin-activated
MMP-3 in a cell-free system using purified components. A purified
preparation of pro-MMP-9 (8 nM) was incubated in serum-free
DMEM with increasing concentrations of purified pro-MMP-3 (0-16
nM) in the presence of purified human plasmin (1 µg/ml).
The incubations were carried out in the absence (A) or
presence (B) of purified TIMP-1 (4 nM). The
reaction mixtures were incubated at 37 °C for 24 h and then
analyzed by gelatin substrate zymography.
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The requirements of the proteolytic cascade for MMP-9 activation and
the contribution of the individual components was evaluated by the
addition of specific inhibitors to the supplemented MDA-MMP-9 cell
cultures. Pro-MMP-3 and plasminogen were employed at concentrations that would yield 50-75% conversion of pro-MMP-9, and samples of culture supernatants were analyzed by zymography for conversion of
pro-MMP-9 and solution phase gelatinase activity for appearance of
active enzyme (Fig. 6). Only background
levels of gelatinase activity were generated when either plasminogen or
pro-MMP-3 was added. Interestingly, some processing of pro-MMP-9 to an
intermediate 84-86-kDa form was present (zymograph, lanes 2 and 3), but this form was not enzymatically active in
solution (bar graph, lanes 2 and 3).
Substantial gelatinase activity was generated only in plasminogen-containing cultures with added pro-MMP-3 (bar graph, lane 4) and was accompanied by near full conversion of pro-MMP-9 to the 82-kDa form of the enzyme (zymograph, lane 4). The
generation of plasmin from plasminogen was essential for MMP-9
activation, because the addition of aprotinin resulted in background
levels of gelatinase activity and only partial conversion of pro-MMP-9 to the 86-kDa intermediate form (bar graph and zymograph,
lane 5). The activation of pro-MMP-9 was also blocked by the
addition of TIMP-1 at concentrations (20 nM) in excess of
MMP-3 and MMP-9 (lane 6). The most effective inhibitor of
pro-MMP-9 processing and activation was an anti-MMP-9 monoclonal
antibody 7-11C. The purified 7-11C IgG completely blocked the
plasmin-plus MMP-3-dependent activation of MMP-9
(lane 7), while control IgG allowed for full processing and
activation (lane 8). A partner monoclonal antibody, 6-6B,
isolated from the same fusion as 7-11C, had been shown previously to
block organo-mercurial mediated activation of pro-MMP-9 (46).

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Fig. 6.
Effect of specific inhibitors and a
neutralizing antibody on the plasmin/MMP-3-mediated activation of
pro-MMP-9. Cultures of MDA-MMP-9 (1 × 105
cells/cm2) were incubated for 48 h in serum-free DMEM
in the absence and presence of the indicated components. Aprotinin (40 µg/ml), TIMP-1 (40 nM), and anti-MMP-9 IgG (30 µg/ml)
were added as indicated. The conditioned medium was collected and
analyzed for pro-MMP-9 processing by gelatin substrate zymography
(inset) and pro-MMP-9 activation by gelatinase activity in
solution (bar graph).
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The presence in the cultures of both plasmin and pro-MMP-3 are required
for active MMP-9 to be generated, and neither one alone is sufficient
(Fig. 6). Plasmin efficiently activates pro-MMP-3 (Fig. 2), and
presumably the active MMP-3 directly converts 92-kDa pro-MMP-9 to
82-kDa active MMP-9. However, it was possible that plasmin, in addition
to activating pro-MMP-3, could proteolytically modify the pro-MMP-9,
thereby allowing it to be efficiently processed and activated by MMP-3.
Plasmin thus could have a dual requirement in the system. To address
this possibility, serum-free conditioned medium from MDA-MMP-9 cells
containing endogenous pro-MMP-9 (8 nM) was preincubated in
the absence or presence of plasminogen (2 µg/ml). After 16 h of
incubation, the plasminogen-containing sample had a measured
concentration of active plasmin of 0.9 µg/ml (10 nM), a
significant level of active enzyme. Aprotinin was added to the samples,
completely inhibiting any further action of plasmin, and then 5 nM active MMP-3 was added to both samples and incubation at
37 ° was continued. At various times, aliquots were withdrawn from
both samples and analyzed by gelatin zymography to monitor the
pro-MMP-9 conversion process and by radiolabeled gelatin degradation to
monitor the appearance of active MMP-9. Fig.
7 demonstrates that the processing of
pro-MMP-9 by MMP-3 is nearly identical for plasmin-pretreated or
-untreated samples (Fig. 7, A and B). Plasmin
pretreatment also did not enhance the rate of appearance of active
MMP-9 and in fact slightly retarded the appearance of gelatinase
activity (Fig. 7C). These results also illustrate that prior
to MMP-3 addition, plasmin alone at physiological concentrations does
not generate any detectable gelatinase activity or conversion to 82-kDa
forms (Fig. 7B, lane 1). Furthermore, the
requirement for plasmin in the activation process appears to be solely
to activate pro-MMP-3, since 5 nM active MMP-3 alone can
replace both plasminogen and the 16 nM pro-MMP-3 in
generating full conversion to the 82-kDa gelatinase (Fig.
7A, lane 6). Interestingly, 2.5 nM
MMP-3, in contrast to 5 nM MMP-3, was unable to generate
any active gelatinase in the treated or untreated conditioned media (data not shown), consistent with the need to overcome the 3-4 nM TIMP present in the conditioned medium.

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Fig. 7.
Conversion and activation of pro-MMP-9 by
active MMP-3 following pretreatment with plasmin. Serum-free
conditioned medium was collected after 24 h from 1.5 × 107 MDA-MMP-9 cells yielding a concentration of pro-MMP-9
of 8-10 nM. The cell-free conditioned medium (3 ml) was
incubated at 37 °C for 16 h in the absence (A) or
presence (B) of plasminogen (2 µg/ml). Plasminogen
conversion to plasmin was monitored, and at the end of the
preincubation, active plasmin was present at 0.9 µg/ml (10 nM). Aprotinin (40 µg/ml) was added to the conditioned
media to block any further activity of plasmin, and then 5 nM active MMP-3 was added along with radiolabeled gelatin
(20 µg/ml, 2000 cpm/µg), and incubation was continued at 37 °C.
At the indicated times, 1-µl aliquots were removed for gelatin
zymography and 100-µl aliquots removed for measuring
[3H]gelatin degradation (C).
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Activation of the Cell-associated MMP-9 Appears to Have Similar
Plasmin/MMP-3 Requirements--
It has been reported that MMP-2 and
MMP-9 are associated with the cell surface and can be activated
independently of other MMPs directly via a cell surface uPA/plasmin
cascade (52). The association of pro-MMP-9 with cells, and its
activation requirements were examined in the overexpressing MDA-MMP-9
cultures. The cells were incubated for 24 h in serum-free medium
in the absence or presence of either plasminogen, pro-MMP-3, or both
together. The conditioned medium (5 ml) was harvested from the four
cultures, and a total cell and membrane extract was prepared from the
same harvested cultures. The extract was passed over and eluted from gelatin-Sepharose in order to concentrate all the cell-associated MMP-9
free of any extraneous cellular proteins and inhibitors (0.15 ml).
Aliquots (1 µl) from each conditioned medium and extract were
examined by zymography, and aliquots (75 µl) from each were tested
for active gelatinase (Fig. 8). The total
gelatinase activity in each sample was calculated to provide a measure
of how much active MMP-9 was cell-associated compared with the amount
of secreted, soluble MMP-9. The zymographic analysis indicates that a
lower molecular mass form of MMP-9 (~80-85 kDa) exists in the cells from the untreated cultures (lane 5), but it would not appear to be the
82-kDa active gelatinase, since no enzymatic activity can be detected
assaying as much as 50% (75 µl) of the total cell extract. The
80-85-kDa band may be a variant glycosylated form of pro-MMP-9 found
associated only with cells and not secreted (53). Such a
cell-associated MMP-9 variant would exist as a zymogen and thus would
manifest little or no soluble enzymatic activity but yield a zone of
lysis in the zymograph. Alternatively, the 80-85-kDa species could be
an active form of MMP-9 that is tightly complexed with a
cell-associated inhibitor and thus in solution unable to express
gelatinase activity, but following dissociation in SDS-polyacrylamide
gel electrophoresis would yield a zone of lysis in the zymograph. This
band is present in all of the cell extracts; however an enhanced
zymographic signal at 80-85 kDa appears in the extracts prepared from
cells treated with pro-MMP-3 and plasminogen (Fig. 8, lane
8). This zymograph enhancement is accompanied by a small but
significant increase in the soluble gelatinase activity of the sample.
The cell-associated gelatinase activity (659 cpm) is much lower than
the activity (14,056 cpm) manifested by a similar 82-kDa band generated
in the corresponding conditioned medium (lane 4), possibly
indicating the presence of an inhibitor in the cell extracted sample.
When the gelatinase activity present in the total cell extract is
calculated, the amount of active enzyme present (0.30 unit) represents
only 0.15% of the total activity of the culture: 99.8% of the
activity (197.6 units) is present in the conditioned medium. Thus only a very small level of active MMP-9 is associated with the tumor cells,
possibly on the cell surface. However, even that small level of active
enzyme appears to require the combined action of plasmin and pro-MMP-3
for its generation. With no pro-MMP-3 present, the presence of plasmin
alone causes little or no increase in cell-associated active MMP-9
(Fig. 8, lane 7).

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Fig. 8.
The cell-associated MMP-9 appears to require
the same proteolytic cascade for generating a low level of activated
gelatinase. Cultures of MDA-MMP-9 (2 × 107
cells/plate) were incubated in serum-free DMEM in the absence or
presence of the indicated components. After 24 h, the conditioned
media (5 ml) were harvested, and cell extracts were prepared and
concentrated by gelatin-Sepharose chromatography (to 0.15 ml) as
described under "Materials and Methods." All samples were analyzed
for pro-MMP-9 processing by subjecting 1 µl to gelatin zymography.
Samples were analyzed for pro-MMP-9 activation by subjecting 75 µl to
a 44-h [3H]gelatin degradation assay. Total gelatinase
activity in enzyme units was calculated for each conditioned medium and
cell extract. Gelatinase units are defined as 100 cpm released per
hour.
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MMP-9 Activation via the Plasmin/MMP-3 Cascade Enhances ECM
Degradation--
The unique reactivity of the monoclonal antibody,
7-11C, to human MMP-9 (46) and that it could prevent the generation of active MMP-9 (Fig. 6) indicates that the antibody could be used to
assess the specific involvement of MMP-9 activation in human tumor cell
invasive behavior. To assess the functional activity of the MMP-9
generated in the MDA-MMP-9 tumor cell culture system, progressive
matrix degradation by these cells was analyzed in the absence and
presence of the monoclonal antibody. The ECM is coated with
glycoproteins that protect the fibrillar collagens from the catalytic
activity of MMPs; thus examination of the role of specific MMPs in ECM
degradation requires removal of this glycoprotein layer (54). To
evaluate the role of MMP-9 activity in ECM degradation, MDA-MMP-9 cells
were cultured on a smooth muscle cell matrix that was radiolabeled and
partially depleted of glycoproteins by trypsin treatment (Fig.
9). Incubation of the cells alone for 3 days resulted in a low level of ECM degradation (Fig. 9, condition
A), and addition of plasminogen to this system resulted in a
small but significant increase of released radiolabeled ECM fragments
over the three days (condition B). The addition of
plasminogen and pro-MMP-3, however, enhanced the ECM degradation 3-fold
over the culture of cells alone (C). This enhanced ECM
degradation was due to MMP-9 activation as shown by the resulting
inhibition using the specific anti-MMP-9 monoclonal antibody (condition
D) and TIMP-1 (condition E). The enhanced
solubilization of radiolabeled ECM that is sensitive to the anti-MMP-9
antibody is apparently the result of active MMP-9 degrading the
collagenous components of the ECM. Since the smooth muscle ECM does not
contain type IV or type V collagen, the known native substrates of
MMP-9, the observed degradation is likely the result of MMP-9 acting
catalytically on unfolded or partially denatured collagens I and III,
the major collagens found in this ECM.

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Fig. 9.
Degradation of ECM by MDA-MMP-9 cells is
enhanced upon induction of the protease cascade that activates
endogenous pro-MMP-9. MDA-MMP-9 cells were plated onto
radiolabeled, modified ECM (see "Materials and Methods") and
incubated in the absence or presence of plasminogen (plg.,
2.5 µg/ml), pro-MMP-3 (16 nM), anti-MMP-9 IgG (30 µg/ml), and TIMP-1 (40 nM) singly or in the indicated
combinations. Samples of conditioned medium (50 µl) were collected on
days 1, 2, and 3, and the soluble 3H-labeled peptides
released from the ECM were measured in a scintillation counter. The
bar graphs represent the mean counts/min ± S.D. released
from triplicate samples in a single experiment.
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MMP-9 Activation Results in Enhanced Tumor Cell Invasion of
Basement Membrane--
Transmigration of cells across a complex
basement membrane is often used as an indicator of the invasive
behavior of cells. The migratory and invasive ability of MDA-MMP-9
cells were examined in a series of Matrigel invasion assays to
determine the relative contribution of MMP-9 activation to this
translocation process (Fig. 10). MMP-9
conversion and the generation of MMP-9 gelatinase activity were
monitored using samples of culture medium that were removed directly
from the upper compartment of the invasion chamber during the assay
(Fig. 10, top). In the absence of plasminogen and pro-MMP-3,
2600 cells, approximately 5% of the inoculated MDA-MMP-9 cells,
transmigrated across the filter in 48 h (Fig. 10, column
1). In the presence of either plasminogen or pro-MMP-3, 7-8% of
the inoculated cells transmigrated across the filter (columns 2 and 3). These levels of cellular invasion were
accompanied by a small generation of gelatinase activity (418 and 595 cpm, respectively) above background levels (267 cpm). When plasminogen
and pro-MMP-3 were both added to the culture chamber, 6000 cells or
~12% of the inoculated cells transmigrated across the filter in
48 h (column 4). This enhanced transmigration was
accompanied by conversion of pro-MMP-9 to the 82-kDa form of the enzyme
(Fig. 10, zymograph) and a substantial increase in gelatinase activity
(3056 cpm). The cellular transmigration and gelatinolytic activity were
inhibited by the anti-MMP-9 monoclonal antibody (column 5)
and TIMP-1 (column 6), indicating a specific role for MMP-9
and its activation in the invasion process.

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Fig. 10.
Enhanced matrigel invasion is coincident
with activation of pro-MMP-9 via a plasmin/MMP-3 cascade.
MDA-MMP-9 cells (5 × 104) were added to the upper
compartments of Matrigel invasion chambers supplemented with the
indicated (+) components. After a 48-h incubation the total number of
cells that invaded and migrated to the underside of the filters was
counted (bar graph). After 24 h of invasion, an aliquot
of the conditioned medium from the upper chambers was removed and
analyzed by gelatin substrate zymography (zymograph inset).
The conditioned medium also was assayed for gelatinase activity by
monitoring the degradation of 3H-labeled gelatin (mean
counts/min released is indicated above each lane of the
zymograph).
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DISCUSSION |
In the present study, we have utilized a cell culture model in an
attempt to recapitulate some of the biochemical events that might take
place in an invasive tumor when active MMP-9 is generated. A critical
feature of the model is that the natural regulators of MMP function,
the TIMPs, are present endogenously in nanomolar amounts. A second
feature of the model is that it has the capacity to generate an
additional protease system, namely the uPA/plasmin cascade, that has
been closely linked to the migratory, invasive phenotype (7) and also
linked functionally to the activation of a number of MMPs including
MMP-9 (55). Our initial analysis of this culture system (Figs. 1 and 2)
illustrates the inherent stability of the pro-MMP-9 zymogen and its
apparent resistance to activation even when the levels of MMP-9 are
increased by cytokine treatment, when plasminogen is added to the
cultures and when active MMP-3 is generated. Since active plasmin was
demonstrated to be present in the cultures and at sufficient catalytic
levels to activate pro-MMP-3, plasmin did not appear to be an effective activator of pro-MMP-9. However, MMP-3 had been shown in
vitro to be an efficient pro-MMP-9 activator (32), and the
question arose as to why the newly generated MMP-3 did not activate the available pro-MMP-9. Determining the total TIMP (4 nM)
present in the cultures and comparing it with the amount of available pro-MMP-9 (0.25 nM) indicated that sufficient TIMP-1 was
present to complex all of the pro-MMP-9 and retard its activation.
Furthermore, excess TIMP-1 and TIMP-2 also would be present to inhibit
the nanomolar levels of the plasmin-activated MMP-3. Even if the newly generated MMP-3 exceeded the TIMP levels, it was possible that pro-MMP-9 at subnanomolar levels tightly complexed with TIMP-1 and in
equilibrium with other TIMPs in the cellular milieu was unable to
interact efficiently with the excess MMP-3 to progress through
activation. Thus the dominating molar excess of the TIMPs was one
compelling reason why MMP-9 activation could not progress even in the
presence of a potential activator.
In order to overcome the suppressive effect of the TIMPs, transfection
of MDA-MB-231 cells was employed to up-regulate the MMP-9 levels. The
transfected cells were unchanged in terms of expression of all the
proteases and protease inhibitors tested (Fig. 3) with the exception of
MMP-9, which was increased 200-fold over the parental cultures and
30-fold over that of the PMA-treated cultures. However, overriding the
TIMP levels with excess MMP-9 expression was not sufficient by itself
to initiate activation of pro-MMP-9. The MMP-9-overexpressing cultures
needed to be supplemented with both plasminogen and pro-MMP-3 (Figs. 4
and 6). Supplementation with plasminogen alone did not bring about
activation, even though active plasmin was generated via the endogenous
tumor cell uPA. This result documents the inherent stability of the
pro-MMP-9 zymogen in a cellular setting and further indicates that
plasmin is not a direct activator of pro-MMP-9 under the cellular
conditions employed herein. Studies from other laboratories also had
concluded that plasmin was not an efficient activator of pro-MMP-9 (31, 37), although a number of reports had implicated plasmin as a direct
activator of pro-MMP-9 (38, 57, 58, 62). Supplementation of the
transfected cultures with pro-MMP-3 alone also did not yield activated
MMP-9, as the pro-MMP-3 in the absence of plasminogen remained a 55-kDa
zymogen, further demonstrating that plasmin was responsible for the
conversion of 55-kDa pro-MMP-3 to the 45-kDa active MMP-3. When both
plasminogen and pro-MMP-3 were added to the cultures, the tumor cell
uPA activated the plasminogen, the generated plasmin activated the
pro-MMP-3, and then active MMP-3, when it exceeded the concentration of
TIMP, converted 92-kDa pro-MMP-9 to 82-kDa active MMP-9.
In studies of zymogen activation by proteases, it would seem important
to distinguish true enzyme activation from proteolytic conversion of
the proenzyme to smaller but catalytically inactive forms. Enzyme
activity measurements in solution were critical in the present study,
since distinct lower molecular mass forms of MMP-9 were generated upon
addition of plasminogen alone or pro-MMP-3 alone (Fig. 6). These
processed forms were active in gelatin substrate gels (zymographs) but
were not active in solution, and thus true activation of MMP-9 did not
occur under these conditions. In addition, a lower molecular mass form
of MMP-9 was observed in tumor cell extracts (Fig. 8), and its similar
zymographic position to the 82-kDa active MMP-9 suggested that it
represented a cell-associated form that had been activated. However,
enzyme activity measurements on the isolated cellular MMP-9 indicated
that this lower molecular mass form was not an active enzyme but
behaved like a zymogen. Other laboratories also have shown that certain
lower molecular weight forms of MMP-9 are not enzymatically active (41,
42, 53). Toth et al. (53) demonstrated that a
cell-associated 85-kDa species of MMP-9 was active in zymographs but
enzymatically inactive in solution. The 85-kDa form represented a
different glycosylated variant of pro-MMP-9. Ogata et al.
(41) showed that an 86-kDa processed form of MMP-9 was zymographically
active but not enzymatically active in solution. This 86-kDa form
represented a species that had been proteolytically processed at the
Glu41-Met42 site in pro-MMP-9, 47 residues
upstream of the actual activation site. A number of studies (38,
56-58), however, have relied mainly on zymographic or immunoblot
detection methods and implied that the appearance of distinct lower
molecular mass forms of MMP-9 represented activation of the zymogen. In
some of these studies plasmin was implicated as a direct activator of
pro-MMP-9 (57, 58). In the culture system described herein, plasmin
indeed can generate lower molecular mass forms of MMP-9 but does not yield activated enzyme. The plasmin involvement in MMP-9 activation in
this system is only indirect; it functions within an interacting cascade to generate active MMP-3. This indirect role of plasmin and the
apparent requirement for plasminogen in the cascade is more clearly
illustrated when already activated MMP-3 is added directly to the tumor
cell culture medium (Fig. 7). The requirement for plasminogen and
pro-MMP-3 in the cascade is circumvented as active MMP-3 directly
generates the 82-kDa MMP-9 and in turn generates significant gelatinase activity.
In contrast to the present studies, Mazzieri et al. (52),
using HT1080 cultures, concluded that plasmin could process and activate pro-MMP-9 (and also pro-MMP-2) in the absence of any detectable MMP-3. The plasmin that appeared to be responsible for this
MMP activation was cell surface-activated plasmin, and the gelatinases
that were activated were apparently associated with the cell surface.
Since most of our studies involve the secreted, soluble pro-MMP-9,
which could be readily activated by MMP-3 and could not be activated at
all by plasmin, the apparent contrasting results may reflect
cell-associated activation versus solution phase activation.
However, when the tumor cell-associated MMP-9 was examined (Fig. 8), it
was found that little if any active MMP-9 was associated with the
cells, that plasmin did not enhance the level of active MMP-9 that
became cell-associated, and that when a detectable level of active
MMP-9 was found associated with the cells (<1%), the specific
requirements for its activation appeared to be the same as that for
activation of the secreted, soluble MMP-9. Thus it is not yet resolved
whether significant levels of pro-MMP-9 become bound to the cell
surface and whether such cell-associated zymogen has a distinct mode of activation.
A possible limitation of this cell culture model for MMP-9 activation
is that the source of active MMP-3 is via exogenous addition of
purified pro-MMP-3. The cultured breast carcinoma cells do not produce
MMP-3 (Fig. 3), and indeed a majority of human tumor cells may not
manifest elevated expression of MMP-3 (59). The major source of MMP-3
in tumor tissue appears to be not from the tumor cells but from stromal
cells, namely fibroblasts and macrophages (60, 61). In a study of
breast tumor tissue, Heppner et al. (19) indicated that the
enhanced production of a number of MMPs, including MMP-3, was not from
the tumor cells but from adjacent inflammatory and stromal cells
responding to the presence of tumor. Therefore we envision the
exogenous addition of pro-MMP-3 as representing a stromal source of
this zymogen. Indeed the concentration of pro-MMP-3 (2-16
nM) employed in the model system was higher than that which
might be found in normal stromal tissue or in standard cultures of
fibroblasts and macrophages. However, the elevated levels of MMP-3
often found with inflammation, wound repair, and tumor infiltration
(1-3) might approach nanomolar levels. Thus the concentration of MMP-3
employed in the present study may not be physiologic but rather pathologic.
It should be emphasized that MMP-3 may not be the sole natural
activator of pro-MMP-9. In the injured arteries of homozygous MMP-3-deficient mice, some MMP-9 activation occurs approximately equal
to the level observed in the injured arteries of wild type mice, and it
was concluded that MMP-9 activation does not depend solely on MMP-3
(62). It should be noted, however, that MMP-9 activation in (62) was
monitored only by the appearance of lower molecular mass forms of
MMP-9. Nevertheless, MMP-3 probably will not be found in all the
tissues where MMP-9 activation occurs. However, since very favorable
catalytic parameters describe MMP-3's efficient MMP-9 activating
ability in vitro (32), and since active MMP-3 can be
generated by a widely distributed serine protease cascade (Fig. 6), and
since MMP-3 can effectively generate a processed form of MMP-9 that
functions catalytically in tissue invasion processes (Figs. 9 and 10),
MMP-3 would appear to be a prime candidate as a natural activator of
pro-MMP-9 in vivo. Other pro-MMP-9 activators also may
function in vivo (and in MMP-3-deficient mice).
Procollagenase-3 (MMP-13), like MMP-3, exhibits favorable kinetics of
pro-MMP-9 activation, can itself be activated by a widely distributed
protease system and is co-expressed with MMP-9 in a number of
situations that involve tissue and skeletal remodeling (40, 63).
The TIMPs are clearly an important controlling element in any
MMP-mediated function. Their regulatory capacity in the MMP-9 activation model described in this study is multifold. They can inhibit
MMP-3, the activator of the process; one of them, TIMP-1, can bind
specifically to the MMP-9 zymogen and dampen its activation; and
finally the TIMPs can inhibit the activated MMP-9 from functioning catalytically. The regulatory ability of TIMPs appears to be based mainly on stoichiometry, and thus a mechanism for circumventing TIMP
control of cellular MMP-9 activation would be to titer out the
endogenous TIMP. In our model system this was accomplished by inducing
overexpression of pro-MMP-9 in the tumor cells and by providing
nanomolar levels of pro-MMP-3. In vivo such titering or
saturation of TIMPs may occur when MMP-9, MMP-3, or other MMP expression in tissues is induced by endogenous cytokines, growth factors, or oncoproteins. The titering of TIMPs by secreted MMPs may
occur only in the tissues in very focalized areas such as in regions of
active wound repair, or at sites of inflammatory infiltrates, or at the
edge of the tumor/stromal interface where locally a high concentration
of secreted enzymes can develop. However, such upward shifts in the
overall MMP/TIMP balance in the case of MMP-9 may not be sufficient to
initiate zymogen activation. MMP-9, being a relatively stable zymogen,
appears to require exogenous catalytic intervention. Based on the model
system we have described, that exogenous intervention involves the
convergence of a highly regulated serine protease cascade with that of
a distinctly different but also highly regulated metalloproteinase cascade.