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INTRODUCTION |
The MMP family is composed of at least 25 zinc-dependent extracellular endopeptidases whose
activities are regulated predominantly by expression as inactive
precursors, or zymogens (1-3). Although precise physiological
activators of MMPs1 are
unknown, a variety of serine proteinases and other MMPs in the
extracellular milieu execute the initial propeptide cleavage events
in vitro (2). An exception to serine protease activation is
pro-MMP-2 (72-kDa gelatinase A), which lacks the necessary basic amino
acid cleavage sites in its pro-domain (4). A primary mechanism of
pro-MMP-2 activation involves zymogen association with the cell surface
via formation of a ternary complex containing tissue inhibitor of
metalloproteinase (TIMP)-2 and membrane type 1-MMP (MT1-MMP, MMP-14)
(3-6). Following trimeric complex formation, it is hypothesized that a
neighboring MT1-MMP molecule that is not associated with TIMP-2 cleaves
pro-MMP-2 at the Asn37-Leu38 peptide bond
within the pro-domain (7). Intermediately processed MMP-2
(Leu38-MMP-2) undergoes further
concentration-dependent autolytic cleavage(s) to generate
mature enzymes that can be released into the soluble phase or remain
surface-associated (8). Although biological mechanisms of active MMP-2
release from the cell surface are not well characterized and
dissociation kinetics provide little insight, cellular binding
affinities may shift following pro-MMP-2 cleavage.
Culturing a variety of cell types within a three-dimensional gel of
type I collagen stimulates cellular activation of pro-MMP-2 (9-12).
Although MT1-MMP is implicated in MMP-2 processing, regulation of
cellular events that promote MMP processing are poorly understood. As
cellular interaction with type I collagen is mediated largely through
integrin receptors, it has been postulated that collagen stimulation
occurs either directly or indirectly through integrin signaling
(12-16). In support, we have previously demonstrated that culturing
DOV13 ovarian cancer cells in a three-dimensional collagen gel elicits
a strong pro-MMP-2 activation response that can be mimicked by
clustering of
1 integrin receptors (12). Furthermore,
pro-MMP-2 activation coincides with the processing of MT1-MMP into
truncated 55- and 43-kDa forms on the cell surface. In this study, we
utilize a variety of approaches to elucidate the biochemical
requirements of type I collagen stimulation of MMP zymogen activation,
characterize processed forms of MT1-MMP that are generated in this
response, and examine the proteinase requirements for cellular invasion
of type I collagen gels. Our findings illustrate a general mechanism by
which cells may regulate cell surface-associated MMP activity via
interactions with pericellular collagen matrix.
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EXPERIMENTAL PROCEDURES |
Materials--
Bovine serum albumin, gelatin, cell culture
reagents, human placental type I collagen, aminophenylmercuric acetate,
anti-(rabbit IgG)-peroxidase conjugates, purified mouse
immunoglobulins, 2.97-µm diameter latex beads, concanavalin A (ConA),
and ortho-phenanthroline were all purchased from Sigma.
Anti-human
1 integrin mAb clones 21C8 and P5D2,
anti-human
2 mAb clones P1E6 and AK7, anti-human
3 integrin mAb clones P1B5 and ASC-6, anti-human
3
1 heterodimer integrin mAb clone
M-KD102, anti-human (carboxyl domain) TIMP-2 mAb clone 67-4H11, and
anti-human MT1-MMP polyclonal antibody (AB815, hinge domain) were
all obtained from Chemicon (Temecula, CA). Hydrobond-P:polyvinylidene
difluoride membrane was obtained from Amersham Pharmacia Biotech.
SuperSignal-enhanced chemiluminescence reagents were purchased from
Pierce. The general hydroxamic acid MMP inhibitor INH-3850-PI (MMPI)
was purchased from Peptides International (Louisville, KY). Purified
TIMP-2 and TIMP-1 and anti-MT1-MMP (raised against amino acids
160-173/catalytic domain) polyclonal antibody MTK3 were generous gifts
of Dr. Hideaki Nagase (Kennedy Institute of Rheumatology, Imperial
College School of Medicine, UK). Recombinant MMP-2 type II fibronectin
domain repeats (rCBD123) and hemopexin carboxyl domain
(Gly417-Cys631) were generated as described
previously (17, 18). Collagenase-resistant (CR) murine type I collagen
(
1(I) chain mutations, Gln774
Pro774, Ala777
Pro777, and
Ile776
Met776) and wild type murine type I
collagen were the generous gifts of Dr. Stephen Krane (Harvard
University) (19).
Cell Culture--
The ovarian carcinoma cell line DOV13 was
provided by Dr. Robert Bast, Jr. (M.D. Anderson Cancer Center, Houston,
TX). Cell culture was maintained under standard conditions in
75-cm2 cell culture flasks (20).
Quantification of Cell Adhesion--
96-well cluster plate
chambers were coated with 50 µl of 10 µg/ml type I collagen,
3/4,1/4 collagen fragments (prepared as described below),
or type I gelatin (1.6 µg/cm2) in sterile
phosphate-buffered saline (PBS) for 4 h at 25 or 37 °C, blocked
with 3% BSA in minimal essential medium for 1 h at 37 °C,
washed with PBS, and air-dried. Cells (1 × 105
cells/ml in serum-free medium) were incubated for 20 min at 37 °C
with specific integrin function-blocking antibodies or nonspecific control antibodies, plated at a density of 2 × 104
cells/well, and allowed to adhere for 75 min. Unbound cells were removed by washing with PBS, and adherent cells were fixed in ethanol
(10 min), stained with 0.5% crystal violet (20 min), washed extensively with water, and solubilized with 100 µl 1% SDS. Relative adhesion was quantified by monitoring the absorbance of released dye at
540 nm (n = 5).
Isolation of Collagen Fragments and Preparation of
Collagen-containing Surfaces--
Human placental collagen (7 mg) was
cleaved into 3/4 and 1/4 fragments by incubating with 2 µg of MMP-1 for 16 h at 25 °C in Tris-buffered saline (TBS)
(pH 7.4) containing 60 nM CaCl2. Cleaved
collagen was isolated using a modified ammonium sulfate precipitation
protocol (21). Ammonium sulfate (12%) was added to collagen suspension on ice at 4 °C under constant stirring for 1 h and then
centrifuged (15,000 × g, 1 h, 4 °C). The 12%
pellet containing intact collagen was washed with ice-cold TBS
containing 12% ammonium sulfate, re-centrifuged (15,000 × g, 1 h, 4 °C), solubilized with 0.2 M acetic acid, and dialyzed against PBS. To isolate the 3/4 and
1/4 fragments, ammonium sulfate was added to the 12%
supernatant to a final concentration of 25%, incubated for 1 h,
and centrifuged (15,000 × g, 1 h, 4 °C). The
25% pellet containing 3/4 and 1/4 collagen fragments was
washed with ice-cold TBS containing 25% ammonium sulfate,
re-centrifuged (15,000 × g, 1 h, 4 °C),
solubilized, and dialyzed as described above. Type I gelatin was
produced by thermal denaturation of type I collagen for 20 min at
60 °C. Protein concentrations were determined using the Bio-Rad
DC kit and bovine albumin as a standard.
Assays using thin deposits of type I collagen were performed by
dialyzing acid-solubilized collagen against PBS, diluting to 10 µg/ml
in 100 mM sodium carbonate (pH 9.6), and coating 24-well cluster plate chambers with 200 µl (10 µg/ml, 1.1 µg/cm2) of collagen. Chambers were incubated for 1 h
at 37 °C, washed with sterile PBS, and air-dried. For
three-dimensional collagen gel experiments, dialyzed type I collagen
was diluted to 1.5 mg/ml with cold minimum essential medium containing
20 mM Hepes (pH 7.4). Diluted collagen (200 µl, 158 µg/cm2) was added to 24-well plates and allowed to gel at
37 °C for 30 min before the addition of cells (2.5 × 105) to wells. Cells were incubated for 48-72 h in
serum-free medium at 37 °C before collection of conditioned media.
Integrin Clustering--
Anti-integrin subunit-specific
antibodies or control IgG were passively adsorbed onto 2.97-µm
diameter latex beads as described previously using the following
modifications (12, 24). Latex beads were incubated at a final
suspension of 0.1% in 100 mM MES buffer pH 6.1), with 25 µg/ml appropriate antibody in 1-ml volumes overnight at 4 °C under
agitation, and then blocked with 10 mg/ml BSA for 2 h at room
temperature. Blocked beads were pelleted (3,000 × g, 3 min, 25 °C), washed twice with 1 ml of serum-free media, and
resuspended to 1% by volume. Protein concentration assays using a BCA
detection kit (Sigma) indicated 60-70% adsorption of immunoglobulins.
Cells were plated at a density of 2.5 × 105
cells/well in 24-well cluster plates (Becton Dickinson) overnight in
serum-containing medium, incubated for 2 h in serum-free medium prior to the addition of fresh medium containing soluble antibodies (10 µg/ml), concanavalin A (20 µg/ml), (25) or antibody-adsorbed latex
beads (3-4 µg/ml; 0.02% beads by final volume) for 18-20 h. All
final volumes were 500 µl/well.
Gelatin Zymography--
Gelatinase activities in conditioned
media were determined using SDS-polyacrylamide gel electrophoresis
zymography. Conditioned media (20 µl) from an equivalent number of
cells were electrophoresed without reduction on SDS-polyacrylamide gel
electrophoresis gels prepared with 9% acrylamide containing 0.1%
gelatin (23). SDS was removed through a 1-h incubation in 2.5% Triton
X-100, and gels were incubated in 20 mM glycine, 10 mM CaCl2, 1 µM ZnCl2 (pH 8.3), at 37 °C for 24 h prior to staining for gelatin with Coomassie Blue. Enzyme activity was visualized as zones of gelatin clearance within the gels.
MMP-2 Competition Experiments--
Cells were grown to
confluency in 24-well chamber plates and incubated for 2 h in
serum-free medium. Fresh medium containing various concentrations of
rCBD123 (17) or recombinant hemopexin carboxyl domain (18) were added
to cells in a 500-µl volume. After 2 h, ConA was added to a
final concentration of 20 µg/ml, and cells were incubated for an
additional 18 h. Conditioned media and cell lysates were collected
and processed as described above.
MT1-MMP Immunoblots--
Cells were incubated under various
conditions, collected with lysis buffer, and protein concentration of
lysates was analyzed using the Bio-Rad DC detection kit and
bovine albumin standards. Cell lysates (5-15 µg) were
electrophoresed on 9% SDS-polyacrylamide gels, transferred to
polyvinylidene difluoride membrane, and blocked with 3% BSA in 50 mM Trizma (Tris base) (pH 7.5), 300 mM NaCl, 0.2% Tween 20 (TBST). Membranes were incubated for 1 h at room temperature with a 1:4000 dilution of anti-human MT1-MMP polyclonal antibody in 3% BSA/TBST. Immunoreactive bands were visualized with a
peroxidase-conjugated anti-(rabbit-IgG) (1:5000 in 3% BSA/TBST) and
enhanced chemiluminescence.
Isolation of Biotin-labeled Cell Surface Proteins--
Cells
were grown to confluency in 6-well cluster dishes, washed with PBS, and
incubated for 2 h in serum-free medium. Fresh serum-free medium (1 ml) containing a 0.06% antibody-coated bead suspension or 20 µg/ml
concanavalin A was added, and cells were incubated for 20 h.
Conditioned medium was removed; cells were washed with 2 × 2 ml
of PBS, and surface proteins were labeled with a non-cell-permeable
sulfo-NHS biotin analog (500 µl at 500 µg/ml PBS, Pierce) under
gentle shaking at 4 °C for 30 min. After washing, cells were
incubated with 1 ml of 100 mM glycine/PBS for an additional
20 min under gentle shaking at 4°C. Washed cells were lysed with 500 µl of lysis buffer (50 mM sodium phosphate buffer (pH
8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% Triton X-100, 1 µg/ml aprotinin, 1 µM pepstatin, 10 µM
leupeptin, and 10 µM E64), collected with a cell scraper,
and clarified by centrifugation (10,000 × g, 10 min,
4 °C). Protein concentrations were calculated as described. To
precipitate biotin-labeled cell surface proteins, lysate (1 ml, 750 µg/ml) was added to either ImmunoPure immobilized monomeric avidin or
multimeric streptavidin gel (40 µl) (Pierce) and incubated overnight
at 4 °C on a rotator. Gels were washed 5× with lysis buffer. In
some experiments, 10 mM free D-Biotin/PBS (Pierce) was added to aliquots of monomeric avidin gel-immobilized protein to compete off bound protein at 4 °C overnight. Eluates (30 µml) were analyzed by gelatin substrate zymography or immunoblotting. Control conditions utilized non-biotinylated cell lysates.
Isolation of Plasma Membranes--
DOV13 cells were grown to
confluency in 15-cm culture dishes, washed with PBS, switched to
serum-free medium for 2 h, and incubated in 5 ml of fresh
serum-free medium containing 20 µg/ml ConA for 20 h. Cells were
washed twice with 5 ml of PBS, collected with a cell scraper, and
pelleted by centrifugation (1500 × g, 10 min,
4 °C). Cells were resuspended in ice-cold 5 mM Tris/HCl (pH 7.8) and incubated for 10 min prior to homogenization by 30 passages through a 261/4-gauge needle (8). Crude membrane
preparations were isolated by centrifuging whole cell lysates
(10,000 × g, 10 min, 4 °C), retaining the
supernatant, and centrifuging the supernatant (100,000 × g, 1 h, 4 °C). The recovered pellet was washed in 20 mM Tris/HCl (pH 7.8), 10 mM CaCl2,
0.05% Brij 35 and recentrifuged (100,000 × g, 45 min,
4 °C). The plasma membrane-containing pellet was resuspended in
washing buffer, and protein concentration was assessed as described above.
Cross-linking and Immunoprecipitation--
To isolate
TIMP-2-binding MT1-MMP species, cell surface proteins were
cross-linked, and TIMP-2 complexes were isolated through immunoprecipitation. Confluent cells were incubated for 16 h in 5 ml of serum-free medium containing 20 µg/ml ConA and 125 ng/ml TIMP-2. After washing (2 × 5 ml of PBS), cell surface proteins were cross-linked with 2 mM
3,3'-dithiobis(sulfosuccinimidylpropionate) (Pierce) in PBS at 4 °C
for 25 min under gentle shaking. Cells were washed (2 × 10 ml of
PBS), and lysates were collected in cold lysis buffer. Clarified cell
lysates (700 µg/ml) were incubated with 5 µg of anti-TIMP-2
(carboxyl-terminal) mAb clone 67-4H11 or murine IgG (
) for 2.5 h at 4 °C on a rotator prior to the addition of a 50% slurry of
protein A-agarose (20 µl) for 90 min. Immunoprecipitates were
centrifuged (10,000 × g, 3 min, 4 °C), washed with
lysis buffer (5 × 1 ml), and protein solubilized with 50 µl of
5× Laemmli buffer and processed for MT1-MMP immunoblotting as
described above. To evaluate MT1-MMP activity, immunoprecipitations using anti-MT1-MMP hinge antibodies were performed using lysates from
cells that were not exposed to cross-linking agent. Lysates were
evaluated by gelatin zymography as described above.
Stable Transfection of MT1-MMP--
The eukaryotic plasmid
vector pCR3.1-Uni (Invitrogen, Carlsbad, CA) containing the MT1-MMP
gene under a cytomegalovirus promoter was the generous gift of Dr.
Duaniqng Pei (University of Minnesota). A 6 histidine repeat was
incorporated 3' of the carboxyl terminus using a polymerase chain
reaction based approach with a 5' primer of ATgggCAgCgATgAAgTC
and 3' primer of CgTCTAgATCAgTgATgATggTggTgATggACCTTgTCCAgCAgggA. The
amplified polymerase chain reaction product was cloned back into the
vector using a unique Fse1 restriction site and the
XbaI restriction site in the 3' polylinker region of the
vector. Plasmid DNA was sequenced and then isolated for transfection
experiments using a Qiafilter maxi-kit (Qiagen, Valencia, CA). The
vector control used in experiments is the pCR3.1-Uni vector driving the chloramphenicol transferase gene (pCR3.1/CAT, Invitrogen).
pCR3.1/MT1-His6 and control pCR3.1/CAT were transfected
with LipofectAMINE (Pierce) as a delivery vehicle and isolated
transformants screened for neomycin resistance selection (450 µg/ml
G418). Soluble recombinant MT1-MMP lacking the linker from the
carboxyl-terminal hemopexin domain through the cytoplasmic tail was
expressed in CHO cells using the pW1HG vector. Soluble MT1-MMP purified
from CHO serum-free conditioned medium was examined by electrophoresis
on SDS-polyacrylamide gels and silver staining to visualize proteins.
Immunocytochemistry--
Cells were cultured on glass coverslips
overnight prior to addition of a 1-ml volume of serum-free medium
containing 0.005%
1 integrin mAb-coated beads. Cells
were fixed with 3.7% formaldehyde without permeabilization and
incubated with anti-MT1-MMP polyclonal antibody (MTK3) or normal rabbit
serum in PBS. Immunoreactivity was visualized with a
fluorescein-conjugated anti-rabbit secondary antibody previously
purified against cross-reactivity to mouse immunoglobulins (Chemicon).
Phase contrast and indirect fluorescent images were collected using a
Zeiss fluorescence microscope (model II) and Adobe PhotoShop Software
(version 4.0).
Cellular Migration and Invasion--
Type I collagen was
dissolved in 0.5 M acetic acid at a concentration of 2 mg/ml. For invasion experiments, the collagen stock was neutralized
with 100 mM Na2CO3 (pH 9.6) to a
final concentration of 0.4 mg/ml. Transwell inserts (0.8 µm, Becton
Dickinson, Bedford, MA) were coated on the underside with 500 µl of
collagen diluted to a concentration of 100 µg/ml at 37 °C for
1 h. Collagen gels were prepared in the inner well by adding 50 µl of collagen (20 µg) at room temperature and allowing gels to
air-dry overnight. Collagen-coated inserts were then washed with
minimum essential medium three times to remove salts and used
immediately. In some experiments, transwell inserts were coated with
collagenase-resistant (19), rather than wild type, collagen. Cells were
trypsinized, washed with serum-free medium, and 1 × 105 cells were added to the inner invasion chamber in a
volume of 200 µl. The outer wells contained 400 µl of culture
medium (serum-free, except in experiments using murine collagen). To
evaluate the MMP dependence of invasion, after a 2-h incubation, the
MMP inhibitors TIMP-1 (10 nM), TIMP-2 (10 nM),
or MMPI (10 µM) were added to the inner and outer
chambers as indicated. Control wells for MMPI contained the solvent
Me2SO. Cells were allowed to invade for 24-48 h as
indicated; non-invading cells were removed from inner wells using a
cotton swab, and invading cells adherent to the bottom of membrane were
fixed and stained using a Diff-Quick staining kit (DADE AG, Miami, FL).
Invading cells were enumerated by dividing membranes into 4 quadrants
and counting the number of cells in 3 distinct areas for each quadrant
under a 10× objective using an ocular micrometer. Assays were
performed in triplicate. To evaluate the integrin dependence of
invasion, after a 2-h incubation anti-integrin antibodies (15 µg/ml)
or control IgG (15 µg/ml) were added to the inner and outer chambers,
as indicated. To evaluate the collagen structural requirements of
invasion, wells were coated with thermally denatured collagen (gelatin,
50 µg/well in a 50-µl volume) and allowed to invade for 24 h
in the presence of TIMP-1 (10 nM), TIMP-2 (10 nM), or aprotinin (20 µg/ml), as indicated. Invasion of
native and denatured collagen was also evaluated in the presence of the
MMP-2 carboxyl hemopexin-like domain (rCD, 100 nM) (18) or
the fibronectin type II-like domain (rCBD, 100 nM) (17)
under the conditions described above.
To assess cell motility, migration through transwell membranes coated
with a thin layer of collagen (100 µg/ml, 37 °C, 1 h) on both
the upper and lower surfaces was evaluated as described above using an
incubation time of 5.0 h. Haptotactic motility was assessed as
described previously by plating cells on coverslips coated with
colloidal gold overlaid with type I collagen (100 µg/ml) (22). Cells
were allowed to migrate for 18 h, and phagokinetic tracks were
monitored by visual examination using a Zeiss microscope with
dark-field illumination. Semi-quantitative analysis of phagokinetic tracks was performed by measuring track area using computer-assisted image analysis and NIH Image.
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RESULTS |
Collagen Structure Regulates Cell Adhesion--
To evaluate the
relative contribution of
2
1 and
3
1 integrins to collagen binding,
attachment of DOV13 cells to type I collagen in the presence of
integrin function-blocking antibodies was evaluated. DOV13 cells adhere
rapidly to type I collagen-coated microtiter wells in an
integrin-dependent fashion (Table
I). Addition of anti-
1
integrin function-blocking monoclonal antibodies (15 µg/ml, clone
P5D2) significantly reduced binding to collagen relative to a
nonspecific IgG control (75%), whereas equivalent amounts of either
2 (P1E6) or
3 (P1B5) integrin-blocking
antibodies inhibited adhesion by 35-40% (Table I). Combination of
integrin blocking antibodies (7.5 µg/ml each) together reduced
adhesion nearly 60%, supporting the conclusion that both
2
1 and
3
1 integrin heterodimers contribute to DOV13 cell attachment to type I
collagen.
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Table I
Rapid attachment of DOV13 cells requires 2 1 and
3 1 integrins
DOV13 cells (2 × 104/well) were preincubated with the
indicated concentration of antibodies for 20 min under standard cell
culture conditions prior to addition to collagen-coated wells. Cell
attachment was measured as described under "Experimental
Procedures." DOV13 cells utilize both 2 1 and
3 1 integrins to bind native type I collagen.
Values were corrected for control binding to BSA-coated wells.
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To assess the collagen structural requirements for integrin-mediated
binding, adhesion to both collagenase-cleaved and thermally denatured
collagen was analyzed. Native type-I collagen was incubated with
collagenase-1 (MMP-1) and the 3/4 and 1/4 digestion
products isolated through ammonium sulfate precipitation (21). Intact collagen heterofibrils, also isolated through this approach, were gelatinized by thermal denaturation. Intact collagen, 3/4 and
1/4 collagen fragments, or gelatin were coated onto microtiter
wells at either 25 or 37 °C, and adhesion was evaluated as described above. Both intact collagen and 3/4 and 1/4 fragments
support adhesion of DOV13 cells when matrices are coated at 25 °C;
however, adhesion to 3/4 and 1/4 fragments was
significantly reduced when matrices were coated at physiologic
temperature (Table II). Although cells
will slowly adhere to thermally denatured collagen (3-4 h), little adhesion to this matrix was observed under any coating condition over
the course of the assay (75 min) (Table II). These results indicate
that 3/4 and 1/4 collagen fragments coated at 25 °C
retain a triple helical conformation that is required for recognition by
2
1 and
3
1 integrins, whereas coating at 37 °C
results in destabilization of fragment helices (21). Together, these
data suggest that collagenase activity at the cell/matrix interface can
reduce the efficiency of integrin-mediated adhesion on the surface of
DOV13 cells, thereby potentially affecting matrix-induced signaling
events that are involved in cellular invasion.
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Table II
Intact collagen is required for DOV13 attachment
Type I collagen, 3/4 and 1/4 collagen fragments, and type
I gelatin were coated at the indicated temperature (1.6 µg/cm2), blocked with BSA, and cell attachment (2 × 104/well) measured as described under "Experimental
Procedures." Values are reported as the percent absorbance at 540 nm
in comparison to intact type I collagen, coated at the specified
temperature.
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Integrin Clustering Promotes Cell Surface MMP-2 Processing--
To
evaluate the matrix structural requirements for collagen induction of
pro-MMP-2 activation, cells were cultured in the presence of native or
thermally denatured collagen. Processing of pro-MMP-2 was not observed
by cells cultured on type I gelatin (Fig.
1, lanes 4 and 5).
However, culturing cells with collagenase-resistant collagen (19)
stimulated pro-MMP-2 processing as efficiently as wild type collagen
(Fig. 1, lanes 6 and 7), indicating that collagen
processing is not necessary to promote a cellular gelatinase activation
response. Similar to the wild type protein, gelation of
collagenase-resistant collagen abrogated the ability to elicit pro-MMP-2 activation (Fig. 1, lane 5). In conjunction with
cell adhesion studies, these data indicate that integrin interaction with intact triple-helical collagen is necessary to stimulate pro-MMP-2
activation and that destabilization of the collagen matrix following
collagenase activity will reduce the ability of pericellular collagen
to elicit an MMP response.

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Fig. 1.
Collagen stimulation of pro-MMP-2
activation. DOV13 cells (2.5 × 105 cells/well)
were incubated on plastic or a thin deposit of type I collagen (1.1 µg/cm2), in the presence of thermally denatured wild type
or collagenase-resistant (CR) collagen (Gelatin or CR
Gelatin, respectively, 158 µg/cm2), or within wild type
or CR type I collagen gels (Collagen or CR Collagen, respectively, 158 µg/cm2), as indicated, in serum-free medium for 48 h. Conditioned media were analyzed by gelatin zymography. The relative
migration positions of pro- and active MMP-2 are indicated.
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We have demonstrated previously that
1 integrin
clustering stimulates a pro-MMP-2 processing event that correlates with
enhanced gelatinolytic activity in conditioned media, indicating that
integrin signaling is sufficient to elicit pro-MMP-2 activation (12). As adhesion of DOV13 cells to type I collagen is supported by both
2
1 and
3
1
integrins (Table I), integrin clustering was induced through the
-subunit to evaluate the effects of clustering each specific
integrin heterodimer on pro-MMP-2 activation. Consistent with adhesion
blocking assays,
2,
3, and
1 integrin antibody-coated beads attached with similar
efficacy (data not shown); however, analysis of conditioned media by
zymography demonstrates that clustering
3 or
1 integrins elicits a stronger pro-MMP-2 activation response than
2 integrins (Fig.
2A, lower panel). The observed difference in
integrin subunit specificity was independent of the
antibody clone used to promote clustering, and utilization of
antibodies recognizing an
3
1
heterodimer-specific epitope also resulted in a pro-MMP-2 activation
response (Fig. 2A, lower panel, lane 8). Soluble integrin
antibodies did not influence pro-MMP-2 expression or activation (Fig.
2A, upper panel), supporting the hypothesis that
multivalent integrin aggregation is necessary for proteinase
induction.

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Fig. 2.
3 1
integrin clustering promotes MMP-2 processing and surface
association. A, DOV13 cells were treated with the
indicated soluble (10 µg/ml) (upper panel) or
bead-immobilized (4 µg/ml) (lower panel) antibodies for
20 h, and conditioned media were analyzed by gelatin zymography.
Antibody clone numbers are given in parentheses. The
relative migration positions of pro- and active MMP-2 are indicated.
B, cells were untreated (Control) or treated with
the indicated bead-immobilized antibodies (12 µg/ml) for 20 h
prior to biotinylation of surface proteins using a non-cell-permeable
biotin and cell lysis. Labeled protein (750 µg) was captured with
monomeric avidin gels, eluted with D-biotin, and eluates
analyzed by gelatin zymography. The lack of activity eluted from
non-biotinylated ConA-treated (20 µg/ml) cell lysates
( Biotin) demonstrates the specificity of the system for
cell surface proteins, as shown in the biotinylated, ConA-treated (20 µg/ml) control (+Biotin).
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As the transmembrane proteinase MT1-MMP is predicted to catalyze cell
surface integrin-mediated pro-MMP-2 activation, MMP-2 association with
the plasma membrane was evaluated by surface labeling with
cell-impermeable s-NHS-biotin and isolating biotinylated proteins with
monomeric avidin-agarose gels. Proteins were eluted from avidin-agarose
gels using 10 mM free D-biotin and analyzed by
zymography for MMP activity. Clustering of both
3 and
1 integrins promoted a significant association of MMP-2
to the cell surface (Fig. 2B, lanes 3 and 5)
relative to cells subjected to
2 integrin clustering
(Fig. 2B, lane 4) or controls (Fig. 2B, lanes 1 and 2), confirming that
3
1
integrins elicit a more robust pro-MMP-2 processing response.
Release of MMP-2 from the Cell Surface--
Secreted pro-MMP-2
binds to the cell surface through the carboxyl-terminal hemopexin
domain, and an intermediately processed form is generated through
MT1-MMP-mediated cleavage of the MMP-2 pro-domain at the
Asn37-Leu38 peptide bond (7). The MMP-2
intermediate is then predicted to undergo maturation to an active
enzyme through concentration-dependent autolysis at the
cell surface (8). Although formation of the trimeric
MT1-MMP·TIMP-2·MMP-2 activation complex at the cell surface is well
established (reviewed in Ref. 3), the mechanism by which active MMP-2
is released from the plasma membrane is presently unclear. Saturation
binding studies are not technically feasible due to the instability of
the intermediate and mature forms of the activated enzyme in
concentrated solution. Thus, the ability of the MMP-2 hemopexin domain
to dissociate surface-bound MMP-2 was evaluated. In the presence of
increasing concentrations of recombinant hemopexin domain, active MMP-2
is not associated with the cell surface (Fig.
3A). In control experiments,
competition of MMP-2 from the cell surface was not observed with
rCBD123 at the same molar concentrations (data not shown).

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Fig. 3.
Competition of MMP-2 from the cell
surface. Cells were cultured for 20 h in the presence or
absence of ConA (20 µg/ml) and recombinant MMP-2 carboxyl hemopexin
domain (rCD, 0-125 nM), as indicated.
Conditioned media (C.M.) and cell lysates (5 µg of total
protein) were analyzed by gelatin zymography for processed forms of
MMP-2. Pro, pro-MMP-2 (66 kDa); Int,
intermediately processed MMP-2 (64 kDa); Mat, mature MMP-2
(62 kDa).
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Integrin Clustering Promotes Cell Surface MT1-MMP
Processing--
The appearance of active MMP-2 on the cell surface
implicates the involvement of MT1-MMP in integrin stimulation of
pro-MMP-2 processing. To evaluate the effect of
integrin clustering
on events further upstream in the zymogen activation pathway, cells were treated with integrin antibody-coated beads, and cell lysates were
immunoblotted with an antibody reactive against the hinge domain of
MT1-MMP. Consistent with previous observations (12), stimulation of
DOV13 cells with ConA promotes accumulation of 55- and 43-kDa forms of
MT1-MMP in cell lysates (Fig. 4A,
lane 6). Clustering of
1 integrins, and to a lesser
extent
3 integrins, promoted processing of MT1-MMP to a
43-kDa form (Fig. 4A, lanes 3 and 5,
respectively), whereas little or no change in MT1-MMP expression was
observed following clustering of
2 integrins (lane 4) or under control conditions with nonspecific IgG (lane
2). Furthermore,
1 integrin clustering results in a
small, but reproducible, accumulation of 55-kDa MT1-MMP in cell lysates
(lane 3).

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Fig. 4.
Integrin clustering promotes MT1-MMP cell
surface expression and processing. A, integrin-induced
MT1-MMP processing. Cells were treated for 20 h with
bead-immobilized antibodies (4 µg/ml) as indicated, and lysates (10 µg for all samples except ConA, 7 µg) were electrophoresed on 8%
polyacrylamide gels and Western-blotted using an antibody to the
MT1-MMP hinge region. Blots were developed using enhanced
chemiluminescence. The migration position of molecular weight standards
is indicated in the left margin, and the migration positions of the 55- and 43-kDa species of MT1-MMP are indicated in the right
margin. B, cells were treated with ConA (20 µg/ml) or
the indicated bead-immobilized antibodies (12 µg/ml) for 20 h
prior to biotinylation of surface proteins using a non-cell-permeable
biotin and cell lysis. Labeled protein (750 µg) was captured with
monomeric avidin gels, eluted with D-biotin, and eluates
analyzed by electrophoresis and Western blotting for MT1-MMP. The lack
of protein eluted from non-biotinylated ConA-treated cell lysates (lane
designated ConA-Biotin Control) demonstrates the specificity
of the system. C, to evaluate what fraction of the total
pool of 55- and 43-kDa MT1-MMP species resides on the cell surface,
cells were stimulated with ConA (20 µg/ml) for 20 h,
surface-biotinylated, lysed, and the lysate (750 µg, + Biotin)
depleted of biotin-labeled proteins through precipitation using
multimeric streptavidin-conjugated agarose gels (+ Streptavidin). Streptavidin-depleted or control lysates (7.5 µg)
were analyzed by electrophoresis and Western blotting for
MT1-MMP.
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To address whether integrin clustering stimulates MT1-MMP expression on
the cell surface, biotin-labeled cell surface proteins were isolated
and immunoblotted for MT1-MMP species. ConA treatment promoted a strong
accumulation of 55- and 43-kDa MT1-MMP species on the cell surface
(Fig. 4B, lane 1). Higher molecular weight species that were
detected in immunoblots of cell lysates (brackets, Fig.
4A) were absent in cell surface preparations
(brackets, Fig. 4B), indicating that these
proteins are likely intracellular. Consistent with cell lysate results,
clustering of
1 integrins promoted cell surface
expression of 55- and 43-kDa forms of MT1-MMP (Fig. 4B, lane
5), whereas clustering of either
integrin subunit was not
sufficient to promote detectable changes in the cell surface expression
profile of MT1-MMP (Fig. 4B, lanes 6 and 7).
Together, these data suggest that clustering of
1
integrins promotes pro-MMP-2 cell surface binding and activation
through increased expression of cell surface-localized MT1-MMP.
Furthermore, cell surface gelatinolytic profiles indicate that
3
1 integrins potentiate a stronger MMP response than
2
1 integrins (Fig.
2B). The ability to detect enhanced levels of cellular, but
not cell surface, MT1-MMP following
3 integrin
clustering likely reflects the technical limitations of the assay, as
1 integrin clustering induces a more robust overall
response. To evaluate further the pool of MT1-MMP species that
reside on the cell surface, cells were stimulated with ConA, surface
biotin-labeled, and lysates were depleted of biotinylated cell surface
proteins using multimeric streptavidin-conjugated agarose. A selective
depletion of only the 55- and 43-kDa forms of MT1-MMP was observed in
streptavidin-treated samples compared with non-treated controls (Fig.
4C, lane 2), supporting the conclusion that these species,
but not the higher molecular weight immunoreactive material present in
Fig. 4A, are stably expressed on the cell surface.
Characterization of Cell Surface MT1-MMP--
As MT1-MMP has been
reported to have weak gelatinase activity (30, 31), plasma membrane
preparations of ConA-treated DOV13 cells were analyzed by gelatin
zymography to determine whether either of the surface-associated
MT1-MMP species are proteolytically active. In addition to
surface-bound MMP-2, an ortho-phenanthroline-sensitive gelatinolytic band that co-migrated with the non-reduced 55-kDa form of
MT1-MMP was observed (Fig.
5B), whereas no gelatinase activity was attributable to the 43-kDa species. Control immunoblots demonstrate that both the 55- and 43-kDa species were prevalent in the
experimental sample (Fig. 5A). To confirm that the observed 55-kDa gelatinolytic activity is a property of MT1-MMP, cell lysates were immunoprecipitated with an anti-MT1-MMP-specific antibody and the
immunoprecipitates were analyzed by gelatin zymography. A 55-kDa
gelatinolytic activity was recovered together with MMP-2, suggesting
that both proteinases co-precipitate as components of the ternary
complex (Fig. 5C). The relative ratio of MT1-MMP to MMP-2
gelatinase activity is enhanced by the immunoprecipitation approach
(Fig. 5, C versus B). Similar to
results obtained in whole plasma membrane preparations, there was no
observable gelatinolytic activity attributable to the 43-kDa form of
MT1-MMP. As these results indicated that the 55-kDa form of MT1-MMP is
an active species on the cell surface, the ability to bind TIMP-2 was
assessed. Exogenous TIMP-2 was added directly to ConA-treated cells,
followed by cross-linking with a reducible, cell-impermeable
cross-linker. Cell lysates were then immunoprecipitated with an
antibody specific to the carboxyl-terminal domain of TIMP-2, reduced,
and analyzed by electrophoresis and immunoblotting for MT1-MMP. The
55-kDa species of MT1-MMP was specifically precipitated through
TIMP-2 (Fig. 5D, lane 3), providing additional evidence that
it is an active, TIMP-2-binding protein.

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Fig. 5.
Identification of active MT1-MMP
species. DOV13 cells were cultured in the presence of ConA (20 µg/ml) for 20 h, lysed, and homogenized. Plasma membranes were
isolated by centrifugation, and aliquots (20 µg) were analyzed for
MT1-MMP protein (A) and activity (B) by
immunoblotting and gelatin zymography, respectively. A,
Western blot of plasma membranes showing proteins cross-reactive with
MT1-MMP antibody. The arrows indicate the migration
positions of the 55- and 43-kDa MT1-MMP species. B, zymogram
depicting MT1-MMP gelatinolytic activity associated with the 55-kDa
species (upper arrow). (Note that the prevalent gelatinase
activity in this sample is due to plasma membrane-bound MMP-2.) All
gelatinase activity is inhibited by the zinc chelating agent
ortho-phenanthroline (O-PA). C,
immunoprecipitation (IP) of DOV13 cell lysates. Lysates
(non-denatured) were immunoprecipitated with either IgG control or
anti-MT1-MMP antibodies as indicated and analyzed by gelatin
zymography. Although the immunoprecipitating antibody recognizes both
the 43- and 55-kDa forms of MT1-MMP by immunoblotting (A),
gelatinase activity corresponding to only the 55-kDa species is
observed (arrow). MMP-2 co-precipitated as a component of
the ternary complex, as evidenced by the co-migration with an MMP-2
standard (bracket). Lane designated conditioned
media, DOV13 conditioned medium to designate migration
position of MMP-2 (not subjected to immunoprecipitation). D,
cross-linking and immunoprecipitation. Cells were treated with ConA (20 µg/ml) and an excess of free TIMP-2 (125 ng/ml) for 18 h. After
washing, cell surface proteins were cross-linked with a reducible
cross-linking agent (2 mM
3,3'-dithiobis(sulfosuccinimidylpropionate), 25 min, 4 °C).
Clarified cell lysates (700 µg) were incubated with 5 µg of
anti-TIMP-2 (carboxyl-terminal) mAb clone 67-4H11 or murine IgG ( )
as indicated and precipitated using protein A-agarose.
Immunoprecipitates were solubilized with Laemmli sample buffer,
electrophoresed under reducing conditions on 8% polyacrylamide gels,
and immunoblotted with the MT1-MMP hinge polyclonal antibody. The
arrow designates the migration position of 55-kDa MT1-MMP,
and the left margin indicates the migration position of
molecular weight standards.
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MT1-MMP Processing in the Absence of Cell Surface MMP-2--
As
reported previously (12), active 55-kDa MT1-MMP is converted to an
inactive 43-kDa form through MMP-dependent proteolysis in
DOV13 cells (Fig. 6A, compare
lanes 3 and 6) (12). In a regulated cellular
system generating an endogenous MMP-2/MT1-MMP activation response, it
is unclear whether this cleavage is mediated by activated MMP-2 or
through concentration-dependent autolysis of MT1-MMP. As active
MMP-2 is effectively removed from the cell system with 125 nM hemopexin domain (Fig. 3), cellular MT1-MMP processing in the absence of MMP-2 was assessed by immunoblotting. Conversion of
55-kDa MT1-MMP to the 43-kDa form was not affected by the loss of
active MMP-2 from the cell surface (Fig. 6A, lanes 3 and
4), indicating that MMP-2 is not required for proteolytic
processing of endogenous 55-kDa MT1-MMP in DOV13 cells.

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Fig. 6.
MT1-MMP processing in the absence of cell
surface MMP-2. A, cells were cultured in the presence
or absence of MMPI (10 µM), ConA (20 µg/ml), or rCD
(125 nM) as indicated. Lysates (7.5 µg) were
electrophoresed on 8% polyacrylamide gels and immunoblotted for
MT1-MMP. B, stable DOV13 transfectants expressing
polyhistidine-tagged MT1-MMP (MT1-6xHis) or control vector
(CAT) were isolated, cultured in the presence or absence of
ConA (20 µg/ml) as indicated, and 7.5 µg of cell lysates analyzed
by electrophoresis and immunoblotting for MT1-MMP (lanes
1-4) or polyhistidine (lanes 4-8). Lane 9 contains soluble recombinant MT1-MMP (rMT1) lacking the
transmembrane and cytoplasmic domain purified from CHO cell-conditioned
medium and visualized by silver staining. C, DOV13 stable
transfectants expressing polyhistidine-tagged MT1-MMP
(MT1-6xHis) or vector controls (CAT) were
isolated, cultured in the presence or absence of ConA (20 µg/ml) or
MMPI (10 µM) as indicated, and conditioned media analyzed
for pro-MMP-2 activation by gelatin zymography. The relative migration
positions of the pro-, intermediate (Int), and activated
mature (Mat) forms of MT1-MMP are indicated in the
left margin.
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To determine whether MT1-MMP degradation proceeds through autolysis or
through activity of an unidentified metalloprotease, DOV13 cells that
stably overexpress full-length histidine-tagged MT1-MMP were generated.
Overexpression of MT1-MMP resulted in accumulation of 40-45-kDa
species of recombinant enzyme as detected by Western blotting with
anti-MT1-MMP (Fig. 6B, lane 4) and confirmed using an
antibody against the polyhistidine label (Fig. 6B, lanes 7 and 8). Similar results were obtained following expression
of soluble recombinant MT1-MMP lacking the transmembrane and
cytoplasmic domain, in which autolytic processing to 40-45-kDa species
was also observed (Fig. 6B, lane 9, labeled rMT1),
supporting the conclusion that the 55-kDa MT1-MMP-processing activity
is directly attributable to the level of cell surface MT1-MMP activity
(32). Together, these data indicate that active 55-kDa MT1-MMP
undergoes concentration-dependent autolysis to an inactive
43-kDa form on the surface of DOV13 cells. Control experiments
demonstrated MMP-dependent processing of pro-MMP-2,
confirming surface expression of the recombinant enzyme in DOV13 cells
(Fig. 6C).
Localization of Cell Surface MT1-MMP--
In previous studies,
MT1-MMP has been localized to membrane protrusions, termed invadopodia,
which are rich in matrix proteinases and integrins, including
3
1 integrins (27-29). Immunocytochemical analysis of MT1-MMP surface staining in non-permeabilized DOV13 cells
demonstrates MT1-MMP localization to distinct cell surface projections (Fig. 7D),
characteristic of integrin-rich sites of cell-matrix contact. Following
1 integrin clustering using antibody-coated beads,
MT1-MMP immunoreactivity is substantially redistributed to the
periphery of the aggregated integrins (Fig. 7H). These data
indicate that MT1-MMP can be actively recruited to membrane sites
containing clustered
1 integrins on the surface of DOV13 cells.

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Fig. 7.
MT1-MMP localizes to cellular processes and
redistributes to clustered 1
integrins. Cells were cultured on glass coverslips in the absence
(A-D) or presence (E-H) of
anti- 1 integrin (clone 21C8)-coated beads for 8 h
and then processed for immunofluorescence as described under
"Experimental Procedures." Cells were incubated with normal rabbit
serum (B and F) or anti-MT1-MMP antibody
(D and H). Immunoreactivity was visualized with
fluorescein-conjugated anti-rabbit secondary antibody previously
purified against cross-reactivity to mouse immunoglobulins. Phase
contrast images A, C, E, and G correspond with
B, D, F, and H, respectively, and identify
cellular processes and 2.97 µm diameter latex beads.
Arrowheads denote MT1-MMP immunoreactivity in cell surface
projections. Magnification bar is 5 µm (A-D)
or 10 µm (E-H), and images were collected with a 63×
objective using a Zeiss fluorescence microscope.
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MMP Dependence of Migration and Collagen Gel
Invasion--
Three-dimensional collagen culture and clustering of
collagen binding integrins up-regulate surface MMP activity in DOV13 cells. To assess the functional consequences of matrix-enhanced proteolytic potential, the ability of cells to migrate and to invade a
three-dimensional collagen matrix was evaluated. MMP activity was not
required for general cell motility, as cells migrated through uncoated
transwell filters with equal efficiency in the presence or absence of a
broad spectrum MMP inhibitor (MMPI), TIMP-1, or TIMP-2 (Fig.
8A). Haptotactic motility over
collagen-coated colloidal gold surfaces is also unaffected by a broad
spectrum MMP inhibitor (Fig. 8B). Semi-quantitative analysis
of phagokinetic tracks from 30 cells in the absence and presence of
MMPI using computer-assisted image analysis gave relative migration
areas of 1.4 ± 0.1 and 1.5 ± 0.3, respectively, indicating
that MMP activity does not contribute to collagen-driven migration of
DOV13 cells.

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Fig. 8.
Migration of DOV13 cells is
MMP-independent. A, cells (2.5 × 105)
were seeded onto Transwell filters (8 µm pore) coated with a thin
layer of collagen (as described under "Experimental Procedures") in
the presence or absence of MMPI (10 µM),
Me2SO (DMSO, vehicle for MMPI), TIMP-1 (10 nM), or TIMP-2 (10 nM) as indicated and
incubated for 5 h to permit migration. Non-migrating cells were
removed from the upper chamber; filters were stained, and migrating
cells adherent to the underside of the filter were enumerated using an
ocular micrometer. Data are expressed as % of control migration
(Me2SO for MMPI and PBS for TIMP-1 and -2). There is no
significant difference in migration of inhibitor-treated samples
relative to respective controls (p > 0.05).
B, haptotactic motility of DOV13 cells. Cells (1000) were
plated onto coverslips coated with colloidal gold overlaid with type I
collagen (100 µg/ml) in the presence or absence of MMPI (10 µM) as indicated, allowed to migrate for 24 h, and
phagokinetic tracks visualized using dark field illumination.
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DOV13 cells efficiently penetrate three-dimensional collagen gels via
2
1 and
3
1
integrin receptors, and obstructing collagen-induced integrin
clustering using integrin function blocking antibodies inhibits
invasion (Fig. 9A). Invasion
correlates with collagen-induced pro-MMP-2 processing (Fig. 9B,
inset), and in contrast to cellular migration, invasion of
collagen is MMP-dependent, as both a broad spectrum MMPI
and exogenous TIMP-2 abrogate invasion and reduce or eliminate
pro-MMP-2 processing (Fig. 9B, lanes 2 and 5).
TIMP-1, a poor inhibitor of MT1-MMP (26), failed to reduce either
collagen invasion or MT1-MMP-mediated pro-MMP-2 processing (Fig.
9B), suggesting that MT1-MMP collagenolytic activity may
potentiate invasion. The serine proteinase inhibitor aprotinin had no
effect on invasion of intact collagen gels (not shown). Furthermore,
although collagenase-resistant collagen is sufficient to stimulate
pro-MMP-2 activation (Fig. 1), cellular penetration of this matrix is
significantly inhibited relative to wild type collagen (Fig.
9C), providing further support for the hypothesis that
collagenolysis is required for invasive activity. Disrupting the triple
helical structure of collagen by thermal denaturation removes the
requirement for collagenase activity, as no difference in invasion of
cells through gelatin derived from either wild type or
collagenase-resistant collagen is observed (Fig. 9C). This
is supported by data using wild type gelatin, in which invasion is
effectively blocked by both TIMP-1 and -2 as well as by the serine
proteinase inhibitor aprotinin (Fig. 9D), indicating that
additional (non-MMP) gelatinolytic proteinases potentiate invasion
following destabilization of collagen triple helical structure.
Collagen invasion is unaltered in the presence of either the rCD or
rCBD of MMP-2 (Fig. 9E). However, both the rCD, which
inhibits MMP-2 cell surface activation (Fig. 3) (6, 8), and rCBD, which
prevents MMP-2 binding to native and denatured collagen (17),
effectively inhibit the MMP-2-dependent component of
gelatin invasion (Fig. 9F). Together these data suggest that
the stimulation of MT1-MMP collagenolytic activity (31) through
collagen-binding integrins is a rate-limiting step for invasion of
native type I collagen-rich matrices.

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Fig. 9.
Characterization of the proteinase
requirements for invasion. A-C and E,
invasion of collagen. Cells (1.0 × 105) were added to
collagen-coated Transwell chambers (20 µg) in serum-free medium for
2 h prior to addition of reagents indicated below and allowed to
invade for 48 h under the indicated conditions. Following
invasion, cells were removed from the top chamber with a cotton swab;
membranes were stained, and invading cells were enumerated using an
ocular micrometer. C, D, and F, invasion of
gelatin. Cells (1.0 × 105) were added to
gelatin-coated Transwell chambers (50 µg) in serum-free medium for
2 h prior to addition of reagents indicated and allowed to invade
for 24 h under the indicated conditions. Invading cells were
quantified as described. A, integrin subunit-specific
antibodies block invasion of collagen. Integrin subunit-specific
antibodies or control IgG (15 µg/ml) were added as indicated. Results
are expressed as % of control invasion (IgG), normalized to 100%. (*,
p < 0.05 relative to control.) B, effect of
MMP inhibitors on collagen invasion. Invasion was quantified in the
presence or absence of MMPI (10 µM), TIMP-1 (10 nM), or TIMP-2 (10 nM), as indicated. Results
are expressed as % of control invasion. MMPI data are normalized to
Me2SO controls (designated 100%), and TIMP-1 and -2 data
are normalized to PBS controls (designated 100%). (*,
p < 0.05 relative to control.) Inset,
analysis of conditioned media from invasion chambers by gelatin
zymography. C, invasion of wild type and CR collagen
gels. Cells were allowed to invade gels composed of wild type or CR
collagen or wild type and CR gelatin, as indicated. Invasion of CR
collagen is expressed as % of control invasion (with wild type
collagen designated 100%). Invasion of gelatin is designated as fold
increase relative to wild type collagen control. (*, p < 0.05 relative to control.) D, effect of proteinase
inhibitors on invasion of gelatin. Invasion was evaluated in the
presence or absence of TIMP-1 (10 nM), TIMP-2 (10 nM), or aprotinin (20 µg/well), as indicated. Invasion of
gelatin is expressed as % of control invasion (designated 100%). (*,
p < 0.05 relative to control.) E, effect of
MMP-2 domains on collagen invasion. Invasion was quantified in the
presence or absence or rCD (100 nM) or rCBD (100 nM), as indicated. Results are expressed as % of control invasion (designated 100%). F, effect
of MMP-2 domains on gelatin invasion. Invasion was quantified in the
presence or absence or rCD (100 nM) or rCBD (100 nM), as indicated. Results are expressed as % of control
invasion (designated 100%). (*, p < 0.05 relative to
control.)
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DISCUSSION |
MT1-MMP is a cell surface activator of pro-MMP-2 and has been
implicated in collagen invasion and turnover (33-36). In this study,
DOV13 ovarian cancer cells activate MT1-MMP as a consequence of culture
in type I collagen gels and display MMP-dependent invasion of type I collagen, indicating that MMP activity is required for removal of collagen matrix constraints during invasion. However, migration over two-dimensional collagen is not impeded by MMP inhibitors. Although TIMP-1 does not interact with MT1-MMP, TIMP-2 specifically binds the proteinase, functioning in both inhibition and
stabilization of the enzyme on the cell surface (26, 32). The ability
of exogenous TIMP-2, in contrast to TIMP-1, to inhibit DOV13 collagen
gel invasion implicates a cell surface proteolytic cascade initiated by
MT1-MMP. This is further supported by data demonstrating that
inhibition of MMP-2 cellular activation or collagen binding using rCD
and rCBD (17, 18), respectively, has no effect on collagen invasion. As
the cellular events that govern the collagen-induced MMP-2/MT1-MMP
response are unclear and technically difficult to assess in
three-dimensional collagen gel systems, a variety of biochemical
approaches were employed in this study to dissect the interplay between
collagen-cell interactions and regulation of cell surface MMP activity.
DOV13 cells bind type I collagen via
2
1
and
3
1 integrins. Recognition of collagen
by these integrins depends on retention of the triple helical
conformation, as thermal gelation of collagen abrogates cellular
adhesion. Collagenase-cleaved type I collagen produces 3/4 and
1/4 fragments that display a lower Tm than intact fibrils (37). Adhesion data in the current study demonstrate that the triple helical conformation of the collagen fragments is
stabilized at low coating temperatures but is lost
under physiological coating conditions (21).
Together, these data suggest that pericellular type I collagenolysis
will reduce
2
1 and
3
1 integrin-mediated cell-matrix
contacts. By using a similar approach, the appearance of cryptic
V
3 integrin-binding sites (RGD) in
collagenase-generated collagen fragments was reported (21). However,
DOV13 cells adhere weakly to type I gelatin or 3/4 and
1/4 fragments, and aggregation of
V
3 integrins on the surface of DOV13
cells does not elicit a cellular MMP processing response (12, 20).
Nevertheless, the exposure of cryptic
V
3-
or
V
5-binding sites in
collagenase-cleaved collagen may further influence MMP expression in a
cell type-specific manner. In support of this observation, we have
previously demonstrated that vitronectin-induced aggregation of
melanoma cell
V
3 integrins up-regulates
MMP-2 expression (38). Relative to collagen gel penetration, DOV13
cells rapidly invade a gelatin matrix. Although pro-MMP-2 activation is
not up-regulated over basal levels under these conditions, additional
proteinases of other mechanistic classes can provide gelatinase
activity (1, 39, 40). Together these data indicate that collagenase
activity provided by MT1-MMP is critical to invasion of an intact
collagen matrix. Subsequent clearance of resultant fragments can then
proceed by activation of cell surface MMP-2 along with contributions
from other cell surface proteinases including seprase (39) and the
components of the plasminogen activator/plasmin system (40), which have been implicated in DOV13 cellular invasion of Matrigel (22).
Stimulation of pro-MMP-2 activation does not require collagenolysis, as
collagenase-resistant collagen is as efficacious as wild type type I
collagen at inducing pro-MMP-2 processing. However, thermal
denaturation of either collagen abolishes the ability to enhance MMP
activation, suggesting that
2
1 and/or
3
1 integrin binding to intact
triple-helical collagen mediates the MMP activation response in DOV13
cells. By using subunit-specific antibodies to dissect integrin
requirements for MMP processing, our data demonstrate that clustering
of
3 integrins promotes a stronger cellular MMP
processing response than
2 integrin aggregation. A
potential role for
integrin-specific regulation has been
demonstrated previously for type I collagen-induced cellular responses,
including those involving MMP-1 expression (41, 42). Administration of
function blocking antibodies against either
integrin subunit reveals a role for both receptors during invasion. Although this study
implicates the
3
1 heterodimer in
mediating the MT1-MMP response, it is likely that both
2
1 and
3
1
integrins provide a mechanical advantage to the migration component of
invasion. Furthermore, it is unclear at this level of investigation
whether
2
1 integrins are required for
effective dispersal of
3
1 into focal
adhesions (43, 44). Interestingly,
3
1
integrins have recently been hypothesized to play a major
organizational role in the formation of invadopodia in response to
cellular engagement of type I collagen (45). As MT1-MMP also localizes
to invadopodia (27-29), it is possible that
3
1 clustering in DOV13 cells selectively initiates cellular events that mimic formation of invadopodial projections, which in turn regulate MT1-MMP activity. Moreover, our
data demonstrate localization of MT1-MMP immunoreactivity to the
periphery of clustered
1 integrins, indicating that
MT1-MMP redistribution occurs during integrin clustering events. This observation, together with previous reports of MT1-MMP localization to
integrin-rich cellular protrusions, suggests a cellular regulatory mechanism for MT1-MMP aggregation, thereby promoting effective pro-MMP-2 processing and efficient matrix degradation. As MT1-MMP can
function as a collagenase (31) and MT1-MMP null mice exhibit severe
deficiencies in collagen remodeling (33), localization of the enzyme to
cellular collagen receptors could clearly influence physiologic events
such as collagen gel contraction, adhesion, and invasion. In addition,
pro-MMP-2 bound to intact peri-cellular collagen may readily infiltrate
the MT1-MMP activation pathway, resulting in a switch from a
collagenase to a gelatinase environment as pro-MMP-2 activation and
collagen triple helix denaturation ensues (17).
It has recently been demonstrated that exogenous MT1-MMP overexpressed
in a MMP-2 null background undergoes autolysis to a 43-kDa form, the
rate of which is regulated by TIMP-2 (32). Similarly, endogenously
expressed MT1-MMP in DOV13 cells exists in two major forms of 55 and 43 kDa (12). The current data indicate that the 55-kDa form of MT1-MMP is
the active TIMP-2-binding species, whereas the 43-kDa form is an
inactive autolysis product. This result is consistent with the
amino-terminal sequences of similar MT1-MMP species obtained from
overexpression systems, which demonstrate a loss of essential amino
acids in the zinc-binding consensus sequence (30, 32). Although
catalytically inactive, the 43-kDa form of MT1-MMP is nevertheless
retained on the cell surface. As this 43-kDa species contains the
carboxyl-terminal domains necessary for invadopodial localization and
enzyme aggregation, it is interesting to speculate that
MT1-MMP-mediated proteolysis may be down-regulated through the dilution
of active enzyme with truncated proteinase.
In summary, our data support the hypothesis that as DOV13 cells
interact with type I collagen, integrin receptors cluster on the cell
surface, resulting in up-regulation of MT1-MMP and pro-MMP-2
processing, recruitment of MT1-MMP to sites of cell-matrix contact,
MMP-2 surface association, and MT1-MMP-dependent collagen gel invasion. As a consequence of MT1-MMP collagenolysis, the resulting
collagen cleavage products thermally denature, providing a substrate
for a number of proteinases. In addition, MMP-2 is released from the
cell surface to further advance matrix clearance through directed
gelatinase activity on denatured collagen fragments. As
2
1 or
3
1
integrin occupancy is reduced, collagen matrix stimulation of
proteolysis is attenuated. Furthermore, MT1-MMP activity can be
down-regulated by autolytic processing to a stable, inactive 43-kDa
form that may functionally dilute productive enzyme-substrate interactions. Together, these data support an hypothesis wherein matrix
status influences cell surface matrix-degrading potential to facilitate
cellular functions including migration, invasion, and matrix remodeling.