The initial site of melanoma cell metastasis is
frequently the regional lymph nodes, and the appearance of lymph node
metastasis correlates with poor prognosis. Lymph node adhesion is
mediated by an interaction between the tumor cell integrin
v
3 and
lymph node vitronectin. In this study, we explored the relationship between adhesion and proteolysis by examining the direct effect of
vitronectin receptor ligation on matrix metalloproteinase-2 (MMP-2)
production by B16F1 and B16F10 melanoma cells. We report a
dose-dependent increase in secretion of both MMP-2 and
tissue inhibitor of metalloproteinases-2 (TIMP-2) in response to
vitronectin. Cellular invasiveness was also enhanced by vitronectin, as
shown by the increased ability of vitronectin-treated cells to invade a
synthetic basement membrane (Matrigel). Both the vitronectin-induced MMP-2 production and vitronectin-enhanced invasion were blocked by the
peptide ligand Arg-Gly-Asp-Ser (RGDS). Furthermore, neither plasmin-degraded vitronectin nor the peptide ligand RGDS stimulated MMP-2 secretion or invasiveness, indicating that a multivalent ligand-receptor interaction rather than simple receptor occupancy was
required for MMP-2 induction. MMP-2 and MMP-2/TIMP-2 interaction with
the plasma membrane of melanoma cells resulted in enhanced catalytic
activity against 14C-labeled gelatin, suggesting that
membrane association may function in posttranslational regulation of
MMP-2 activity. This is supported by data showing increased cellular
invasion by cells containing membrane-bound MMP-2. Binding of proMMP-2
and proMMP-2/TIMP-2 to melanoma cells was not inhibited by RGDS, and
melanoma cell adhesion to vitronectin was unaffected by pro- or active
MMP-2, indicating that MMP-2 did not interact with the murine
vitronectin receptor. Together, these data provide evidence for a
functional link between adhesion and proteolysis and suggest a
potential mechanism whereby adhesion of an invasive cell to the
extracellular matrix regulates subsequent invasive behavior.
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INTRODUCTION |
Adhesion of tumor cells to specific extracellular matrix
macromolecules is an initial component of the metastatic process (reviewed in Refs. 1 and 2). In metastatic melanoma, tumor cell
adhesion to the regional lymph nodes, which correlates with poor
prognosis, is mediated via interaction of specific integrins on the
melanoma cell surface with lymph node vitronectin (3). Previous data
have demonstrated a relationship between elevated levels of
vitronectin-binding integrins and increased melanoma cell invasiveness
(4-6). Furthermore, ligation of the
v
3 integrin on melanoma
cells by anti-
v
3 antibodies enhances secretion of matrix
metalloproteinase-2 (MMP-2, gelatinase A, 72-kDa type IV collagenase),
resulting in increased cellular invasiveness (7). Together, these
data suggest that integrin-mediated binding of tumor cells to a
specific matrix-associated protein, such as vitronectin, can
promote tumor cell invasion by increasing the levels of a matrix-degrading proteinase.
The majority of integrins recognize multiple extracellular matrix
ligands (1, 2), and precise biologic responses may be regulated by
differential integrin ligation with distinct extracellular matrix
proteins. In this study, we have explored the direct effect of the
matrix-associated ligand vitronectin on production of MMP-2 by melanoma
cells. We report a dose-dependent increase in secretion of
both MMP-21 and tissue
inhibitor of metalloproteinases-2 (TIMP-2), as well as enhanced
cellular invasiveness, in response to vitronectin. Intact vitronectin
is required for MMP-2 induction because neither plasmin-treated
vitronectin nor a peptide ligand (Arg-Gly-Asp-Ser (RGDS)) alters MMP-2
secretion, indicating the requirement for a multivalent ligand-receptor
interaction. Furthermore, MMP-2 interacts with the plasma membranes of
melanoma cells, exhibits enhanced catalytic activity relative to the
solution phase enzyme, and increases cellular invasive activity.
Membrane binding of MMP-2 is unaffected by RGDS, and melanoma cell
adhesion to vitronectin is not inhibited by MMP-2, indicating that
MMP-2 does not bind the vitronectin receptor on murine melanoma cells.
These data suggest a potential physiologic mechanism whereby the
relative integrity of the adhesive substratum may differentially
regulate secretion and activity of a matrix-degrading proteinase and
subsequent cellular invasive behavior.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The murine B16F10 and B16F1 melanoma cell
lines were obtained from Dr. I. J. Fidler (The University of Texas
M. D. Anderson Hospital) and were cultured in Eagle's minimal
essential medium supplemented with 5% fetal calf serum, nonessential
amino acids, L-glutamine, and vitamins. Prior to each
experiment, cells were washed with calcium and magnesium-free
Dulbecco's phosphate-buffered saline (PBS) and incubated for 2 min at
25 °C with 1 mM EDTA in serum-free Eagle's minimal
essential medium to release cells from the culture flask. Cells were
seeded in 1 ml of serum-free Eagle's minimal essential medium at a
density of 1 × 106 cells/ml and incubated 18 h
at 37 °C in the presence of vitronectin, peptide, or both as
described below. In some experiments, colchicine (0.2 µM)
was included for inhibition of secretion. Conditioned media were
removed and concentrated 4-fold at 4 °C using a Centricon 10 (Amicon) concentrator and analyzed immediately as described below.
Proteins and Antibodies--
Native vitronectin was purified
from pooled human plasma according to the procedure of Dahlback and
Podack (8). Briefly, pooled plasma was subjected to salt fractionation
followed by ion exchange, dye affinity, and gel filtration
chromatography. Presence and purity of vitronectin in each fraction was
assessed by both dot blot and electrophoretic analysis on 5-15%
gradient SDS-polyacrylamide gels. ProMMP-2 and proMMP-2/TIMP-2 complex were purified as described previously (9, 10). Plasminogen was purified
from human plasma by affinity chromatography on
L-lysine-Sepharose and separated into isoforms 1 and 2 as
described previously (11). Plasmin was generated by incubating 100 µg
of plasminogen with 100 µl of urinary-plasminogen activator-Sepharose
(Calbiochem) in 10 mM Hepes, pH 7.4, at 25 °C for
12 h followed by centrifugation to remove the resin. Protein
concentrations were determined spectrophotometrically at 280 nm using
an A1 cm1 % value of 16.8 and molecular
masses of 92 and 81 kDa for plasminogen and plasmin, respectively.
Endoproteinase Glu-C from Staphylococcus aureus strain V8
(endoproteinase V8) and soybean trypsin inhibitor were purchased from
Sigma and were coupled to cyanogen bromide-activated Sepharose 4B
(Sigma) according to the manufacturer's specifications. The synthetic
peptide Arg-Gly-Asp-Ser (RGDS), which blocks
v
3 and
v
5-mediated adhesion to vitronectin (12), and the noninhibitory control peptide Gly-Asp-Glu-Ser (RGES) were purchased from Sigma.
Analysis of MMP-2 and TIMP-2 Secretion--
Latent MMPs in
concentrated conditioned medium were activated by incubation at
37 °C for 60 min in the presence of 1.0 mM amino-phenylmercuric acetate (APMA). Zymographic analysis of secreted MMPs was performed using 9% SDS-polyacrylamide gels containing co-polymerized gelatin as described previously (13). Reverse zymography
for detection of TIMPs was performed using 15% SDS-polyacrylamide gels
containing co-polymerized gelatin and reagents purchased from
University Technologies International (Calgary, Alberta, Canada)
according to the manufacturer's specifications. Inactivation of the
TIMP(s) in conditioned medium by reductive carboxymethylation was
carried out as described by Salvesen and Nagase (14). Briefly, conditioned medium was treated with 2 mM dithiothreitol for
1 h at 37 °C followed by incubation with iodoacetamide (5 mM) for 15 min at 37 °C.
In Vitro Invasion Assays--
In vitro invasive
activity was assessed by determining the ability of cells to invade a
synthetic basement membrane (Matrigel, Becton Dickinson, Bedford, MA).
Polycarbonate filters (8-µm pore size, Becton Dickinson) were coated
with Matrigel (11 µg/filter) and placed in modified Boyden chambers.
Cells (1 × 105) were added to the top of the chamber
in serum-free medium in the presence or absence of vitronectin,
plasmin-treated vitronectin, RGDS, or RGES. Following incubation for
8-24 h, noninvading cells were removed from the top surface of the
membrane filter with a cotton swab, and filters were removed and
stained with Diff-Quik (Fisher). Cells on the lower surface of the
filter were enumerated using an ocular micrometer and counting a
minimum of six high-powered fields. In some experiments, untreated
cells (1 × 105) were preincubated for 90 min with
MMP-2 (50 nM) in serum-free culture medium containing 3%
bovine serum albumin. Cells were pelleted, washed twice with PBS to
remove unbound MMP-2, and enumerated, and invasiveness was assayed as
described above.
Limited Proteolysis of Vitronectin--
Limited plasmin
proteolysis of vitronectin was carried out by incubating vitronectin
and plasmin in a 20:1 (w/w) vitronectin:plasmin ratio for various time
periods from 10 min to 3 h at 37 °C. After incubation, plasmin
was removed from the reaction by the addition of soybean trypsin
inhibitor-Sepharose, followed by centrifugation to remove the
plasmin-soybean trypsin inhibitor-Sepharose complex. Complete removal
of plasmin was confirmed by addition of the synthetic plasmin substrate
D-Val-Leu-Lys-p-nitroanilide (Sigma) and
monitoring absorbance at 405 nm. Limited proteolysis of vitronectin by
endoproteinase V8 was performed by incubating vitronectin (200 µg)
with 200 µl of endoproteinase V8-Sepharose for various time periods
from 30 min to 18 h at 25 °C, followed by centrifugation to
remove the proteinase resin. Cleavage of vitronectin by MMP-2 was
assessed by incubating vitronectin with activated MMP-2 in a 20:1
vitronectin:MMP-2 (w/w) ratio in 50 mM Tris-HCl, 0.2 M NaCl, 10 mM CaCl2, pH 7.6 at
37 °C for 18 h. Intact vitronectin and vitronectin treated with
plasmin, endoproteinase V8, or MMP-2 were analyzed by electrophoresis on 5-15% SDS-polyacrylamide gradient gels followed by staining with
Coomassie Blue.
Adhesion Assay--
Vitronectin was passively adsorbed to
24-well culture plates as described previously (15), blocked by
incubating with PBS containing 2% bovine serum albumin for 2 h at
25 °C, and utilized for cell attachment assays as described by
Pierschbacher and Ruoslahti (16). Briefly, cells were plated at a
constant of 1.5 × 105 cells/ml in a total volume of
0.5 ml and incubated for 30-60 min on vitronectin-coated plates. After
incubation, plates were washed twice with PBS, and bound cells were
enumerated using an ocular micrometer and counting six high-powered
fields. The effect of RGDS, RGES, proMMP-2, and active MMP-2 on
adhesion to vitronectin was determined by addition of cells in the
presence of various concentrations of peptide or proteinase to
vitronectin-coated wells.
Analysis of MMP-2 Membrane Association--
To analyze binding
of proMMP-2 or proMMP-2/TIMP-2 to intact melanoma cells, proteins were
labeled with 125I using Iodogen (Pierce) according to the
manufacturer's specifications. Labeled protein (100 nM in
minimal essential medium containing 0.1% bovine serum albumin) was
added to B16F10 cells (5 × 104) for 1 h at
4 °C in the presence of increasing concentrations of RGDS or RGES
peptide (0-1000 µg/ml). Cells were washed three times with Hanks'
balanced salt solution, and cell associated radioactivity was measured
in a
-counter. To assess the interaction of proMMP-2 or
proMMP-2/TIMP-2 with isolated melanoma cell membranes, melanoma cells
were cultured overnight in serum-free medium, released from the flask
with EDTA, and washed three times with PBS. The plasma membrane
fractions of B16F1 (2 × 108) and B16F10 (4 × 108) cells were isolated using nitrogen cavitation (350 p.s.i., 20 min) and differential centrifugation as described previously
(17). Cell lysates were treated with soybean trypsin inhibitor (20 µg/ml), leupeptin (25 µg/ml), elastatinal (25 µg/ml), 3,4 dichloroisocoumarin (0.05 mM), and
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64, 0.01 mM) to prevent proteolysis of membrane
proteins. B16F1 and B16F10 plasma membranes were incubated (2 h,
37 °C) with purified proMMP-2/TIMP-2 or proMMP-2 (100 nM) in 20 mM Tris, 5 mM
CaCl2, 25 mM sucrose, pH 7.4 (Tris-Ca-sucrose).
Control samples contained buffer alone. The membranes were recovered by
centrifugation (50,000 × g for 30 min), supernatants
were discarded, and pellets were washed twice in Tris-Ca-sucrose.
Membrane extracts were prepared by incubating aliquots of membrane
fractions with 1% Triton X-100 at 4 °C for 16 h followed by
clarification by centrifugation (50,000 × g for 30 min) as described previously (17). After resuspension in
Tris-Ca-sucrose, aliquots of membranes were analyzed for bound activity
by gelatin zymography and by 14C-gelatin degradation (17,
18). 14C-Gelatin was incubated for 22 h at 37 °C
with treated membrane aliquots in Tris-Ca-sucrose containing 50 mM NaCl and 0.5 mM APMA. Parallel experiments
contained 1 mM o-phenanthroline. Reactions were
stopped by trichloroacetic acid precipitation, and soluble radioactivity was determined by scintillation counting. Gelatin degradation by membrane-associated proteinase was determined relative to membrane-free controls. The effect of membrane association on the
gelatinolytic activity of pre-activated MMP-2 was also determined as
described previously (17).
 |
RESULTS |
It has been previously reported that treatment of A375M melanoma
cells with antibodies directed against the vitronectin-binding integrin
v
3 enhanced MMP-2 secretion and stimulated cellular invasion
through Matrigel (7). To analyze the direct effect of vitronectin on
melanoma MMP-2 secretion, B16F1 and B16F10 cells were incubated with
increasing concentrations of vitronectin, and secretion of
gelatin-degrading MMPs was analyzed by zymography. A
dose-dependent increase in secretion of a gelatinolytic
metalloproteinase was observed in the conditioned medium of
vitronectin-treated B16F1 cells (Fig. 1)
and B16F10 cells (Fig. 2A).
The gelatinolytic enzyme was activated by treatment with APMA,
inhibited by the zinc chelator o-phenanthroline (data not
shown), and co-migrated with authentic MMP-2, suggesting its identity
as MMP-2. MMP-2 secretion was not detectable by zymography in untreated
B16F1 or B16F10 cells (Fig. 1, lane 1, and Fig. 2,
lane 1), but it was observed in cells treated with as little
as 10 µg/ml vitronectin. Addition of vitronectin in the presence of
RGDS peptide inhibited the vitronectin-induced MMP-2 secretion,
demonstrating that direct interaction of vitronectin with its cellular
receptor is required for MMP-2 induction (Fig. 1). Furthermore,
vitronectin-induced MMP-2 secretion was inhibited by colchicine,
demonstrating that vitronectin does not displace MMP-2 from a common
cell surface receptor such as
v
3 (data not shown). Analysis of
TIMP-2 activity by reverse zymography revealed a metalloproteinase
inhibitor that co-migrated with TIMP-2 (Mr
21,000) in the conditioned medium of untreated cells (Fig. 2B,
lane 1), which was increased in vitronectin-treated cells (Fig.
2B, lanes 2-4). Incubation of the conditioned medium samples with dithiothreitol and iodoacetamide, a procedure shown to
denature TIMP to a noninhibitory form (14), removed the TIMP-2 activity
from the conditioned medium (Fig. 2C). Vitronectin treatment also enhanced cell surface-associated MMP-2. B16F1 and B16F10 cells
cultured with vitronectin (50 µg) displayed a 17 and 20% increase in
cell surface MMP-2, respectively, as determined by cell surface
enzyme-linked immunosorbent assay using an anti-MMP-2 antibody and an
alkaline-phosphatase-conjugated secondary antibody (15). To assess the
functional effect of MMP-2 induction, the ability of melanoma cells to
penetrate a synthetic basement membrane (Matrigel) was analyzed. Both
B16F1 and B16F10 cells displayed enhanced in vitro invasive
behavior when cultured in the presence of vitronectin (Fig.
3).

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Fig. 1.
Zymogram depicting MMP-2 activity in
B16F1-conditioned medium. B16F1 cells (1 × 106)
were cultured for 18 h in serum-free medium containing increasing amounts of vitronectin (0-200 µg/ml), and conditioned media were analyzed for MMP activity by gelatin zymography on 9%
SDS-polyacrylamide gels. The lane designated 100+ contained
conditioned medium from cells cultured in the presence of 100 µg/ml
of vitronectin and 500 µg/ml of RGDS.
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Fig. 2.
MMP and TIMP activity in B16F10-conditioned
medium. B16F10 cells (1 × 106) were cultured for
18 h in serum-free medium containing 0 (lane 1), 100 (lane 2), 200 (lane 3), or 300 (lane
4) µg/ml vitronectin, and conditioned media were analyzed for
MMP activity by gelatin zymography on 9% SDS-polyacrylamide gels
(A) and for TIMP activity by reverse zymography on 15%
SDS-polyacrylamide gels (B and C). Samples in
C were subjected to reductive carboxymethylation to inactivate TIMPs as described under "Experimental Procedures" prior
to reverse zymography. Lane S, MMP-2 standard.
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Fig. 3.
Effect of vitronectin on Matrigel
invasion. B16F1 or B16F10 cells (1 × 105) were
added to 8-µm pore size polycarbonate filters coated with Matrigel
basement membrane extract as described under "Experimental Procedures." Following incubation for either 8 (B16F10) or 17 (B16F1)
h, membranes were removed and stained, and the invading cells were
enumerated. Open bars, control B16F1 (designated
F1( )) or B16F10 (designated F10( ));
solid bars, B16F1 (designated F1(+)) or B16F10
(designated F10(+)) in the presence of 100 µg/ml
vitronectin. Hatched bar (designated F1(*)),
B16F1 cells in the presence of 100 µg/ml V8-degraded vitronectin.
Experiments were performed in triplicate, and error bars
represent S.D.
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In addition to metalloproteinases, B16 melanoma cells also secrete the
serine proteinase tissue-type plasminogen activator, which converts the
plasma zymogen plasminogen to the active proteinase plasmin (13).
Plasmin is a broad spectrum serine proteinase that degrades numerous
extracellular matrix proteins, including vitronectin (19). To determine
the effect of plasmin-degraded vitronectin on melanoma cell MMP-2
secretion, vitronectin was subjected to limited proteolysis with
plasmin (Fig. 4C, lane 2) prior to incubation with melanoma cells. In contrast to results observed with intact vitronectin (Fig. 4A, lane 2),
plasmin-degraded vitronectin did not induce MMP-2 secretion (Fig.
4A, lane 3). Decreasing the extent of proteolysis did not
reinstate the stimulatory effect (data not shown). Because plasmin has
trypsin-like specificity and therefore may cleave within the RGD site
utilized by melanoma cells for integrin-mediated vitronectin binding,
limited proteolysis of vitronectin with endoproteinase V8 (which has
Glu specificity (20)) was also performed (Fig. 4C, lane 4).
Similar to results obtained with plasmin, endoproteinase V8-cleaved
vitronectin also failed to induce melanoma cell MMP-2 production (Fig.
4B, lane 3). Furthermore, Matrigel invasion was not
stimulated by endoproteinase V8-cleaved vitronectin (Fig. 3,
hatched bar). To determine whether MMP-2 itself may initiate
vitronectin degradation, vitronectin was incubated with APMA-activated
MMP-2 for 18 h at 37 °C at a 1:20 MMP-2:vitronectin ratio and
analyzed by electrophoresis on a 5-15% SDS-polyacrylamide gel. No
change in the electrophoretic migration of MMP-2-treated vitronectin
was observed (Fig. 4D, lane 2), demonstrating that
vitronectin is not susceptible to proteolysis by MMP-2.

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Fig. 4.
Limited proteolysis of vitronectin.
Vitronectin (200 µg) was subjected to limited proteolysis with
plasmin (A and C) or endoproteinase V8
(B and C) and incubated with B16F10 cells (1 × 106) in serum-free medium (1 ml) for 18 h.
Conditioned media were analyzed for MMP activity by gelatin zymography
on 9% SDS-polyacrylamide gels. A, lane 1, cells only;
lane 2, cells + intact vitronectin; lane 3, cells + plasmin-degraded vitronectin; lane 4, MMP-2 standard. B, lane 1, cells only; lane 2, cells + intact
vitronectin; lane 3, cells + endoproteinase V8-degraded
vitronectin; lane 4, MMP-2 standard. Panel C,
control showing limited proteolysis of vitronectin. Vitronectin was
incubated with plasmin (37 °C for 30 min) (lane 2) or
endoproteinase V8 (37 °C for 2 h) (lane 4) as
described under "Experimental Procedures," and reaction products
were analyzed by electrophoresis on 5-15% gradient SDS-polyacrylamide
gels and stained with Coomassie Blue. Lanes 1 and
3, intact vitronectin (20 µg); lane 2,
plasmin-degraded vitronectin (20 µg); lane 4, endoproteinase V8-degraded vitronectin (20 µg). D, effect
of MMP-2 on vitronectin. Vitronectin (20 µg) was incubated with
purified APMA-activated MMP-2 (1 µg) for 18 h at 37 °C.
Reaction products were analyzed by electrophoresis on 5-15% gradient
gels and stained with Coomassie Blue. Lane 1, vitronectin;
lane 2, vitronectin + MMP-2.
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To analyze further the effect of ligating the melanoma cell vitronectin
receptor on MMP-2 secretion, the peptide RGDS, which interacts with
both the
v
3 and
v
5 vitronectin receptors (12), was
utilized. Control experiments demonstrated binding of RGDS to
vitronectin receptors on B16F1 and B16F10 cells, as evidenced by the
ability of the peptide to inhibit melanoma cell adhesion to vitronectin
in a concentration-dependent manner (Table
I). Incubation of either B16F1 or B16F10
cells with up to 1 mg/ml of RGDS or RGES did not induce MMP-2 secretion
(data not shown). However, simultaneous addition of vitronectin and
RGDS inhibited vitronectin-induced MMP-2 secretion as shown in Fig. 1
(lane designated 100+). Furthermore, inhibition of
vitronectin-enhanced Matrigel invasion was 50% greater using RGDS
peptide as compared with RGES (Fig. 5),
further demonstrating that interaction of the RGD sequence of
vitronectin with a cellular integrin(s) is necessary for MMP-2 induction.
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Table I
Effect of RGDS on adhesion of B16F1 and B16F10 cells to vitronectin
Increasing concentrations of RGDS were added to melanoma cells, and
adhesion to vitronectin-coated wells was quantitated as described under
"Experimental Procedures." Results are expressed relative to wells
containing no RGDS.
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Fig. 5.
Effect of RGD peptides on
vitronectin-enhanced Matrigel invasion. B16F1 cells (1 × 105) were added to 8-µm pore size polycarbonate filters
coated with Matrigel in the presence (hatched bars) or
absence (open bars) of 100 µg/ml vitronectin. RGDS or RGES
(100 µg/ml) was added to control or vitronectin-containing samples as
indicated. Following incubation for 23 h, membranes were removed
and stained, and invading cells were enumerated. Experiments were
performed in triplicate, and error bars reflect S.D.
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Because recent data indicate that MMP activity may be regulated
posttranslationally by interaction with the cell surface (17, 22-27),
the ability of MMP-2 to associate with the plasma membrane of B16F1 or
B16F10 cells was determined. Analysis of MMP-2 binding to the membranes
of intact melanoma cells was assessed using 125I-labeled
proMMP-2 or proMMP-2/TIMP-2. Attempts to determine an equilibrium
dissociation constant for binding of either proMMP-2 or proMMP-2/TIMP-2
to intact cells were unsuccessful because saturation of binding sites
was not achieved in the presence of 125I-labeled ligand
concentrations up to 500 nM (data not shown). These data
suggest that proMMP-2 and/or proMMP-2/TIMP-2 interacts with a prevalent
cell-associated protein such as collagen or fibronectin (25-26) or
that binding is mediated by a receptor present in low abundance such
that specific binding is obscured by nonspecific interactions. To
determine whether proMMP-2 or proMMP-2/TIMP-2 may associate with
vitronectin binding integrins, binding experiments were performed in
the presence of RGDS. No significant change in the amount of bound
proMMP-2 or proMMP-2/TIMP-2 was observed in the presence of up to 1 mg/ml (2.3 mM) RGDS or control peptide RGES (Table
II), suggesting that interaction of
proMMP-2 and proMMP-2/TIMP-2 with intact murine melanoma cells is not
mediated by vitronectin-binding integrins. This was confirmed by
adhesion experiments that demonstrated that MMP-2, whether in proenzyme
or active form, was unable to inhibit melanoma cell adhesion to
vitronectin (Table III).
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Table II
Effect of RGDS on binding of proMMP-2 and proMMP-2/TIMP-2 to B16F10
cells
125I-labeled proMMP-2 or proMMP-2/TIMP-2 (100 nM)
was added to wells containing 5 × 104 B16F10 cells in the
presence of increasing concentrations of RGDS or RGES peptide. Bound
ligand was determined as described under "Experimental Procedures."
Results are expressed as % bound relative to control wells containing
no added peptide (designated 100%). ProMMP-2 binding was analyzed in
triplicate, whereas proMMP-2/TIMP-2 data are the average of duplicate
experiments.
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Table III
Effect of MMP-2 on melanoma cell adhesion to vitronectin
B16F10 cells (1.5 × 105) were added to 24-well culture
plates coated with vitronectin in the presence or absence of pro- or active MMP-2, as indicated. After incubation for 40 min at 37 °C, plates were washed with PBS and fixed, and bound cells were enumerated using an ocular micrometer. Experiments were performed in triplicate, and S.D. is indicated.
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In addition to binding to intact cells, exogenous proMMP-2 (data not
shown) and proMMP-2/TIMP-2 bound to isolated plasma membranes from both
B16F1 and B16F10 cells and retained hydrolytic activity against a
macromolecular gelatin substrate (Fig. 6,
A and B). Gelatinolytic activity was not detected
in either clone of B16 plasma membranes prior to the addition of
exogenous proMMP-2 or proMMP-2/TIMP-2 (Fig. 6), which is consistent
with the lack of MMP-2 secretion observed in unstimulated cells (Figs.
1 and 2). The membrane-associated gelatinolytic activity was fully
inhibited by addition of o-phenanthroline (Fig.
6B). Furthermore, membrane association enhanced the
catalytic activity of activated MMP-2/TIMP-2. Incubation of
MMP-2/TIMP-2 with either plasma membranes or detergent soluble extracts
from B16F1 and B16F10 cells resulted in a 33-76% increase in
gelatinolytic activity (Fig. 6C), suggesting that membrane
association may function as a mechanism for posttranslational regulation of MMP-2/TIMP-2 activity in melanoma cells. To assess the
potential functional consequence of MMP-2 cellular association on
invasive behavior, cells were incubated with 50 nM MMP-2 in serum-free culture medium containing 3% bovine serum albumin, washed
to remove unbound MMP-2, and analyzed in a Matrigel invasion assay. A
3-fold increase in invasiveness was observed in cells containing bound
MMP-2 relative to controls (Table IV),
demonstrating that cellular association of MMP-2 contributes
functionally to enhanced invasiveness.

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Fig. 6.
MMP-2 association with melanoma cell plasma
membranes. A, B16F1 (2 × 108) and B16F10
(4 × 108) cells were fractionated as described under
"Experimental Procedures," and plasma membranes (P.M.)
were incubated for 2 h at 37 °C in the absence
(control) or presence (+MMP-2) of purified
proMMP-2/TIMP-2 (100 nM). Membranes were washed to remove
unbound complex, treated with APMA, and analyzed by gelatin zymography
to detect bound enzyme. B, plasma membranes (mem)
from B16F1 or B16F10 cells were incubated in the presence (+) or
absence ( ) of proMMP-2/TIMP-2 as indicated, and bound enzyme was
analyzed by incubating membrane aliquots with 14C-gelatin
in Tris-Ca-sucrose containing 50 mM NaCl and 0.5 mM APMA in the presence (solid bars) or absence
(hatched bars) of 1 mM
o-phenanthroline. Reactions were terminated by
trichloroacetic acid precipitation, and soluble radioactivity was
determined by scintillation counting. Experiments were performed in
quadruplicate, and error bars represent S.D. C, effect of membrane association on
MMP-2 catalytic activity. 14C-gelatin cleavage by
APMA-activated MMP-2/TIMP-2 (10 nM) was determined in the
absence of membranes or in the presence of B16 plasma membranes (1 µg) (open bars) or Triton X-100 detergent extracts (100 ng) (hatched bars). Data are presented as the percent increase in gelatin hydrolysis relative to the activity of control MMP-2/TIMP-2 determined in the absence of membranes. Experiments were
performed in triplicate, and error bars represent S.D. In additional controls, the membrane preparations were incubated with the
gelatin substrate in the absence of enzyme (data not shown).
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Table IV
Effect of cell-associated MMP-2 on Matrigel invasion
B16F1 cells were incubated for 90 min in serum-free medium with 3% BSA
containing 50 nM proMMP-2 and washed twice with PBS to
remove unbound proMMP-2, and cells (1 × 105) were added
to 8 µm pore size polycarbonate filters coated with Matrigel.
Following incubation for 17 h, membranes were removed and stained,
and invading cells were enumerated.
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DISCUSSION |
The initial site of melanoma cell metastasis in vivo is
frequently the regional lymph nodes, and the appearance of lymph node metastases is correlated with poor prognosis (3). Studies of experimental metastasis using B16F1 and B16F10 cells demonstrated similar incidence of lymph node metastases, although animals injected with B16F10 cells were more likely to develop pulmonary metastases (28). Expression of the vitronectin-binding integrin
v
3 is enhanced in metastatic melanoma cells relative to parental
nonmetastatic variants and recent experiments have demonstrated that
adhesion of metastatic melanoma cells to lymph node sections is blocked by either anti-
v
3 or RGD-containing peptides (3). In related experiments, ligation of melanoma cell
v
3 using an anti-integrin antibody was shown to enhance cellular invasiveness in
vitro, and conditioned medium from cells treated with
anti-
v
3 displayed increased MMP-2 activity (7). This is
particularly interesting in light of previous data that indicate that
MMP-2 expression by melanoma cells correlates with increased
invasiveness, and it provides a biochemical mechanism whereby invasion
may be enhanced (29-31).
These observations are supported by data from the present study that
demonstrate a direct dose-dependent increase in MMP-2 secretion in vitronectin-treated B16F1 and B16F10 melanoma cells. As a
functional consequence of increased MMP-2 levels, cellular invasiveness
is also enhanced. The vitronectin-induced increase in MMP-2 secretion
and invasive activity was abolished by RGDS peptide, providing evidence
that vitronectin interaction with cellular integrins regulates invasive
behavior. Concomitant with MMP-2 secretion, levels of TIMP-2 were also
increased by vitronectin treatment. Previous studies have shown that
MMP-2 is secreted as a proenzyme in complex with TIMP-2 by melanoma
cells and other cell types, and additional reports suggest that the
presence of TIMP-2 in this proenzyme-inhibitor complex is required for
cellular activation of the MMP-2 zymogen by membrane-type MMPs (24,
32-36).
Recent evidence indicates that cell surface association may function as
a mechanism for posttranslational regulation of MMP activity (17,
21-26). The present data demonstrate that MMP-2/TIMP-2 associates with
the plasma membrane fraction of murine melanoma cells and exhibits
enhanced catalytic activity against macromolecular substrates relative
to the solution phase enzyme supporting the role of membrane
association in MMP regulation. The mechanism of interaction of proMMP-2
and proMMP-2/TIMP-2 with B16F1 and B16F10 cells is currently unknown.
However, it has recently been reported that recombinant chick MMP-2
binds to hamster melanoma cells transfected with the
3 integrin
subunit via a direct interaction with
v
3, which is inhibited by
RGD peptides, as well as to purified
v
3 immobilized on microtiter
wells (37). This is in contrast to the results of the present study,
which show that binding of human proMMP-2 and proMMP-2/TIMP-2 to intact
murine melanoma cells is not RGD-mediated. This is supported by the
observation that MMP-2 secretion by vitronectin-treated cells was
inhibited by colchicine, demonstrating that the enhanced proteinase
levels observed were not due simply to displacement of MMP-2 by
vitronectin from a common cell surface receptor. Furthermore, neither
the peptide RGDS nor endoproteinase V8-treated vitronectin, both of which bind the cellular vitronectin receptor, increased the level of
MMP-2 in conditioned medium. In addition, neither pro- nor active MMP-2
inhibited cell adhesion to vitronectin. The discrepancy between results
reported in the current study and those of Brooks et al.
(37) may reflect differences in the distinct model systems employed.
Alternatively, differences in the activation state of the enzymes may
also influence cellular association because proteolytic activity may
alter (or confer) cell binding ability.
The complex array of functions attributed to integrins, including
cell-matrix adhesion, cytoskeletal organization, and signal transduction, results from a cellular ability to discriminate functionally between transmembrane signals induced by simple ligand occupancy, receptor aggregation, or simultaneous occupancy and aggregation (38). In the present study MMP-2 secretion was induced by
intact vitronectin in both B16F1 and B16F10 cells; however, limited
proteolysis of vitronectin removed the stimulatory effect, regardless
of whether the RGD site was disrupted (plasmin) or remained intact
(endoproteinase V8). Furthermore, simple ligation of the vitronectin
receptor with the peptide RGDS also failed to induce MMP-2 activity at
concentrations well in excess of that required for inhibition of cell
adhesion. However, antibody ligation of melanoma cell
v
3, which
can induce receptor aggregation, stimulated MMP-2 production (7).
Together, these data indicate that a multivalent ligand-receptor
interaction, rather than simple ligand occupancy, is required for
induction of MMP-2. In light of these results, it is interesting to
consider recent biophysical data that demonstrate that under
physiologic conditions, vitronectin can exist in both monomeric and
multimeric forms (39). Vitronectin in extravascular sites
(i.e. matrix and tissue-associated) is predominantly in the
multimeric form, suggesting that multivalent ligand-receptor
interactions may prevail in vivo (39, 40). Furthermore, the
current data suggest a biologic control mechanism whereby the stimulus
for MMP-2 induction, i.e. intact vitronectin, may be
removed. It is interesting to speculate that vitronectin-adherent melanoma cells, which also catalyze tissue-type plasminogen
activator-mediated plasmin generation (13), may initiate
plasmin-dependent proteolysis of vitronectin, thereby disrupting the
multivalent signal necessary for MMP-2 induction.
We thank Dr. C. N. Rao (Department of
Dermatology, Northwestern University) for assistance with the in
vitro invasion assay.