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INTRODUCTION |
During metastasis, invasive cells must traverse tissue barriers
comprised largely of type I collagen. This process depends on the
ability of tumor cells to degrade the surrounding collagen matrix and
then migrate through the matrix defects (1-3). The actin dynamics
regulated by Rho family of small GTPases play a critical role in cell
migration (4). The initiation of cell migration is characterized by
actin polymerization at the leading edge and extension of a lamella in
the direction of motion. Rac1, a member of the Rho family proteins that
regulates the assembly of a meshwork of actin filaments at the cell
periphery to produce lamellipodia (5), has been implicated in oncogenic
transformation (6-8) and metastasis induction (9). Recent studies have
demonstrated the direct role of Rac1 activity in cell invasiveness in
type I collagen matrix (10, 11). Transfection of non-invasive mammalian epithelial cells T47D with active Rac1 induces cell invasion through type I collagen (10). Similarly, overexpression of adaptor proteins p130Crk-associated substrate
(CAS)1/c-CrkII (Crk) induces
Rac1-dependent COS-7 cell invasiveness in three-dimensional
collagen (3D-col) culture (11). However, the proteolytic activities
during Rac1-promoted cell invasion through type I collagen barriers
remain undefined.
Increasing evidence has suggested that proteolytic activities at cell
surface promote cell invasion (12). Matrix metalloproteinase (MMP)-2
(type IV collagenase; gelatinase A) is a cell surface-associated type I
collagen-degrading MMP (13, 14). Overexpressed in different types of
tumor (15), MMP-2 is involved in tumor metastasis, primary tumor
growth, and angiogenesis (2, 16-19). Although considerable attention
has been focused on the role of MMP-2 as a type IV collagenase in cell
invasion across basement membrane, recent evidence has implicated MMP-2
in type I collagen remodeling by tumor cells (20) and the tubular
organization of endothelial cells in 3D-col (21). In common with all
MMPs, MMP-2 is synthesized and secreted as a latent precursor,
requiring proteolytic removal of the propeptide for activation. The
physiological mechanism that accounts for MMP-2 activation is under
intense study. It is suggested that MMP-2 is activated in a
membrane-associated mechanism after a two-step process that involves an
initial cleavage of the zymogen followed by an autocatalytic conversion
of the intermediate into a fully active enzyme (22-25). MT1-MMP, the
best characterized member of MT-MMPs, is believed to carry out the initial cleavage of MMP-2 after the binding of MMP-2 proenzyme to an
MT1-MMP·TIMP-2 complex at the cell surface (23, 26, 27).
Interestingly, MT1-MMP becomes degraded to an inactive 43-kDa form
during the MMP-2 activation (28-31). MT1-MMP is also overexpressed in
several tumor tissues where activated MMP-2 is found (19). In addition
to its ability to activate MMP-2, MT1-MMP has intrinsic ECM degrading
activity (32-36). Therefore MT1-MMP and MMP-2 activities at the cell
surface provide a powerful combination for the localized ECM remodeling
(33, 37).
To evaluate the hypothesis that MT1-MMP/MMP-2 proteolytic cascade might
play a functional role in Rac1-induced tumor cell invasion through type
I collagen-rich tissue barrier, we examined an invasive HT1080
fibrosarcoma cell line that showed elevated level of active MMP-2
during cell-fibrillar collagen interaction (38). In this report, we
provide evidence that Rac1 is a mediator of collagen-stimulated MMP-2
activation and MT1-MMP expression/processing, collagenolytic activity,
and cell invasion through 3D-col. Furthermore, active MMP-2 contributes
to Rac1-induced collagen invasive activity. Our findings suggest that
Rac1 mediates MMP-2 activation and MT1-MMP expression/processing during
the encounter between invading tumor cells and type I collagen-rich
stroma, thereby facilitating collagenolysis and cell invasion.
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MATERIALS AND METHODS |
Cell Culture and Plasmids--
HT1080 fibrosarcoma and HEp3
epidermoid carcinoma cell lines were maintained in DMEM containing 10%
heat-inactivated fetal bovine serum (Hyclone) supplemented with
2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin and streptomycin (Life Technologies, Gaithersburg,
MD). Stably transfected cell lines were maintained in medium that
included 200 µg/ml G418 in addition to the above mentioned
supplements. Plasmids RSV-neo-
-galactosidase, c-Myc-tagged
Rac1V12N17, and Rac1V12 were kindly provided by Dr. Lorne Taichman,
SUNY at Stony Brook, and Dr. Alan Hall, University College London,
London, United Kingdom. Plasmids containing cDNAs for MT1-MMP,
MMP-2, TIMP-2, and 36B4 were purchased from ATCC.
Stable Transfection of HT1080 Cell Line--
Subconfluent cell
culture was co-transfected with one of testing plasmids (Rac1V12N17,
Rac1V12, and vector) and a plasmid construct containing the
neomycin-resistant gene by the calcium phosphate precipitation
technique as previously described (39). After transfection, individual
colonies from Rac1V12N17- and Rac1V12-transfected cells were isolated
after 2-3 weeks of 500 µg/ml G418 selection. As control,
vector-transfected clones were pooled. All stably transfected cell
lines were maintained in growth medium containing 200 µg/ml G418.
Treatment of Cells with Concanavalin A--
Cells grown on
tissue culture plates in growth medium (10% heat-inactivated fetal
bovine serum/DMEM) were washed twice with serum-free medium before the
addition of serum-free medium supplemented with 0.2% heat-inactivated
lactoalbumin and 50 µg/ml concanavalin A (Sigma). Medium was then
collected after 16-24 h for zymography.
Three-dimensional Collagen and Fibrin Cell Culture--
Type I
collagen or fibrin gel cultures were prepared according to a procedure
previously described with some modification (40). Pepsin-solubilized
bovine dermal collagen dissolved in 0.012 M HCl was 99.9%
pure containing 95-98% type I collagen and 2-5% type III collagen
(Vitrogen 100, Collagen Corp). Briefly, cells detached from tissue
culture plates in growth medium were washed with warm DMEM twice before
being seeded into a serum-free solution that contained 2 mg/ml vitrogen
or 3 mg/ml fibrinogen (Calbiochem-Novabiochem). Subsequently,
cell-collagen or cell-fibrinogen suspension (5 × 105
cells/ml) was plated onto 24-well plates at 250 µl/well or 35-mm plastic dishes at 1.5 ml/dish. Cell-collagen cultures were incubated at
37 °C to form gel. The gelling of cell-fibrin cultures occurred in
less than 5 min at room temperature after the addition of thrombin to a
final concentration of 0.2 unit/ml. After both collagen and fibrin cell
cultures formed gel, serum-free DMEM supplemented with 0.2%
heat-inactivated lactoalbumin was added. Gels remained attached to the
plastic dish for the duration of incubation.
Substrate Zymography--
Conditioned medium was collected from
cells cultured for 18-24 h in serum-free medium. To analyze the
activity of cell-associated MMP-2, cells (7.5 × 105)
in 35-mm plates were released from collagen or fibrin gel after digestion with bacterial collagenase D (Roche Molecular Biochemicals, Indianapolis, IN) for 10 min or dispase (Becton Dickinson, Bedford, MA)
for 5 min, respectively. Cells were washed gently with ice-cold phosphate-buffered saline 10-12 times and suspended in 150 µl of
1 × SDS sample buffer. Aliquots (total cell lysates) were
immediately processed for enzymatic assay using zymography. Substrate
zymography was performed as described previously with some
modifications (41, 42). SDS-polyacrylamide (12% unless indicated in
the figure legend) gels were co-polymerized with 1 mg/ml gelatin or 0.5 mg/ml type I collagen (Sigma). Samples (conditioned medium or total
cell lysates) were resolved under nonreducing conditions. Gels were
washed twice in 2.5% Triton X-100 for 30 min and incubated overnight
in a buffer containing 50 mM Tris-HCl, pH 7.5, 5 mM CaCl2, and 0.02% NaN3 (gelatin
substrate) or 100 mM Tris-HCl, pH 8.0, 5 mM
CaCl2, 0.005% Brij-35, and 0.02% NaN3 (type I
collagen substrate). At the end of the incubation, gels were stained
with Coomassie Blue and destained.
Staining of Actin Cytoskeleton--
Actin organization was
visualized by staining with Texas Red-conjugated phalloidin (Molecular
Probes, Eugene, OR). Cells in serum-free vitrogen solution were plated
in 8-well chamber slides to form gel. After 24 h, cells in
collagen gel were fixed in 4% paraformaldehyde, permeabilized with
acetone at
20 °C, and stained with Texas Red-conjugated phalloidin
for 30 min at room temperature. The images were captured using an
epifluorescence microscope by the University Microscopy Imaging Center,
SUNY at Stony Brook.
Northern Blot Analysis--
Total RNA was isolated from cells
cultured in collagen or fibrin gel for 10 h and Northern analysis
was performed as previously described (40). RNA was detected with
-32P-labeled cDNA probes for MT1-MMP and TIMP-2.
Control probe was 36B4 cDNA.
Western Blot Analysis--
Cell extracts were prepared using a
detergent extraction method described by Lee et al. (30)
with a few modifications. Briefly, cells cultured in collagen or fibrin
gel were washed gently with cold phosphate-buffered saline three times,
transferred into a 1.5-ml microcentrifuge tube, and subject to
centrifugation to remove as much solution as possible. The pellet was
suspended in a Triton X-100 lysis buffer (2% Triton X-100 in 100 mM Tris buffer, pH 7.5, and 150 mM NaCl in the
presence of protein inhibitors: 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and forced
through a syringe with a 26-gauge needle several times. The supernatant
after centrifugation was designated as cell extracts for Western
analysis. Western blotting analysis was performed as previously
described (43). After detection by monoclonal antibodies against Rac1
(Upstate Biotechnology, Lake Placid, NY), MMP-2 (Chemicon, Temecula,
CA), TIMP-2, and MT1-MMP (Calbiochem-Novabiochem), the blots were
visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).
Collagen Fibril Dissolution--
The capacity of cells to
degrade type I collagen fibrils was assessed based on the modification
of a procedure as described (35). Briefly, 300 µl of vitrogen
solution at 1 mg/ml was added to each well of a 24-well plate and
allowed to air-dry overnight. The collagen fibril film was washed with
several changes of distilled water and serum-free DMEM before a pellet
of 4 × 104 cells in 25 µl of 10% fetal bovine
serum/DMEM was dotted onto the center of each well. Cells were allowed
to attach for 5 h, washed twice with serum-free DMEM, and then
incubated for 3 days in serum-free DMEM supplemented with 0.2%
lactoalbumin. Following the incubation, cells were removed with
trypsin/EDTA and collagen remaining in the wells was visualized by
staining with Coomassie Blue.
For quantification of fibrillar type I collagen degradation, type I rat
tail collagen (Becton Dickinson) was labeled with [3H]acetic anhydride (Amersham Pharmacia Biotech)
according to the protocols described (44, 45). 150 µl of
3H-labeled type I collagen (0.5-1 × 106
cpm/mg protein) was allowed to polymerize in individual wells of a
48-well plate. Cells at 1.5 × 104/300 µl were added
to each well and incubated at 37 °C. To follow the progressive
degradation of collagen fibrils, aliquots (50 µl) of the medium were
collected after 24 and 48 h. The soluble radioactivity was
quantified in a liquid scintillation counter.
Expression and Preparation of MMP-2 Domains--
A cDNA
clone K-121 that contains the coding sequence for MMP-2 with a
partially truncated propeptide domain (46) was used as a template for
amplification with polymerase chain reaction. The regions corresponding
to residues Gly417-Cys631 (C-terminal
hemopexin-like domain; CTD) or Val191-Gln364
(fibronectin type-II-like modules; CBD) of MMP-2 proenzyme,
respectively, were amplified. The resulting fragments were inserted
into a pGEX-3X plasmid, respectively (Amersham Pharmacia Biotech,
Uppsala, Sweden). The glutathione S-transferase fusion
proteins were purified on Sepharose 4B-coupled glutathione beads
(Amersham Pharmacia Biotech) based on the manufacturer's instructions.
Collagen Invasion Assay--
The assay was performed using the
modification of procedures previously described (47). 50 µl of
vitrogen solution at 1 mg/ml was applied to the upper compartment of
each well in a 24-well Transwell plate (8-µm pore size; Costar) and
allowed to gel at 37 °C. Cells at 5 × 104 in 200 µl of serum-free DMEM were added to the upper chamber. Culture medium
was added to the lower compartment. In indicated experiments,
inhibitors or controls were added to both upper and lower chambers at
the following final concentrations: 25 µM SC68180 (formerly SC44463,
N-[3-(N'-hydroxycarboxamido)-2-(2-methylpropyl)propanoyl]-O-methyl-L-tyrosine-N-methylamide; a generous gift of Dr. W. C. Parks, Washington University, St. Louis), 100 µg/ml aprotinin (Sigma), 500 ng/ml recombinant TIMP-1 (Chemicon), 500 ng/ml recombinant TIMP-2 (Chemicon), 100 µM furin inhibitor Dec-Arg-Val-Lys-Arg-CH2Cl
(Bachem Biochemicals), 500 ng/ml CTD
(Gly417-Cys631), and 500 ng/ml CBD
(Val191-Gln364). The invasion proceeded for
24 h at 37 °C. After incubation, the filters were fixed and
stained with Diff-Quick staining kit (Fisher Scientific). The cells
that reached the underside of the filter were counted. For each filter,
the number of cells in 10 randomly chosen microscope fields was
determined and averaged. Three invasion chambers were used per
condition. The final values were the average of triplicates.
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RESULTS |
HT1080 Cells Stably Expressing Rac1 Mutants Exhibit Distinct
Morphology and Invasive Properties in Three-dimensional Collagen
Gel--
A human fibrosarcoma cell line, HT1080, was transfected with
dominant negative Rac1V12N17, constitutively active Rac1V12, or a
vector control. Ectopic Rac1 expression was examined by Western analysis in G418-resistant clones. In a representative experiment, a
monoclonal antibody against Rac1 detected both ectopic Rac1 tagged with
c-Myc (upper band) and endogenous Rac1 (lower band) in lysates of cells
expressing Rac1V12N17 (HN) and Rac1V12 (HV), but only endogenous Rac1
in vector-transfected cell extracts (HW) (Fig.
1A). Clones that express
comparable levels of Rac1 were selected for further study.

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Fig. 1.
Stable expression of dominant negative and
active Rac1 changes morphology, organization, and invasiveness of
HT1080 fibrosarcoma cells in three-dimensional collagen
matrix. A, HT1080 cells stably expressing vector
only (HW), Myc-tagged Rac1V12N17 (HN), and
Myc-tagged Rac1V12 (HV) were examined for expression of Rac1
protein. Lower band corresponds to endogenous Rac1 that
migrates faster than tagged ectopic Rac1 (upper band).
B, three HT1080-derived cell lines were cultured in 3D-col
in serum-free medium for 1 day. Changes in cell morphology were
visualized with epifluorescence microscope viewing of rhodamine
phalloidin staining (a-c) and with phase-contrast microscope
(d-f). a and d, HW cells; b
and e, HN cells; c and f, HV cells.
C, three HT1080-derived cell lines were added to the upper
compartments of type I collagen invasion chamber. After 24 h cells
that invaded to the underside of the filters were counted. Results
shown are the mean of cell number in 10 randomly selected fields in a
single representative experiment of six performed. D, the
invasion of three HT1080-derived cell lines through 3D-col was examined
in the presence of either 25 µM SC68180, a general
inhibitor of MMP, or 100 µg/ml aprotinin, an inhibitor of PA/plasmin
system. The number of invasive HW cells in the absence of inhibitors
was set as 100%. The results are representative of four independent
experiments.
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Cells were embedded in 3D-col in serum-free medium for 24 h and
subject to morphological examination. To view single cells in type I
collagen matrix, the entire cell-containing 3D-col was stained with
rhodamine phalloidin. As shown in Fig. 1B, HW and HV cells
developed long protrusions extending from cell body, whereas HN cells
possessed a compact morphology (Fig. 1B, panels a-c).
Interestingly, HV cells displayed multiple membrane protrusions (Fig.
1B, panel c). Cell organization in 3D-col was visualized by
phase-contrast microscopy (Fig. 1B, panels d-f).
Consistent with the single cell morphology, HV cells were
assembled into complex branching tubular networks (Fig. 1B, panel
f). In contrast, HN cells failed to demonstrate organized
structure (Fig. 1B, panel e).
Because the branching phenotype of carcinoma cells has been associated
with their metastatic capacity (47), whether Rac1 mediates HT1080 cell
invasion was assessed. Rac1V12 substantially increased, whereas
Rac1V12N17 reduced, HT1080 cell invasion across 3D-col (Fig.
1C). To assess whether MMPs or plasminogen activator (PA)/plasmin system is responsible for this process, we monitored cell
invasion in the presence of inhibitors for MMPs and PA/plasmin system,
SC68180 and aprotinin, respectively. SC68180, formally known as
SC44463, is a hydroxymate compound that has been shown to inhibit human
keratinocyte migration on native type I collagen as a general MMP
inhibitor (48). As shown in Fig. 1D, invasion of HW and HV
cells through 3D-col was inhibited by SC68180. Interestingly, the
active Rac1-induced cell invasion was reduced ~90% by the MMP
inhibitor. In contrast, aprotinin did not impact on the collagen invasiveness. These data indicate that invasion of HT1080 cells across
collagen depends on both Rac1 and MMP activities and that Rac1 requires
MMP activities to promote cell invasion.
MMP-2 Is Differentially Activated in HT1080 Cells Stably Expressing
Rac1 Mutants Cultured in 3D-col--
To investigate the Rac1-mediated
MMP activity in HT1080 cells surrounded by collagen matrix, we
monitored MMP production by gelatin zymography in these cell lines when
cultured in 3D-col for 24 h (Fig.
2A). The conditioned medium of
HEp3, an epidermoid carcinoma cell line that secretes MMP-1, MMP-2, and
MMP-9 as detected by gelatin zymography (49), was used as a control
(Fig. 2A, lane 1). Four major gelatinolytic bands with
different intensity were detected in the serum-free medium of HW cell
culture (Fig. 2A, lane 2). These bands corresponded to the
inactive MMP-9 proenzyme (92 kDa) and three MMP-2 species, latent (L),
intermediate (i), and active (A). Interstitial collagenase MMP-1 was
undetectable in HT1080-derived cell lines in gelatin (Fig.
2A) as well as type I collagen zymography (Fig.
2B), a method that more sensitively detects MMP-1 than does
MMP-2 (42). The distribution of three MMP-2 species was distinctly
altered by the stable expression of Rac1V12N17 (HN) and Rac1V12 (HV).
Active and latent MMP-2 became the major species in HV and HN cells,
respectively (Fig. 2A, lanes 2-4), suggesting Rac1
specifically mediates MMP-2 proenzyme processing cells cultured in
3D-col. Active MMP-9 was not detected in this system; instead, latent
MMP-9 at 92 kDa was detected at low level in both HW and HV cells, but
nearly undetectable in HN cells (Fig. 2A).

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Fig. 2.
Stable expression of dominant negative and
active Rac1 in HT1080 fibrosarcoma cells mediates MMP-2 activation in
three-dimensional collagen matrix. Conditioned medium of cells
cultured in three-dimensional collagen or fibrin was analyzed by
zymography as described under "Materials and Methods."
A, gelatin zymography. Lane 1, epidermoid
carcinoma cell lines HEp3 in 3D-col; lanes 2-4,
HT1080-derived cell lines in 3D-col; lanes 5-7,
HT1080-derived cell lines in 3D-fibrin; lane 8, purified
MMP-2 standard. A top gelatenolytic band as marked with the
asterisk was considered the MMP-2 dimer by the supplier
(Chemicon). The results are representative of six individual
experiments. L, latent; I, intermediate;
A, active. B, type I collagen zymography.
Lanes 1-3, HT1080-derived cell lines in 3D-col; lanes
4-6, HT1080-derived cell lines in 3D-fibrin; lane 7,
purified MMP-2 standard. C, serum-free medium of cells grown
on tissue culture plastic plates in the presence or absence of 50 µg/ml ConA was analyzed by gelatin zymography. Results are
representative of three individual experiments.
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Cells cultured in fibrin gel were used as control. Unlike 3D-col that
induced MMP-2 processing, fibrin gel did not promote MMP-2 activation
(Fig. 2A, compare lanes 2 and 5).
Predominantly latent MMP-2, as well as low level of the intermediate
form, were detected in HW and HN cells in fibrin gel (Fig. 2A,
lanes 5 and 6). However, Rac1V12 induced MMP-2
activation by cells cultured in fibrin gel (Fig. 2A, compare
lanes 5 and 7). Similar MMP-2 activation pattern
was also observed using type I collagen zymography (Fig.
2B). Taken together, these results suggest that Rac1 is required for maximal activation of MMP-2 by 3D-col and that active Rac1
is sufficient to induce MMP-2 processing in the absence of collagen signal.
To determine the specificity of Rac1 in mediating collagen-induced
MMP-2 activation, MMP-2 activating signals that are independent of Rac1
activity were sought. Concanavalin A (ConA) is known to activate MMP-2
(50). Stimulation of Rac1V12- or Rac1V12N17-transfected cells with ConA
had similar effects on MMP-2 activation (Fig. 2C),
suggesting that Rac1 is specifically involved in MMP-2 activation in
response to type I collagen stimulation. Latent MMP-9 level remained
similar in all three cell lines under this culture condition (Fig.
2C).
Rac1 Mediates Cell-associated Activation of MMP-2 by Type I
Collagen--
The activation of MMP-2 is thought to occur on cell
membrane (51). To determine whether Rac1-dependent MMP-2
activation is associated with cells, cell-bound MMP-2 activity was
assessed. Consistent with results from secreted MMP-2, the
collagen-induced level of cell-associated active MMP-2 was greatly
enhanced by Rac1V12, but reduced by Rac1V12N17 (Fig.
3A, lanes 4-6). Active MMP-2
was also detected in cell extracts prepared from Rac1V12-expressing cells grown in fibrin gel, a condition that did not stimulate MMP-2
activation (Fig. 3A, lanes 1-3). Western blotting of cell extracts detected both proenzyme and processed MMP-2 in response to
collagen or Rac1V12 (Fig. 3B), confirming the zymographic
results (Fig. 3A). Therefore, we conclude that Rac1 mediates
cell-associated activation of MMP-2.

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Fig. 3.
Rac 1-mediated MMP-2 activation is associated
with cells. A, three HT1080-derived cell lines were
cultured on a thin layer of 3D-col or fibrin gel in serum-free medium
for 18-22 h. Gelatin zymography analysis of total cell lysates
prepared as described under "Materials and Methods" was performed.
B, three HT1080-derived cell lines were cultured in 3D-col
or fibrin gel in serum-free medium for 18-24 h. Western analysis of
cell extracts detected by affinity-purified monoclonal antibody against
MMP-2 was performed as described. The experiments have been performed
at least three times.
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Rac1 Mediates MT1-MMP Expression and
Processing--
Cell-associated activation of MMP-2 requires MT-MMPs
(51). Among several MT-MMPs identified to data, MT1-MMP is the best documented in its ability to activate MMP-2. To assess whether Rac1
mediates MMP-2 activation through MT1-MMP, we first determined whether
MMP-2 activation by Rac1V12 requires MMP activities. The synthetic MMP
inhibitor SC68180 abrogated MMP-2 activation (Fig. 4, lanes 2 and 5),
whereas the inhibition of serine proteinases/plasmin system by
aprotinin did not (Fig. 4, lanes 3 and 6),
suggesting that MMP activities were responsible for the cell membrane
activation of MMP-2. The inhibitory effect of SC68180 on MMP-2
activation was observed in both cell and conditioned medium (Fig.
4).

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Fig. 4.
Rac 1-mediated MMP-2 activation requires MMP
activity. Rac1V12-expressing HT1080 cells were cultured on a thin
layer of 3D-col or fibrin gel in serum-free medium for 18-22 h in the
presence of 25 µM SC68180, a general inhibitor of MMP,
and 100 µg/ml aprotinin, an inhibitor of PA/plasmin system. Total
cell lysates (upper panel) and conditioned medium
(lower panel) were analyzed by gelatin zymography. The
results are representative of four individual experiments.
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Next, we assessed whether Rac1-mediated MMP-2 activation coincides with
MT1-MMP expression. MT1-MMP mRNA was increased modestly by 3D-col
(Fig. 5A, compare lanes
1 and 4). This induction was further enhanced by
Rac1V12, but attenuated by Rac1V12N17 (Fig. 5, lanes 5 and
6). Rac1V12 had the ability to increase MT1-MMP mRNA in
cells cultured in fibrin gel (Fig. 5A, lane 3). As shown in
Fig. 5B, Western blotting detected two bands corresponding to 60 and 43 kDa, the two MT1-MMP species thought to represent the
mature protein and its processing product, the N-terminal truncated
protein, respectively (28, 52). Two unspecified bands of high molecular
weight were also detected by monoclonal antibody against MT1-MMP,
presumably representing an artifact of the antibody. Although the level
of the 60-kDa form was enhanced by Rac1V12, HW and HN cells
demonstrated comparable level of the 60-kDa MT1-MMP (Fig.
5B). Interestingly, the level of 43-kDa protein, the
truncated MT1-MMP that was suggested to reflect the consumption of
MT1-MMP in the activation of MMP-2 proenzyme (28), mirrored the MMP-2
activation pattern as observed earlier (Fig. 2A). As shown
in Fig. 5B, both collagen and Rac1V12 induced the level of
43-kDa MT1-MMP (lanes 3, 4, and 6). Therefore,
Rac1 mediated both MT1-MMP expression/processing and the accumulation
of the 43-kDa MT1-MMP processing product in correlation with MMP-2
activation.

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Fig. 5.
Stable expression of dominant negative and
active Rac1 in HT1080 fibrosarcoma cells mediates MT1-MMP expression
and processing in three-dimensional collagen matrix. Three
HT1080-derived cell lines were cultured in 3D-col or fibrin gels. Total
RNA or cell extracts were prepared after 10 or 18-24 h, respectively,
for analysis by Northern (A) or Western (B)
blotting. A, Northern blots were detected with cDNA
probes for MT1-MMP or 36B4 mRNA, a ubiquitously expressed gene
product (79). B, Western blots were detected with affinity
purified monoclonal antibody against MT1-MMP. Two bands corresponding
to molecular mass 60 or 43 kDa were indicated. Results are
representative of at least three individual experiments.
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Tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), an effective
inhibitor of MT1-MMP and membrane-associated MMP-2, is proposed to be a
necessary component for MMP-2 activation at low concentration (23, 27).
The role of Rac1 in TIMP-2 expression was also determined. Results from
Northern and Western blotting indicated that TIMP-2 mRNA and
protein levels were not obviously changed by either collagen or Rac1V12
(data not shown). An increase in the amount of MT1-MMP and little
change in the level of TIMP-2 protein by collagen or active Rac1 may
imply a shift to increased cellular capability of activating MMP-2 proenzyme.
Rac1 Mediates Type I Collagen Fibril Degradation--
Because both
MMP-2 and MT1-MMP have the ability to degrade type I collagen (14, 33,
34), we wondered whether Rac1V12 might increase collagenolytic activity
by HT1080 cells. To examine this possibility, fibrillar collagen
degradation was assessed. Cells were seeded in a pellet on a
reconstituted type I collagen fibril film and incubated in serum-free
medium. Cells expressing vector control and Rac1V12 readily degraded
the underlying collagen film to a different extent (Fig.
6A). Cells expressing
Rac1V12N17, however, did not degrade as much the underlying collagen
matrix. Interestingly, like 3D-col, the collagen fibril film also
induced the processing of a fraction of MMP-2 proenzyme to active form in a Rac1-dependent manner (Fig. 6B). This
process coincided with the modulation of collagenolytic activity.

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Fig. 6.
Stable expression of dominant negative and
active Rac1 in HT1080 fibrosarcoma cells mediates collagenolytic
activity. A, three HT1080-derived cell lines were
plated in the center of each well in a 24-well plate coated with a thin
film of air-dried polymerized type I collagen. Cells were removed with
typsin/EDTA after 3 days in serum-free medium and the residual collagen
fibril film was stained with Coomassie Blue. Results are representative
of three experiments. B, three HT1080-derived cell lines
were plated in each well of a 24-well plate coated with a thin film of
air-dried polymerized type I collagen. Serum-free medium was collected
after 24 h and analyzed by gelatin zymography. C, three
HT1080-derived cell lines were incubated in wells of a 48-well plate
coated with 3H-acetylated rat tail type I collagen. Samples
of conditioned medium (50 µl) were collected after 1 and 2 days, and
the released radioactive degraded collagen was measured in a liquid
scintillation counter. Net collagen degradation was quantified by
subtracting the counts/min contained in 50 µl of cell-free medium
from each sample. The bar graphs represent the mean counts
of triplicate samples in a single experiment. The similar experiment
was performed four times with triplicate wells for each
condition.
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To quantify the collagenolytic activity, cells were plated onto
radioactive collagen fibril film. Released radioactivity indicated that
collagenolytic activity in HW and HV cells was 1-5-fold higher than
that in HN cells (Fig. 6C). Together these results
demonstrated that Rac1 mediates collagenolytic activity in accordance
with cell invasiveness (Fig. 1C), MMP-2 activation (Fig.
2A and Fig. 3), and MT1-MMP expression/processing (Fig. 5,
A and B).
Rac1-induced Cell Invasiveness in 3D-col Is Reduced by MMP-2
Inhibitors--
The C-terminal hemopexin domain (CTD) of MMP-2 has
been extensively used to specifically inhibit cell-associated MMP-2
activation (22, 53, 54) and to study MMP-2
activity-dependent cellular functions (20, 36, 55). In
contrast, the collagen-binding domain (CBD) of MMP-2 that contains
fibronectin type II-like modules does not have inhibitory effect on
MMP-2 activation as stimulated by ConA (54, 56). The recombinant CTD
(Gly417-Cys631) and CBD
(Val191-Gln364) were synthesized and tested for
their ability to inhibit MMP-2 activation in HW and HV cells embedded
in 3D-col (Fig. 7A). CTD inhibited MMP-2 activation in both cell lines in a
concentration-dependent manner (Fig. 7A, lanes
1-6), whereas CBD did not have inhibitory effect (Fig. 7A,
lanes 7 and 8). Therefore, CTD and CBD were used as a
specific inhibitor of MMP-2 and a non-inhibitory control, respectively,
in the collagen invasion assay.

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Fig. 7.
Rac1-promoted cell invasion through type I
collagen requires MMP-2 activity. A, HT1080 cells
expressing vector (HW) and Rac1V12 (HV) were
cultured in 3D-col in the presence of CTD or CBD at the indicated
concentrations. After 24 h, medium was collected and subjected to
gelatin zymography using a 10% SDS-polyacrylamide gel electrophoresis.
The results were representative of three individual experiments.
B, Rac1V12-expressing HT1080 cells were added to the upper
compartments of type I collagen invasion chamber in the presence or
absence of the indicated components. After 24 h, an aliquot of the
conditioned medium from the upper chamber was removed and analyzed by
gelatin zymography. Cells that invaded to the underside of the filters
were counted. Three invasion chambers were used per condition. Results
shown are the mean of cell number in 10 randomly selected fields in a
single representative experiment of six performed. FI, furin
inhibitor; CTD, C-terminal domain; CBD,
fibronectin type II-like modules.
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To evaluate whether MMP-2 activity contributes to the invasive behavior
of Rac1V12-expressing cells, we analyzed cell transmigration across
3D-col in the presence of inhibitors of MMP-2 activation. Serum-free
medium was taken from the upper chamber for analysis of proteolytic
activities released by cells during invasion. CTD at 500 ng/ml
completely inhibited Rac1V12-promoted MMP-2 activation during invasion
(Fig. 7B, lane 5), whereas the control CBD at the same
concentration did not impact on the process (lane 6), supporting the specificity of CTD inhibition of MMP-2 activation. Other
positive and negative controls were also used. MMP-2 activation in
active Rac1-expressing cells was not affected by TIMP-1 at 500 ng/ml
(Fig. 7B, lane 2). In contrast, TIMP-2, a preferential inhibitor of MMP-2 (57) and MT1-MMP (24), blocked MMP-2 activation at
the same concentration (Fig. 7B, lane 3). A furin inhibitor, Dec-Arg-Val-Lys-Arg-CH2Cl (58), is reported to reduce
MT1-MMP processing and MMP-2 activation in HT1080 cells (59). Indeed, furin inhibitor at 100 µM reduced Rac1-mediated MMP-2
activation (Fig. 7B, lane 4), further confirming the role of
MT1-MMP in Rac1-mediated MMP-2 activation.
The inhibition of MMP-2 activation by various inhibitors coincided with
the reduced cell invasiveness (Fig. 7B). As much as 80%
inhibition of cell invasion was observed in the presence of TIMP-2. The
presence of CTD resulted in ~60% reduction in cell invasion.
However, TIMP-1 and CBD, two controls that did not inhibit MMP-2
activation, did not affect Rac1-induced cell invasiveness. Therefore,
our results support the notion that modulation of MMP-2 activation is
at least one of mechanisms by which Rac1 mediates cell invasion through
type I collagen matrix barrier.
 |
DISCUSSION |
The Rho group of small GTPases and MMPs are two protein families
that play key roles in cell movement. Here we report that Rac1-regulated cellular MMP-2 activation by type I collagen and that
MMP-2 activity is required for Rac1-induced cell invasion across type I
collagen barrier. In correlation with MMP-2 activation, Rac1 also
modulated collagen-induced MT1-MMP expression/processing and
collagenolytic activity. This suggests that Rac1 mediates the
collagen-dependent MT1-MMP/MMP-2 activity as one of
mechanisms by which it induces cell invasion.
Cells expressing dominant negative Rac1 reduced MMP-2 activation by
3D-col, implying that collagen fibril-cell interaction initiates a
proteolytic cascade to cleave MMP-2 proenzyme through Rac1. To
determine that MMP-2 proenzyme processing coincided with Rac1
activation in cells cultured in 3D-col, we performed the pulling down
assay that selectively detects the GTP-bound Rac1. However, Rac1
activity was unchanged in vector-expressing cells stimulated by type I
collagen (data not shown). It has been recently proposed that the
GTP-bound small GTPases stimulated by selective ECM signals may be
difficult to detect by the pulling down assay because the activated
forms probably appear very transiently and are rapidly down-regulated
(60). An alternative approach was thus taken to confirm the role of
active Rac1 in MMP-2 activation by 3D-col. The stable expression of
active Rac1 enhanced 3D-col-induced MMP-2 activation, confirming the
results showing the suppression of MMP-2 activation by dominant
negative Rac1. Furthermore, Rac1V12 conferred cells with the ability to
induce MMP-2 activation and MT1-MMP expression/processing when cells
expressing active Rac1 were cultured in fibrin gel, a matrix that does
not induce MMP-2 activation. Therefore, we conclude that active Rac1 is
necessary and sufficient for collagen-induced MMP-2 activation.
Our finding is in line with the study showing that MMP-2 activation is
induced by 3D-col via
1 integrin (61). Cellular interaction with 3D-col has been shown to result in
1
integrin aggregation (62). This mode of
1 integrin
modulation promotes MMP-2 activation/MT1-MMP processing (29) as well as
phosphorylation of focal adhesion kinase (63). Activated focal adhesion
kinase is linked to Rac1 by stimulating adaptor protein CAS (64), a molecule that promotes Rac1 activation after the complex formation with
CrkII and DOCK180 (65-67). As expected from this chain of events
(
1 integrin aggregation
focal adhesion kinase
CAS/CrkII/DOCK180
Rac1), the role of Rac1 in MMP-2 activation may
reflect that of
1 integrin aggregation. Indeed, we found
that similar to
1 integrin aggregation, active Rac1 was
sufficient for induction of MMP-2 activation. Furthermore, a blocking
antibody against
1 integrin failed to inhibit
Rac1V12-promoted MMP-2 activation in cells cultured in 3D-col, whereas
1 integrin aggregation-induced MMP-2 activation was
attenuated by the stable expression of Rac1V12N17 (data not shown).
MMP-2 activation is unique among secreted MMPs in that it occurs on
cell membrane (51). Membrane-bound MT1-MMP is known to initiate MMP-2
activation (24, 26). It has been hypothesized that MT1-MMP and TIMP-2
form a "receptor" complex that binds MMP-2, resulting in the
proteolysis of bound MMP-2 by an adjacent free MT1-MMP (23, 27, 68).
This model predicts that the balance between TIMP-2 and MT1-MMP is of
critical importance in determining the activation status of MMP-2. A
variation in either TIMP-2 or MT1-MMP status could result in the
modulation of MMP-2 proenzyme processing. Indeed, the culture of
several cell types on or in 3D-col induces MT1-MMP expression and,
correspondingly, an increase in MMP-2 activation (21, 69). In line with
these studies, our data showed that Rac1 induced cell-associated MMP-2
activation in correlation with altered level of MT1-MMP mRNA, but
not TIMP-2 expression, implying that Rac1 activity facilitates a shift
in balance toward increasing proteolytic activity of MMP-2. How might Rac1 mediate MT1-MMP expression that leads to MMP-2 activation? Active
Rac1 has been previously reported to increase interstitial collagenase
(MMP-1) expression in rabbit synovial fibroblasts through a series of
events that include the induction of H2O2 production, NF-
B activation, and interleukin-1
secretion (70). Inflammatory cytokines including interleukin-1
and tumor necrosis factor-
can induce MT1-MMP expression in human endothelial cells (71). It is thus likely that in our cell system, Rac1 may regulate MT1-MMP expression and MMP-2 activation by modulating interleukin-1
production. However, the exact mechanism by which Rac1 mediates MT1-MMP
expression and MMP-2 activation remains to be elucidated.
In addition to MT1-MMP expression, we also detected Rac1-mediated
MT1-MMP processing as judged by Western analysis. MT1-MMP proteins were
detected as two major bands, 60 and 43 kDa. The accumulation of the
43-kDa MT1-MMP mirrored the status of MMP-2 activation mediated by
active or negative Rac1. Recently, increasing evidence has indicated
that the generation of a 43-kDa truncated MT1-MMP directly correlates
with MMP-2 activation by signals such as phorbol 12-myristate
13-acetate (28, 52, 72), ConA (29), fibronectin (31), and 3D-col (30).
In contrast, the level of 60-kDa MT1-MMP, most likely corresponding to
the processed mature form of MT1-MMP (32), was not modulated by these
stimulators. It is suggested that the 43-kDa form of MT1-MMP represents
an inactive byproduct during the activation of MMP-2 proenzyme by active MT1-MMP, reflecting the consumption of MT1-MMP in the activation and release of MMP-2 (28). The spatial localization of MT1-MMP at
invadopodia, a special membrane protrusion that is analogous to
lamellipodia and makes contacts with the underlying ECM surface, is
essential for its function in degrading ECM substrates (37). It is
probable that MMP-2 activation by MT1-MMP also depends on the
invadopodia localization (73). Therefore, the proper function of
MT1-MMP may depend on both cellular expression and specific membrane
localization. Rac1 may be involved in both processes. While it is
conceptually unclear at this stage about the invadopodia structure in
our three-dimensional tissue culture model, striking morphological
differences among HW, HN, and HV cell lines were observed. We speculate
that the Rac1-mediated spatial arrangement in 3D-col may be a
functional switch for MT1-MMP in our cell system. This could explain,
at least partially, that as reflected by the 43-kDa form level, only a
fraction of cellular MT1-MMP is accessible to MMP-2. This possibility
will be the subject of future investigation.
A number of experimental approaches have been taken to determine the
function of MMP-2 in cell motility. These approaches include the
specific inhibition with neutralizing antibody against MMP-2 and the
C-terminal hemopexin domain of MMP-2 (36, 74, 75) and the indirect
inhibition with furin inhibitor and antisense oligonucleotides to
prevent processing and synthesis of MT1-MMP, respectively (36, 59, 76).
We assessed the functional role of MMP-2 in Rac1-induced cell invasion
by inhibiting MMP-2 activity with reagents that target at the membrane
activation of MMP-2 (the C-terminal hemopexin domain of MMP-2), MT1-MMP
processing (furin inhibitor), activity of both MMP-2 and MT1-MMP
(TIMP-2), and activity of general MMPs (SC68180). The inhibition of
MMP-2 activation correlated with the reduction of Rac1-mediated cell invasiveness, suggesting that MMP-2 activity contributes to this process. It should be pointed out that MT1-MMP and MMP-2 may mediate cell invasion as components of a proteolytic activity cascade as well
as two individual type I collagen-degrading MMPs. This notion is
consistent with our finding that suppression of both MT1-MMP and MMP-2
activity by furin inhibitor or TIMP-2 more effectively reduced cell
invasion than the inhibition of MMP-2 activation alone by its C-domain.
However, it is also possible that these two inhibitors might target at
a broader range of molecules involved in cellular invasion than the
specific MMP-2 inhibitor C-domain does. While the function of MMP-2 is
studied in the context of its activator MT1-MMP, the direct
contribution of MT1-MMP in Rac1-mediated cell invasion may be studied
in a cell system that does not express MMP-2. By using MMP-2-negative
cell lines, Koshikawa et al. (36, 77) identified the
MMP-2-independent function of MT1-MMP in cleaving laminin-5, a
substrate of MMP-2. Whether native helical type I collagen can directly
serve as a substrate of MMP-2 remains controversial (14, 78). Whereas
Aimes and Quigley (14) have shown the evidence that suggests MMP-2 as
an interstitial collagenase, Seltzer and Eisen (78) have indicated that
the helix-relaxed, but not native helical, type I collagen is
susceptible to digestion by purified MMP-2. In our cell systems, it is
yet to be defined whether MMP-2 is sufficient to degrade native type I
collagen or requires the helix relaxation by another MMP to initiate
type I collagen cleavage process.
In summary, we have shown that Rac1 mediated collagen-induced MMP-2
activation and a shift in balance between MT1-MMP and TIMP-2 toward
collagenolytic phenotype. Evidence was also presented that MMP
activities, largely MT1-MMP and MMP-2, were necessary for Rac1-promoted
cell invasion through 3D-col. These findings suggest a mechanism by
which the encounter between type I collagen and invading tumor cells
could stimulate elevated level of MMP-2/MT1-MMP activities, leading to
increased collagenolysis. This reciprocal regulation between collagen
and MT1-MMP/MMP-2 is at least partially responsible for the
Rac1-mediated cell invasion across collagen barrier. Therefore, the
block of an activity chain, type I collagen
Rac1
MT1-MMP
MMP-2, may impair the invasiveness of certain tumor cells in
interstitial stroma.