From The Burnham Institute, La Jolla, California
92037 and the § Vascular Biology Department, The Scripps
Research Institute, La Jolla, California 92037
Received for publication, August 30, 2000, and in revised form, April 20, 2001
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ABSTRACT |
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Membrane type-1 matrix
metalloproteinase (MT1-MMP) is a key enzyme in the activation pathway
of matrix prometalloproteinase-2 (pro-MMP-2). Both activation and
autocatalytic maturation of pro-MMP-2 in trans suggest that
MT1-MMP should exist as oligomers on the cell surface. To better
understand the functions of MT1-MMP, we designed mutants with
substitutions in the active site (E240A), the cytoplasmic tail (C574A),
and the RRXR furin cleavage motifs (R89A, ARAA, and R89A/ARAA) of the
enzyme. The mutants were expressed in MCF7 breast carcinoma cells that
are deficient in both MMP-2 and MT1-MMP. Our results supported the
existence of MT1-MMP oligomers and demonstrated that a disulfide bridge
involving the Cys574 of the enzyme's cytoplasmic tail
covalently links MT1-MMP monomers on the MCF7 cell surface. The
presence of MT1-MMP oligomers also was shown for the enzyme naturally
expressed in HT1080 fibrosarcoma cells. The single (R89A and ARAA) and
double (R89A/ARAA) furin cleavage site mutants of MT1-MMP were
processed in MCF7 cells into the mature proteinase capable of
activating pro-MMP-2 and stimulating cell locomotion. This suggested
that furin cleavage is not a prerequisite for the conversion of
pro-MT1-MMP into the functionally active enzyme. A hydroxamate class
inhibitor (GM6001, or Ilomastat) blocked activation of MT1-MMP in MCF7
cells but not in HT1080 cells. This implied that a matrixin-like
proteinase sensitive to hydroxamates could be involved in a
furin-independent, alternative pathway of MT1-MMP activation in breast
carcinoma cells. The expression of the wild type MT1-MMP enhanced cell
invasion and migration, indicating a direct involvement of this enzyme in cell locomotion. In contrast, both the C574A and E240A mutations render MT1-MMP inefficient in stimulating cell migration and invasion. In addition, the C574A mutation negatively affected cell adhesion, thereby indicating critical interactions involving the cytosolic part
of MT1-MMP and the intracellular milieu.
MT1-MMP1 (MMP-14) is a
member of a large family of zinc endoproteinases, matrixins or
matrix metalloproteinases (MMPs) (1, 2). There are several structural
features such as the modular domain structure and the existence of an
N-terminal propeptide domain, a zinc-coordinating active site domain,
and a C-terminal hemopexin-like domain that are characteristic for most
MMPs (1-3). A subfamily of membrane type (MT)-MMPs including MT1-MMP
is distinguished by a relatively short transmembrane domain and a
cytoplasmic tail, which associate these enzymes with discrete regions
of the plasma membrane and the intracellular compartment. MT1-MMP
expression has been documented in many tumor cell types and strongly
implicated in malignant progression (3, 4). In addition to its ability to directly cleave certain components of the extracellular matrix (5,
6), MT1-MMP initiates the activation pathway of the most widespread
MMP, MMP-2, by converting pro-MMP-2 into an activation intermediate
that further undergoes autocatalytic conversion to generate the mature
enzyme of MMP-2 (7-9). Structure-function relationships of MT1-MMP
(10-16) and the mechanisms of pro-MMP-2 activation to the mature
enzyme (9, 17-20) are not understood in detail (21-23). An immediate
proximity of at least two molecules of MT1-MMP (an "activator" and
a "receptor") on the cell surface is required for in
trans activation of MMP-2 to the mature form (17, 19, 20,
24). However, there is no direct biochemical evidence to support the
existence of MT1-MMP oligomers on cell surfaces. In addition,
mechanisms involved in activation and trafficking of MT1-MMP are not
well elucidated and remain controversial (10, 13-15, 25-28). Thus,
furin, a serine proteinase of the trans-Golgi network, has been earlier
assumed to function as a unique activator of MT1-MMP (25). However,
evidence is emerging that there could be alternative pathways of
MT1-MMP activation (27, 28). In this respect, it is not possible to
rule out certain autocatalytic steps in MT1-MMP activation such as
those involved in the activation pathway of pro-MMP-2 and pro-MMP-9 (8,
29-31).
To better understand functions of MT1-MMP, we constructed mutant
MT1-MMPs and evaluated cell surface expression of the wild type and
mutant enzymes in MCF7 breast carcinoma cells deficient in MT1-MMP and
MMP-2. This allowed us to specifically identify the direct effects of
MT1-MMP on cell locomotion. Here, we report novel mechanisms that may
control dimerization, processing, and self-inactivating proteolysis of
MT1-MMP in breast carcinoma cells.
Proteins, Antibodies, and Inhibitors--
Pro-MMP-2, essentially
free from tissue inhibitor of metalloproteases (TIMP)-2, was isolated
from medium conditioned by p2AHT2A72 cells (a derivative of HT1080
fibrosarcoma cell line doubly transfected with E1A and pro-MMP-2) (8).
The total levels of TIMP-2 existing in pro-MMP-2 samples and produced
by MCF7 cells were sufficient to support activation of exogenous
pro-MMP-2 in our cell system (8, 9, 22). Control rabbit IgG and murine
IgG were from Sigma. Rabbit antibodies AB815 against a hinge region
(anti-hinge; residues 285-318) of MT1-MMP were from Chemicon
(Temecula, CA). Human TIMP-2 and TIMP-2-specific mAb T2-101 were from
Fuji Chemical Industries (Tokyo, Japan) and Calbiochem, respectively. A
general hydroxamate inhibitor of MMP activity,
N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (Ilomastat or GM6001) (32) was from AMS Scientific (Concord, CA). Protease inhibitors (phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin) were from Sigma. A furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, was from Bachem
(King of Prussia, PA).
MT1-MMP Constructs--
The full-length human wild type MT1-MMP
(MT1-MMP-wt) (GenBankTM accession number U41078) was cloned
into the pcDNA3-zeo plasmid (Invitrogen, San Diego, CA).
Mutagenesis was done by using the QuickChange system (Stratagene, San
Diego, CA) and the following mutagenic primers:
5'-GGTGGCTGTGCACGCGCTGGGCCATGCCC-3' and
5'-GGGCATGGCCCAGCGCGTGCACAGCCACC-3' for the E240A mutant, 5'-GAGATCAAGGCCAATGTTGCAAGGGCGGCCTACGCCATCCAGGG-3' and
5'-CCCTGGATGGCGTAGGCCGCCCTTGCAACATTGGCCTTGATCTC-3' for the ARAA mutant,
5'-CACCATGAAGGGCATCGCGCGCCCCCGATGTGGTG-3' and
5'-CACCACATCGGGGGCGCGCCATGGCCTTCATGGTG for the R89A mutant,
and 5'-CGACTGCTCTACGCCCAGCGTTCCCTGC-3' and
5'-GCAGGGAACGCTGGGCGTAGAGCAGTCG-3' for the C574A
mutant (nucleotides shown in boldface type indicate the altered
codons). Mutants of MT1-MMP were recloned into the pcDNA3-zeo
plasmid, and their structure was confirmed by sequencing. To construct
the double R89A/ARAA mutant, the R89A mutation was inserted in the
MT1-MMP-ARAA template.
Cell Transfection--
Human MCF7 breast carcinoma cells were
stably transfected with MT1-MMP-wt, -C574A, -E240A, and -ARAA using
LipofectAMINE according to the manufacturer's recommendations (Life
Technologies, Inc.). Cell clones resistant to 0.6-0.8 mg/ml of zeocin
were further selected for cell surface MT1-MMP by flow cytometry as
described (9, 22, 33). Briefly, cells were incubated with 5 µg/ml control rabbit IgG or rabbit anti-hinge and further with a fluorescein isothiocyanate-conjugated F(ab')2 fragment of goat
anti-rabbit IgG (Sigma). Viable cells were analyzed on a FACStar flow
cytometer (Becton Dickinson, Mountain View, CA). To avoid any
clone-specific effects, transfected cell lines were generated as
corresponding pools of positive cell clones (3-5 clones for each cell
line). Control cells transfected with the original pcDNA3-zeo
plasmid were generated as a pool of zeocin-resistant cells. Transfected cells were routinely grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 0.2 mg/ml zeocin.
In addition, a series of cells transiently transfected with the control
zeo-plasmid, the wild type, and R89A and R89A/ARAA mutant MT1-MMPs were
constructed to facilitate the studies involving the role of furin in
MT1-MMP processing. For these purposes, MCF7 cells were seeded at
1 × 106 cells/well of a six-well plate in DMEM
supplemented with 10% FCS. After incubation for 18 h, cells were
transfected with the respective recombinant plasmids (2 µg each)
mixed with LipofectAMINE Plus reagent (Life Technologies, Inc.)
according to the manufacturer's recommendations. After incubation for
48 h, the efficiency of transiently transfected cells was analyzed
by gelatin zymography and immunocapture to evaluate MMP-2 activation
and the levels and processing of MT1-MMP, respectively (see
"Activation of MMP-2").
Assay of Gelatinolytic Activity--
To measure the proteolytic
activity of MMP-2, we used biotin-labeled gelatin as a substrate (34).
For these purposes, MT1-MMP-wt, -C574A, -E240A, and -ARAA cells were
plated at 2.5 × 105 cells/well of a 24-well cluster
in 0.5 ml of serum-containing DMEM. After an overnight incubation,
cells were washed with serum-free DMEM and then incubated for 1 h
with 750 ng of pro-MMP-2 in 0.15 ml of serum-free DMEM to fully
saturate the TIMP-2·MT1-MMP complexes existing on cell surfaces.
Next, cells were extensively washed with DMEM and 0.1% BSA-DMEM to
remove soluble unbound pro-MMP-2, and biotinylated gelatin was added to
each well (75 pg in 0.5 ml of 0.1% BSA-DMEM). After incubation at
37 °C for 4 h, aliquots were taken from each well and mixed
with EDTA (final concentration of 10 mM) to stop the
reaction. Further, the amounts of degraded gelatin were quantified in
each sample as described earlier (34).
Activation of MMP-2--
To evaluate MT1-MMP constructs in
pro-MMP-2 activation, 2 × 105 cells of each stably
transfected MT1-MMP-wt, -C574A, -E240A, and -ARAA cell lines were
plated per well of a 24-well cluster. After an overnight incubation,
serum-containing DMEM was replaced by 0.3 ml of serum-free DMEM
supplemented with purified pro-MMP-2 (10 ng/ml). In cell cultures
transiently transfected with the mock, MT1-MMP-wt, MT1-MMP-R89A, and
MT1-MMP-R89A/ARAA plasmids in 48 h after the transfection,
DMEM-FCS was replaced with serum-free DMEM (1.5 ml/well, each
containing 1 × 106 cells) supplemented with purified
pro-MMP-2 (10 ng/ml). After incubation for 0.5-24 h, aliquots of each
medium were mixed with equal volumes of 2× SDS sample buffer and
analyzed by zymography on 10% acrylamide, 0.1% gelatin gels (8).
Immunocapture of MT1-MMP--
Cells were surface-biotinylated
for 1 h on ice with 0.1 mg/ml
sulfo-N-hydroxysuccinimide-LC-biotin (Pierce). Where
indicated, cells were incubated with protease inhibitors for 48 h
prior to labeling. Labeled cells were solubilized at 5 × 106 Flow Cytometry Analysis of MT1-MMP
Expression--
Fluorescence-activated cell sorting analyses were
performed as previously described (9). All staining procedures were
done on ice in Dulbecco's PBS supplemented with 1 mM
CaCl2, 1 mM MgCl2, and 1% BSA
(DPBS/BSA), pH 7.2. Cells were stained with 2 µg/ml anti-hinge
antibody. Further, cells were incubated with a fluorescein isothiocyanate-conjugated F(ab')2 fragment of goat
anti-rabbit IgG (Sigma). Population gates were set by using cells
incubated with normal rabbit IgG.
Flow Cytometry Analysis of TIMP-2 Binding--
Cells were first
incubated for 1 h with or without 5 µg/ml TIMP-2, washed with
DPBS/BSA, and then incubated with 10 µg/ml TIMP-2-specific mAb T2-101
for 2 h followed by incubation with fluorescein
isothiocyanate-conjugated F(ab')2 fragment of sheep anti-murine IgG (1:100) for 30 min. After removal of unbound
antibodies, cells were resuspended in DPBS/BSA supplemented with 3 µg/ml propidium iodide (Sigma), and viable cells were analyzed on a
FACScan flow cytometer (Becton Dickinson). Population gates were set by
using cells incubated with normal murine IgG.
Cell Adhesion--
Cell adhesion was performed in the wells of a
high binding 96-well plate (Corning Glass) precoated with 1 µg/ml
collagen type I (Vitrogen 100; Cohesion, Palo Alto, CA) overnight at
4 °C. Plates were washed with PBS and blocked for 1 h at
37 °C with 1% BSA in DMEM supplemented with 10 mM
HEPES, pH 7.2 (DMEM/BSA). Cells were incubated overnight in
serum-containing DMEM, detached with enzyme-free buffer (Specialty
Media, Lavalette, NJ), washed, and resuspended in DMEM/BSA. Cells were
plated at 5 × 104/0.1 ml in DMEM/BSA for 1 h at
37 °C. After three washes with DPBS, adherent cells were fixed and
stained with Crystal Violet in 10% ethanol. Following washing with
DPBS, the incorporated dye was extracted with a 1:1 mixture of 100 mM sodium phosphate and 50% ethanol, pH 4.5, and the
absorbance was measured at 540 nm.
Cell Migration in Transwells--
The directional migration of
cells in Transwells (Costar, Cambridge, MA) was analyzed under
serum-free conditions as previously described (9, 22, 33). The
undersurface of a 6.5-mm insert membrane with an 8-µm pore size was
coated overnight at 4 °C with 20 µg/ml collagen type I, washed
with PBS, and blocked with 1% BSA. Cells were cultured overnight in
DMEM plus 10% FCS and then detached with enzyme-free buffer. A total
of 7.5 × 105 cells were plated in 0.15 ml of AIM-V
medium (Life Technologies) per insert. The outer chamber was filled
with 0.6 ml of AIM-V medium. Following incubation for 48 h, cells
that migrated to the membrane's undersurface were detached with
trypsin/EDTA and counted.
In Vitro Cell Invasion--
Cell invasion assays were performed
in serum-free AIM-V in 6.5-mm Transwells with the 8-µm pore size
membranes. The undersurface of the Transwell membrane was precoated
with collagen type I at 20 µg/ml overnight at 4 °C. After washing
with PBS, 50 µl of PBS containing 3 µg of Matrigel (Becton
Dickinson, Bedford, MA) were dried overnight on the upper surface of
the membrane at room temperature. Matrigel was reconstituted in PBS at
37 °C for 2 h. Cells were seeded at 3 × 105
in 0.1 ml of AIM-V medium into the inner chamber of the Transwell. The
outer chamber was filled with 0.5 ml of AIM-V medium. Cells were
allowed to invade Matrigel for 44-48 h at 37 °C in a
CO2 incubator. Then the upper surface of the inserts was
wiped. To harvest cells that migrated onto the membrane's
undersurface, the inserts were incubated in 0.5 ml of trypsin/EDTA.
Cells were collected, pelleted, resuspended in trypan blue solution,
and counted with a hemocytometer.
Immunofluorescence--
To visualize MT1-MMP, cells (4 × 104 cells/well) were plated onto Lab-Tek II chamber slides
(Nalge Nunc International, Naperville, IL) precoated with 10 µg/ml
fibronectin at 4 °C overnight. Following incubation for 48 h,
cells were fixed for 20 min with 4% paraformaldehyde in PBS and
permeabilized with 0.2% Triton X-100 for 5 min. Cells were stained
with 10 µg/ml rabbit anti-hinge and further with 10 µg/ml goat
anti-rabbit IgG conjugated with Alexa Fluor 568 (Molecular Probes,
Inc., Eugene, OR). Following washing with PBS, the slides were mounted
in SlowFade Light Antifade solution (Molecular Probes). Confocal images
were collected with differential-interference contrast and
epifluorescence optics on a Bio-Rad confocal microscope. The recorded
images were processed with Adobe Photoshop software (San Jose, CA).
Dimerization of MT1-MMP on Cell Surfaces--
To identify cell
surface forms of MT1-MMP, MCF7 breast carcinoma cells were stably
transfected with MT1-MMP-wt. We specifically selected these
cells for our studies, since the parental cell line is deficient in
both MT1-MMP and MMP-2 (Fig.
1A, zeo; Fig. 4A, lane 1). High levels of MT1-MMP
expression in transfected cells relative to those of mock-transfected
cells were verified by flow cytometry employing rabbit polyclonal
antibodies directed against the hinge region of MT1-MMP (Figs.
2 and
3A). Since the hinge region is
localized between the catalytic domain and the C-terminal
hemopexin-like domain of MT1-MMP, the anti-hinge antibodies permit the
detection of the full-length proenzyme and the active enzyme as well as
its inactive forms lacking the catalytic domain.
The immunocapture studies revealed that reduced MT1-MMP-wt was
represented by the 63-kDa (the proenzyme), 60-kDa (the enzyme), 42-kDa,
and 39-kDa protein bands (degradation products; these bands are the
most prominent). Under nonreducing conditions, a relatively low amount
of the protein material in the 39-42-kDa region was present, while the
significant quantities of several high molecular weight MT1-MMP-wt
forms (78-85 kDa and 120 kDa (the two major forms) and 170 kDa and 220 kDa (the two minor forms)) were observed (Fig. 1A,
wt). MT1-MMP-wt was undetectable in the lysates of cells
transfected with the original pcDNA3-zeo plasmid (Fig.
1A, zeo). No specific bands were detected in the
samples immunocaptured with control rabbit IgG (data not shown).
To further address a question of whether the oligomers of MT1-MMP-wt
preexisted on the cell surface or formed in cell lysates, cells
expressing MT1-MMP-wt were surface-biotinylated and lysed in buffer
containing 10 mM iodoacetamide. Incubation was carried out
for 1 h to complete the modification of free cysteines. Residual iodoacetamide was blocked by excess cysteine (100 mM). When
MT1-MMP-wt was immunocaptured and analyzed, the pattern of alkylated
MT1-MMP was identical to that shown on Fig. 1A. Since
alkylation during lysis had no effect, these findings confirmed that
the observed MT1-MMP-wt oligomers preexisted on the cell surface.
To verify that the 78-85-kDa species of MT1-MMP-wt revealed under
nonreducing conditions contained the 39- and 42-kDa MT1-MMP-wt monomers, we excised the 78-85-kDa band from the nonreducing gel and
extracted the protein in PBS plus 0.1% SDS. Streptavidin-coated Dynabeads (Dynal, Lake Success, NY) were used to capture the
biotin-labeled proteins from the extract. After washings, the captured
proteins were eluted with 1% SDS, reduced with DTT, and rerun on the
gel followed by Western blotting and developing by avidin-horseradish peroxidase (Fig. 1A, eluate). Two protein forms
with apparent molecular masses of 39 and 42 kDa were identified
after these procedures. These findings confirmed that the 39- and
42-kDa degradation products of MT1-MMP-wt form dimers and explained the
broad width of the 79-85-kDa MT1-MMP-wt band that might include three
combinations of disulfide-linked 39- and 42-kDa species. Accordingly,
the 120-kDa MT1-MMP-wt form observed under nonreducing conditions
probably corresponds to a dimer consisting of the 60- or 63-kDa
MT1-MMP-wt monomers. Low nanogram (or high picogram) amounts of the
120-, 170-, and 220-kDa MT1-MMP-wt forms greatly complicate their
direct isolation and analysis similar to that performed with the
78-85-kDa species.
Pretreatment of cells with Ilomastat, a specific hydroxamate inhibitor
of MMPs (32), abrogated both activation and proteolysis of MT1-MMP-wt.
Consequently, the 63-kDa band of the MT1-MMP proenzyme was the major
species observed in reduced samples. Under nonreducing conditions,
treatment with the inhibitor revealed increased levels of the 170- and
220-kDa MT1-MMP-wt bands (Fig. 1A; wt + Ilomastat). In contrast, if cells were pretreated with the
inhibitors of serine, aspartic, and cysteine proteinases
(phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin) or
a competitive inhibitor of furin (50 µM
decanoyl-Arg-Val-Lys-Arg-chloromethylketone), the pattern of MT1-MMP-wt
remained unchanged relative to that of untreated MT1-MMP-wt cells (data
not shown).
To evaluate whether the same forms of MT1-MMP exist in another cell
type, we analyzed HT1080 fibrosarcoma cells (Fig. 1B). The
HT1080 cell line is widely used in MMP studies and known to naturally
express substantial levels of MT1-MMP (7-9, 13, 20, 35). To avoid any
misinterpretation of the MT1-MMP forms, HT1080 and MCF7 samples were
run side-by-side on the same gel (Fig. 1B). Under reducing
conditions, the 60-kDa MT1-MMP was the only form of the enzyme observed
in HT1080 cells (Fig. 1B; +DTT,
HT1080). No 39-42-kDa proteolyzed forms were observed. This
correlates well with earlier reports indicating stabilization of the
activated MT1-MMP enzyme by the relatively high levels of TIMP-2
existing in HT1080 cells (14).
MCF7 cells transfected with the wild type MT1-MMP exhibited the 60-kDa
enzyme and significant levels of the 39- and 42-kDa fragments (Fig.
1B; +DTT, MCF7-wt). Under nonreducing
conditions, only high molecular weight forms of MT1-MMP (120, 170, and
220 kDa) were present in HT1080 cells (Fig. 1B;
Although the relative amount of MT1-MMP in HT1080 cells was lower than
in MT1-MMP-transfected MCF7 breast carcinoma, the same 60-kDa (reduced)
and 120-kDa (nonreduced) mature enzyme forms were present in both cell
lines. These findings suggest that a significant fraction of naturally
expressed MT1-MMP may exist on the cell surface as dimer and/or multimers.
Site-directed Mutagenesis of MT1-MMP--
The data on transfected
MCF7 cells showed that extensive maturation, dimerization, and
degradation of cell surface MT1-MMP occurred. To specifically address
the processing and dimerization of this proteinase, we designed and
expressed mutant MT1-MMPs in MCF7 cells.
The expression plasmids encoding cDNA for mutant MT1-MMPs were
constructed using a polymerase chain reaction-based QuickChange mutagenesis system (Stratagene) and cloned into the pcDNA3-zeo plasmid. The first mutant carries the sequence
Ala108-Arg109-Ala110-Ala111
(MT1-MMP-ARAA) that modifies the
Arg108-Arg109-Lys110-Arg111
site susceptible to cleavage by furin, a reported activator of MT1-MMP
(25). In the second mutant, the Arg89 of the another
putative furin cleavage motif
(Arg89-Arg90-Pro91-Arg92-Cys93)
(36) was replaced by alanine (MT1-MMP-R89A). The third mutant carried
mutations in both furin cleavage motifs (MT1-MMP-R89A/ARAA). In the
fourth mutant, the Glu240 residue of the active site domain
of MT1-MMP was replaced by alanine to create inactive MT1-MMP
(MT1-MMP-E240A). Finally, to evaluate the possible role in
oligomerization of the unique cysteine residue of the MT1-MMP's
cytoplasmic portion, the Cys574 residue of MT1-MMP was
replaced with alanine (MT1-MMP-C574A). The structure and relative
positions of the mutations are schematically illustrated in Fig. 2.
MCF7 cells were transfected with mutant MT1-MMPs. According to flow
cytometry (Fig. 3A) and efficiencies of the respective transfectant cells in MMP-2 activation (Fig.
4) and immunocapture (Figs. 1A
and 3B), the levels of MT1-MMP-wt, -ARAA, -R89A, -R89A/ARAA, -E240A, and -C574A expression were highly similar. Further, these results were supported also by immunofluorescence and TIMP-2 binding studies (Fig. 5, A and
B; Table I).
C574A Mutation Affects a Disulfide Bridge That Covalently Links
MT1-MMP Monomers--
The immunocapture analysis of MT1-MMP-C574A
demonstrated the existence of the 60-kDa enzyme and its 39- and 42-kDa
degradation forms in reduced samples. In contrast to MT1-MMP-wt, no
78-85-kDa or 120-kDa forms of MT1-MMP-C574A were observed in
nonreduced samples (Fig. 3B, C574A). Evidently, a disulfide
bridge covalently links the wild type enzyme monomers on the cell
surface, thereby creating stable dimers of MT1-MMP-wt. The mutation of
the Cys574 residue of the cytoplasmic tail abrogated the
ability of MT1-MMP-C574A monomers to form this disulfide bridge.
Catalytically Inactive MT1-MMP-E240A Is Incapable of
Self-proteolysis--
Next, we analyzed molecular forms of the
catalytically inactive MT1-MMP-E240A mutant. Immunocapture and the
subsequent analysis of the E240A protein showed the 60-kDa mature
MT1-MMP as the major band in reduced samples (Fig. 3B,
E240A). The existence of the dominant 60-kDa MT1-MMP-E240A protein
excludes autocatalytic mechanisms of MT1-MMP activation. Since there is
a complete absence of degraded forms (39-42-kDa reduced, 78-85-kDa
non-reduced), we concluded that the E240A construct is incapable of
self-proteolysis (Fig. 4A; lane
1).
Under nonreducing conditions, the E240A mutant showed relatively
significant amounts of the 170- and 220-kDa MT1-MMP-specific bands
(Fig. 3B, E240A). Similar high molecular weight forms were also observed in the nonreduced wild type plus Ilomastat samples from
MCF7 cells (Fig. 1, A and B) and in the MT1-MMP
samples from HT1080 fibrosarcoma cells (Fig. 1B).
Furin Cleavage Is Not Essential for Activation of MT1-MMP in Breast
Carcinoma Cells--
Recent controversial studies implicated furin, a
serine protease of the trans-Golgi network, in the processing of the
latent 63-kDa MT1-MMP proenzyme to the active enzyme by cleaving either the 108RRKR111, the
89RRPR92, or both sequences in the propeptide
domain (25-28, 36). To evaluate the effects of furin motif cleavage in
the processing and dimerization of MT1-MMP, we constructed and analyzed
MT1-MMP-R89A and MT1-MMP-ARAA mutants, each exhibiting a single
respective modified furin motif, and the double MT1-MMP-R89A/ARAA
mutant with no sites susceptible to furin cleavage. Immunocapture
demonstrated that the pattern of MT1-MMP-ARAA, -R89A, and -R89A/ARAA on
cell surfaces was highly similar to that of MT1-MMP-wt (Fig.
3B). Specifically, the mutants showed the 60-, 42-, and
39-kDa bands when reduced and the 78-85-kDa (major) and 120-kDa
(minor) MT1-MMP-specific bands under nonreducing conditions. There is
no evidence of a 63-kDa MT1-MMP proenzyme. A complete conversion of
MT1-MMP-R89A/ARAA to the 60-kDa enzyme demonstrated that there is a
furin-independent alternative pathway of MT1-MMP activation in these
breast carcinoma cells. Since Ilomastat inhibited the processing of the
63-kDa MT1-MMP-wt to the 60-kDa mature forms in MCF7 cells (Fig.
1A, wt + Ilomastat), a putative
pro-MT1-MMP-processing enzyme appears to be a matrixin-like metalloproteinase.
To additionally support our findings, we evaluated the effects of
Ilomastat on MT1-MMP expressed in HT1080 cells. The samples of HT1080
and MCF7 cells were run side-by-side to facilitate the comparison of
MT1-MMP forms (Fig. 1B). Ilomastat induced the accumulation of the 63-kDa proenzyme in MT1-MMP-wt cells (Fig. 1B,
compare +DTT, MCF7-wt with MCF7-wt + Ilomastat). This correlated with the presence of higher
levels of the 170- and 220-kDa species of MT1-MMP revealed under the
nonreducing conditions (Fig. 1B, MT1-MMP-E240A Is Incapable of MMP-2 Activation and TIMP-2
Binding--
Further, we assessed whether MT1-MMP mutants were capable
of pro-MMP-2 activation. Since MCF7 cells are deficient in MMP-2 (Fig.
4A, lane 1), purified pro-MMP-2 (Fig.
4A, lane 2) was added to the cultures.
After incubation for 24 h, aliquots of medium were analyzed by
gelatin zymography to follow the conversion of the 68-kDa proenzyme of
MMP-2 into the 64-kDa intermediate and the 62-kDa mature enzyme. As
expected, mock-transfected cells and MT1-MMP-E240A cells both failed to
process pro-MMP-2 (Fig. 4A, lanes 3 and 5, respectively). The other mutants, including MT1-MMP-C574A, -ARAA, -R89A, and -R89A/ARAA (Fig. 4A,
lanes 6-9, respectively), as well as MT1-MMP-wt
(Fig. 4A, lane 4) were able to convert
the 68-kDa proenzyme into the mature 62-kDa enzyme via the 64-kDa
activation intermediate. Transiently mock- and MT1-MMP-wt-transfected
cells showed similar results in zymography studies as compared with the
respective stably transfected cells (data not shown).
To evaluate the mutants in more detail, we assessed the time course of
pro-MMP-2 activation by cells expressing the wild type, ARAA, and C574A
constructs. Aliquots of medium were withdrawn in 0.5-8 h and analyzed
by gelatin zymography. Fig. 4B shows that the wild type,
ARAA, and C574A constructs (upper, middle, and bottom panels, respectively) were similarly
efficient in activating pro-MMP-2.
To quantitatively confirm that activation of pro-MMP-2 by cells
expressing mutant MT1-MMP results in gelatinolytic activity, we
employed activity assay using biotinylated gelatin as a substrate (34).
Since MCF7 cells do not produce any detectable gelatinolytic activity
in serum-free conditions (Fig. 4A), cells were supplemented with exogenous pro-MMP-2. For these purposes, cells were incubated with
excess pro-MMP-2 to fully saturate the available MT1-MMP·TIMP-2 surface receptors. Next, cells were washed to remove unbound soluble pro-MMP-2 and any traces of the MMP-2 enzyme and free TIMP-2 that might
have preexisted in the proenzyme samples. This significantly reduced
the background activity and allowed us to follow the activation of
pro-MMP-2 associated with the MT1-MMP·TIMP-2 surface receptors. Biotin-labeled gelatin was added to cells to examine the gelatinolytic activity of MMP-2 converted into the active enzyme by the
MT1-MMP·TIMP-2 complexes. The gelatinolytic activity of MMP-2
generated by the cells expressing MT1-MMP-wt, -ARAA, and -C574A (Fig.
4C) correlated well with the results of zymography (Fig.
4B). Thus, the C574A mutant was almost as efficient in
generating MMP-2's gelatinolytic activity as the ARAA mutant. As
expected, MT1-MMP-E240A failed to demonstrate any gelatinolytic
activity (Fig. 4C).
To evaluate whether MT1-MMP mutants were capable of TIMP-2 binding,
transfected MCF7 cells were pretreated with excess TIMP-2 followed by
staining with anti-TIMP-2 mAb T2-101 and flow cytometry. Without
TIMP-2 pretreatment, none of the cells were capable of binding
anti-TIMP-2 mAb (Table I). In turn, if wild type, ARAA, and C574A cells
were pretreated with TIMP-2, the levels of cell-associated TIMP-2
significantly increased relative to those of mock-transfected cells
(Table I). These findings agreed with the results of gelatin zymography
(Fig. 4, A and B), activity measurements (Fig.
4C), and immunocapture studies (Fig. 3B),
confirming that there were no significant differences in the levels of
active MT1-MMP expressed on the surface of wild type, ARAA, and C574A
cells. Evidently, ARAA and C574A mutations did not affect TIMP-2
binding. In contrast, the E240A mutation in the enzyme's active site
abolished the ability of MT1-MMP to bind TIMP-2. It is clear from the
crystal structure of the MT1-MMP·TIMP-2 complex (38) and TIMP-1
binding studies with the Glu mutant of ministromelysin-1 (39) that the
interaction of TIMPs with active MMPs does not rely on the Glu in the
active site. Accordingly, our data suggest that the E240A mutation
abolished TIMP-2 binding by significantly perturbing the overall
structure of the enzyme's active site. The immunocapture of a 60-kDa
form of the E240A mutant (Fig. 3B) suggests that the
mutation did not affect the N-terminal processing of MT1-MMP. The
recent data of Valtanen et al. (40), who have experimentally
documented the proper processing of MT1-MMP-E240A mutant, support our suggestion.
Cell Surface Localization of Mutant MT1-MMP--
To analyze the
localization of MT1-MMP, cells expressing wild type, ARAA, C574A, and
E240A constructs were plated on fibronectin-coated glass slides, fixed,
and subjected to immunofluorescence staining with rabbit anti-hinge
antibodies followed by fluorescence and confocal microscopy. We
specifically employed permeabilized cells in these experiments to
identify if there was any difference in both the intracellular and
plasma membrane pools of mutant MT1-MMPs as compared with MT1-MMP-wt. A
comparison of the phase contrast and fluorescence images indicated that
endogenous expression of MT1-MMP in mock-transfected cells was not
sufficient to generate any detectable specific fluorescence (Fig.
5A, zeo; upper right panel). Staining of any tested cells with control rabbit IgG
was also negative (data not shown). In cells transfected with
MT1-MMP-wt and MT1-MMP-C574A, the protein products were mainly
localized to the cell surface. Cell localization and distribution
across the plasma membrane of MT1-MMP-C574A was similar to that of the wild type enzyme (Fig. 5A). Cells expressing MT1-MMP-ARAA
and -E240A exhibited a pattern of MT1-MMP staining similar to that of
the wild type or C574A constructs (data not shown). ZX sections of
stained cells (Fig. 5B) confirmed the cell surface
localization of MT1-MMP in cells expressing the wild type enzyme and
the C574A mutant.
However, there was a significant difference in the morphology of cells
expressing MT1-MMP-C574A relative to cells expressing the wild type
MT1-MMP. Under routine cell culture conditions, MT1-MMP-wt cells plated
on plastic were well spread and demonstrated cell protrusions and
ruffling, i.e. displaying a motile phenotype. C574A
cells remained more round and appeared as cell clusters with smooth
edges and almost no ruffling or spreading (Fig. 5C), thereby
suggesting a lower migratory potential and indicating alterations in
the cytoskeleton.
MT1-MMP-C574A Does Not Support Cell Adhesion, Migration, and
Invasion--
To analyze the effects of mutant MT1-MMPs on cell
locomotion, we evaluated cells expressing the wild type construct and
the mutants in a series of adhesion, migration, and invasion assays (Fig. 6). Expression of the wild type
enzyme or MT1-MMP-E240A did not affect adhesive characteristics of
cells. In contrast, expression of the C574A mutant significantly
reduced the adhesive efficiency of cells onto type I collagen (Fig.
6A). Similar results were obtained when fibronectin and
vitronectin were used as the substrates for cell attachment (data not
shown).
Further, we evaluated the migratory efficiency of cells expressing wild
type, E240A, and C574A constructs on collagen-coated surfaces. The
expression of the wild type enzyme increased collagen-mediated migration of cells at least 2.5-fold compared with that of
mock-transfected cells (Fig. 6B). The C574A and the
catalytically inactive E240A mutants failed to facilitate cell migration.
To analyze the effects of mutant MT1-MMPs on the ability of cells to
invade through basement membranes, we employed the Transwell cell
invasion assay. Relative to mock-transfected cells, cell invasion
through Matrigel was strongly enhanced by the expression of either
MT1-MMP-wt or MT1-MMP-ARAA (Fig. 6C). Significant inhibition of cell invasion by Ilomastat additionally supported a direct role of
MT1-MMP in cell locomotion. In contrast, both the C574A and
catalytically inactive E240A mutants did not stimulate cell invasion.
Since the C574A mutation affected the adhesive efficiencies of the
transfected cells, low migration and invasion of the C574A mutant were
not surprising. These findings indicate a significant functional role
of the cytosolic portion of MT1-MMP in stimulating cell motility.
Given the central role of MT1-MMP in diverse aspects of malignancy
(41-44), the localization of this enzyme to specific cell surface
sites such as the invasive front and invadopodia (13, 23, 45-48) can
efficiently regulate matrix proteolysis in the vicinity of cell
surfaces. MT1-MMP has transmembrane and cytoplasmic domains, which
target the enzyme to invasive front (3, 13, 45, 47, 49). In addition to
its ability to directly degrade the extracellular matrix (3), MT1-MMP
initiates activation of MMP-2 and MMP-13 (8, 50). These activation
mechanisms are not understood in detail (9, 21, 51, 52). The in trans mechanisms of pro-MMP-2 activation implicate at least
two molecules of MT1-MMP, a "receptor" molecule in a complex with TIMP-2 and an "activator" TIMP-2-free molecule. Accordingly, these two molecules of MT1-MMP should be co-localized in immediate proximity on the plasma membrane in order to bring together the binding and the
activation of pro-MMP-2 (12, 17, 19, 20). Dimerization of MT1-MMP could
accomplish this co-localization. However, direct evidence for
dimerization of MT1-MMP has been missing.
To better understand functions of MT1-MMP, we designed MT1-MMP proteins
with mutations in the active site, the two furin cleavage motifs and
the cytoplasmic tail (MT1-MMP-E240A, -R89A, -ARAA, -R89A/ARAA, and
-C574A, respectively). The wild type and mutant MT1-MMPs were expressed
in MCF7 cells that are deficient in both MMP-2 and MT1-MMP. The absence
of any MMP-2 and MT1-MMP activities in the parental cells facilitated
the analysis of MT1-MMP in the transfected cells.
Our observations suggest that MT1-MMP is capable of oligomerization on
cell surfaces. Homodimerization was most evident for the enzyme's
autolytic ectodomain forms. These inactive forms of MT1-MMP, 39 kDa
(presumably, starting from Gly285) and 42 kDa (presumably,
starting from Ile256) both lacking the zinc-binding
catalytic site domain were identified and characterized in previous
reports of other groups (14, 49). While our manuscript was in
preparation, dimerization was demonstrated for MT1-MMP naturally
expressed by platelets (53). Apparently, the Cys574 residue
of the cytoplasmic tail is involved in an intermolecular disulfide bond
linking monomers of the wild type MT1-MMP.
Since the C574A mutant was quite proficient in MMP-2 activation, we
suggest that this mutation and, accordingly, the absence of a covalent
link between monomers, does not completely abolish dimerization of the
enzyme. The existence of self-proteolyzed forms as well as efficacy of
the mutant in pro-MMP-2 activation indirectly supports the presence of
non-S-S dimers on the surface of cells expressing the MT1-MMP-C574A
construct. Association of the homodimer is likely to be initiated by
the motif involving the
PRXXLYC574XRSXXXXV
sequence of the cytoplasmic tail. This motif is fully conserved in
MT1-, MT2-, and MT3-MMPs while MT5-MMP lacks several essential residues
of this motif (54). MT4- and MT6-MMPs are entirely missing the motif
(55-57). It cannot be excluded that protein-disulfide isomerase
activity (58) is involved in the mechanisms that facilitate a disulfide
bridge formation and stabilization of MT1-MMP dimers.
Further, there is evidence of the extensive self-proteolysis of
MT1-MMP-wt, -R89A, -ARAA, -R89A/ARAA, and -C574A in our cell system
that is devoid of MMP-2 (22). The catalytically inactive MT1-MMP-E240A
protein was incapable of self-proteolysis. In agreement, a hydroxamate
inhibitor, Ilomastat, blocked autolytic cleavage of MT1-MMP. Autolysis
of MT1-MMP that occurs under deficiency of TIMP-2 (14) is likely to be
a mechanism for negative regulation of MT1-MMP. Our observations
suggest that the soluble activity of MMP-2 is not a prerequisite for
the degradation of MT1-MMP on cell surfaces (11, 13, 49, 59).
Recent studies suggested that furin might be a physiologically relevant
activator of MT1-MMP (25-28). However, evidence is emerging that there
could be alternative pathways of MT1-MMP activation (27, 28). Our data
confirmed the hypothesis that furin cleavage of both putative
RRXR motifs of MT1-MMP is not necessary for the processing
of MT1-MMP and the subsequent activation of pro-MMP-2 in breast
carcinoma cells. In our experiments, MT1-MMP-ARAA, -R89A, -R89A/ARAA,
and -wt displayed similar, if not identical pattern in immunocapture
and MMP-2 activation studies. Resistance of the double
MT1-MMP-R89A/ARAA mutant to furin cleavage did not cause any
accumulation of the respective proenzyme in MCF7 cells. However, MCF7
cells accumulated the MT1-MMP proenzyme in the presence of Ilomastat. A
putative matrixin-like proteinase involved in activation of MT1-MMP
remains to be identified. These findings extend the physiological
implications of the recent report that furin-independent pathway of
MT1-MMP activation exists in rabbit dermal fibroblasts (27). In
addition, we expressed MT1-MMP in furin-deficient LoVo lung carcinoma
cells. Our studies correlate well with the observations of Yana and
Weiss (36) and indicated that LoVo cells were fully capable of MT1-MMP
activation (data not shown). These data support the existence of
furin-independent cellular pathways involved in the processing of the
full-length membrane-anchored MT1-MMP proenzyme.
MT1-MMP-wt, -ARAA, and -C574A were efficient in TIMP-2 binding and,
with the exception of MT1-MMP-C574A, facilitated migration and invasion
of the respective cells through basement membrane-like matrices. The
catalytically inactive E240A construct failed to promote cell
locomotion. In agreement, Ilomastat inhibited invasion of cells
expressing MT1-MMP-wt. Thus, our results indicate that MT1-MMP is
directly involved in cell invasion and migration and support our
earlier report that, in functional cooperation with integrin
Intriguingly, the proteolytically active mutant MT1-MMP-C574A failed to
stimulate migration and invasion of transfected cells. In contrast to
all other MT1-MMP constructs, the expression of C574A also negatively
affected the adhesive ability of the respective cells. Poor adhesion of
C574A cells may result in their inefficient migration and invasion.
Immunofluorescence, flow cytometry, TIMP-2 binding, and MMP-2
activation studies demonstrated that the expression levels of this
mutant were similar to those of MT1-MMT-wt. However, MT1-MMP-C574A
cells were unable to efficiently accomplish adhesion and locomotion.
Similarly, a chimeric MT1-MMP protein containing the interleukin-2
receptor Apparently, there are two distinct mechanisms that affect cell
locomotion and involve MT1-MMP: the first where the proteolytic activity of MT1-MMP facilitates cell motility and the second where the
cytoplasmic tail of the enzyme communicates with the putative intracellular components. Thus, the expression of the C574A mutant is
likely to modify specifically the interactions of the MT1-MMP's cytoplasmic tail with the intracellular milieu, thereby affecting cell
morphology, adhesion, and migration.
Hypothetically, translocations across the cell surfaces in migrating
versus stationary cells indicate the direct critical interactions of MT1-MMP with the intracellular milieu (13, 23, 48, 61,
62). Since the putative cytoplasmic components that associate with cell
surface MT1-MMP would not be biotinylated, they are not seen in our
immunocapture experiments. Our most recent results indicate that the
peptide derived from the cytoplasmic tail of MT1-MMP is capable of
binding specifically with the p32/gC1q-R multifunctional protein (63).
This protein may be a compartment-specific partner of MT1-MMP. The p32
is likely to be involved in directional trafficking of MT1-MMP from the
Golgi network to the plasma
membrane.2 Further studies
are needed to confirm direct interactions of MT1-MMP with the
intracellular milieu via the enzyme's cytoplasmic domain.
In summary, we would like to emphasize that the existence of dimers and
possibly, higher oligomers of MT1-MMP on cell surfaces correlates well
with the mechanisms of pro-MMP-2 activation. Further, our data point to
an important function of the cytosolic portion of the MT1-MMP molecule
in modulating cell adhesion and locomotion. There is growing evidence
that MT1-MMP is a key enzyme involved in cancer cell invasion. Mutant
MT1-MMPs characterized in this report may find further applications in
structure-function analyses of MT-MMPs and other cancer-related studies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 × 107 cells/ml in PBS, pH 7.4, containing 50 mM
n-octyl-
-D-glucopyranoside or 1% Triton
X-114, 1 mM CaCl2, 1 mM
MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin for
1 h on ice. Insoluble material was removed by centrifugation. Next, supernatants were precleared for 2 h on ice with Protein A-agarose beads (Calbiochem). Aliquots (each containing 1 mg of total
protein) of precleared supernatants were incubated overnight at 4 °C
with 1-3 µg of anti-hinge and 30 µl of a 50% Protein A-agarose slurry. Following washes, the beads were boiled with 2× SDS sample buffer with or without 50 mM DTT for 5 min. Eluted proteins
were separated by electrophoresis on 10% acrylamide gels and
transferred onto an Immobilon-P membrane (Millipore Corp., Bedford,
MA). Bands containing biotin-labeled proteins were visualized by using
avidin-horseradish peroxidase (Sigma) and TMB/M (Chemicon) as a substrate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of the wild type and mutant
MT1-MMPs in MCF7 breast carcinoma cells and HT1080 fibrosarcoma
cells. A, dimerization and proteolysis of MT1-MMP-wt in
MCF7 cells. MCF7 cells stably transfected with the original
pcDNA3-zeo plasmid (zeo) and MT1-MMP-wt (wt)
were incubated in serum-free DMEM for 24-48 h, labeled with biotin,
and lysed with n-octyl- -D-glucopyranoside.
Labeled MT1-MMP was immunocaptured in cell lysates with anti-hinge and
Protein A-agarose. The MT1-MMP samples were separated by
SDS-polyacrylamide gel electrophoresis under reducing (+DTT)
and nonreducing (
DTT) conditions, transferred onto an
Immobilon-P membrane, and detected with avidin-horseradish peroxidase.
To identify the composition of the nonreduced 78-85-kDa form, this
band was excised from the gel, and the biotin-labeled proteins were
extracted and captured by streptavidin-coated Dynabeads. The captured
proteins were eluted with 1% SDS, reduced with DTT, and rerun on the
gel (eluate). To evaluate the effect of a hydroxamate inhibitor,
MT1-MMP-wt cells were incubated with 50 µM Ilomastat for
48 h (wt + Ilomastat) and further analyzed
as above. B, oligomerization of naturally expressed MT1-MMP
in HT1080 cells. HT1080 cells were surface-labeled with biotin and
lysed with 1% Triton X-114. MT1-MMP was immunocaptured in cell lysates
with anti-hinge and Protein A-agarose and analyzed under nonreducing
(
DTT) and reducing conditions (+DTT) as in
A.
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Fig. 2.
Schematic representation of site-directed
mutagenesis of MT1-MMP. Relative positions of mutations (R89A,
ARAA, R89A/ARAA, the furin cleavage sites; E240A, the catalytic site;
C574A, the cytoplasmic tail) and the binding site of anti-hinge
antibody AB815 within MT1-MMP are indicated. The numbering of amino
acids includes the signal peptide sequence.
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Fig. 3.
Expression and analysis of mutant
MT1-MMPs. A, flow cytometry analysis of MT1-MMP
expression. Parental and mock-transfected (zeo) MCF7 cells
as well as MCF7 cells expressing the wild type (wt) and
mutant MT1-MMPs (E240A, C574A, and ARAA) were stained with control
rabbit IgG (open histograms) and anti-hinge
(shaded histograms). x axis, mean
fluorescence intensity; y axis, cell number. Profiles are
representative of several independent experiments. B,
dimerization and proteolysis of mutant MT1-MMPs. MCF7 cells expressing
MT1-MMP-wt and mutant E240A, C574A, R89A, R89A/ARAA, and ARAA
constructs were lysed in 1% Triton X-114 and further analyzed by
immunocapture as described in the legend to Fig. 1A. The
apparent molecular weights of MT1-MMP forms are shown on the
left. The positions of the molecular weight markers are on
the right. Note the absence of C574A dimers and the presence
of the 60-kDa protein and the major bands of the 39- and 42-kDa
degradation products under nonreducing conditions.
DTT, HT1080), whereas mainly the 120- and
78-85-kDa species of MT1-MMP were immunocaptured from the MCF7 cells
transfected with MT1-MMP-wt (Fig. 1B;
DTT, MCF7-wt).
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Fig. 4.
Activation of pro-MMP-2 by the wild type and
mutant MT1-MMPs. A, MT1-MMP-E240A is incapable of MMP-2
activation. MCF7 cells transfected with the original pcDNA3-zeo
(zeo) plasmid (lanes 1 and
3) and MT1-MMP-wt, -E240A, -C574A, -ARAA, -R89A, and
-R89A/ARAA (lanes 4-9, respectively) were incubated for 24 h in
serum-free DMEM in the presence of 10 ng/ml purified pro-MMP-2
(lanes 3-9; lane 2,
pro-MMP-2 alone, no cells). Aliquots of conditioned medium were
analyzed by gelatin zymography. The molecular masses of the proenzyme,
the intermediate, and the active enzyme of MMP-2 (68, 64, and 62 kDa,
respectively) are shown on the right. B, the time
course of pro-MMP-2 activation by cells expressing the wild type, ARAA,
and C574A constructs. MCF7 cells stably expressing MT1-MMP-wt, -ARAA,
and -C574A (upper, middle, and lower
panels, respectively) were incubated for 0.5-8 h in
serum-free DMEM in the presence of pro-MMP-2 (20 ng/ml). Aliquots of
conditioned medium were analyzed by gelatin zymography. The molecular
weights of the proenzyme, the intermediate, and the active enzyme of
MMP-2 are shown on the right. C, gelatinolytic
activity of MMP-2. Pro-MMP-2 (750 ng) was incubated for 1 h with
2.5 × 105 MCF7 cells stably expressing MT1-MMP-wt,
-ARAA, -C574A, and -E240A and mock cells (zeo) in 0.15 ml of
serum-free DMEM. Further, cells were washed to remove unbound pro-MMP-2
and incubated with 75 pg of biotinylated gelatin in 0.5 ml of 0.1%
BSA-DMEM for 4 h. Afterward, the gelatinolytic activity of MMP-2
was quantified as described earlier (33). Without pro-MMP-2, cells
demonstrated low or no gelatinolytic activity. Data are mean ± S.E. of three independent experiments.
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Fig. 5.
MT1-MMP localization on the cell
surface. A, immunofluorescence of MCF7 cells.
Mock-transfected cells (zeo) and cells stably expressing
MT1-MMP-wt and -C574A were plated on slides precoated with 10 µg/ml
fibronectin, incubated for 48 h, and then fixed and stained with
10 µg/ml rabbit anti-hinge. Goat anti-rabbit IgG conjugated with
Alexa Fluor 568 (10 µg/ml) was used to visualize bound antibodies.
The images were collected and analyzed by confocal microscopy. The
image obtained with the transmitted light is shown for mock-transfected
cells (zeo, upper left
panel). The pattern of immunofluorescent staining of cells
expressing MT1-MMP-ARAA and MT1-MMP-E240A constructs was
indistinguishable from that of MT1-MMP-wt. Note clustered localization
of MT1-MMP on the plasma membrane, on cell protrusions, and at
cell-cell junctions (arrows). B, ZX sections of
stained cells expressing the wild type and C574A constructs. The
sections confirm cell surface localization of MT1-MMP-wt
(left panel) and MT1-MMP-C574A (right
panel). C, phase-contrast micrographs of MCF7
cells expressing MT1-MMP-wt and -C574A. Cells were cultured on plastic
for 2 days in serum-containing DMEM. Note the difference in spreading
and the number of protrusions between cells expressing MT1-MMP-wt
(wt, left panel) and cells transfected
with the MT1-MMP-C574A construct (C574A, right
panel).
TIMP-2 binding by mutant MT1-MMP
DTT,
MCF7-wt + Ilomastat). In contrast, Ilomastat
failed to affect naturally expressed MT1-MMP in HT1080 (Fig.
1B). These results are not surprising, since activation and
processing of intrinsic MT1-MMP in HT1080 cells were specifically shown
to involve furin (37). However, this does not rule out an existence of
the furin-independent pathway(s) of MT1-MMP processing in other cell
types (27, 28) including breast carcinomas. The furin-independent
mechanisms involved in MT1-MMP activation in cancer cells are yet to be elucidated.
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Fig. 6.
Effects of the wild type and mutant MT1-MMPs
on cell adhesion (A), migration (B),
and invasion (C). Data are presented as a
percentage relative to invasion of mock-transfected cells (100%) and
are mean ± S.E. from three experiments performed in triplicate.
A, mock-transfected cells (zeo) and cells
expressing MT1-MMP-wt, -E240A, and -C574A (5 × 104
cells/well) were allowed to adhere for 1 h to the wells of a
96-well plate coated with 1 µg/ml collagen type I. Adherent cells
were fixed and stained with Crystal Violet in 10% ethanol. The
incorporated dye was extracted, and the absorbance was measured at 540 nm. B, mock-transfected cells (zeo) and cells
expressing MT1-MMP-wt, -E240A, and -C574A were plated into the
Transwells (7.5 × 105 cells/insert) with the undersurface
membranes coated with 20 µg/ml collagen type I. After 48 h,
cells that migrated onto the membrane undersurface were detached and
counted. C, mock-transfected cells (zeo) and
cells stably expressing MT1-MMP-wt, -E240A, ARAA, and -C574A were
plated in Transwell inserts coated with Matrigel. Cells were allowed to
invade Matrigel and to transmigrate for 48 h to the membrane's
undersurface precoated with 20 µg/ml collagen type I. Transmigrated
cells were detached and counted. Where indicated, MT1-MMP-wt cells were
pretreated for 2 days with 50 µM Ilomastat and then
allowed to migrate in the presence of the same concentration of the
inhibitor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
v
3, MT1-MMP facilitated migration of MCF7
cells devoid of MMP-2 (9, 22). In addition, our studies extend the
recent observations that MT1-, MT2-, and MT3-MMP confer
invasion-incompetent Madin-Darby canine kidney cells with the ability
to penetrate collagen type I matrices (43). Hence, the previously
underestimated function of MT1-MMP to support cell locomotion appears
to be a general phenomenon (44, 60).
chain transmembrane and cytoplasmic domains failed to
localize to invadopodia and to facilitate invasion of melanoma cells
(45). Recent results of Lehti et al. (13), who reported that
a truncation of 10 amino acids that included the Cys574
decreased the invasion activity of melanoma cells by 30%, have pointed
out that the middle portion of the cytoplasmic tail had an important
role in cell invasion. In contrast, Hotary et al. (43) observed that truncation of the MT1-MMP cytoplasmic domain did not affect the invasive phenotype of Madin-Darby canine kidney cells stimulated with hepatocyte growth factor. However, the assays of
Hotary et al. (43) were not strictly quantitative.
Alternatively, Urena et al. (15) and Nakahara et
al. (45) demonstrated that the cytoplasmic tail is critically
involved in trafficking of MT1-MMP to discrete regions of the cell
surface. In addition, our most recent finding clearly indicates that
expression of either the C574A construct or the MT1-MMP mutant missing
the entire cytoplasmic tail does not affect the locomotion of extremely
migratory U-251 glioma cells (data not shown). Thus, although our
results are not identical to what has been observed previously, we used
a significantly different cell system that could account for the apparent disparity in findings.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA83017 and CA77470, California Breast Cancer Program Grant 5JB0094, and Susan G. Komen Breast Cancer Foundation Grant 9849 (to A. Y. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence and reprint requests should be addressed: The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-713-6271; Fax: 858-646-3192; E-mail: strongin@burnham.org
Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M007921200
2 D. V. Rozanov, E. I. Deryugina, B. I. Ratnikov, and A. Y. Strongin, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: MT, membrane type; MMP, matrix metalloproteinase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; DPBS, Dulbecco's phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; TIMP, tissue inhibitor of metalloproteinases; mAb, monoclonal antibody; anti-hinge, rabbit antibody AB815 to hinge domain of MT1-MMP; FCS, fetal calf serum; DMEM/BSA, DMEM supplemented with 1% BSA and 10 mM HEPES, pH 7.2; DPBS/BSA, Dulbecco's PBS supplemented with 1 mM CaCl2, 1 mM MgCl2, and 1% BSA, pH 7.2; MT1-MMP-wt, wild type MT1-MMP; DTT, dithiothreitol.
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