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INTRODUCTION |
Remodeling of the extracellular matrix
(ECM)1 is critical for cancer
cell invasion and tumorigenesis (1-5). Membrane type matrix
metalloproteinases (MT-MMPs) localized to the invasive front of highly
motile cancer cells (6, 7) were shown to be directly involved in matrix
breakdown (8-13). A cooperation involving MT-MMPs and cell adhesion
receptors is likely to be essential to migrating cells (3, 14-16). So
far, six members of the MT-MMP subfamily have been identified and
partially characterized (2, 17-22). MT1-, MT2-, and MT3-MMP strongly
contribute to tumor cell invasion (12). Recent studies demonstrated a
functional significance and a direct role of MT1-, MT2-, and MT3-MMP in
cell locomotion on laminin-5 (11) and three-dimensional collagen type I
lattice (8, 9, 12). In addition, MT-MMPs contribute indirectly to cell
invasion by activating soluble secretory MMP-2 (23) and MMP-13 (24),
which further cleave multiple matrix substrates (2, 5, 25-29).
Integrin adhesion receptors dynamically regulate cell-matrix
interactions by the binding to matrix proteins and inside-out signaling
(30, 31). This allows cells to discriminate any subtle alteration of
the environment and to adjust cell locomotion accordingly. Direct
interactions with multiple transmembrane and cell surface proteins (32)
including integrin-associated protein-50 (33), TM4SF proteins
(tetraspanins) (34) and tTG (35) further attenuate adhesive and
signaling efficiency of integrins.
Cell surface tTG (protein-glutamine
-glutamyltransferase, EC
2.3.2.13) promotes integrin-dependent adhesion and
spreading of cells. By both direct associations with multiple
1 and
3 integrins and the binding with
Fn, tTG independently mediates the interactions of integrins with Fn
(35). The high affinity binding of tTG with Fn specifically involves
the 42 kDa gelatin-binding domain of the Fn molecule, which consists of
modules I6II1,2I7-9 (36). The
enhancement of integrin-mediated adhesion and spreading of cells on Fn
is independent from the enzymatic activity of surface tTG (35).
Intriguingly, reduced expression of tTG has been linked to
aggressiveness and high metastatic potential of tumors, whereas overexpression of tTG in fibrosarcomas inhibited primary tumor growth
(37, 38). Proteolysis of tTG at the normal tissue/tumor boundary was
observed in invasive tumors (38).
Here, we report that depending on the structure of the ECM,
MT-MMPs are capable of both positively and negatively regulating locomotion of cancer cells. Matrix-dependent proteolysis of
surface tTG by MT1-MMP occurs on tumor cells of a diverse tissue
origin, thereby representing a general phenomenon and a novel MT-MMP
function. Our data suggest an existence of an unexpected link between
tumor cell locomotion, the ECM and membrane-anchored MMPs. Regulatory proteolysis of cell surface adhesion proteins by the adjacent MT-MMP
molecules is likely to play a significant functional role in cancer
cell invasion.
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MATERIALS AND METHODS |
Proteins, Antibodies, and Cell Lines--
The tTG protein was
purified from human red blood cells (39). GM6001 (Ilomastat), tissue
inhibitors of metalloproteinases TIMP-1 and TIMP-2, the individual
catalytic domains of MT1-MMP, MT2-MMP, MT3-MMP, and MT4-MMP and
anti-MT1-MMP rabbit polyclonal antibody AB815 were purchased from
Chemicon (Temecula, CA). Anti-
1 integrin mAb 9EG7 and
sulfo-NHS-biotin were from Pharmingen (San Diego, CA) and Pierce
(Rockford, IL), respectively. Fn and its proteolytic fragments were
obtained as reported earlier (35). Vector- and MT1-MMP-transfected
human HT1080 fibrosarcoma and U251 glioma cell lines were selected and
maintained as described previously (27-29, 32). Where indicated,
protein synthesis in cells was blocked with 20 µg/ml cycloheximide.
Rabbit polyclonal antibody against the full-length tTG protein, as well
as rabbit function-blocking antibody against the
NH2-terminal 1-165 Fn-binding fragment of tTG inhibitory
to the tTG-Fn interactions, and anti-tTG mAbs TG100 and CUB7402 were
characterized earlier (35). Radioactive labeling of cells with
Tran35S-label (ICN Biochemicals, Irvine, CA) was performed
as described earlier (35).
Proteolysis of tTG in Vitro and the NH2-terminal
Sequencing of the Proteolytic Fragments--
Purified tTG (10-20
µg) was incubated with the individual catalytic domain of MT-MMPs
(0.2 µg each) for 0.5-12 h at 37 °C in 0.05 M
Tris-HCl buffer, pH 7.5, containing 50 mM NaCl, 1 mM CaCl2, and 10 µM
ZnCl2. The reaction was stopped by SDS-sample buffer. After
electrophoresis in 12% gels and transfer to a polyvinylidene difluoride membrane, protein bands were stained, excised, and subjected
to the NH2-terminal microsequencing at the Protein
Chemistry Facility of Washington University (St. Louis, MO).
Flow Cytometry--
To determine surface levels of MT1-MMP, live
untreated or GM6001-treated HT-vector and HT-MT cells were
stained with 10 µg/ml rabbit anti-MT1-MMP antibody followed by
fluorescein-labeled goat anti-rabbit IgG. For measurements of cell
surface tTG, live nonpermeabilized transfectants were each incubated
for 2 h at 37 °C without or with 20 µM GM6001, 1 µg/ml TIMP-1, and 1 µg/ml TIMP-2 and then stained with 10 µg/ml
rabbit polyclonal anti-tTG antibody and secondary
fluorescein-conjugated IgG (35). Cells were analyzed by a FACScan flow
cytometer (Becton Dickinson). At least three independent experiments
were performed for each cell line.
Zymography--
To evaluate the status of MMP-2, nonreduced
aliquots of medium conditioned with HT-vector and HT-MT cells were
analyzed by gelatin zymography. Zymography was performed in 0.1%
gelatin and 10% polyacrylamide gels as described previously (29).
After electrophoresis, SDS was replaced by Triton X-100, followed by incubation in a Tris-based buffer overnight. Gels were stained with
Coomassie Brilliant Blue, and gelatinolytic activity was detected as
clear bands in the background of uniform staining.
Measurement of Transglutaminase Activity--
Transglutaminase
activity expressed on the surface of HT-vector and HT-MT cells was
determined as reported earlier (35). For these purposes, cells were
detached by EDTA. Live cells (2 × 106) were
resuspended in 0.5 ml of phosphate-buffered saline containing 2 mM Ca2+ and 10 mg/ml
N,N-dimethylcaseine. Further, cells were
incubated with 10 µCi of [3H]putrescine (35.7 Ci/mmol,
PerkinElmer Life Sciences) for 1 h at 37 °C on a rotator. To
evaluate the incorporation of [3H]putrescine,
N,N-dimethylcaseine was precipitated from
cell-free supernatants with ice-cold 10% trichloroacetic acid. Excess
label was removed by successively washing the pellets with 5%
trichloroacetic acid, ethanol, and acetone. Finally, the pellet was
dried and redissolved in 100 µl of 1% SDS. Protein-incorporated
radioactivity was determined by scintillation counting.
Binding of the 42-kDa Fragment of Fn to Cells--
EDTA-detached
HT-vector and HT-MT cells resuspended at 2 × 106
cells/200 µl of Tyrode's buffer, 10 mM HEPES, 150 mM NaCl, 2.5 mM KCl, 2 mM
NaHCO3, 2 mM MgCl2, 2 mM CaCl2, 1 mg/ml bovine serum albumin, 1 mg/ml
dextrose, pH 7.4, were incubated with 1 µM
125I-labeled 42 kDa Fn fragment (specific activity 1.6 × 106 cpm/µg) for 1 h at 37 °C on a rotator.
Further, cells were layered on 0.5 ml of 20% sucrose in Tyrode's
buffer and centrifuged for 5 min at 10,000 rpm. 125I
radioactivity associated with the cell pellets was quantified in a
gamma counter. Measurements were performed in triplicates. Values were
corrected for nonspecific binding determined in presence of excess (10 µM) unlabeled 42-kDa Fn fragment.
Immunoprecipitation of Cell Surface MT1-MMP and tTG--
To
determine the expression and the status of surface MT1-MMP and tTG,
EDTA-detached cells were surface biotinylated for 15 min in
phosphate-buffered saline containing 0.1 mg/ml sulfo-NHS-biotin. MT1-MMP and tTG were immunoprecipitated from the lysates of
surface-biotinylated cells with MT-MMP- and tTG-specific polyclonal
antibodies, respectively. The resulting immune complexes were separated
by SDS-PAGE and transferred to a membrane support. Biotin-labeled
proteins were visualized by probing the membrane with
neutravidin-horseradish peroxidase.
Coprecipitation Studies--
To analyze coprecipitation of tTG
with
1 and
3 integrins, HT-MT cells were
plated for 12 h in serum-free medium supplemented with 20 µg/ml
cycloheximide on plastic coated with Fn or collagen type I. Where
indicated, 20 µM GM6001 was added to the medium. Next,
cells were lysed in RIPA buffer, containing 1% Triton X-100, 0.55 sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris-Cl, pH 7.5, with 0.5 mM
phenylmethylsulfonyl fluoride, 0.5 mM benzamidine, 10 µg/ml leupeptin, and 10 µg/ml aprotinin (35).
1
integrins,
3 integrins, and tTG were each
immunoprecipitated from the aliquots (0.5 mg of total protein) of the
RIPA lysates with mAb P4C10, mAb LM609, and rabbit polyclonal anti-tTG
antibody, respectively. The precipitated samples were separated by
SDS-PAGE and transferred to a membrane. To identify specifically tTG in
the precipitated samples the membrane was probed with mAb TG100 against
tTG.
Immunofluorescence--
To protect surface tTG from proteolysis,
cells were plated for 24 h in serum-containing medium on Fn-coated
coverslips. Next, live nonpermeabilized cells were double stained for
tTG/MT1-MMP, tTG/
1 integrin, and
1
integrin/MT1-MMP by using a respective combination of murine anti-tTG
mAbs CUB7402 or TG100 (20 µg/ml), rat anti-
1 integrin
mAb 9EG7 (10 µg/ml), and rabbit anti-MT1-MMP antibody AB815 (10 µg/ml). After incubation with antibodies for 40 min, cells were
washed and fixed with 3% paraformaldehyde in phosphate-buffered
saline. Cell surface proteins were visualized using secondary
species-specific IgG conjugated with rhodamine or fluorescein (Chemicon).
To induce proteolytic degradation of surface tTG by MT1-MMP, cells were
detached by EDTA and then incubated in serum-free medium at 37 °C
for 2 h. Then, cells were plated for 3 h on Fn in serum-free
medium containing 20 µg/ml cycloheximide. Three antibodies (murine
anti-tTG, rat anti-
1 integrin, and rabbit anti-MT1-MMP)
were used to visualize simultaneously tTG,
1 integrin, and MT1-MMP on live nonpermeabilized cells. After fixation, the proteins were detected with goat anti-rabbit IgG, goat anti-mouse IgG,
and goat anti-rat IgG conjugated with blue Alexa-Fluor 350, green
Alexa-Fluor 488, and red Alexa-Fluor 594 (Molecular Probes, Eugene, OR)
with nonoverlapping emission spectra, respectively. The cells were
analyzed and photographed by using Nikon Eclipse E800 epifluorescence
microscope and Spot RT digital camera (Molecular Diagnostics).
Cell Adhesion and Migration Assays--
For adhesion studies, a
suspension of 35S-labeled U-vector or U-MT cells in
serum-free medium was incubated for 2 h without or with 20 µM GM6001, 1 µg/ml TIMP-1, or 1 µg/ml TIMP-2 in the presence of 20 µg/ml cycloheximide. 1 h before plating on
plastic coated with 10 µg/ml Fn, the 110-kDa or the 42-kDa Fn
fragments and blocked with bovine serum albumin, function-blocking
anti-tTG antibody (10 µg/ml) or control nonimmune IgG (10 µg/ml)
was added to the corresponding cell samples. The cells were plated for
1 h without or with GM6001 in serum-free medium supplemented with 20 µg/ml cycloheximide (35). Adherent cells were washed with phosphate-buffered saline and lysed in 1% SDS. The bound radioactivity was counted and converted in the number of adherent cells by referring to the levels of 35S incorporation/103 cells.
Directional migration of cells (5 × 104 cells/insert)
in Transwells (Costar, Cambridge, MA) with the membrane undersurface coated with collagen I, Fn, the-42 kDa or 110-kDa Fn fragments (10 µg/ml each), was analyzed under serum-free conditions (27, 32).
U-vector and U-MT cells were incubated for 2 h without or with 20 µM GM6001, 1 µg/ml TIMP-1, and 1 µg/ml TIMP-2 in
serum-free AIM-V medium (Life Technologies, Inc.). 1 h prior to
plating, function-blocking anti-tTG antibody (10 µg/ml) or control
nonspecific IgG (10 µg/ml) was added to the respective samples. Both
the upper and the lower chambers of the inserts were supplemented with
10 µM GM6001, 1 µg/ml TIMP-1, 1 µg/ml TIMP-2, 10 µg/ml function-blocking anti-tTG antibody, or 10 µg/ml
control IgG. After incubation for 12 h, cells transmigrated to
the membrane undersurface were detached and counted. At least three
independent experiments were performed with each reagent and cell type.
 |
RESULTS |
tTG Is Degraded and Inactivated on the Surface of Cancer Cells
Expressing MT1-MMP--
HT1080 fibrosarcoma and U251 glioma cells that
naturally express relatively low levels of MT1-MMP were transfected
with the MT1-MMP cDNA (GenBank U41078) to up-regulate MT1-MMP and
create HT-MT and U-MT pools of cells, respectively (27-29, 32). To
avoid any undesirable clonal effects, our studies were performed with the corresponding pools of stable transfectants. Highly elevated levels
of MT1-MMP in U-MT cells relative to U-vector control were confirmed
previously by immunoprecipitation, flow cytometry, and functional
assays (27-29, 32, 40). To corroborate these findings further, we
evaluated the levels of MT1-MMP in HT-MT cells by immunoprecipitation,
flow cytometry, and by analysis of the ability of cells to initiate the
activation of the secretory MMP-2 proenzyme. Immunoprecipitation and
flow cytometry studies employed rabbit polyclonal AB815 antibody
directed against the hinge region of MT1-MMP. Immunoprecipitation
confirmed an increase in the amounts of 60-kDa enzyme and the 42-kDa
ectodomain form of MT1-MMP expressed on the surface of HT-MT cells,
relative to those of HT-vector cells (Fig.
1a). Earlier, it has been
shown that TIMP-2 regulates the amount of active 60-kDa MT1-MMP enzyme
on the cell surface, whereas in the absence of TIMP-2 MT1-MMP undergoes
autocatalysis to the 42-kDa form (41, 42). Apparently, our
immunoprecipitation experiments showing high levels of the 42-kDa form
point to an insufficiency of TIMP-2 relative to MT1-MMP in HT-MT
transfectants (43).

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Fig. 1.
Characterization of MT1-MMP and MMP-2 in
HT-MT transfectants. Panel a, analysis of
cell surface MT1-MMP by immunoprecipitation. MT1-MMP was
immunoprecipitated from the lysates of surface-biotinylated cells with
anti-MT1-MMP antibody and protein A-Sepharose. After SDS-PAGE under
reducing conditions and transfer to a membrane, MT1-MMP was detected
with neutravidin-horseradish peroxidase. Arrows point to the
60-kDa active enzyme and the 42-kDa ectodomain form of MT1-MMP.
Positions of molecular mass markers are shown in kDa on the
right. Panel b, flow cytometry analysis of
MT1-MMP in HT-vector and HT-MT cells. Cells grown without or with
GM6001 were stained with rabbit anti-MT1-MMP antibody and
fluorescein-conjugated secondary IgG. Panel c, MT1-MMP
induces activation of secretory MMP-2 in HT-MT cells. HT-MT and
HT-vector cells were plated in serum-containing medium at 2 × 105 cells/well of a 24-well plate. After overnight
incubation, medium was replaced by serum-free Dulbecco's modified
Eagle's medium with or without GM6001, and cells were incubated for
48 h. Samples of conditioned medium were then analyzed by gelatin
zymography. Arrows (from top to
bottom) point to the proenzyme, the intermediate, and the
mature enzyme of MMP-2. Positions of molecular mass markers are shown
in kDa on the left.
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Flow cytometry confirmed the immunoprecipitation data and demonstrated
high cell surface levels of MT1-MMP expressed in HT-MT cells (Fig.
1b). As determined by gelatin zymography of conditioned medium samples, up-regulation of MT1-MMP correlated directly with the
ability of HT-MT cells to activate the MMP-2 proenzyme (44). The
intermediate and active forms of MMP-2 enzyme were present in the
conditioned medium from HT-MT but not from HT-vector cells (Fig.
1c, middle and bottom arrows).
Apparently, MT1-MMP transfection shifts a balance among the preexisting
levels of TIMP-2, MT1-MMP, and MMP-2, thus inducing the activation of
pro-MMP-2 by the cells (23). GM6001, a hydroxamate inhibitor of MMPs
(45), failed to affect the total amounts of MT1-MMP expressed by HT-MT
and HT-vector cells (Fig. 1b). In contrast, GM6001, by
binding to the active site and, thereby inactivating MT1-MMP,
completely abolished the MT1-MMP-induced activation of pro-MMP-2 (Fig.
1c). These findings correlate well with the results of our
previous studies with U-MT and U-vector control cells (40) and
demonstrate high levels of functionally active MT1-MMP enzyme expressed
on the surface of cancer cells stably transfected with MT1-MMP cDNA.
Flow cytometry analyses demonstrated a 4-fold decrease in surface tTG
expression in HT-MT cells relative to that of HT-vector control cells
(Fig. 2a). GM6001 at 20 µM restored the expression of tTG on the surface of HT-MT
cells to the control levels. Because GM6001 is a general inhibitor of
MMP activity, we employed TIMP-2, an efficient inhibitor of MT1-MMP,
and TIMP-1, which is a poor inhibitor of MT1-MMP, to distinguish the
MT1-MMP effect on tTG. Importantly, TIMP-2 fully restored surface tTG
on HT-MT cells. In turn, TIMP-1 was significantly less potent relative
to TIMP-2 and GM6001 in its ability to restore the tTG expression on
HT-MT cells (Fig. 2a). Any inhibitor had no effect on
surface tTG of HT-vector cells. These findings indicate that MT1-MMP
resistant to TIMP-1 inhibition is primarily involved in proteolysis
affecting cell surface tTG in HT-MT cells.

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Fig. 2.
Proteolytic degradation by MT1-MMP destroys
enzymatic and coreceptor activities of surface tTG. Panel
a, flow cytometry analysis of surface tTG in HT-vector and HT-MT
cells. Untreated cells and cells pretreated with 20 µM
GM6001, 1 µg/ml TIMP-1, or TIMP-2 inhibitors were stained with
anti-tTG antibody followed by secondary fluorescein-labeled IgG. The
level of surface tTG in untreated HT-vector cells was taken as 100%.
Shown are the means of triplicate determinations. Panel b,
immunoprecipitation of surface tTG from HT-vector and HT-MT cells.
Cells were incubated without or with the MMP inhibitors then surface
biotinylated and lysed. tTG was immunoprecipitated from cell lysates
with anti-tTG antibody and protein A-Sepharose. After SDS-PAGE under
reducing conditions and transfer to a membrane, tTG was detected with
neutravidin-horseradish peroxidase. Panel c, measurement of
transglutaminase activity on the surface of HT-vector and HT-MT cells.
Cells were incubated without or with 20 µM GM6001. The
enzymatic activity of tTG expressed on the cell surface was quantified
by measuring cell-mediated incorporation of
[3H]putrescine into
N,N-dimethylcaseine. Bars show the
means of triplicate determinations. Panel d, binding of the
42-kDa gelatin-binding fragment of Fn to HT-vector and HT-MT cells.
Cells in suspension were preincubated without or with 20 µM GM6001 and then incubated with the
125I-labeled 42-kDa Fn fragment (1 µM) for
1 h at 37 °C. Cells were separated from excess labeled fragment
by centrifugation through a layer of 20% sucrose. Cell-bound
radioactivity was counted in a gamma counter. Shown are means of
triplicate measurements. Panel e, immunostaining of surface
tTG in HT-vector and HT-MT cells. Cells were cultured for 24 h in
serum-free medium and then plated for 3 h on coverslips coated
with Fn. Nonpermeabilized cells were stained with polyclonal anti-tTG
antibody. Note the accumulation of tTG at the edge of lamellipodia in
HT-vector cells (arrows) and the lack of tTG staining in
HT-MT cells. Bar, ~50 µm.
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To confirm the proteolytic degradation of surface tTG by MT1-MMP in
cultured cells, the HT-vector and HT-MT transfectants were labeled with
membrane-impermeable sulfo-NHS-biotin. The surface-biotinylated tTG was
immunoprecipitated from cell lysates. In contrast to intact tTG
(molecular mass ~80 kDa) found in HT-vector cells, its
proteolytic fragments of ~70, ~53, ~41, and ~32 kDa were
identified in untreated HT-MT cells (Fig. 2b). Inhibitors
such as GM6001, TIMP-1, and TIMP-2 failed to affect the status of tTG
in HT-vector cells. On the contrary, pretreatment with GM6001
completely abolished the degradation of surface tTG in HT-MT cells. In
these assays, TIMP-2 was slightly less efficient compared with GM6001,
whereas TIMP-1 failed to protect tTG from proteolysis in HT-MT cells
(Fig. 2b). Together, our observations point to the main role
of TIMP-1-resistant MT1-MMP in the proteolysis of cell surface tTG in
HT-MT cells. However, MMP-2 and possibly other MMPs may also contribute
to proteolytic degradation of surface tTG.
To demonstrate that degradation of cell surface tTG in HT-MT cells
abolished its functional activities, we measured both the transglutaminase enzymatic (cross-linking) activity and the ability of
surface tTG to bind specifically the 42-kDa gelatin-binding fragment of
Fn (Fig. 2, c and d). We observed only a low
residual level of transglutaminase activity on the surface of untreated HT-MT cells compared with that of control HT-vector cells. GM6001 restored the enzymatic activity of surface tTG in HT-MT cells to the
level observed in HT-vector cells (Fig. 2c). The fact that the associations of surface tTG with Fn specifically involve the 42-kDa
Fn fragment was established in our earlier work (35). Accordingly, the
efficiency of HT-MT cells to bind the 42-kDa Fn fragment directly was
drastically reduced relative to that of control HT-vector cells. Again,
inhibition of MMP activity by GM6001 fully restored the binding
efficiency of HT-MT cells and failed to affect that of HT-vector cells
(Fig. 2d). In agreement with our previous results (35),
preincubation of HT-vector and HT-MT cells with polyclonal antibody
against tTG abolished the binding of the 42-kDa fragment of Fn to cell
surfaces (data not shown).
Immunofluorescent staining confirmed low levels of surface tTG in
untreated nonpermeabilized HT-MT cells. In contrast, immunofluorescence of surface tTG was far more intense in HT-vector cells, particularly at
the leading edge (Fig. 2e, arrows). Together, our
results show that expression of MT1-MMP by cells strongly contributes
to the degradation of surface tTG. The degradation of tTG correlates with the inactivation of the both enzymatic and coreceptor functions of
this cell surface protein.
Proteolysis of Purified tTG in Vitro--
To support our findings,
we used enzymatically active individual catalytic domains of MT1-MMP
(Fig. 3a) and related MT2-MMP, MT3-MMP, and MT4-MMP (Fig. 3b) to cleave the purified tTG
protein in vitro. MT1-MMP, MT2-MMP, and MT3-MMP efficiently
and specifically degraded tTG in vitro. The proteolytic
fragments of tTG generated in vitro were similar to those
detected on the cell surface (Figs. 2b, 3a, and
4a). This provides a key evidence that the MT1-MMP activity
is essential for the proteolysis of surface tTG observed in cultured
cells.

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Fig. 3.
Proteolysis of tTG in vitro.
Panel a, degradation of purified tTG by the catalytic domain
of MT1-MMP. Purified tTG (20 µg) was incubated for 1-12 h at
37 °C with the catalytic domain of MT1-MMP (0.2 µg; the
enzyme:substrate molar ratio equals about 1:25). The samples were
analyzed further by SDS-PAGE and stained with Coomassie Brilliant Blue.
Molecular mass markers are shown on the right.
Arrowhead points to intact tTG. Arrows indicate
the proteolytic fragments with the molecular masses of ~53, ~41,
and ~32 kDa. These fragments were excised and subjected to
NH2-terminal microsequencing shown in panel
d. [1], [2], and [3]
designate, in the order of appearance, the cleavage sites
H461L, R458A, and P375V,
respectively. Panel b, MT1-MMP, MT2-MMP, and
MT3-MMP, but not MT-4-MMP, degrade purified tTG in vitro.
Purified tTG (20 µg) was incubated for 4 h at 37 °C with the
catalytic domain of MT1-MMP, MT2-MMP, MT3-MMP, and MT4-MMP (0.2 µg
each). The samples were separated by SDS-PAGE and analyzed by blotting
with polyclonal anti-tTG antibody. Panel c, Fn protects tTG
from the degradation by MT1-MMP in vitro. Purified tTG (20 µg) was incubated in a total volume of 100 µl for 4 h at
37 °C with the catalytic domain of MT1-MMP (0.2 µg) either alone
or with 120 µg of Fn, 160 µg of laminin, or 200 µg of collagen I. The samples were separated by SDS-PAGE and analyzed by blotting with
polyclonal anti-tTG antibody. Panel d, sequence of the
cleavage sites and the relative position of the proteolytic fragments.
The major MT1-MMP cleavage sites of tTG (marked [1],
[2], and [3]) were determined by
microsequencing of the 53-, 41-, and 32-kDa fragments.
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Extensive proteolysis of tTG by the catalytic domain of MT1-MMP
resulted in four major (molecular masses of ~70, ~53, ~41, and
~32 kDa) and several minor cleavage products (Fig. 3a,
marked by arrows and numbered). In contrast, the
catalytic domain of MT4-MMP, a
glycosylphosphatidylinositol-linked membrane enzyme that is
structurally and functionally distant from the MT1/MT2/MT3-MMP subfamily (20), failed to degrade tTG in vitro (Fig.
3b).
The 53-, 41-, and 32-kDa fragments were partially sequenced to identify
the MT1-MMP cleavage sites (Fig. 3, b and d,
arrows). The MT1-MMP cleavage at the two close
FTR458-AN and ANH461-LN sites generated
the 53- and 32-kDa fragments that represented the NH2- and
the COOH-terminal parts of tTG, respectively. The NH2
terminus of both the intact tTG polypeptide and the 53-kDa fragment is
blocked by acetylation. The cleavage of tTG at PVP375-VR
splits the protein in half providing the COOH-terminal 41-kDa fragment
with the NH2-terminal V376RAIK sequence. The
PVP375-VR sequence partially fits the PXP(G)L
consensus cleavage site of MT1-MMP elucidated by using phage display
libraries (46). Importantly, MT1-MMP cleavage at any of these three
sites should eliminate both the receptor and enzymatic activity of tTG
by separating its NH2-terminal Fn binding and the
COOH-terminal integrin binding domains (35, 47) and by inactivating its
second (catalytic) domain (Fig. 2, c and d).
Thus, the mapping of cleavage sites on tTG in vitro is in
agreement with the results of our enzymatic and coreceptor binding
studies of surface tTG in HT-MT cells.
Fn Inhibits Proteolysis of tTG--
We next assessed whether Fn, a
matrix ligand of cell surface tTG, and laminin and collagen type I,
other common ECM proteins but not the ligands of tTG (35), affect the
degradation of tTG by MT1-MMP. Excess Fn blocked proteolysis of tTG
in vitro. In contrast, excess laminin or denatured collagen
type I had no effect on the tTG proteolysis (Fig. 3c). To
confirm these effects in cultured cells, we plated HT-MT and HT-vector
cells in serum-free medium on plastic coated with Fn and collagen type
I. Further, cells were detached, labeled with biotin, and surface tTG
was analyzed by immunoprecipitation (Fig.
4a). Without GM6001, HT-vector cells on collagen expressed intact surface tTG. On the contrary, surface tTG was degraded in HT-MT cells plated on collagen type I but
intact in cells cultured on Fn (Fig. 4a). To elaborate
further on the effects of Fn, we explored our earlier findings that
cell surface tTG coprecipitated with
1 and
3 integrins (35). For these purpose, we
immunoprecipitated
1 and
3 integrins from RIPA lysates and analyzed the precipitated samples for tTG. Large amounts of undegraded (~80 kDa) tTG, which comprised ~15-25% of total cellular tTG, coprecipitated with
1 and
3
integrins from the lysates of HT-MT cells cultured on collagen in the
presence of GM6001 or on Fn without this hydroxamate inhibitor (Fig.
4b, middle and bottom panels,
respectively). In contrast, only low levels of undegraded tTG, which
accounted for ~1.8-2% of total cellular tTG, coprecipitated with
1 or
3 integrins if HT-MT cells were
grown on collagen I without GM6001 (Fig. 4a,
top panel, arrow). These results show that Fn
specifically inhibited proteolysis of its own adhesion receptor, cell
surface tTG, by MT1-MMP. A failure of collagen type I, a substrate more
susceptible to MT1-MMP degradation than Fn (8, 45), to inhibit
proteolysis of tTG, emphasizes a functional significance of the
specific inhibitory effect of Fn.

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Fig. 4.
Fn specifically protects surface tTG from
proteolysis by MT1-MMP. HT-vector and HT-MT cells were plated for
12 h in serum-free medium supplemented with 20 µg/ml
cycloheximide on plastic coated with Fn or collagen type I. Where
indicated, GM6001 was added to the medium. Panel a, Fn
inhibits proteolysis of tTG. 12 h after plating on substrates,
cells were detached with EDTA, surface biotinylated, and lysed. tTG was
immunoprecipitated from the cell lysates, separated by SDS-PAGE, and
analyzed by blotting with neutravidin-horseradish peroxidase. Note the
degradation of surface tTG in HT-MT cells on collagen I and the
inhibition of the tTG proteolysis by Fn and GM6001. Panel b,
Fn increases the levels of tTG coprecipitated with 1 and
3 integrins in HT-MT cells. 12 h after plating on
substrates, cells were lysed with RIPA buffer. 1
integrins, 3 integrins, and tTG were immunoprecipitated
from aliquots of cell lysates (0.5 mg of total protein) with specific
mAb P4C10, mAb LM609, and mAb TG100, respectively. The precipitated
samples were separated by SDS-PAGE and transferred to a membrane.
Precipitated tTG was detected in the samples by probing the membrane
with anti-tTG polyclonal antibody. Note a reduction of
integrin-associated surface tTG in cells plated on collagen I.
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Localization of tTG,
1 Integrins, and MT1-MMP on
Cell Surfaces--
Our earlier results indicated that tTG was
colocalized with
1/
3 integrins at
discrete regions of the cell surface (35). Apparently, surface tTG
could be degraded by MT1-MMP only if this membrane-anchored proteinase
is proximal to the tTG·integrin clusters. To demonstrate that
tTG degradation by MT1-MMP might occur on cancer cells of a diverse
tissue origin, we examined tTG, MT1-MMP, and
1 integrins
on the surface of U-MT and U-vector cells derived from parental U251
human glioma cells. The status of MT1-MMP and MMP-2 in U-MT and
U-vector cells has been characterized extensively in our previous work
(39). When U-MT cells were plated for 24 h in serum-containing
medium on Fn, that was shown to prevent proteolysis of tTG (Fig. 4,
a and b), an extensive colocalization of tTG,
MT1-MMP, and
1 integrins became evident at the periphery of U-MT cells (Fig. 5a, merge
in yellow).

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Fig. 5.
Colocalization of tTG and
1 integrins with MT1-MMP.
Panel a, both surface tTG and 1 integrins are
colocalized with MT1-MMP in U-MT cells. To protect surface tTG from
proteolysis, cells were plated in serum-containing medium for 24 h
on Fn-coated coverslips. Next, live nonpermeabilized cells were double
stained with anti-tTG mAbs CUB7402/TG100 and polyclonal anti-MT1-MMP
antibody AB815 (left panels), anti- 1 integrin
mAb 9EG7 and polyclonal anti-MT1-MMP antibody AB815 (middle
panels), and anti-tTG mAbs CUB7402/TG100 and anti- 1
integrin mAb 9EG7 (right panels). Note the colocalization
(lower panels, merge in yellow) of these proteins
at the edge of lamellipodia. Bar, ~40 µm. Panel
b, MT1-MMP expression causes a disappearance of surface tTG
resulting from proteolytic degradation. U-vector and U-MT cells were
detached with EDTA and incubated for 2 h prior to plating in
cycloheximide-containing serum-free medium without or with GM6001
(left and two right panels, respectively).
Because cycloheximide by significantly blocking de novo
protein synthesis did not allow cells to restore the surface tTG pool,
degradation of surface tTG by MT1-MMP became evident in the absence of
the MMP inhibitor. 3 h after plating on Fn, nonpermeabilized cells
were triple labeled with rabbit anti-MT1-MMP antibody AB815
(blue), murine mAbs CUB7402/TG100 against tTG
(green), and rat mAb 9EG7 against 1 integrin
(red). Note the colocalization of tTG with 1
integrins at the periphery of untreated U-vector and GM6001-treated
U-MT cells (left and right panels,
arrows). MT1-MMP degradation caused the loss of tTG on U-MT
cells cultured without GM6001 (middle panels,
arrowheads). Bar, ~25 µm.
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To corroborate these findings, we preincubated U-MT and U-vector cells
in suspension for 2 h without or with GM6001 and then plated the
cells for 3 h on Fn in serum-free medium containing cycloheximide.
No MT1-MMP was detected on U-vector cells, whereas
1
integrins and tTG were colocalized at the cell periphery (Fig. 5b, left panels, arrows). In U-MT cells, MT1-MMP
accumulated at the edges of lamellipodia (Fig. 5b, top
panels, arrow, arrowhead). In the absence of
GM6001 the peripheral staining of tTG in U-MT cells was consistently
very weak or absent, pointing to degradation of the cell surface
protein (Fig. 5b, middle panels,
arrowhead). Localization of
1 integrins at
the periphery of untreated U-MT cells was partially disrupted (Fig.
5b, arrowhead). In contrast, GM6001 restored both
the peripheral staining and the colocalization of tTG with MT1-MMP and
1 integrin at the leading edge of U-MT cells (Fig.
5b, right panels, arrows).
Functional Significance of the Regulatory Proteolysis of Cell
Surface tTG--
To evaluate the functional significance of the
proteolytic degradation of surface tTG by MT1-MMP, we employed cell
function in vitro tests such as adhesion and migration
assays. Surface tTG interacts with Fn via binding to its 42-kDa gelatin
binding domain (35). Accordingly, we used intact Fn and its proteolytic gelatin-binding fragment of ~42 kDa in our adhesion and migration experiments. As a control, we employed the ~110-kDa Fn fragment that
is incapable of binding tTG but associates with integrins (35).
Adhesion of untreated U-MT cells to Fn was ~30% lower relative to
that of control U-vector cells (Fig.
6a). In the presence of MMP
inhibitors such as GM6001 or TIMP-2, adhesion of U-MT cells was
restored to the control levels. In contrast, TIMP-1, a poor inhibitor
of MT1-MMP activity, showed only a weak effect in restoring adhesion of
U-MT cells to Fn. Because the interaction of U-251 glioma cells with Fn
involves multiple integrin receptors (27-29), elimination or blocking
of a single receptor such as tTG cannot affect the overall adhesive
properties of cells strongly. Indeed, we observed a moderate effect of
tTG proteolysis on the adhesion of U-MT cell to Fn. To emphasize
specifically the effect of tTG degradation on cell adhesion, we
evaluated further the adhesion of U-vector and U-MT cells to the 42-kDa
fragment of Fn (Fig. 6, b and d).

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Fig. 6.
Degradation of surface tTG by MT1-MMP
decreases cell adhesion on Fn and its 42-kDa fragment. Cells were
detached with EDTA, incubated where indicated with 1 µg/ml TIMP-1, 1 µg/ml TIMP-2, 20 µM GM6001, or 10 µg/ml
function-blocking anti-tTG antibody, or both GM6001 and anti-tTG. Then
cells in serum-free medium in the presence of 20 µg/ml cycloheximide
were allowed to adhere for 1 h on plastic wells coated with 10 µg/ml Fn (panels a and c) or the 42-kDa
gelatin-binding fragment of Fn (panels b and d).
Shown are the means of triplicate measurements. Panels a and
b, effects of the inhibitors of MMP activity on adhesion of
U-vector and U-MT cells to Fn (panel a) and the 42-kDa
fragment of Fn (panel b). Panels c and
d, effects of GM6001 and function-blocking anti-tTG antibody
on the adhesion of U-vector and U-MT cells to Fn (panel c)
or the 42-kDa Fn fragment (panel d). Note that in U-MT cells
anti-tTG antibody reversed the stimulatory effect of GM6001 on both
substrates.
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A more striking difference between U-MT and U-vector cells was
identified when cells were allowed to adhere on this fragment of Fn.
U-MT cells were about 7-fold less efficient in adhesion to this
fragment compared with U-vector control. GM6001 and TIMP-2 each
restored adhesion of U-MT cells to U-vector control levels, whereas
TIMP-1 displayed a weak effect (Fig. 6b). All of the
inhibitors of MMP activity had no effect on U-vector cells (Fig. 6,
a and b).
A direct role of tTG in facilitating cell adhesion was confirmed in
experiments that employed function-blocking antibody against tTG. When
this antibody was used in adhesion assays without GM6001, it did not
affect adhesion of U-MT cells to Fn or its 42-kDa fragment (Fig. 6,
c and d). In contrast, the use of antibody
against tTG abolished stimulatory effects of GM6001 on adhesion of U-MT
cells to both of these substrates. Neither MMP inhibitors nor anti-tTG affected adhesion of U-MT and U-vector cells to the 110-kDa fragment of
Fn (data not shown).
Further, we evaluated migration of U-vector and U-MT cells in
Transwells with the undersurface coated with Fn (Fig.
7a). The stimulatory effects
of GM6001, TIMP-2 and TIMP-1 on U-vector cells were insignificant. In
contrast, GM6001 and TIMP-2 each strongly enhanced migration of U-MT
cells whereas TIMP-1 had relatively low effect (Fig. 7a).
Finally, we assessed migration of U-vector and U-MT cells in Transwells
with the undersurface coated with collagen type I, Fn, or its 42- and
110-kDa fragments. GM6001 did not alter migration of control U-vector
cells on Fn and collagen I significantly (Fig. 7a). Compared
with U-vector cells, U-MT cells displayed significantly higher levels
of migration on collagen type I, but much lower migration on Fn. GM6001
caused an opposite effect on U-MT cells migrating on collagen type I
and Fn. Thus, this inhibitor of MMP activity reduced migration of U-MT
cells on collagen but enhanced migration of these cells on Fn to the levels characteristic of U-vector cells (Fig. 7, a and
b). Migration of U-MT cells on the 42-kDa Fn fragment also
strongly increased in the presence of GM6001 (Fig. 7d).
Blocking anti-tTG antibody abolished stimulatory effect of GM6001 on
U-MT cells migrating on Fn or its 42-kDa fragment, whereas control
antibody had little or no effect (Fig. 7, c and
d). As expected, GM6001 and anti-tTG antibody did not alter
migration of cells on the 110-kDa fragment of Fn (data not shown).

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Fig. 7.
Proteolysis of surface tTG by MT1-MP affects
cell migration on Fn and collagen type I differentially. U-vector
and U-MT cells (5 × 104 cells/insert) migrated in
serum-free medium for 12 h onto the insert undersurface coated
with 10 µg/ml Fn (panels a, b, and
c, the 42-kDa Fn fragment (panel d), or collagen
type I (panel b). Transmigrated cells were detached and
counted. Shown are the means of triplicate determinations. Panel
a, proteolysis of surface tTG by MT1-MMP decreases migration of
U-MT cells on Fn. Where indicated, cells were treated with 20 µM GM6001, 1 µg/ml TIMP-1, or 1 µg/ml TIMP-2.
Panel b, MT1-MMP expression affects migration of U-MT cells
on Fn and collagen I differentially. The migration
efficiency of U-MT cells was expressed in a percent relative to that of
untreated U-vector cells on Fn (3.2 ± 0.3 × 104
cells) and collagen type I (1.9 ± 0.2 × 104
cells). Panels c and d, function-blocking
anti-tTG antibody abolishes the stimulatory effect of GM6001 on
migration of U-MT cells on Fn (panel c) and the 42-kDa
gelatin-binding fragment of Fn (panel d). Where indicated,
cells were incubated with 20 µM GM6001 in the presence of
either 10 µg/ml function-blocking anti-tTG antibody or 10 µg/ml
control nonspecific IgG.
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DISCUSSION |
MT-MMP activity that is mostly expressed in aggressive tumors (18,
19) greatly contributes to invasion of many tumor cell types (3, 12,
13). MT1-MMP has been thought to be exclusively involved in the
breakdown of the ECM components including collagens (8) and laminin-5
(11), and in the activation pathway of soluble MMPs, i.e.
MMP-2 (9, 23) and MMP-13 (24). However, our report presents evidence
that membrane-anchored MT-MMPs in addition to the breakdown of the ECM
may be directly engaged in the regulatory proteolysis of adhesion
receptors on tumor cell surfaces.
Here we report that MT1-MMP and its structural homolog (MT2-MMP and
MT3-MMP) specifically degrade tTG, a ubiquitous integrin-binding cell
surface coreceptor for Fn (35). Our studies with inhibitors of MMP
activity including a hydroxamate inhibitor GM6001 (45), TIMP-2, and
TIMP-1, showed that MT-MMPs are likely to have a primary role in the
degradation of surface tTG under the conditions of cell culture.
Although our results were obtained with cancer cells overexpressing
MT1-MMP, they demonstrate that tTG is a novel substrate for MT1-MMP and
a potential target for cancer cell MT1-MMP in vivo. Further
work is needed to assess a relative contribution of individual MMPs to
the proteolytic degradation of surface tTG in vivo.
Proteolysis of tTG by MT1-MMP specifically decreases adhesion and
migration of cells on Fn. This underscores the significance of the
coreceptor function of surface tTG on cancer cells. Our findings
identified an unexpected function of MT-MMPs and a novel means of
regulating cell locomotion. Trafficking and subsequent clustering of
MT1-MMP with the tTG·integrin receptor complexes at the invasive
front of tumor cells might be involved in the temporal and spatial
control of cell locomotion. Importantly, Fn inhibited the proteolytic
degradation of its own receptor, cell surface tTG by MT1-MMP, thereby
supporting and enhancing cancer cell locomotion. This suggests that
individual components of the ECM may reciprocally regulate proteolysis
of their specific adhesion receptors on cell surfaces.
Recently, we reported that up to 40% of total cellular
1 integrins including
1
1
and other collagen-binding integrins, are directly associated with tTG
on cell surfaces (35). Apparently, proteolysis by MT1-MMP of the tTG
component of the cell surface tTG·integrin complexes is likely to
alter the pattern of cell matrix recognition and to switch cells from
the binding to Fn to a more efficient interaction with collagens or
other ECM proteins. These versatile adjustment mechanisms may regulate
migration of cells within composite matrices including connective
tissue and tissue barriers such as the basement membrane.
Why do cancer cells, instead of aggressively invading the tissue,
down-regulate their locomotion? Degradation of adhesion proteins
including surface tTG by MT1-MMP may allow cells to avoid invasion of
deficient, degraded matrices and, on the contrary, to trigger
detachment and subsequent metastasis. In this regard, our data explain
previous findings that the reduced expression and proteolysis of tTG
observed in vivo are linked to high metastatic potential of
tumor cells (37, 38, 48).
A coordinated interplay of adhesion receptors, proteolytic enzymes, the
ECM, and the cytoskeleton is likely to control spatially and temporary
invasion and the focalized proteolysis by tumor cells (3). Our work
identifies a novel functional link between cell motility and the ECM
composition. This link establishes a dual regulation of cell locomotion
imposed by MT-MMPs. Evidently, depending on the structure and
composition of the ECM, these proteinases may serve as both positive
and negative regulators of cell motility. In accordance with the
continually changing environment, MT-MMPs might be capable of
differentially regulating cell locomotion by shifting molecular gears
on the surface of migrating cancer cells. The regulatory proteolysis
involving membrane-anchored MT-MMPs is likely to be critical to the
status of adhesion proteins such as tTG and, possibly integrins (32).
Reciprocally, proteolysis of ECM proteins and a deposition of
tumor-specific components can modify the ECM structure. Jointly, these
complex mechanisms are likely to control cancer cell invasion efficiently.
The existence of the ancestral membrane-anchored MMPs in plants (49)
that have no ECM similar to that of mammals also indicates that matrix
breakdown may not be a primary function of MT-MMPs. The cleavage
specificity of MT-MMPs is not unique enough (2, 8, 9) to define an
exquisite role of these membrane-anchored proteinases in cell
locomotion (11-13).
In contrast, targeting of MT-MMPs and the tTG·integrin complexes to
the leading edge of migrating cancer cells where new transient cell
matrix adhesive contacts are formed and most of the protrusive activity
occurs, can modulate the efficiency of the regulatory proteolysis of
adhesion proteins by MT-MMP. This localized proteolytic control of cell
adhesion and migration is not possible for soluble MMPs. This
hypothesis may partially explain a unique functional role of MT1-MMP,
MT2-MMP, and MT3-MMP in tumor cell invasion (12).