(Received for publication, September 14, 1994; and in revised form, November 28, 1994)
From the
Matrix metalloproteases are secreted by mammalian cells as
zymogens and, upon activation, initiate tissue remodeling by
proteolytic degradation of collagens and proteoglycans. Activation of
the secreted proenzymes and interaction with their specific inhibitors
determine the net enzymatic activity in the extracellular space. We
have previously demonstrated that 72T4Cl can be activated by a plasma
membrane-dependent mechanism specific for this enzyme. Here, we report
purification of the membrane activator of 72T4Cl, which is a new
metalloprotease identical to a recently cloned membrane-type matrix
metalloprotease (MT-MMP). We demonstrate that activated MT-MMP acts as
a cell surface tissue inhibitor of metalloprotease 2 (TIMP-2) receptor
with K = 2.54
10
M. The activator
TlMP-2 complex in
turn acts as a receptor for 72T4Cl (K
= 0.56
10
M),
binding to the carboxyl-end domain of the enzyme. Activation of 72T4Cl
on the cell membrane provides a basic mechanism for spatially regulated
extracellular proteolysis and presents a new target for prognosis and
treatment of metastatic disease. The activator, purified as a
tri-molecular complex of MT-MMP
TIMP2
carboxyl-end domain of
72T4Cl, is itself an activated form of MT-MMP, posing the following
question: what is the mechanism of the activator's activation?
Normal physiological processes such as morphogenesis, tissue
repair, angiogenesis, uterine involution, and bone resorption are
dependent upon spatial and temporal regulation of extracellular
proteolysis. Metalloproteases secreted by eucaryotic cells can initiate
tissue remodeling by degradation of ECM ()macromolecules
such as collagen and proteoglycans(1, 2, 3) .
Malignant cells can exploit these same proteases to promote invasion
and metastasis(4, 5, 6) . All enzymes of this
group, identified so far, are secreted in a soluble proenzyme form.
Proenzyme activation(7, 8, 9, 10, 11, 12, 13, 14) and
interaction with the specific inhibitors TIMP-1 (14, 15, 16, 17, 18) and
TIMP-2 (19, 20, 21) determine the net
activity of secreted enzymes. The understanding of the mechanisms
determining the fate of metalloproteases after secretion and providing
for a spatial regulation of their activity has remained an elusive
goal.
We (22) and others (23, 24, 25, 26, 27) have
demonstrated a ``plasma membrane-dependent'' mechanism,
specific for activation of 72T4Cl, that can be induced in normal and
tumorigenic human cells by treatment with TPA or concanavalin A.
Activation of 72T4Cl in this system results in the cleavage of the
amino-terminal propeptide generating a 64-kDa conversion intermediate
with an amino terminus at Leu, which in turn is converted
to a Tyr
62-kDa active form (22) . TIMP-2 and a
26-kDa peptide derived from the carboxyl domain of the 72T4Cl
competitively inhibit membrane-dependent enzyme activation (22, 23) . Here, we report the isolation of an active
form of a membrane-bound activator of 72T4Cl, which is a new
membrane-associated metalloprotease, MT-MMP. The activator acts as a
cell surface TIMP-2 receptor. The resulting activator
TIMP-2
complex serves as a receptor for the carboxyl-end domain of 72T4Cl, the
binding of which causes enzyme activation. While this report was in
preparation, Sato et al.(28) cloned MT-MMP, using
degenerate polymerase chain reaction, and demonstrated that it causes
activation of 72T4Cl upon transfection into Cos-1 cells.
Anti-TIMP-2 rabbit antibodies (a generous gift from Dr. H. Birkedal-Hansen) were affinity purified on immobilized TIMP-2 as described(34) . After extensive washing of unbound proteins, affinity-purified antibodies were eluted with 0.2 M glycine-HCl buffer, dialyzed against phosphate-buffered saline, and stored at -80 °C.
The cleavage of 72T4Cl, isolation of the carboxyl-end domain fragment, reduction-alkylation, and CNBr cleavage were performed as described (22) .
Zymogram analysis, Western blot, gel electrophoresis, and plasma membrane activation of 72T4Cl were performed as described(22) .
The inhibitory activity of the carboxyl-end peptide
is dependent on its native conformation(22) . Reduction of the
disulfide bridge Cys-Cys
or partial
cleavage with CNBr led to a loss of its inhibitory activity. The 26-kDa
fragment was shown to interact with purified TIMP-2 or to form a
dimer(22) . Formation of the complex with the inhibitor and
dimerization of the fragment are mutually exclusive. This is
demonstrated by the fact that an excess of the 26-kDa fragment is able
to compete the formation of the complex with TIMP-2 with concomitant
appearance of the 52-kDa carboxyl-end domain dimer(22) , and
the formation of this dimer is abolished in the presence of a molar
excess of TIMP-2. These observations provide a sensitive way to
establish whether native and recombinantly expressed fragments of
72T4Cl have a similar conformation and activity. Purified recombinant
FLAG-CT was assayed for (i) the presence of a disulfide bridge between
2 Cys residues in the domain; (ii) its ability to inhibit
membrane-dependent activation of 72T4Cl; and (iii) its ability to bind
TIMP-2. All three parameters were similar or identical to those of
native carboxyl-terminal domain peptide obtained from the 72T4Cl
enzyme. SDS-PAGE analysis of the purified FLAG-CT fusion under reducing
and non-reducing conditions demonstrates the shift in migration
characteristic of the presence of the disulfide bridge (Fig. 1A). Competitive inhibition of membrane
activation of 72T4Cl and binding of TIMP-2 by FLAG-CT protein was as
efficient as the carboxyl-end domain fragment derived from the enzyme (Fig. 1, B and C). Increasing the
concentration of
I-labeled 26-kDa CT fragment leads to
the formation of a 52-kDa dimer (Fig. 1C, lanes1 and 2), as previously described(22) .
Based on these results, we used the FLAG-CT fusion protein in place of
the proenzyme-derived peptide for cross-linking experiments, as well as
for affinity chromatography of the HT1080 plasma membrane extract (see
below).
Figure 1:
Characterization of the FLAG-CT fusion
protein. A, the FLAG-CT fusion protein was purified from
periplasm extract of E. coli TOPP5 host (Stratagene) on red
and anti-FLAG-M1 monoclonal antibody columns, as described under
``Materials and Methods,'' and analyzed by SDS-PAGE either
before (A, lane1) or after (A, lane2) reduction in 10 mM dithiothreitol. B, 72T4Cl (lane4, 15 ng) was activated with
plasma membranes (20 µg) from HT1080 cells (lane1) as described under ``Materials and Methods''
with the addition of 400 ng of either 72T4Cl-derived 26-kDa CT (lane2) or recombinant FLAG-CT fusion protein (lane3) prior to the addition of 72T4Cl. C,
either I-labeled 26-kDa CT (200,000 cpm, 10
cpm/µg, lanes1-4) or
I-labeled FLAG-CT fusion (160,000 cpm, 0.5
10
cpm/µg, lane5) were cross-linked
with BS
(2 mM) for 1 h at 0 °C in 25 mM HEPES buffer, pH 7.5, containing 150 mM KCl in the
presence of 100 ng of unlabeled 26-kDa CT (lane1),
FLAG-CT (lane2), FLAG-CT and TIMP-2 (lane3), 26-kDa CT and TIMP-2 (lane4), or
TIMP-2 (lane5). The numbers show molecular
masses of the markers (A), 72T4Cl and conversion intermediates (B), and 26-kDa CT dimer and complex with TIMP-2 (C).
Cross-linking experiments using I-labeled
26-kDa CT and HT1080 plasma membranes or their Lubrol extract
demonstrate the presence of two specific products with apparent
molecular masses of 105 and 44 kDa (Fig. 2). The formation of
both cross-linking products is competitively inhibited in the presence
of the excess of unlabeled 26-kDa CT (Fig. 2, lane4). Reduction-alkylation of the 26-kDa CT abolished its
inhibitory activity in the cross-link assay (not shown). An identical
result was obtained after the 26-kDa CT was cleaved with CNBr (Fig. 2, lane5). Both the internal fragment
(not shown), produced by cleavage at Met
and
Met
, and a fragment containing a single cleavage at
Met
alone lacked the ability to inhibit the formation of
the specific cross-linking products. These findings demonstrate that
modifications of the 26-kDa CT that abolish its ability to interact
with TIMP-2 also abolish its inhibitory activity (22) in the
72T4Cl membrane activation assay and its ability to interact with the
membrane components.
Figure 2:
Cross-linking of radiolabeled 26-kDa CT to
plasma membrane extracts from TPA-treated HT1080 and p2AHT2a cells. The I-labeled 26-kDa CT (1.2
10
cpm,
10
cpm/µg, lanes1-7 or 1.5
10
cpm, 0.8
10
cpm/µg, lanes8-10) in 25 mM HEPES-KOH buffer,
pH 7.5, containing 150 mM KCl and 0.1 mM CaCl
were incubated with lubrol (1%) extracts of plasma membranes from
HT1080 cells (30 µg of protein, lanes2-5)
or from p2AHT2a cells (20 µg, lane8, or 40
µg, lanes9 and 10) with (lanes1 and 3-10) or without 2 mM of
BS
(lane2) for 1 h at 0 °C.
Unlabeled ligands, 26-kDa CT (300 ng, lanes4, 7, and 10), or CNBr-cleaved 26-kDa CT (400 ng, lanes5 and 6) were added to the reaction
for competition. After incubation, the reactions were analyzed by
SDS-PAGE under reducing conditions followed by autoradiography. The numbers show molecular masses of the specific cross-linked
products.
The molecular mass of the 44-kDa cross-linking
product is indistinguishable from that of the 26-kDa CT-TIMP-2
cross-link(22) . The p2AHT2a cells, expressing the adenovirus
E1A gene, despite transcribing TIMP-2 mRNA, do not secrete appreciable
amounts of TIMP-2. ()Thus, to ascertain whether membrane
associated TIMP-2 may be an essential element in membrane-dependent
activation, we investigated the ability of the plasma membrane
preparations from p2AHT2a cells (29) to yield the 44-kDa
cross-linking product with FLAG-CT and activate 72T4Cl proenzyme.
Cross-linking experiments using plasma membranes prepared from these
cells (Fig. 2, lanes 8-10) demonstrate no
specific cross-link products except the 52-kDa dimer of
I-labeled 26-kDa CT (Fig. 2, lanes
8-10), which is indistinguishable from the dimer found in
the presence of the 26-kDa CT ligand alone (Fig. 2, lane7).
The results presented in Fig. 3demonstrate
that plasma membranes derived from these cells are not very efficient
in activation compared with the parental HT1080 cell line (Fig. 3, lane2). However, the addition of
carefully titrated amounts of TIMP-2 to the extracts from p2AHT2a cells (Fig. 3, lanes 3-6) stimulated the conversion of
proenzyme into the activated form, while further increase of TIMP-2
concentration (Fig. 3, lanes 7-9) caused complete
inhibition of the reaction as was demonstrated earlier(22) .
Conversely, the addition of increasing amounts of affinity-purified
anti-TIMP-2 antibodies to the activation reaction prevents conversion
of the 72T4Cl into activated forms (Fig. 4). The results of
these experiments demonstrate that in addition to the carboxyl-terminal
domain of the enzyme, the membrane-associated TIMP-2, represented by
the 44-kDa cross-linking product with FLAG-CT is an essential element
in the assembly of the membrane-associated activation complex. We
therefore investigated the cross-linking pattern of I-TIMP-2 to the proteins of the plasma membranes from
HT1080 and p2AHT2a cells (Fig. 5). Cross-linking of radiolabeled
TIMP-2 to membrane extracts from either cell type yielded a specific
product with an apparent molecular mass of 82 kDa (Fig. 5, lanes4-6 and 9-12), the
formation of which was competitively inhibited by an excess of
unlabeled TIMP-2 (Fig. 5, lanes 6-8 and 13), suggesting that a 60-kDa membrane-associated protein can
interact with TIMP-2. Taken together with the results of cross-linking
experiments using
I-labeled 26-kDa CT (Fig. 2,
105- and 44-kDa specific products), these findings indicate a
possibility that the carboxyl-end domain of 72T4Cl interacts with
membrane-associated TIMP-2 (44-kDa cross-linking product), which in
turn interacts with a putative 60-kDa membrane activator to make a
tri-molecular activation complex represented by a 105-kDa specific
cross-linking product.
Figure 3: Effect of TIMP-2 on activation of 72T4Cl by plasma membranes isolated from p2AHT2A cells. The 72T4Cl (15 ng) was incubated for 2 h alone (lane1) or activated with p2AHT2A plasma membranes (30 µg of protein, lanes2-9). Purified TIMP-2 (3, 10, 15, 30, 60, 120, and 240 ng) was added to the reaction (lanes3-9, respectively), and the mixture was incubated an additional 30 min at 0 °C prior to the addition of the enzyme.
Figure 4:
Inhibition of 72T4Cl activation by
affinity-purified anti-TIMP-2 antibodies. The 72T4Cl (15 ng) was
incubated for 2 h at 37 °C in 25 mM HEPES-KOH buffer, pH
7.5, containing 0.1 mM CaCl, alone (lane1) or with HT1080 plasma membranes (8 µg, lanes2-8). Affinity-purified anti-TIMP-2 rabbit
antibodies were added to the samples in lanes3-8 in the amounts indicated.
Figure 5:
Cross-linking of radiolabeled TIMP-2 to
plasma membrane extracts from phorbol 12-myristate 13-acetate-treated
HT1080 and p2AHT2a cells. The I-labeled TIMP-2 (3
10
cpm, 4
10
cpm/µg, lanes1-13) in 25 mM HEPES-KOH buffer, pH 7.5,
containing 150 mM KCl and 0.1 mM CaCl
was
incubated with Lubrol (1%) extracts of plasma membranes from p2AHT2a
cells (20 µg of protein, lane4; 40 µg of
protein, lanes3, 5, 6, 7,
and 8) or from TPA-induced p2AHT2a cells (20 µg, lane11; 40 µg, lane 12) or from TPA-induced
HT1080 cells plasma membranes (20 µg, lanes9 and 13 or 40 µg, lane 10) with (lanes2 and 4-13) or without (lanes1 and 3) 0.5 mM of BS
for 1 h at 37 °C.
Unlabeled ligand, TIMP-2, was added to the reaction for competition (10
ng, lane6; 30 ng, lane7; 100 ng, lanes8 and 13). After incubation, the
reactions were analyzed by SDS-PAGE under reducing conditions, followed
by autoradiography.
The fact that an excess of TIMP-2 inhibits the reaction suggests that assembly of such a complex may be blocked when both the activator and the carboxyl-end domain of the proenzyme are occupied with the inhibitor. Two alternative possibilities exist regarding the assembly of such complex. Either free putative activator binds TIMP-2 and then inhibitor-free enzyme sequentially or it can bind a preformed enzyme-inhibitor complex. We previously demonstrated (22) that at best only partial activation of a purified stoichiometric enzyme-inhibitor complex can be achieved in the presence of plasma membranes from HT1080 cells, and this residual activation can be abolished in the presence of additional amounts of TIMP-2. An examination of the activation reaction using TIMP-2-deficient p2AHT2a plasma membranes revealed that addition of a titrated amount of TIMP-2 prior to the addition of the enzyme improves the activity of these membranes (Fig. 4), while the results presented in Fig. 6show that a preformed enzyme-inhibitor complex is still resistant to activation. These results favor the sequential model of complex assembly in which membrane-bound free activator interacts with TIMP-2 prior to binding of the free enzyme for activation to occur.
Figure 6:
Plasma membrane activation of 72T4Cl and
the 72T4ClTIMP-2 complex. The 72T4Cl (15 ng, lanes1-9) or 72T4Cl
TIMP-2 complex (lanes
10-18) were incubated in 25 mM HEPES-KOH buffer, pH
7.5, containing 0.1 mM CaCl
alone (lanes1 and 10) or with plasma membranes from either
HT1080 (20 µg, lanes2-5 and 11-14) or p2AHT2A cells (40 µg, lanes6-9 and 15-18) for the indicated
times. An extra amount of TIMP-2 (50 ng) was added prior to the
addition of plasma membranes to the samples in lanes5, 9, 14, and 18. The
72T4Cl
TIMP-2 stoichiometric complex was prepared from purified
TIMP-2 and 72T4Cl and separated by gel filtration chromatography as
previously described(22) .
Figure 7:
Binding of radiolabeled TIMP-2 and
FLAG-26-kDa CT fusion to HT1080 and p2AHT2a cells. The cells were
washed with serum-free media containing 25 mM HEPES buffer, pH
7.3, and 1 mg/ml bovine serum albumin and incubated on ice for 20 min
with (background) or without unlabeled TIMP-2 or FLAG-CT fusion ligands
prior to addition of I-radiolabeled TIMP-2 or FLAG-CT to
reach indicated concentrations. The cells were incubated for 40 min on
ice, washed five times with serum-free media, and lysed in 0.4 M NaOH, 0.3% SDS; radioactivity was then counted. One well of each
cluster was used to count the number of cells. The nonspecific
background binding was determined using a 500-fold excess of unlabeled
over highest concentration of
I ligand. The binding is
displayed as a function of ligand concentration. The solidcurves are the binding curves determined by a fit to the
Scatchard equation. B and C, the Scatchard plots of
data presented in A. The dissociation constants and saturation
binding were obtained from a least squares (solidline) fit to the data.
If, as proposed above, the binding of the carboxyl-end domain of the enzyme represented by the 105-kDa specific cross-linking product is mediated through membrane-associated TIMP-2, then such interdependent interaction of two ligands with the cell surface can be described by the following model.
A membrane-associated activator (A) acts as a receptor for exogenous or endogenous TIMP-2 (T). The activator-TIMP-2 complex (AT) in turn acts as a receptor for 72T4Cl (C) to form the complex ATC, which causes activation of the enzyme. The latter binding is mediated through the interaction between the carboxyl-end domain of 72T4Cl and TIMP2 present in the AT complex and has the same dissociation constant as that of the pro-72T4Cl-TIMP-2 complex (38) in solution. These steps are described by the following reactions where lower and uppercasesymbols represent free and total concentrations of each reactant, respectively,
where at equilibrium, K = a
t/at, K
= at
c/atc, and K
= t
c/tc. Solutions of the , , and for at and atc (total cell surface-bound 26-kDa CT) in terms of c and t are
assuming that K = K
, total cell surface-bound TIMP2 is
where c and t are calculated by
The dissociation constants of TIMP-2 and the 26-kDa CT for the
HT1080 cell surface, K and K
,
were determined above (Fig. 7). The constant K
is the dissociation constant of the CT complex in solution that
is identical to K
( Fig. 7and (38) ) within error of measurement. The total number of binding
sites for both ligands were determined independently in this experiment
(not shown) since the total number of such sites and the ratio of
occupied (AT) to unoccupied (A) receptor can vary
significantly between experiments. This is due to the fact that TPA
induction stimulates the expression of receptor A (Fig. 7) while
inhibiting the expression of TIMP-2(39) .
and were used to model the
competition kinetics of I-labeled 26-kDa CT cell surface
binding by exogenously added TIMP-2 (Fig. 8A, solidline) and
I-TIMP-2 binding by exogenously
added 26-kDa CT (Fig. 8B, solidline), respectively. The experimental data (open and solidsquares) obtained in such competition
experiments are in good agreement with computer-generated predictions (solidlines) and thus support a tri-molecular
complex model outlined above.
Figure 8:
Competition for binding of the
radiolabeled FLAG-CT fusion with TIMP-2 (A) and the
radiolabeled TIMP-2 with FLAG-CT fusion (B). The competition
of binding of radiolabeled ligands with the unlabeled counterpart were
performed as described in Fig. 7. The cells were preincubated
with increasing concentrations of unlabeled ligand prior to addition of I-labeled 72-kDa CT (2.2
10
M) or
I-TIMP-2 (4.4
10
M). Competition of
I-labeled 26-kDa CT with TIMP-2 (A) and of
I-TIMP-2 with 26-kDa CT (B) were performed in
the same experiment. Binding is displayed as a function of competing
ligand concentration. The solidlines are the
computer-generated model predictions, as described in the
text.
The pattern of I-labeled
26-kDa CT binding and competition with TIMP-2 suggest the presence of
membrane-bound activator in two forms, a free form available for
binding of TIMP-2 and 72T4Cl and a form occupied with endogenous TIMP-2
that can bind 72T4Cl only. Availability of the first form on the
surface of TPA-induced HT1080 cells explains the increase in binding of
26-kDa CT in the presence of small quantities of exogenously labeled
TIMP-2, which is necessary for the 26-kDa CT to bind to free activator.
Further increase in the concentration of TIMP-2 leads to complete
inhibition of binding due to the formation of the 26-kDa CT
TIMP-2
and activator
TIMP-2 complexes. The competition experiments
described here do not discriminate as to whether the assembly of the
ATC complex can be accomplished by binding of TC to A. To determine
whether this reaction takes place, we conducted a separate binding
experiment using a preformed CT complex with a radiolabeled TIMP-2
component and demonstrated that the K
of its
binding to cell surface is 3.7 times lower then that of TIMP-2 alone in
the same experiment (data not shown). This result can explain why the
72T4Cl
TIMP-2 complex is resistant to activation in most cases and
is activated very slowly in others (see ``Discussion'' and (26) ).
The anti-FLAG-M1 antibody resin (0.5-ml bed
volume) was saturated with an excess of purified recombinant FLAG-CT
fusion protein. The Lubrol extract of HT1080 membranes (100 mg, total
protein) was applied on the FLAG-CT-M1 column, and bound proteins were
eluted in 25 mM HEPES buffer, pH 7.5, containing 2 mM EDTA. The SDS-PAGE analysis of the eluted material (Fig. 9)
revealed the presence of four protein bands. In addition to protein
bands corresponding to the FLAG-CT and the 21-kDa band co-migrating
with the TIMP-2, two bands, major and minor, with a molecular mass of
about 60 kDa, are apparent on the gel. These proteins were
electroblotted onto a polyvinylidene difluoride membrane and subjected
to amino-terminal sequence analysis. The 21-kDa protein had the
amino-terminal sequence XSXSPVHPQQAFXNADVVI,
identifying this protein as TIMP-2 (the corresponding TIMP-2 sequence
is CSCSPVHPQQAFCNADVVIRA). The amino-terminal amino acid
sequences of the major and minor 60-kDa bands were identical. This
sequence (Fig. 9B) identified a novel protein with
substantial homology to sequences of secreted metalloproteases. No
identical sequence was found in a GeneBank search. While this work was
in progress, Sato et al.(28) isolated a cDNA clone
for MT-MMP from human placenta RNA using degenerate polymerase chain
reaction priming. The isolated cDNA upon transfection into Cos-1 cells
caused activation of the co-transfected 72T4Cl(28) . The
sequence of the 60-kDa putative metalloprotease isolated in a
tri-molecular complex with 26-kDa CT and TIMP-2 in our experiments is
identical to the sequence of MT-MMP (residues 111-130) and thus
represents its activated form with a loss of 105 amino acid residues of
the propeptide, including the conserved propeptide Cys residue.
Figure 9:
Purification of the activator in complex
with TIMP-2 and FLAG-CT fusion from Lubrol extract of TPA-induced
HT1080 cell plasma membranes. A, affinity chromatography of
the membrane extract on FLAG-CT, immobilized on an M1 anti-FLAG
monoclonal antibody column. A fresh portion of 0.5 ml of the M1
monoclonal antibody resin (IBI, 0.5-ml bed volume) in 10 mM HEPES buffer, pH 7.5, containing 150 mM KCl, 1 mM Ca, and 0.002% Brij 35, was saturated with 1 mg
of purified FLAG-CT fusion protein and washed with 20 ml of the same
buffer, containing 1% Lubrol. Plasma membranes from HT1080 cells in the
above buffer were extracted with 1% Lubrol for 1 h at 0 °C. The
extract (100 mg of protein) was loaded on the column, washed with 50 ml
of the same buffer and 10 ml of the same buffer containing 0.1% Lubrol,
and eluted with the same buffer containing 2 mM EDTA and no
Ca
. Ten fractions of 500 µl each were collected
and analyzed by SDS-PAGE. Fractions 3 and 4 are shown (lanes1 and 2). B, alignment of
amino-terminal sequence of the 60-kDa protease with known secreted
metalloproteases.
Cell translocation during both normal and pathological processes of tissue remodeling requires spatially regulated degradation of the existing ECM macromolecules. Two proteolytic systems are apparently involved in this process: (i) plasminogen activator of the urokinase type, which is sequestered on the cell surface and is activated via interaction with its receptor(40, 41, 42, 43, 44, 45, 46, 47, 48, 49) and (ii) secreted metalloproteases that initiate degradation of collagens and proteoglycans(1, 2, 3, 4, 5, 6) . All known members of the latter group of enzymes are secreted as soluble zymogens and require activation in the extracellular space. Their activation can be accomplished by a proteolytic cascade initiated by serine proteases such as plasmin that invariably leads to processing of the amino-terminal pro-peptide of the enzyme(7, 8, 9, 10, 11, 12, 13, 14) . This causes a structural transition unlocking the active center of the partially processed enzyme by the cysteine switch mechanism (11) and is followed by further processing of the amino terminus by auto-proteolysis or with the help of a related metalloprotease such as stromelysin(8, 10, 12, 13) . Although activation mechanisms in solution have been worked out in considerable detail, the mechanistic understanding of spatial regulation of metalloprotease activity has remained an elusive goal.
Recently, we (22) and others (23, 24, 25, 26, 27) demonstrated
a cell membrane-dependent mechanism of activation specific for 72T4Cl.
In the presence of plasma membranes from TPA-induced HT1080 cells, the
activation of exogenously added, inhibitor-free 72T4Cl generates an
active enzyme of 62 kDa. Formation of the 72T4ClTIMP-2 complex
inhibits activation at the level of the initial pro-peptide
cleavage(22) . Membrane-dependent activation of 72T4Cl is
competitively inhibited in the presence of a 26-kDa peptide derived
from the carboxyl domain of the enzyme (22) and the inhibitor,
TIMP-2, which forms a complex with the same domain. These results were
interpreted to mean that interaction of the carboxyl-end domain with a
cell membrane-associated protein is essential for activation of the
enzyme.
Cross-linking and activation experiments described in this report demonstrate that membrane-associated TIMP-2 interacts with the carboxyl-end domain of 72T4Cl and is an essential part of the cell surface activation complex. Cross-linking of radiolabeled FLAG-CT with extracts of cell membrane yielded two specific products with molecular mass of 105 and 44 kDa, the latter representing the cross-link with TIMP-2. Chemical modifications abolishing the interaction of the carboxyl-end domain with the inhibitor also abolish its ability to competitively inhibit membrane activation and the appearance of the 44-kDa cross-linking product. The activity of plasma membranes from p2AHT2a cells that do not secrete appreciable amounts of TIMP-2 can be improved by an addition of titrated amounts of the purified inhibitor. Finally, anti-TlMP-2 antibodies inhibited the activation of the 72T4Cl. Cross-linking of radiolabeled TIMP-2 with either preparation of cell membranes yielded an 82-kDa specific product. These results suggested that activation of 72T4Cl requires an assembly of a tri-molecular stoichiometric complex represented by the 105-kDa specific cross-link and involving a 60-kDa membrane-associated activator, the inhibitor, TIMP-2, and the carboxyl-end domain of 72T4Cl.
This model implies
that the enzyme does not bind to the cell surface independently of
TIMP-2. The binding experiments presented here support this model.
Kinetic equations describing this model predict competition curves that
are in agreement with the experimental data. The results demonstrate
the presence of occupied and unoccupied receptor of TIMP-2 on the cell
surface of the TPA-induced HT1080 cells. The occupied form can serve as
a receptor for the carboxyl-end domain of 72T4Cl, the binding of which
increases in the presence of small amounts of TIMP-2, as the unoccupied
receptor becomes saturated with the inhibitor. Further increase of the
TIMP-2 concentration completely inhibits the binding of the 26-kDa CT
since the receptor saturated with TIMP-2 fails to bind the 26-kDa
CTTIMP-2 complex. Thus, the competition experiments demonstrate
the existence of TIMP-2-dependent carboxyl-end domain binding site but
do not exclude the possibility of a TIMP-2-independent 26-kDa CT
receptor. The linearity of Scatchard plots for FLAG-CT binding,
however, suggests no distinguishable second binding component.
The
model presented here can explain the apparent discrepancy between the
results of several laboratories showing that TIMP-2 inhibits activation
of 72T4Cl (22, 23) and the observation of Brown et
al.(26) where the radiolabeled complex of
72T4ClTIMP-2 was activated using concanavalin A-treated
fibroblasts. In the former case, relatively short incubation times (1
h) were employed, while long incubation times (22 h) were used to
observe the latter. Thus, while inhibition of activation of 72T4Cl in
complex with TIMP-2 was not observed during short incubations, longer
incubations could allow for rearrangement of components to occur,
producing the observed activation. Our results are also in agreement
with the cell surface binding experiments using 72T4Cl observed by
Emonard et al.(50) in two human breast adenocarcinoma
cells, MDA-MB231 and MCF-7, although some what lower affinity (K
= 2
10
M) was observed in these experiments.
Purification of the activator was achieved here by isolating its occupied form in a tri-molecular complex with the recombinant FLAG-CT fusion protein. The purified complex contained the carboxyl-end domain fragment, TIMP-2, and a 60-kDa protein whose amino-terminal sequence is homologous to sequences of other secreted metalloproteases. The isolated preparation of such a complex can not be used in cross-linking experiments to demonstrate specificity of interaction since all the involved components are present in a ratio defined by the properties of the affinity column. The activation assay is also not feasible for the same reason. To positively demonstrate activity of the isolated protease, transfection experiments with its cDNA clone are necessary. While this work was in progress, Sato et al.(28) isolated a cDNA clone of a membrane-associated metalloprotease, MT-MMP, using degenerate polymerase chain reaction primers, that is identical to the activator isolated here. Co-transfection of the expression vectors with this cDNA clone together with cDNA of 72T4Cl caused activation of the expressed enzyme, proving that MT-MMP is indeed the 72T4Cl activator.
The protein sequence of the 60-kDa protease isolated here is
identical to the sequence of the MT-MMP, positions 111-128,
demonstrating that this protein is an activated form of MT-MMP
activator, thus posing the question: how is the activator activated?
The likely answer to this question lies in the amino acid sequence
Arg-Arg-Lys-Arg just upstream from the amino terminus
(Ala) of the activated MT-MMP that makes the pro-peptide
an excellent substrate for serine proteases such as plasmin or
plasminogen activator. This sequence motif is also found in other
members of the metalloprotease family, such as collagenase and
stromelysin, that can be activated by plasmin. This sequence is absent
from the pro-peptide of 72T4Cl, which is resistant to plasmin
activation. Therefore, it is very likely that activation of the 72T4Cl
is the result of a cell surface proteolytic cascade that involves
activation of the membrane metalloprotease by the membrane-associated
plasmin (51, 52) and/or plasminogen activator, which
itself is activated upon binding to a specific receptor. Activated
MT-MMP serves as a receptor for TIMP-2 forming a complex that binds
72T4Cl, leading to its activation.
The data presented here cannot distinguish with certainty whether binding of TIMP-2 requires prior activation of MT-MMP or if the inhibitor can bind the proenzyme, as in the case of 72T4Cl. It is also not clear whether the inhibitor binds to an activated form of MT-MMP through the amino-terminal inhibitory domain (53, 54, 55) while exposing the carboxyl-end domain for interaction with 72T4Cl. We have recently described (13) the isolation of a similar complex between 92T4CI, TIMP, and activated interstitial collagenase(13) , where the activity of the latter was inhibited. Whether MT-MMP retains the proteolytic activity while in complex with TIMP-2 and the carboxyl-end domain of 72T4Cl remains to be elucidated.
The implications of the cell surface activation cascade of 72T4Cl for mechanisms of tissue remodeling are far reaching. 72T4Cl as well as TIMP-2 could be synthesized by a different set of cells than those that express the MT-MMP and utilize the membrane-bound 72T4Cl to accomplish cell translocation. Such a mechanism creates another level of regulation of cell migration in a spatially organized fashion. This model is supported by recent observations (56, 57, 58, 59) that, in most cases, soluble secreted metalloproteases are synthesized in the tumor stroma, but, as in the case of the breast cancer, the enzyme is associated with an epithelial component of the tumor. Moreover, the involvement of TIMP-2 in activation of 72T4Cl can explain a surprising observation of Visscher et al.(60) , where a poor prognosis in breast cancer is correlated with the presence of TIMP-2 in the tumors rests rather than with the amount of 72T4Cl protein. Thus, the model of the 72T4Cl activation described here presents a realistic target for intervention with metastatic disease and other pathological processes of tissue remodeling.