(Received for publication, May 31, 1995; and in revised form, September 28, 1995)
From the
Membrane-type matrix metalloproteinase (MT-MMP) messenger RNA and protein expression were shown to be elevated in human fibroblasts following treatment with concanavalin A, coincident with the induction of the ability to process progelatinase A.
CHO cells transfected with the cDNA for MT-MMP were able to process both wild type progelatinase A and a catalytically inactive mutant, E375A progelatinase A. Both proenzymes were converted to a 68-kDa intermediate (reducing gels) form, but only the wild type enzyme was processed further to a 66-kDa end product. In contrast, both forms of progelatinase were processed via the 68-kDa intermediate to 66 kDa by concanavalin A-stimulated fibroblasts.
Further study of the processing of E375A progelatinase A by plasma membrane preparations from concanavalin A-stimulated fibroblasts showed that addition of active gelatinase A enhanced processing to the mature form. It was concluded that cell membrane-mediated activation of progelatinase A could be via a cascade involving both MT-MMP and intermolecular autolytic cleavage.
The matrix metalloproteinase (MMP) ()family of
enzymes has been strongly implicated in extracellular matrix turnover,
in both physiological and pathological situations. All MMPs are
secreted in proenzyme forms, and much research has been aimed at
elucidating the proteolytic mechanisms required to elicit their full
activities. A number of possible physiological activators have been
described, including plasmin, plasma kallikrein, neutrophil elastase,
and cathepsin G (Nagase et al., 1991). Gelatinase A (MMP-2,
72-kDa gelatinase, type IV collagenase) is of great current interest,
not only because of its possible involvement in tumor invasion and
metastasis (Stetler-Stevenson et al., 1993; Tsuchiya et
al., 1994) but also because the proenzyme cannot be activated by
any of the suggested physiological activators of other MMPs including
serine proteinases (Okada et al., 1990; Nagase et
al., 1991), although activation by other MMPs, especially
matrilysin (Crabbe et al., 1994a) and collagenase (Crabbe et al., 1994b), has recently been described. Furthermore,
there is evidence that activation by self-cleavage can occur at high
progelatinase A concentrations (Crabbe et al., 1993).
Cell-mediated activation of progelatinase A has been the focus of much recent work by the present authors (Ward et al., 1991, 1994) and by others (Strongin et al., 1993, 1995; Brown et al., 1993). We reported that human skin fibroblasts stimulated by concanavalin A (ConA) can bind, proteolytically process, and activate progelatinase A. The importance of the C-terminal domain of the enzyme for the binding to cell membranes and subsequent activation was demonstrated (Murphy et al., 1991; Strongin et al., 1993; Ward et al., 1994). Cell membrane-mediated cleavage of progelatinase A to a 62-kDa active form (Tyr-81) occurs via a 64-kDa intermediate (Leu-38) (Strongin et al., 1993), and exogenous TIMP-2 specifically inhibits the activation process.
Sato et al.(1994) recently reported the cloning of a novel transmembrane member of the matrix metalloproteinase family (MT-MMP) and showed that cells transfected with MT-MMP cDNA can effect the activation of progelatinase A. Questions therefore arise about the precise mechanism of activation of progelatinase A by stimulated fibroblasts, especially whether MT-MMP is an obligatory component, the relative roles of other metalloproteinases, and the involvement of self-cleavage. In this study we have made use of a previously described mutant form of gelatinase A (Crabbe et al., 1994c), in which the active site glutamate was replaced by alanine (E375A). The mutant can be proteolytically processed to remove the propeptide (Crabbe et al., 1994c) but yields a catalytically inactive enzyme that cannot self-process. We have investigated the hypothesis that cell membrane-mediated activation of progelatinase A is, by analogy with other matrix metalloproteinases (Murphy et al., 1987; Nagase et al., 1990), a complex activation cascade. We show that it is likely to involve MT-MMP, which can be induced in fibroblasts by ConA, and bimolecular autolysis.
E375A mutant progelatinase A was constructed, expressed, and purified as described by Crabbe et al. (1994c). Recombinant wild type human progelatinase A, TIMP-1, and TIMP-2 were purified from media conditioned by the relevant transfected mouse myeloma cells as described (Crabbe et al., 1993; Murphy et al., 1991, 1992; Willenbrock et al., 1993). Digoxigenin-11-UTP, nylon membrane, and a Nucleic Acid Detection Kit were obtained from Boehringer Mannheim, East Sussex, UK. Other transcription reagents and pBluescript KS(+) were obtained from Stratagene Ltd., Cambridge, UK. Hybond ECL nitrocellulose membrane, ECL Kit for Western blotting autoradiography, and luminescence detection film were all from Amersham Life Sciences, Amersham, Bucks, UK. Peroxidase-conjugated sheep anti-mouse IgG was from Sigma, and the membrane-type matrix metalloproteinase (MT-MMP) monoclonal antibody (113-5B7) raised against the peptide CDGNFDTVAMLRGEM (residues 310-333) of MT-MMP was a kind gift from Dr. K. Iwata, Fuji Chemical Industries, Toyama, Japan.
A 1.5-kilobase EcoRI-BamHI fragment containing the majority of the coding sequence of MT-MMP (Takino et al., 1995) was subcloned into pBluescript II KS(+), and a labeled antisense riboprobe was generated from the linearized template by transcribing in the presence of digoxigenin-11-UTP.
Apparent molecular masses for the three species of gelatinase A identified in these experiments differed according to the method used to assess them. In this report, molecular masses for the proenzyme, intermediate, and fully active forms, as assessed by nonreducing SDS-PAGE/gelatin zymography followed by autoradiography, were 66, 62, and 59 kDa, respectively. Where SDS-PAGE with reducing conditions was used, i.e. Coomassie-stained gel scanning (Fig. 6), the respective molecular masses were 72, 68, and 66 kDa. Molecular masses reported by other workers (Strongin et al., 1993) referred to in the introduction and under ``Discussion'' are those assessed by gelatin zymography performed under different conditions and are described as 66, 64, and 62 kDa, respectively.
Figure 6:
Enhancement of processing of I-labeled E375A by membrane preparations with active
gelatinase A.
I-labeled E375A progelatinase (14
nM) was incubated at 37 °C for 20 h with increasing doses
of unlabeled APMA-activated gelatinase A from which the APMA had been
removed (see ``Materials and Methods'') and with or without
10-µg aliquots of membrane preparations from ConA-stimulated
fibroblasts. The autoradiograph shows
I-labeled E375A
mutant gelatinase A incubated with: lane 1, 0.1 molar ratio of
APMA-activated gelatinase A; lane 2, 1.0 molar ratio of active
gelatinase A; lane 3, membrane preparation supplemented with
0.1 molar ratio of active gelatinase A; lane 4, membrane
preparation supplemented with 1.0 molar ratio of active gelatinase; lane 5, membrane preparation supplemented with 1.0 molar ratio
of progelatinase A; lane 6, 1.0 molar ratio of progelatinase
A; lane 7, membrane preparation; lane 8,
I-labeled E375A mutant alone.
Figure 1:
MT-MMP is induced by ConA stimulation
of fibroblasts. A, Northern blot of RNA from fibroblast
monolayers. Cells were either untreated(-) or stimulated with 50
µg/ml ConA (+) for 24 h prior to extraction of total RNA.
Samples (5 µg/lane) were fractionated by electrophoresis and
transferred to a nylon membrane. MT-MMP mRNA was detected by
hybridization with a digoxigenin-labeled riboprobe. The positions of
the 28 S and 18 S ribosomal bands are shown to the left.
Ethidium bromide staining of the ribosomal RNA is also shown, as a
measure of loading. B, 10-µl aliquots of membrane
preparations from approximately equal numbers of fibroblasts (2
9 T175 cm
confluent flasks), which had been incubated for
48 h in either DMEM alone(-) or DMEM + 50 µg/ml ConA
(+), were applied to SDS-PAGE. Samples are from two separate
experiments. The electrophoresed proteins were analyzed by Western blot
as detailed under ``Materials and Methods.'' Relative
mobilities of molecular mass markers are indicated on the left.
Figure 2:
Processing of I-labeled
progelatinase A and
I-labeled E375A by CHO cells. CHO
cells transfected with MT-MMP or vector control were incubated with
I-labeled progelatinase A or
I-labeled
E375A (9 nM) as described under ``Materials and
Methods.'' 12-µl aliquots of culture supernatants were
analyzed on 7% polyacrylamide/gelatin zymograms (0.5 mg/ml gelatin)
followed by autoradiography. Lane 1, vector control cells
incubated with
I-labeled progelatinase A; lane
2, vector control cells incubated with
I-labeled
E375A; lane 3, MT-MMP-transfected cells incubated with
I-labeled E375A; lane 4, MT-MMP-transfected
cells incubated with
I-labeled progelatinase A; lane
5, MT-MMP-transfected cells incubated with unlabeled wild type
progelatinase A (100 ng) and
I-labeled E375A; lane
6, vector control cells incubated with unlabeled wild type
progelatinase and
I-labeled
E375A.
Figure 3:
A,
time course of E375A and wild type progelatinase A processing by
ConA-stimulated fibroblasts. Conditioned media from ConA-stimulated
cells which had been incubated for up to 24 h with 9 nMI-labeled E375A mutant or wild type
I-labeled progelatinase A. (i), media from cells
supplemented with
I-labeled wild type progelatinase A and
incubated at 37 °C for 0, 2, 6, and 24 h. (ii), media from
cells supplemented with
I-labeled E375A mutant and
incubated at 37 °C for 0, 2, 6, and 24 h. Relative mobilities of
molecular mass markers are indicated on the right. B,
processing of E375A and progelatinase A by fibroblast membrane
preparations.
I-Labeled E375A mutant and
I-labeled progelatinase A (14 nM) were incubated
with 40 µg of membrane preparation from ConA-stimulated fibroblasts
or with 0.1 mM APMA at 37 °C for up to 20 h. (i), lanes 1-4,
I-labeled E375A incubated alone
for 0, 2, 4, or 20 h, 37 °C; lane 5,
I-labeled E375A incubated with 0.1 mM APMA for
20 h, 37 °C; lanes 6-9,
I-labeled
E375A incubated with membrane preparation for 0, 2, 4, or 20 h, 37
°C; (ii), lanes 1-4,
I-labeled progelatinase A incubated alone for 0, 2, 4, or
20 h, 37 °C; lane 5,
I-labeled progelatinase
A incubated with 0.1 mM APMA for 20 h, 37 °C; lanes
6-9,
I-labeled progelatinase A incubated with
membrane preparation for 0, 2, 4, or 20 h, 37
°C.
Figure 4:
Inhibition of processing by TIMP-1 and
TIMP-2. Cell monolayers were incubated for an initial 20 h in DMEM with
or without 50 µg/ml ConA. The washed cells were then incubated for
an additional 20 h in DMEM with either TIMP-1 or TIMP-2. All wells were
supplemented with I-labeled progelatinase A or
I-labeled E375A mutant (9 nM). 10-µl
aliquots of culture supernatants from unstimulated fibroblasts (lanes 1-5) or ConA-stimulated fibroblasts (lanes
6-10) incubated with
I-labeled progelatinase A
and varying doses of TIMP-2 (A). Lanes 1 and 6, no TIMP-2; lanes 2 and 7, 7.5 nM
TIMP-2; lanes 3 and 8, 15 nM TIMP-2; lanes 4 and 9, 75 nM TIMP-2; lanes 5 and 10, 150 nM TIMP-2. B, as A, except that
I-labeled E375A mutant was used
instead of the wild type enzyme. C, unstimulated fibroblasts (lanes 1-6) or ConA-stimulated fibroblasts (lanes
7-12) incubated with
I-labeled progelatinase A
and varying doses of TIMP-1. Lanes 1 and 7, no
TIMP-1; lanes 2 and 8, 6 nM TIMP-1; lanes 3 and 9, 12 nM TIMP-1; lanes 4 and 10, 24 nM TIMP-1; lanes 5 and 11, 60 nM TIMP-1; lanes 6 and 12,
120 nM TIMP-1. D, as C, except that
I-labeled E375A was used instead of the wild type
enzyme.
Figure 5:
The
processing of E375A progelatinase A and wild type progelatinase A by
active gelatinase A. 2 µM samples of either wild type
progelatinase A (closed symbols) or E375A mutant progelatinase
A (open symbols) were incubated for varying times with either
0.02 µM (,
), 0.2 µM
(
,
), or 2 µM (
,
) APMA-activated
gelatinase A. Progelatinase A is presented as the percentage of
proenzyme remaining after varying times of incubation at 37 °C, as
determined by densitometric scanning.
The activation of progelatinase A by a membrane-mediated
process was studied using ConA-stimulated skin fibroblasts, a model
system that we described previously in our attempts to elucidate the
mechanism of activation of this pro-MMP (Ward et al., 1991,
1994; Murphy et al., 1992). The recent cloning of a putative
membrane-bound metalloproteinase, MT-MMP, from a tumor library, and the
demonstration of the ability of the recombinant protein to process
progelatinase A (Takino et al., 1995; Sato et al.,
1994), was of extreme relevance to the question of the number of
potential gelatinase A activation mechanisms in our model system and in vivo. In this study we have shown that MT-MMP mRNA and
protein are expressed at low levels in normal skin fibroblasts but that
the levels are considerably up-regulated upon treatment with ConA. We
chose gelatin zymography followed by autoradiography to study the fate
of a I-labeled inactive mutant (E375A progelatinase A
(Crabbe et al., 1994c)) or
I-labeled wild type
progelatinase A when incubated in cell cultures or with isolated
membranes. Gelatin zymography was performed so that gelatin degrading
activities could be identified, and autoradiography of the zymograms
was chosen in preference to silver- or Coomassie-stained gels since it
allowed us to detect the relatively low levels of enzyme remaining in
the cell supernatants. For these reasons, the separation of the three
species of gelatinase is less clear than it might have been using
different techniques. Where there is rapid processing to the final
(59-kDa) species, the intermediate (62-kDa) form is only very
transiently present. The intermediate is, however, more evident in
those situations where the processing is slowed either by lack of
sufficient active gelatinase (membrane processing of E375A mutant) or
where active gelatinase or MT-MMP activity is inhibited (TIMP-1). CHO
cells transiently expressing MT-MMP were shown to process wild type
progelatinase A to the fully active species, but could only process the
inactive mutant to an intermediate form. Monolayers of ConA-stimulated
fibroblasts, or membrane preparations derived from them, were, however,
able to process not only wild type progelatinase A but also the E375A
mutant to a 59-kDa (nonreducing gel) form, via a 62-kDa intermediate.
We were unable to prepare sufficient processed material for N-terminal
sequencing and to determine the precise cleavage sites, but the
electrophoretic mobility of the intermediate and final forms of the
wild type and mutant gelatinases appear identical.
We have also
shown that the E375A mutant, which cannot naturally self-process like
the wild type proenzyme in the presence of an organomercurial, can be
processed efficiently at high concentrations in the presence of a high
molar ratio of active gelatinase A. This is in agreement with our
previous data that a truncated mutant of gelatinase A could effect the
same propeptide processing of the E375A mutant as can be seen for the
wild type gelatinase (Crabbe et al., 1994c). Our studies of
proenzyme processing by gelatinase A (Fig. 5) measured the
amount of proenzyme remaining by scanning Coomassie-stained gels. This
method provided a better estimate of progelatinase
``activation'' in this experimental system than attempts to
quantitate the amount of fully processed (66-kDa reducing SDS-PAGE)
enzyme for two reasons. Firstly, the experiment adds increasing amounts
of previously activated 66-kDa gelatinase A to the system. This could
be circumvented by using I-labeled gelatinase, but, for
quantitation experiments, autoradiography is less accurate than
scanning of Coomassie-stained protein bands. Secondly, the 66-kDa form
is itself constantly subject to further degradation at 37 °C at a
rate that is in part determined by the concentration of 66-kDa
gelatinase A (Crabbe et al., 1993). The experiments show that
the rate of proenzyme loss is not only governed by the amount of active
enzyme added to the incubation but by the potential for the generation
of further active enzyme; thus, the processing of wild type proenzyme
at low initial values of added active enzyme accelerates with time,
while the mutant processing rate cannot be modified. These observations
suggest that the processing is by intermolecular reactions.
To investigate the role of endogenous membrane-associated gelatinase A in the processing of the E375A mutant by ConA-stimulated fibroblasts, we took a number of approaches. Initially, we confirmed that the processing was likely to be due to the action of an MMP by demonstrating that the TIMPs were effective inhibitors of processing. As previously noted (Ward et al., 1991), TIMP-2 was a more efficient inhibitor of processing than TIMP-1. This phenomenon was also observed in the inhibition of progelatinase A processing by CHO cells transfected with MT-MMP and has been reported by others (Strongin et al., 1993). It is thought to be due to tighter C-terminal domain interactions of the proenzyme with TIMP-2 (Willenbrock et al., 1993). We therefore proposed this also to be indicative of the necessity for progelatinase to bind to the cell membrane through the C-terminal domain for activation to occur. Such binding and activation can be blocked by TIMP-2, and the isolated C-terminal domain of gelatinase (Murphy et al., 1992; Strongin et al., 1993; Ward et al., 1994). We conclude that TIMP-2 is a more efficient inhibitor of E375A mutant membrane processing for the same reason.
We had noted that the preparation of membrane fractions from ConA-stimulated fibroblasts reduced both the amount of endogenous gelatinase A associated with the system and the ability of the fibroblast membranes to process progelatinases. However, we were unable to reduce further the gelatinase A content of cell membranes using solvents, acid pH etc. (data not shown). The addition of small amounts of activated gelatinase A to the system caused a dose-responsive restoration of the ability to process both the wild type and mutant progelatinases. In processing studies in the absence of membranes, high concentrations of proenzymes and high molar ratios of the active form were shown to be required for this phenomenon to occur (this paper and Crabbe et al., 1993, 1994a). In the presence of membranes, processing can occur at relatively low concentrations (150-fold less) of the reactants. Crabbe et al.(1993) and Ward et al.(1994) have proposed that the binding of gelatinases to the cell membrane may increase its localized concentration such that an intermolecular processing reaction is promoted. We therefore conclude that membrane-associated gelatinase A is involved in the activation of its proform. The differences in the rates of wild type and mutant proenzyme processing by active gelatinase A observed in the absence of cell membranes can be extrapolated to the results obtained in their presence. Thus, because the rate of wild type processing using membranes is only slightly faster than that of the mutant, the amount of active gelatinase A endogenously present on the cell surface of ConA-stimulated fibroblasts is likely to be approximately equal to the amount of bound proenzyme.
Until MT-MMP has been rigorously
characterized, we are not able to assess its importance as a
progelatinase processing enzyme relative to active gelatinase. By
analogy with other MMP activation cascades, it is extremely likely that
MT-MMP and active gelatinase A will act in concert in the cleavage of
the propeptide of progelatinase A. There are numerous examples of this,
such as the case of plasmin activation of stromelysin-1, where the
final propeptide cleavage is autocatalytic (Nagase et al.,
1990) and in the collagenase processing of gelatinase A (Crabbe et
al., 1994b). Our results do not rule out the possibility that ConA
induces another as yet unidentified membrane component to which
progelatinase A can bind such that it becomes concentrated on the cell
surface allowing intermolecular cleavage to take place. Thus, the
up-regulation of MT-MMP in ConA-stimulated fibroblasts could be
entirely coincidental. However, the recent availability of the isolated
catalytic domain of MT-MMP has enabled us to demonstrate conclusively
that the inactive mutant E375A progelatinase A is cleaved only to the
intermediate species by MT-MMP in the absence of catalytically active
enzyme, but can be fully processed by the inclusion of wild type
progelatinase A in the incubation mixture. ()Preliminary
immunohistochemical studies using specific antibodies to MT-MMP and
gelatinase A show that CHO cells transfected with MT-MMP bind exogenous
progelatinase A on the cell surface whereas vector control cells do
not. Confocal microscopy and double labeling techniques have
demonstrated that the bound gelatinase has the same distribution as
MT-MMP. (
)The availability of the E375A mutant which cannot
self-process should prove invaluable in the further unravelling of the
question of the role of different MMPs in membrane processing of
progelatinase A.
While this manuscript was in preparation, Strongin et al. (1995) reported a role for TIMP-2 in the phorbol
ester-induced activation of progelatinase A by HT1080 cells. These
workers demonstrated that MT-MMP acts as a receptor for TIMP-2, forming
a complex capable of binding progelatinase A on the cell surface that
leads to its activation. Our studies using specific antisera to
gelatinase A, TIMP-2, and MT-MMP and confocal microscopy indicate that
in some activating cells, all three are located together on the cell
surface. Levels of TIMP-2 are critical, therefore, in
determining the activation status of gelatinase A in the extracellular
environment, since we have clearly shown that excess TIMP-2 completely
inhibits the activation of progelatinase A by ConA-stimulated
fibroblasts. Further work will be required to understand the nature of
the binding of the three components that leads to activation of
progelatinase A without its inhibition.