(Received for publication, January 25, 1996)
From the
Membrane-type matrix metalloproteinase-1 (MT-MMP-1) has been
proposed to play a critical role in regulating the expression of
tissue-invasive phenotypes in normal and neoplastic cells by directly
or indirectly mediating the activation of progelatinase A. To begin
characterizing MT-MMP-1 structure-function relationships,
transmembrane-deletion mutants were constructed, and the processing of
the zymogens as well as the enzymic activity of the mature proteinases
was analyzed. We now demonstrate that pro-MT-MMP-1 mutants are
efficiently processed to active proteinases following
post-translational endoproteolysis immediately downstream of an
Arg-Arg-Lys-Arg basic motif by a proprotein
convertase-dependent pathway. The secreted form of active MT-MMP-1 not
only displays an N terminus identical with that described for the
processed wild-type enzyme at Tyr
(Strongin, A. Y.,
Collier, I., Bannikov, G., Marmer, B. L., Grants, G. A., and Goldberg,
G. I.(1995) J. Biol. Chem. 270, 5331-5338), but also
directly mediated progelatinase A activation via a two-step proteolytic
cascade indistinguishable from that observed with intact cells.
Furthermore, although the only function previously ascribed to MT-MMP-1
is its ability to act as a progelatinase A activator, purified
transmembrane deletion mutants also expressed proteolytic activities
against a wide range of extracellular matrix molecules. Given recent
reports that MT-MMP-1 ectodomains may undergo intercellular transfer in vivo (Okada, A., Bellocq, J.-P., Rouyer, N., Chenard,
M.-P., Rio, M.-C., Chambon, P., and Basset, P.(1995) Proc. Natl.
Acad. Sci. U. S. A. 92, 2730-2734), our data suggest that
soluble forms of the proteinase confer recipient cells with the ability
to not only process progelatinase A, but also directly degrade
extracellular matrix components.
Members of the matrix metalloproteinase (MMP) ()gene
family have been implicated in the physiologic as well as pathologic
remodeling of the extracellular matrix (ECM) in events ranging from
organogenesis to tumor metastasis (1, 2, 3) .
Very recently, three new members of this family were discovered by
screening cDNA libraries for homologies to conserved regions of the
known MMP genes and named the membrane-type matrix
metalloproteinases-1, -2, and -3 (MT-MMP-1, -2, and -3; (4, 5, 6) ). Based on their predicted amino
acid sequences, each of the MT-MMPs, like almost all previously
characterized MMPs, contains (i) a candidate leader sequence, (ii) a
propeptide region which includes a highly conserved PRCGXPD
sequence that helps stabilize the MMP zymogen in a catalytically
inactive state, (iii) a zinc-binding catalytic domain, and (iv) a
hemopexin-like domain near their respective C
termini(4, 5, 6, 7) . In addition,
in a pattern similar to that described for stromelysin-3, each of the
MT-MMPs contains a short amino acid insert sandwiched between their
pro- and catalytic domains that encodes a potential recognition motif
for members of the proprotein convertase
family(4, 5, 6, 7, 8) .
Despite their considerable similarity to other MMP family members, (
)however, only the MT-MMPs contain
75-100 amino
acid extensions at their C termini, each of which includes a
hydrophobic stretch consistent with the presence of a transmembrane
(TM) domain(4, 5, 6, 9) . Thus, in
contradistinction to all other MMPs, the MT-MMPs are expressed as
membrane-associated ectoenzymes rather than soluble proteins.
Although little is known with regard to the potential functions of
the MT-MMPs, most attention has focused on the ability of MT-MMP-1 as
well as MT-MMP-3 to induce the processing of the MMP zymogen,
progelatinase A, to its activated form (i.e. [Tyr]gelatinase A) via a
[Leu
]gelatinase A
intermediate(4, 6, 10) . Given the ability of
activated gelatinase A to cleave a wide range of ECM substrates
(including native types I, IV, V, VII, and XI collagen, denatured
collagens, elastin, proteoglycans, laminin, and fibronectin) as well as
the association of gelatinase A activation with the expression of
tissue-invasive
phenotypes(1, 2, 3, 11, 12, 13, 14, 15) ,
MT-MMPs have been dubbed as possible ``master switches'' that
control ECM remodeling(16) . Nonetheless, the processes that
regulate the activation of the MT-MMP zymogens themselves to mature
forms remain undefined as does the mechanism by which MT-MMPs mediate
progelatinase A
activation(4, 5, 6, 9, 10) .
In large part, further progress in characterizing MT-MMP activities has
been hindered by the technical problems associated with isolating and
purifying membrane-associated molecules. Given that similar
difficulties with other transmembrane enzymes have been negotiated by
generating TM-deleted soluble mutants (17, 18, 19) , we noted that, with the
exception of the extended C-terminal domain, the modular organization
of the MT-MMPs is identical with that of the secreted MMPs(7) .
Hence, two TM-deletion mutants of MT-MMP-1 were constructed by either
truncating the molecule (i) immediately upstream of the start site of
the TM domain (i.e. MT-MMP-1
; herein
referred to as MT-MMP-1
) or (ii) at the conserved
cysteinyl residue found at, or near, the terminus of all hemopexin
domain-containing MMPs (i.e. MT-MMP-1
or
MT-MMP-1
). Utilizing these constructs, we now
demonstrate that TM-deleted MT-MMP-1 (
MT-MMP-1) mutants undergo
efficient post-translational endoproteolysis between
Arg
-Tyr
by a proprotein
convertase-dependent pathway to generate fully active proteinases.
Furthermore, the purified
MT-MMP-1 mutants not only activate
recombinant progelatinase A directly via a two-step activation cascade
identical with that described for the membrane-associated enzyme, but
they also express heretofore unsuspected ECM-degrading activities.
Figure 1:
Expression and characterization of
MT-MMP-1. A, domain alignments of MT-MMP-1 and the
MT-MMP-1 mutants. Wild-type MT-MMP-1 contains 582 amino acids
arranged as a series of pre- (shaded box), pro-, catalytic,
hemopexin (bounded by a pair of highly conserved cysteinyl residues
indicated as C), TM (shaded in black and marked TM), and cytosolic (residues 564 to 582) domains.
MT-MMP-1
is truncated at the edge of the TM domain
while MT-MMP-1
ends at the conserved cysteinyl residue
that marks the extreme C terminus of the hemopexin domain. B,
Western blot analysis of
MT-MMP-1-transfected cells. Serum-free
conditioned medium from COS-7 cells transiently transfected with
control (lane 1), MT-MMP-1
(lane 2),
MT-MMP-1
(lane 3), or wild-type MT-MMP-1 (lane 4) expression vectors were analyzed by immunoblotting
with MT-MMP-1-specific polyclonal antisera. The dark and clear arrowheads indicate the positions of the putative pro-
and processed forms of
MT-MMP-1. In wild-type MT-MMP-1-transfected
cells, immunoblots of cell lysates identified the 63-kDa form of the
enzyme as the major product (data not shown). C, gelatin
zymography of MT-MMP-1-transfected cells. Serum-free conditioned medium
from COS-7 cells transiently transfected with control (lane
1), MT-MMP-1
(lane 2),
MT-MMP-1
(lane 3), or MT-MMP-1 (lane
4) expression vectors were depleted of endogenous progelatinase A
by gelatin affinity chromatography and analyzed by zymography. In lane 5, the MT-MMP-1
zymogram was developed
in the presence of 1.0 µM BB-94. Identical results were
obtained with
-casein or
-elastin zymograms (D. Pei and S. J.
Weiss, unpublished observation).
In intact cell
systems, MMPs can be recovered in conditioned medium as a mixture of
zymogens, processed active enzymes, zymogen-inhibitor complexes, or
enzyme-inhibitor complexes(1, 2, 3) . In the
case of almost all MMP family members, many of these forms can be
detected following SDS-PAGE in substrate-impregnated
gels(1, 2, 3) . Thus, serum-free conditioned
media from control, MT-MMP-1,
MT-MMP-1
, or MT-MMP-1 transfected cells were depleted
of endogenous gelatinases by gelatin affinity chromatography
(
MT-MMP-1 does not bind to gelatin; see below) and electrophoresed
in gelatin- containing gels. Proteinases were then allowed to renature
following the removal of SDS and then incubated overnight at 37 °C.
As shown in Fig. 1C, supernatants recovered from
MT-MMP-1
- or MT-MMP-1
-transfected
cells each revealed the presence of a single band of gelatinolytic
activity whose relative mobility matched that of the major form of
MT-MMP-1 detected by Western blotting. Significantly, identical
results were obtained when zymograms were performed with either
-casein- or
-elastin-impregnated gels as well (data not
shown). Regardless of substrate used, the band of proteolytic activity
attributed to either
MT-MMP-1 mutant was completely inhibited when
zymograms were performed in the presence of the MMP-specific inhibitor,
BB-94 (Fig. 1C).
Figure 2:
Purification of MT-MMP-1. A and B, fractionation of MT-MMP-1
on Q-Sepharose and heparin-Sepharose, respectively. Conditioned
media from MDCK cells stably transfected with MT-MMP-1
were loaded onto a Q-Sepharose column and eluted with a NaCl
gradient. The protein content of each fraction was monitored at A
(dark squares) while
MT-MMP-1
content in each fraction was monitored by
immunoblot analysis and reported as the percent recovered relative to
the fraction containing the highest concentration of
MT-MMP-1
(open squares). Fractions 9-16
were combined, dialyzed against buffer A, loaded onto a
heparin-Sepharose column, and eluted with a NaCl gradient. C,
characterization of the isolated MT-MMP-1
products.
Conditioned media (lane 1), a pool of fractions 9-16
eluted from Q-Sepharose (lane 2), flow-through of fractions
9-16 that did not bind to heparin-Sepharose (lane 3),
pool of fractions 10-15 eluted from heparin-Sepharose column (lane 4), and final purified form of MT-MMP-1
(4.5 pmol; lane 5) were separated by SDS-PAGE and
visualized by Coomassie staining. In lanes 6 and 7,
purified MT-MMP-1
(1.2 pmol) was analyzed by
immunoblotting and gelatin zymography, respectively. D, the N
terminus of MT-MMP-1
as determined after 10 cycles of
sequencing (indicated by bold letters). The open box represents the MT-MMP-1
open reading frame with
the amino acid sequence of MT-MMP-1 from Pro
to
Glu
.
Like stromelysin-3, the only
other membrane of the MMP family to be secreted as a fully processed
active proteinase, MT-MMP-1 contains a motif of basic amino acids (i.e.RRKR) immediately upstream of its
catalytic domain (see Fig. 3A; (4) and (8) ). Recently, the RXKR array in stromelysin-3 (X = nonbasic amino acid) was shown to act as an
endoproteolytic processing signal for an intracellular serine
proteinase belonging to the proprotein convertase family(8) .
Because specific proprotein convertases can display varying
requirements for basic residues at positions -1, -2, and
-4 relative to the scissile bond (i.e. P
, P
, and
P
, respectively; (23) and (24) ), a
potential role for this enzyme class in
MT-MMP-1 processing was
initially assessed by successively substituting each basic residue with
an Ala moiety in transient transfection assays. As shown in Fig. 3B, each of these substitutions almost completely
blocked MT-MMP-1
processing (lanes
2-4). In contrast, a Tyr
Ala substitution at the less
critical P
site (23) did not affect
MT-MMP-1
processing (Fig. 3, lane 5).
Given that COS are known to express only two members of the proprotein
convertase family that recognize RXKR motifs (i.e. furin and PACE4; (24) ), cells were co-transfected with
MT-MMP-1
and the Pittsburgh mutant of
PI (
PI
), a reactive
site variant that inhibits furin (but not PACE4) activity in
situ(25, 26) . Under these conditions,
PI
completely blocked
MT-MMP-1
processing while wild-type
PI exerted no inhibitory effect (Fig. 3C). MT-MMP-1
processing was
similarly inhibited by
PI
in MDCK cells
(data not shown). These results (together with the demonstration that
soluble furin processes pro-
MT-MMP-1 to its active form under
cell-free conditions; see below) indicate that pro-
MT-MMP-1
maturation is regulated by a furin-dependent pathway in an intact cell
system.
Figure 3:
Proprotein convertase-dependent processing
of MT-MMP-1. A, mutational analysis of the
putative proprotein convertase recognition motif. The amino acids
surrounding the cleavage site in MT-MMP-1
are shown
with the basic residue motif underlined. B, expression vectors
for native MT-MMP-1
(lane 1), Arg
Ala (R108A; lane 2), Lys
Ala (K110A; lane 3), Arg
Ala (R111A; lane 4), and Tyr
Ala (Y112A; lane 5) were transfected
into COS-7 cells (2
10
/ml) by LipofectAMINE
treatment, and the secreted products (0.02 ml of the cell-free
supernatant) were analyzed by immunoblotting. C, inhibition of
MT-MMP-1
processing by
PI
. COS-7 cells were transiently
transfected as described above with MT-MMP-1
expression vector alone (lane 1) or co-transfected with
MT-MMP-1
and
PI (lane 2) or
PI
expression vectors (lane
3), and the cell-free supernatants were analyzed by
immunoblotting.
Figure 4:
MT-MMP-1-dependent activation of
recombinant progelatinase A. A, zymography and N-terminal
sequence analysis of progelatinase A. Recombinant progelatinase A (140
nM) was incubated alone (lane 1) or with 14
nM, 28 nM, or 56 nM MT-MMP-1
(lanes 2-4, respectively) for 2 h at 37 °C in
buffer A supplemented with 150 mM NaCl and 0.01% Brij 35 in a
final volume of 0.02 ml. In lanes 5-7, progelatinase was
incubated alone, with MT-MMP-1
or with
MT-MMP-1
and TIMP-2 (350 nM), respectively,
for 16 h at 37 °C. Aliquots of each reaction mixture were analyzed
by gelatin zymography or were electrophoresed and electroblotted for
N-terminal sequence analysis. The bold sequence beginning with
Leu
represents the first 10 cycles of the N terminus of
the
64-kDa form of gelatinase A (indicated by asterisk in lane 4) while the sequence beginning with Tyr
represents the first 5 cycles of the N terminus of the
62-kDa form of gelatinase (indicated by dark arrow in lane 7). The faint bands of gelatinolytic activity detected at
50 kDa are due to MT-MMP-1
(lanes
2-4). In lanes 8-10, progelatinase A (140
nM) was incubated alone (lane 8) with
MT-MMP-1
(14 nM; lane 9) or
MT-MMP-1
(14 nM; lane 10) for 3 h in
20 mM Hepes/KOH (pH 7.5), 0.1 mM CaCl
,
and 0.02% Brij-35 as described (10) . The asterisk, arrowhead, and circle indicate the positions of the
64-kDa,
62-kDa, and
42-kDa forms of gelatinase A,
respectively. B, furin-mediated processing of
pro-MT-MMP-1
. Pro-MT-MMP-1
(100
nM) isolated from
PI
co-transfected cells was incubated alone (lane 1) or
with soluble furin (10 nM; lane 2) for 1 h at 37
°C in a final volume of 0.02 ml and analyzed by Western blotting.
For zymography (lanes 3-5), progelatinase A (140
nM) was incubated alone (lane 3), with
pro-MT-MMP-1
(28 nM; lane 4), or
with furin-processed MT-MMP-1
(28 nM; lane 5) for 16 h at 37 °C. Furin alone did not activate
progelatinase A.
The ability of
mature MT-MMP-1 to mediate progelatinase A
activation is consistent with a model wherein active
MT-MMP-1
directly cleaves the gelatinase zymogen,
but the data do not rule out the possibility that the
MT-MMP-1
zymogen only induces progelatinase A to undergo autocatalytic
processing to its active form. Hence, purified pro-
MT-MMP-1 was
isolated (i.e. from cells co-transfected with
MT-MMP-1
and
PI
), and
its ability to mediate progelatinase A activation was examined. As
shown in Fig. 4B, pro-MT-MMP-1
was
unable to stimulate progelatinase A activation. However, when
pro-MT-MMP-1
was processed to its active form ex
situ with a TM-deleted soluble form of furin (Fig. 4B, lane 2) and then incubated with
progelatinase A, the gelatinase zymogen was readily activated (lane
5). Thus, only the processed active form of
MT-MMP-1 is able
to mediate progelatinase A activation.
Figure 5:
Substrate specificity of
MT-MMP-1. Type I collagen (3 µg; lane 1),
type IV collagen (2 µg; lane 3), and type V collagen (2
µg; lane 5) were incubated alone or with 45 nM MT-MMP-1
(lanes 2, 4, and 6, respectively) at 25 °C for 16 h. Type I gelatin (3
µg; lane 7), fibronectin (4 µg; lane 10),
laminin (4 µg; lane 13), vitronectin (4 µg; lane
16), or dermatan sulfate proteoglycan (5 µg; lane 19)
were incubated alone, with MT-MMP-1
(45 nM; lanes 8, 11, 14, 17, and 20, respectively) or with MT-MMP-1
and 1
µM BB-94 (lanes 9, 12, 15, 18, and 21, respectively) at 37 °C in a final
volume of 0.025 ml for 16 h. Reaction mixtures were separated by
SDS-PAGE and Coomassie-stained. The arrowhead by lane 15 indicates the position of the laminin B chain, while the hatch
marks at the margin of lanes 16 and 19 indicate
the positions of the 97-, 69-, 45-, 32-, and 28-kDa molecular mass
markers, respectively. Dermatan sulfate proteoglycan is recorded as DSPG.
Sequence alignments of the 13 human MMPs that have been characterized to date indicate that amino acids 1-508 of the 538-residue-long extracellular domain of MT-MMP-1 contain all of the major structural elements of the secreted members of this gene family (i.e. a propeptide and catalytic domain as well as a hemopexin-like region that is bounded by a pair of highly conserved cysteinyl residues; (4, 5, 6, 7) ). Given that the C termini of virtually all secreted MMPs end at, or extend no more than 8 amino acids beyond, the final cysteinyl residue in the hemopexin domain(7) , we reasoned that TM-deletion mutants of MT-MMP-1 that retained this modular organization would encode functional proteinases. Indeed, as demonstrated, regardless of whether soluble mutants of MT-MMP-1 were truncated either at the edge of the TM domain or at the end of the hemopexin domain, the expressed proteins displayed similar, if not identical, activities as assessed by zymography, progelatinase A processing, or substrate specificity.
By
itself, our work does not rule out the possibility that the TM or
cytosolic domains of MT-MMP-1 convey additional structural information
to the processed proteinase (i.e. beyond acting as a membrane
anchor). However, while our work was in progress, Cao et al.(9) reported that an MT-MMP-1 chimera generated by
exchanging the TM and cytosolic domains of the metalloproteinase with
those of the IL-2 receptor functioned normally in terms of its ability
to mediate progelatinase A activation(9) . Although this result
is consistent with our conclusion that the extracellular domain of
MT-MMP-1 confers the proteinase with its distinct characteristics,
these authors also reported that a TM-deletion mutant encoding residues
1-535 of MT-MMP-1 was unable to process progelatinase
A(9) . In comparing our experimental approaches, it is
important to note that the soluble MT-MMP-1 generated in their study
was not isolated nor were its interactions with progelatinase A
examined directly(9) . Instead, Cao et al.(9) judged their TM-deletion mutant to be inactive on the
basis of its inability to process endogenously derived progelatinase A
secreted by COS-1 cells in a transient transfection assay
system(9) . Under these conditions, however, attempts to assess
the activity of secreted MT-MMP-1 would be complicated by the presence
of cell-derived TIMPs which can interfere with progelatinase A
processing by either inhibiting MT-MMP-1 activity directly, or, in
the case of TIMP-2, by binding to the C-terminal domain of the
gelatinase zymogen(4, 27, 28) . Indeed, when
endogenous levels of TIMP are overwhelmed by co-transfecting COS cells
with MT-MMP-1
and progelatinase A,
gelatinase activation can be readily detected in the intact cell system
as well as our purified system.
Thus, while anchoring a
proteinase to the cell membrane might be predicted to more effectively
shield an active proteinase from soluble inhibitors (and to perhaps
provide a surface more conducive for accelerating processing events) (e.g.(29, 30, 31) ), our data
demonstrate that the TM-deletion mutants retain the key functional
properties of the wild-type enzyme.
As a consequence of our attempts
to characterize the activity of MT-MMP-1, we also discovered that
the TM-deletion mutants are capable of undergoing rapid processing to
their mature forms. This finding is noteworthy since, as a general
rule, MMPs are synthesized and secreted as inactive
zymogens(1, 2, 3, 7) . However, we
recently reported that in a fashion similar to that observed for the
MT-MMP-1 mutants, prostromelysin-3 is secreted as an active enzyme
following its intracellular processing within the constituitive
secretory pathway (8) . In this case, activation was dependent
upon a decapeptide insert that is sandwiched between the pro- and
catalytic domains of stromelysin-3 and encrypted with an extended furin
recognition motif (i.e. RXRXKR). At the time
that these earlier studies were completed, stromelysin-3 was the only
member of the MMPs family known to contain this recognition sequence.
However, with the recent cloning of MT-MMP-1, -2, and -3, it is clear
that all three of these enzymes contain homologous inserts which
include an array of basic residues (i.e. RRKR) that match the
recognition motif of the proprotein convertases (i.e. RX(K/R)-R; (4, 5, 6) ). (
)Consistent with this prediction, (i) the N terminus of
MT-MMP-1 was located at Tyr
on the C-terminal side
of the Arg-Arg-Lys-Arg motif, (ii)
MT-MMP-1 processing could be
inhibited by either inserting point mutations in the RRKR motif or by
co-transfecting cells with the furin-specific inhibitor,
a
PI
, and (iii) the
MT-MMP-1 zymogen
could be processed to its active form ex situ by soluble
furin. While we have not yet identified the intracellular/extracellular
compartments in which the
MT-MMP-1 zymogen undergoes processing in
the intact cell, furin is a membrane-associated endoprotease that not
only cycles between the trans-Golgi network and the cell
surface, but also undergoes processing to a soluble form that
accumulates extracellularly(32, 33, 34) .
Indeed, the possibility that
MT-MMP-1 may undergo extracellular
processing is further supported by our results with the TM-deleted form
of soluble furin. Nonetheless, in spite of the fact that furin is the
most credible MT-MMP-1 activator identified to date, caution should be
exercised in terms of extrapolating processing pathways that are
operative for
MT-MMP-1 to the wild-type enzyme. Indeed, in
contrast to the results obtained with
MT-MMP-1, we and others have
found that COS cells transfected with wild-type MT-MMP-1 route most of
the enzyme to the cell surface as the unprocessed zymogen rather than
the mature enzyme(4, 9, 31) .
(
)Utilizing chimeric constructs between stromelysin-3
and wild-type MT-MMP-1, it appears that while the furin recognition
motif in either of the secreted metalloproteinases can be processed
effectively, the TM domain of MT-MMP-1 appears to ``shield''
the recipient proteinase from undergoing rapid intracellular
processing.
The mechanisms responsible for controlling the
intracellular and extracellular processing of wild-type MT-MMP-1
require further analysis, but the fact that the active form of the
full-length (10) and mutant enzyme display an identical N
terminus directly downstream of the proprotein convertase-recognition
motif strongly suggests a role for furin or, perhaps, a related
proprotein convertase (e.g. PC6; Refs. 24 and 35) in zymogen
activation.
In the presence of purified active MT-MMP-1 (but
not its zymogen), progelatinase A was processed to its mature form (i.e. [Tyr
]gelatinase) via the
formation of the Leu
intermediate. This two-step,
TIMP-2-sensitive activation cascade is identical with that previously
established for crude preparations of plasma membrane-associated
MT-MMP-1 (27) and allows us to conclude that
MT-MMP-1 can
initiate the processing event independently of additional co-factors or
substrates. Interestingly, the ability of
MT-MMP-1 to directly
activate progelatinase A under cell-free conditions contrasts with a
recent report by Strongin et al.(10) wherein an
MT-MMP-1
TIMP-2 complex (rather than MT-MMP-1 alone) was proposed
to function as the membrane-associated activator of the gelatinase
zymogen. We were unable to reproduce this finding with
MT-MMP-1
, but cannot rule out the possibility that
TIMP-2 plays a more complex role on the membrane surface. However, an
interpretation of the data presented by Strongin et al.(10) is complicated by the fact that even in the apparent
absence of TIMP-2, MT-MMP-1 continued to process progelatinase A to the
[Leu
]gelatinase intermediate, but not the final
mature form. Thus, it remains possible that TIMP-2 exerts its
stimulatory effect by accelerating the inter- or intramolecular
autocatalytic conversion of [Leu
]gelatinase to
[Tyr
]gelatinase on the cell surface (31) rather than by stimulating MT-MMP-1 activity directly.
To date, the only function ascribed to MT-MMP-1 has been its ability
to activate progelatinase
A(4, 9, 10, 13, 27, 28) .
However, we have demonstrated that purified MT-MMP-1 can also degrade a number of extracellular matrix components.
These data indicate that the ability of MT-MMP-1-transfected cells to
express a heightened invasive potential may not necessarily be linked
to progelatinase A activation alone(4) . Although our studies
have employed a soluble form of MT-MMP-1, we believe that the modular
organization of MT-MMP-1 strengthens the likelihood that the
membrane-tethered form displays a similar substrate specificity.
Furthermore, the potential physiologic relevance of
MT-MMP-1
mutants have been heightened by recent findings which suggest that
soluble forms of MT-MMP-1 may be generated in
vivo(13) . Thus, while the MT-MMP-1 antigen has been
immunodetected on the surface of cancer cells in
vivo(4) , RNA in situ hybridization studies have
more recently demonstrated that MT-MMP-1 transcripts are confined to
the surrounding stromal cells(13) . Should MT-MMP-1 undergo
solubilization and intercellular transfer in
situ(4, 13) , tumor cells could potentially use
the stroma-derived enzyme to assemble a multicatalytic complex on their
surface that would not only arm them with the ability to catalyze
progelatinase A activation, but also to express an additional
repertoire of proteolytic activities. Additional studies will be
required to directly compare the soluble and membrane-anchored forms of
MT-MMP-1, but the established catalytic activity of the TM-deletion
mutants should provide a useful tool for characterizing the enzymic
properties of this new family of membrane-anchored MMPs.