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Vol. 11, Issue 7, 2387-2401, July 2000
Department of Internal Medicine and the University of Michigan Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan 48109
Submitted December 27, 1999; Revised April 20, 2000; Accepted May 1, 2000  |
ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
SUMMARY
REFERENCES
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Membrane type-1 matrix metalloproteinase (MT1-MMP) is the
prototypical member of a subgroup of membrane-anchored proteinases that
belong to the matrix metalloproteinase family. Although synthesized as
a zymogen, MT1-MMP plays an essential role in extracellular matrix
remodeling after an undefined process that unmasks its catalytic
domain. We now report the existence of a proprotein convertase-MT1-MMP
axis that regulates the processing and functional activity of the
metalloproteinase. Two sets of basic motifs in the propeptide region of
MT1-MMP are identified that potentially can be recognized by the
proprotein convertase family of subtilisin-like proteases. Processing
of proMT1-MMP as well as the expression of its proteolytic activity
were blocked by mutating these recognition motifs or by inhibiting the
proprotein convertases furin and PC6 with the serpin-based inhibitor
1 antitrypsin Portland. Furthermore, both
furin-dependent and furin-independent MT1-MMP processing pathways are
identified that require tethering of the metalloproteinase to the cell
surface. These findings demonstrate the existence of a proprotein
convertase-MT1-MMP axis that can regulate extracellular matrix remodeling.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
SUMMARY
REFERENCES
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Membrane type-1 matrix metalloproteinase (MT1-MMP) is a member of
a family of membrane-anchored matrix metalloproteinases (MMPs)
implicated in tissue-remodeling events that range from tumor invasion
and angiogenesis to growth and development (Sato et al.,
1994
; Hiraoka et al., 1998; Belien et al., 1999;
Holmbeck et al., 1999; Zhou et al., 2000). With
the recent generation of MT1-MMP-deficient mice, it is now clear that
this protease plays an essential role in the formation and maintenance
of skeletal tissues (Holmbeck et al., 1999; Zhou et
al., 2000). Current evidence indicates that MT1-MMP regulates
matrix turnover by means of its ability to degrade matrix-associated
molecules either directly or via the activation of downstream MMPs (Pei
and Weiss, 1996; Ohuchi et al., 1997; Nagase and Woessner,
1999). Like all of the other known members of the human MMP gene
family, MT1-MMP is synthesized as a zymogen that can be processed to
its mature, catalytically active form after the removal of the
regulatory propeptide domain (Nagase and Woessner, 1999). However, the
mechanisms responsible for the processing of proMT1-MMP to its mature
form have remained controversial, and the zymogen itself has been
reported to express enzymic activity (Cao et al., 1996,
1998; Zucker et al., 1998).
Recently, the secreted MMP stromelysin-3 was shown to undergo
intracellular activation after the proteolytic removal of its propeptide domain by furin, a member of the proprotein convertase family (Pei and Weiss, 1995). Activation of the prostromelysin-3 zymogen was controlled by a decapeptide insert located immediately upstream of the N terminus of the catalytically active enzyme that
encodes a proprotein convertase recognition motif, RXRXKR (where R = Arg, K = Lys, and X = a nonbasic amino acid) (Pei and Weiss, 1995). Interestingly, MT1-MMP contains a similar
108RRKR motif upstream of the N terminus of its
mature form (Sato et al., 1994; Nagase and Woessner, 1999).
Because RX(K/R)R sequences can act as general recognition motifs for at
least four members of the proprotein convertase family, i.e., furin,
PACE4, PC6, and PC7 (Nakayama, 1997; Zhou et al., 1999), the
MT1-MMP zymogen could present itself as a substrate for these
processing enzymes.
In this report, we demonstrate that processing and activation of full-length MT1-MMP are controlled by proprotein convertases that recognize one of two potential recognition motifs in the enzyme's prodomain. Although a furin-dependent pathway efficiently processed membrane-anchored proMT1-MMP, a furin-independent route was also identified that required the MMP zymogen to be tethered to the cell surface. Importantly, inhibition of proMT1-MMP processing completely blocked the ability of MT1-MMP-expressing cells to display proteolytic activity. We conclude that cooperative interactions between proprotein convertases and membrane-anchored MMPs play an important role in regulating the remodeling of the extracellular matrix.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
SUMMARY
REFERENCES
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Cell Culture
COS-1, HT-1080, and LoVo cells (all obtained from the American Type Culture Collection, Rockville, MD) were maintained in MEM (Life Technologies, Grand Island, NY), DMEM (Life Technologies), and F-12 nutrient mixture (Life Technologies), respectively. CHO-K1 and RPE.40 cells (kindly provided by T. Moehring, University of Vermont, Burlington, VT) were maintained in RPMI 1640 (Life Technologies). All media were supplemented with 10% FBS (Hyclone, Logan, UT), 4 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Plasmid Constructs and Transfection
Full-length MT1-MMP (Met1 to
Val582), a soluble transmembrane deletion mutant
of MT1-MMP (
MT1-MMP; Met1 to
Ser538), and a cytosolic-domain deletion mutant
of MT1-MMP (Met1 to Phe563)
were generated as described (Pei and Weiss, 1996; Hiraoka et al., 1998) and inserted in the PCR3.1-uni expression vector
(Invitrogen, Carlsbad, CA). Mutagenic primers for substitutions in
MT1-MMP at Arg89, Arg108,
Arg198, Lys110, and
Arg111 or in MT1-MMP at
Glu240 were used as follows (nucleotides in
boldface indicate the altered codons): 5'-ATC AAG GCC AAT GTT GCG
GCC GCT GCC TAC GCC ATC TAG GGT-3' and 5'-CTG GTG GCT GTG CAC
GCT CTG GGC CAT GCC CTG-3' to generate MT1-MMP
Arg108-Arg-Lys-Arg 
Ala-Ala-Ala-Ala and
MT1-MMP Glu240 Ala, respectively, with
the Muta-Gene phagemid in vitro mutagenesis kit (version 2, Bio-Rad,
Richmond, CA). The mutagenic primers 5'-G GCG CGC CCC CGA
TGT GGT GTT CCA GAC A-3' and 5'-GG AAC ACC ACA TCG GGG GCG
GCG CAT GGC CTT CA-3' were used in a sequential PCR-based
method to generate MT1-MMP Arg89 Ala. Primers
encoding the FLAG epitope (5'-GAC TAC AAG GAC GAC GAT GAC AAG-3') were
inserted either between Phe34 and
Ser35 in the MT1-MMP prodomain or after
Val582 at the C terminus of MT1-MMP by PCR-based
methods (see Figures 1 and
2 for schemes). Each MT1-MMP construct
was cloned into PCR3.1-uni and characterized by sequencing. MT1-MMP
expression vectors encoding both a FLAG epitope inserted between
Arg-Arg-Lys-Arg111 and
Tyr112 in the wild-type enzyme or between
Ala-Ala-Ala-Ala111 and
Tyr112 in the MT1-MMP mutant and a chimeric
mutant wherein the transmembrane domain and cytosolic tail were
replaced with that of the interleukin 2 receptor -chain were
provided by M. Seiki and Y. Itoh (University of Tokyo) (Nakahara
et al., 1997). Expression vectors for
1 antitrypsin Portland
(1PDX), full-length furin, and soluble furin
were constructed from cDNAs provided by G. Thomas (Oregon Health
Sciences University, Portland, OR), A. Rehemtulla (University of
Michigan), and R. Kaufman (University of Michigan, Ann Arbor,
MI), respectively (Wesley et al., 1993; Jean et
al., 1998). Cells were transfected with purified plasmid DNA by
LipofectAMINE treatment (Life Technologies) as described (Pei and
Weiss, 1996). After transfection, cells were incubated either alone or
with the synthetic inhibitor BB-94 (5 µM final; British
Biotechnology, Oxford, UK) (Botos et al., 1996),
progelatinase A (see below), or soluble furin.
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Western Blotting and Zymography
Hydrophobic cell-associated proteins were solubilized and
extracted into Triton X-114 (Sigma Chemical, St. Louis, MO) in the presence of a cocktail of proteinase inhibitors as described (Toth et al., 1997). Detergent extracts (normalized to equivalent
cell numbers) were resolved by 10% PAGE under reducing conditions (Pei and Weiss, 1996). For Western blotting, proteins were transferred to
nitrocellulose and probed with anti-MT1-MMP or anti-FLAG antibodies. MT1-MMP was immunodetected with one of the following: 1) a rabbit polyclonal antibody raised against a synthetic peptide in the hemopexin
domain of MT1-MMP (Figure 2A; prepared at Research Genetics, Huntsville, AL) (Toth et al., 1997); 2) a mouse mAb directed
against a synthetic peptide in the catalytic domain of MT1-MMP (Figure 2A; clone 114-2F2, Ab-1; Calbiochem, La Jolla, CA); or 3) a mouse mAb
directed against a FLAG epitope inserted into either the MT1-MMP prodomain or the C terminus (Figures 1 and 2A; Eastman Kodak, Rochester, NY). After incubation of blots with the appropriate HRP-conjugated secondary antibody, the immune complexes were detected with the ECL system (Pierce, Rockford, IL). Densitometric analysis of
ECL-developed immunoblots was performed with a
Nucleovision Imaging workstation (Nucleotech, San Carlos, CA). The
percentage processing of proMT1-MMP to mature MT1-MMP was calculated
(in underexposed gels) with the use of the GelExport software
(Nucleotech, San Carlos, CA).
Progelatinase A processing was assessed by gelatin zymography as
described (Pei and Weiss, 1996). Cells were incubated with human
progelatinase A (generated from COS-1 cells transfected with a
progelatinase A expression vector) under serum-free conditions for
24 h at 37°C. Gelatin zymography of conditioned media was performed on 10% polyacrylamide gels that were cast in the presence of
2 mg/ml gelatin (Sigma Chemical).
Cell Surface Biotinylation
Cell monolayers were washed with ice-cold PBS (pH 8.0) and
incubated with 0.5 mg/ml sulfo-NHS-biotin (Pierce) at 15°C for 40 min
as described (Lehti et al., 1998). Cell lysates were
incubated with strepavidin-Sepharose beads (Pierce) for 1 h at
4°C, and the complexes were resolved under reducing conditions by
SDS-PAGE (10%). The captured biotinylated proteins were then
transferred to nitrocellulose, probed with anti-MT1-MMP hemopexin
domain-specific polyclonal antisera, and visualized with the use of
the ECL system.
Immunofluorescence
After transfection with FLAG-tagged MT1-MMP expression vectors,
CHO-K1 or RPE.40 cells were fixed with 3% paraformaldehyde, blocked
with 5% goat serum and 3% BSA in Tris-buffered saline for 1 h at
25°C, and then reacted with either anti-FLAG M1 mAb (10 µg/ml;
Sigma-Aldrich) in the presence of 1 mM CaCl2 as
described (Itoh et al., 1999) or an anti-MT1-MMP mAb (3H7;
Chenard et al., 1999) for 2 h at 25°C.
Antibody-coated cells were visualized with Texas red-conjugated
anti-mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) by
confocal microscopy with the use of a Bio-Rad MRC 600 laser scanning
microscope with CoMos version 7.0a software (BioRad, Hercules, CA). In
each micrograph, three sequential images were collected and combined in
a Z series with a final resolution of ~6 µm.
Subjacent Proteolysis
The ability of MT1-MMP-transfected cells to express subjacent
proteolytic activity was determined by a modification of previously published protocols (Rice and Weiss, 1990; d'Ortho et al.,
1998). In brief, gelatin (Sigma) was labeled with Texas red (Molecular Probes, Eugene, OR). Labtek slides (ICN Biochemicals, Costa Mesa, CA)
were then coated with poly-L-lysine (Sigma) and
incubated with gelatin for 2 h at 25°C. Gelatin and
poly-L-lysine were then cross-linked with
glutaraldehyde and unreacted aldehyde groups blocked with ammonium
chloride. Wild-type and transfected cells were then seeded atop the
gelatin-coated slides in 10% FBS for 16 h before analysis by
phase-contrast or confocal laser microscopy.
Pulse-Chase Analysis
HT-1080 cells were treated with concanavalin A (20 µM) under
serum-free conditions for 16 h. The monolayers were incubated in
methionine-free DMEM that was supplemented with 500 µCi of [35S]methionine for 15 min at 37°C and chased
as described (Toth et al., 1997). Equivalent cell lysates
were then incubated with anti-MT1-MMP hemopexin domain-specific
polyclonal antisera for 16 h at 4°C, followed by incubation
with protein A-Sepharose beads (Pierce) for 5 h at 4°C.
The beads were harvested by centrifugation, the reduced samples were
resolved by 10% SDS-PAGE, and the radiolabeled proteins were
visualized by autoradiography.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
SUMMARY
REFERENCES
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MT1-MMP Processing in COS-1 Cells
COS-1 cells transfected with MT1-MMP cDNA have been reported to
generate a single immunoreactive product tentatively identified as the
pro form of the enzyme (Sato et al., 1994; Cao et
al., 1996, 1998; Zucker et al., 1998). However, after
transient transfection with wild-type MT1-MMP cDNA, the hemopexin
domain-specific polyclonal antisera detected three immunoreactive
bands at ~63, 60, and 45 kDa (Figure 2, A and B). To determine
whether the 63-kDa band detected in MT1-MMP-transfected COS-1 cells is
the pro form of the enzyme, a mutant MT1-MMP molecule was engineered
with a FLAG epitope inserted in its prodomain (Figure 2A). As shown,
both the wild-type and epitope-tagged forms of MT1-MMP were processed similarly in COS-1 cells (as determined with anti-MT1-MMP polyclonal antisera; Figure 2B). However, when extracts from epitope-tagged MT1-MMP COS cells were developed with the anti-FLAG mAb, only the
63-kDa species was detected (Figure 2B, lane 4).
With the identification of the ~63-kDa species as the MT1-MMP
zymogen, the ~60- and 45-kDa forms of MT1-MMP were noted to comigrate
with those identified in HT-1080 cells (Figure 2C, lanes 2 and 4), a
cell line previously shown to process proMT1-MMP to both an ~60-kDa
mature form and an ~45-kDa truncation product (Lohi et
al., 1996; Lehti et al., 1998; Stanton et
al., 1998). Consistent with this interpretation, a mAb directed at
the catalytic domain of MT1-MMP identified the ~63- and 60-kDa forms
of the enzyme, but not the 45-kDa fragment (Figure 2C, lanes 5-8).
Because the formation of the 45-kDa truncation product in COS-1 cells may arise from the autolytic cleavage of the mature ~60-kDa enzyme (Stanton et al., 1998), MT1-MMP-transfected cells were
incubated with the broad-spectrum MMP inhibitor BB-94. Under these
conditions, the accumulation of the 45-kDa species decreased coincident
with the accumulation of the 63- and 60-kDa products (Figure 2C,
compare lanes 2 and 3).
MT1-MMP Processing Is Regulated by a Pair of Tetrabasic Motifs in the Prodomain
Given that MT1-MMP-transfected COS-1 cells process the zymogen to
its prodomain-deleted form, we assessed the role of the tetrabasic
motif, 108RRKR, which is positioned immediately
upstream of the N terminus of the active enzyme. As described (Pei and
Weiss, 1996), transmembrane-deleted MT1-MMP (i.e., MT1-MMP) was
processed efficiently to its mature form (see Figure 1 for schemes of
mutants and Figure 3A, lanes 1-3),
whereas processing was eliminated when the
108RRKR motif was mutated to
108AAAA (i.e., MT1-MMP/A4). In contrast, a
comparison of the processing of full-length MT1-MMP and MT1-MMP/A4
demonstrated that the 108RRKR 108AAAA mutant continued to undergo partial
processing (Figure 3A, compare lanes 6 and 7). In three paired
experiments, the formation of mature MT1-MMP was inhibited by 83 ± 12% (mean ± SD) when cells were transfected with MT1-MMP/A4.
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The ability of COS-1 cells to process proMT1-MMP in the absence of the
RRKR motif indicated that alternative cleavage sites exist in the
prodomain. Indeed, although previously unrecognized, an overlapping
"triplet" of minimal proprotein convertase recognition motifs is
embedded in the 86KAMRRPR peptide as
86KXXR, 89RXXR, or
89RR (Figure 1) (Zhou et al., 1999).
To simultaneously eliminate all three sites, an
R89 A substitution was inserted in either
wild-type MT1-MMP (i.e., MT1-MMP/AXXR) or MT1-MMP/A4 (i.e.,
MT1-MMP/AXXR-A4). Significantly, whereas the MT1-MMP/AXXR mutant was
processed normally, the double mutant (i.e., MT1-MMP/AXXR-A4) remained
almost entirely in its unprocessed form (Figure 3A, lanes 8-10).
To determine the impact of blocking MT1-MMP processing on enzymic
activity, COS-1 cells were transfected with the wild-type proteinase or
mutants bearing basic motif substitutions and incubated with
progelatinase A as a target substrate (Sato et al., 1994; Pei and Weiss, 1996). As expected, MT1-MMP-transfected COS-1 cells processed the progelatinase A zymogen to its mature form (Figure 3B,
lanes 1 and 2). Under identical conditions, COS-1 cells transfected with MT1-MMP/A4 also processed progelatinase A, but less efficiently than the wild-type enzyme, as reflected in the consistent detection of
higher gelatinolytic activity in the pro form band (Figure 3B, lanes 2 and 3). However, the ability of the AXXR-A4 mutant to process
progelatinase A was almost completely inhibited, whereas the AXXR
mutant functioned normally (Figure 3B, lanes 4-6). The inability of
MT1-MMP/AXXR-A4-transfected COS-1 to process progelatinase A could not
be attributed to altered trafficking of the mutant zymogen to the cell
surface because extracellular biotinylation demonstrated an identical
presentation of each MT1-MMP construct (Figure 3B, lanes 7-10).
Although the mature form of wild-type MT1-MMP cannot be detected by
cell surface biotinylation (the 111-amino acid propeptide contains a
total of 13 Arg and/or Lys residues; Lehti et al., 1998),
these data demonstrate that surface-displayed MT1-MMP/AXXR-A4 neither
undergoes efficient processing nor displays catalytic activity.
1PDX Blocks ProMT1-MMP Processing
Four members of the proprotein convertase family, i.e., furin,
PACE4, PC6, and PC7, have been implicated in the processing of target
molecules in the constitutive secretory pathway (Zhou et
al., 1999). 1PDX is an engineered mutant
of 1 proteinase inhibitor in which the active
site loop has been altered to display an Arg-X-X-Arg motif that acts
specifically as a bait region for the proprotein convertases furin and
PC6 (Benjannet et al., 1997; Cui et al., 1998;
Jean et al., 1998). To determine if these
1PDX-sensitive convertases participate in
MT1-MMP maturation, processing was examined in COS-1 cells
cotransfected with MT1-MMP. As shown in Figure 3,
1PDX inhibited processing of either MT1-MMP
or full-length MT1-MMP (Figure 3, A, lanes 4 and 5, and C, lanes 1 and
2). Similar results were obtained when the processing of a
catalytically inactive form of MT1-MMP (i.e.,
MT1-MMPE
A) was examined
in the absence or presence of 1PDX (Figure 3C,
lanes 3-5). Finally, COS-1 cells cotransfected with MT1-MMP and
1PDX cDNAs were unable to process progelatinase A (Figure 3C, lanes 6-8) despite normal trafficking of
proMT1-MMP to the cell surface (Figure 3C, lanes 9-11).
MT1-MMP Processing and Activity in Furin-deficient Cell Lines
To determine if furin, a ubiquitously expressed proprotein
convertase, plays a predominant role in the
1PDX-sensitive maturation of proMT1-MMP, the
processing of the proteinase was examined in two furin-deficient cell
lines, LoVo and RPE.40 (Inocencio et al., 1997; Zhou
et al., 1999). Significantly, LoVo was unable to process
soluble MT1-MMP unless cotransfected with the furin cDNA (Figure
4A, lanes 1-3). To determine the ability
of LoVo cells to process full-length MT1-MMP, the cells were
transiently transfected with a C-terminal domain, epitope-tagged
zymogen (i.e., MT1-MMP/FLAG), because LoVo cell extracts contained an
immunoreactive product consistent with proMT1-MMP (our unpublished
results). As observed for soluble MT1-MMP, full-length MT1-MMP was
inefficiently processed unless the cells were cotransfected with a
furin cDNA (Figure 4A, lanes 4-6). The ability of furin to mediate
MT1-MMP processing was reversed when LoVo cells were cotransfected with the furin and 1PDX cDNAs (Figure 4A, compare
lanes 6 and 7). Although furin was unable to enhance MT1-MMP processing
in cotransfected COS-1 cells (Figure 4B, compare lanes 1 and 2),
proMT1-MMP displayed on the cell surface could be cleaved after
exposure to soluble furin (Figure 4B, lanes 3-6).
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Furin-deficient LoVo cells were unable to efficiently process
wild-type MT1-MMP, but a role for alternative convertases would depend
on the repertoire of processing enzymes expressed by the host cell.
Consequently, MT1-MMP processing was also examined in the wild-type
cell line CHO-K1 and its furin-deficient strain RPE.40 (Inocencio
et al., 1997) (Figure 5). As
expected, both soluble MT1-MMP (Figure 5A, lanes 1-3) and wild-type
MT1-MMP (lanes 7-10) were processed to their mature forms via an
1PDX-inhibitable process in CHO-K1 cells.
Furthermore, furin-deficient RPE.40 cells were unable to process
MT1-MMP unless cotransfected with a furin cDNA (Figure 5A, lanes
4-6). However, in contrast to LoVo cells, RPE.40 cells processed
full-length MT1-MMP as efficiently as the furin-sufficient CHO-K1 cell
line (Figure 5A, compare lanes 8 and 12). In three paired experiments,
MT1-MMP processing in RPE.40 cells was 82 ± 6% (mean ± SD)
of that observed in CHO-K1 cells.
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To determine whether the furin-independent MT1-MMP processing pathway
operative in RPE.40 cells was dependent on the basic motifs identified
previously in the prodomain, the cells were transfected with the
MT1-MMP/A4, MT1-MMP/AXXR, or MT1-MMP/AXXR-A4 mutant. Compared with
CHO-K1 cells, in which MT1-MMP/A4 processing was 16% of control values
relative to wild-type MT1-MMP processing (n = 2), the
furin-deficient RPE.40 cells were more efficient in processing the A4
mutant (i.e., 35% of control relative to wild-type MT1-MMP [n = 2]; Figure 5A, lanes 15 and 16). However, although the AXXR mutant was
processed comparably to the wild-type enzyme in RPE.40 cells (lane 17),
the AXXR/A4 mutant remained entirely in its pro form when expressed in
the furin-deficient cell line (lane 18; 98% inhibition of processing
relative to wild-type MT1-MMP in RPE.40 cells versus 100% inhibition
in CHO-K1 cells [n = 2]). Because these data are consistent with
a model wherein furin-independent cleavage occurred primarily
downstream of 108RRKR in RPE.40 cells, an MT1-MMP
construct was engineered with a FLAG epitope inserted at the C-terminal
end of the basic motif (i.e., 108RRKR-FLAG).
Transfected cells were then stained with a mAb (anti-FLAG M1) that
specifically recognizes FLAG-tagged proteins only when the epitope is
located at the extreme N terminus of the protein (Itoh et
al., 1999). As shown in Figure 6,
anti-FLAG M1 reacted with furin-sufficient CHO-K1 cells as well as
furin-deficient RPE.40 cells transfected with the
108RRKR-FLAG construct (A-D). Consistent with
the specificity of the antibody, when the FLAG epitope was inserted
downstream of the MT1-MMP/A4 mutant (i.e.,
108AAAA-FLAG), the M1 antibody did not stain
either transfected CHO-K1 or RPE.40 cells (E and F). Negative staining
of the 108AAAA-FLAG construct could not be
attributed to either inefficient expression or surface localization
because MT1-MMP was readily recognized by a mAb (3H7; Chenard et
al., 1999) directed against the extracellular domain of the
proteinase (I and J). Thus, as in furin-sufficient cells, MT1-MMP is
processed by furin-deficient cells after cleavage on the C-terminal
side of the 108RRKR motif.
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Given the selection of the 108RRKR motif as the
primary cleavage site in furin-deficient RPE.40 cells, a potential role
for other proprotein convertases in the processing event was assessed
by cotransfecting these cells with wild-type MT1-MMP and
1PDX cDNAs. As shown in Figure 5A,
1PDX almost completely inhibited MT1-MMP processing to its mature form in RPE.40 cells (compare lanes 13 and
14). In three experiments, 1PDX inhibited
MT1-MMP processing by 92 ± 7% in RPE.40 cells and by 99 ± 2% in CHO-K1 cells (mean ± SD). Because MT1-MMP processing would
be expected to progress until the 1PDX
concentration increased to a level sufficient to inhibit targeted
proprotein convertases, the 45-kDa truncation product of MT1-MMP was
detected occasionally (Figure 5A, lane 14).
The inability of RPE.40 cells to process soluble MT1-MMP while
maintaining the ability to mediate the activation of the full-length proteinase raised the possibility that differential processing is
regulated by membrane anchoring per se and/or by signals embedded in
the MT1-MMP cytosolic tail or transmembrane domains (Nakahara et
al., 1997; Fernandez-Larrea et al., 1999; Urena
et al., 1999). Thus, the processing of a cytosol
tail-deleted form of MT1-MMP or a chimeric MT1-MMP molecule containing
the transmembrane domain and tail of the interleukin 2 receptor was
examined after transfection into CHO-K1 and furin-deficient RPE.40
cells. Significantly, the cytosolic tail-deleted mutant was processed
comparably to the full-length enzyme in both the CHO-K1 and RPE.40 cell
strains (Figure 5B). The transmembrane-substitution mutant was
processed less efficiently than wild-type MT1-MMP, but both cell
strains generated the mature form of the enzyme comparably (Figure 5B, lanes 5 and 6). Thus, membrane anchoring alone dictated the selection of a furin-independent pathway for the processing of MT1-MMP.
Because CHO-K1 cells processed full-length MT1-MMP by either
furin-dependent or furin-independent pathways, the ability of 1PDX to regulate cell-associated proteolytic
activity in this more complex system was assessed. As shown in Figure
5C, the activation of progelatinase A by either MT1-MMP-transfected
CHO-K1 or RPE.40 cells could be blocked almost completely by
1PDX. Finally, because the processing of
progelatinase A only indirectly monitors MT1-MMP activity (Nagase and
Woessner, 1999), the ability of adherent MT1-MMP-transfected cells to
degrade a fluorescently labeled subjacent substrate (i.e., gelatin) was
determined in the absence or presence of 1PDX.
As shown in Figure 7,
MT1-MMP-transfected, but not control, CHO-K1 cells proteolyzed Texas
red-labeled gelatin as they migrated across the coated surface,
leaving dark chemokinetic "tracks" (A-D). In contrast, CHO-K1
cells cotransfected with MT1-MMP and 1PDX were
unable to express proteolytic activity (E and F).
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Proprotein Convertase-dependent Processing of MT1-MMP in HT-1080 Cells
To determine whether endogenously expressed MT1-MMP is
similarly processed by proprotein convertases, metalloproteinase
activation was examined in HT-1080 cells. HT-1080 cells constitutively
process proMT1-MMP on the C-terminal side of
108RRKR to generate mature
112Y-MT1-MMP (Strongin et al., 1995;
Lehti et al., 1998). Consistent with these data, pulse-chase
analysis of MT1-MMP processing in HT-1080 cells demonstrated that the
mature enzyme could be detected within 15 min (Figure
8A). Although epitope-tagged MT1-MMP was also processed to the mature 60-kDa proteinase, both the A4 mutant and
the AXXR-A4 mutant accumulated as their respective pro forms (Figure
8B, lanes 2, 4, and 5). Because other serine proteinases have been
posited to participate in MT1-MMP processing (e.g., trypsin, plasmin,
and urokinase; Will et al., 1996; Okumura et al.,
1997; Kazes et al., 1998), the role of proprotein
convertases was finally assessed by cotransfecting HT-1080 cells with
MT1-MMP/FLAG and 1PDX. Significantly, in the
presence of 1PDX, the mature form of
epitope-tagged MT1-MMP was no longer detected (Figure 8B, lane 6).
MT1-MMP processing was unaffected by serine (aprotinin, soybean trypsin
inhibitor), cysteine (E-64), or aspartate (pepstatin A) proteinase
inhibitors (our unpublished results). Based on the substrate
specificity of 1PDX (Cui et al.,
1998; Jean et al., 1998), we conclude that furin and/or PC6
regulate the processing of endogenously derived MT1-MMP.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
SUMMARY
REFERENCES
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MT1-MMP has been implicated in the remodeling of the extracellular
matrix in events ranging from growth and development to wound healing
and tumor invasion (Okada et al., 1997; Hiraoka et
al., 1998; Belien et al., 1999; Holmbeck et
al., 1999; Zhou et al., 2000). Like other members of
the MMP family, proMT1-MMP is organized into discrete domains,
including a propeptide region that contains a canonical PRCG(V/N)PD
sequence (Nagase and Woessner, 1999). In cooperation with other
portions of the prodomain, the cysteinyl residue in this peptide is
proposed to interact with the Zn2+ atom of the
catalytic site to act as a "lock" to maintain proteinase latency
(Nagase and Woessner, 1999). Given the ability of MT1-MMP to process
other MMP zymogens to their active forms (e.g., progelatinase A and
procollagenase-3), to directly cleave a range of extracellular matrix
molecules (Pei and Weiss, 1996; Ohuchi et al., 1997), and to
regulate collagen turnover in vivo (Holmbeck et al., 1999; Zhou et al., 2000), increased attention has focused on the
means by which proMT1-MMP is itself converted to a catalytically active species (Strongin et al., 1995; Cao et al., 1996,
1998; Zucker et al., 1998).
Until recently, MMPs were thought to undergo activation extracellularly
after either oxidative or proteolytic perturbation of propeptide
domain-catalytic domain interactions (Weiss et al., 1985;
Nagase and Woessner, 1999). However, a precedent for intracellular processing was established when the stromelysin-3 zymogen was reported
to undergo constitutive activation (Pei and Weiss, 1995; Santavicca
et al., 1996). Unlike other secreted MMPs, stromelysin-3 was
cleaved immediately downstream of a tetrabasic motif (RXRXKR) that
served as a recognition site for furin. With the subsequent discovery
of RX(K/R)R sequences at the predicted C-terminal ends of the
prodomains of MT1-, MT2-, MT3-, MT4-, and MT5-MMP (Nagase and Woessner,
1999; Pei, 1999), proprotein convertases have been posited to play a
more prominent role in regulating MMP activation.
Processing of the MT1-MMP Zymogen
The detection of a single MT1-MMP species whose molecular
mass appears consistent with that predicted for the proenzyme has been
widely described in COS cells as well as in endothelial cells and tumor
cell lines (Sato et al., 1994; Cao et al., 1996,
1998; Rajavashisth et al., 1999). However, the detected
species was assumed to represent the pro form of MT1-MMP based solely
on the predicted molecular mass for the unprocessed zymogen. Because mature MT1-MMP migrates anomalously relative to the migration expected from the loss of the 111-amino acid propeptide, positive identification of an immunoreactive band as either proMT1-MMP or mature
MT1-MMP can only be made with prodomain-specific antibodies. In our
study, MT1-MMP transfectants expressed at least three distinct products
(we also note that a glycosylated pro form of MT1-MMP tended to
accumulate under those conditions in which proteinase processing was
inhibited [Maquoi et al., 1998]). By epitope tagging of
the prodomain, COS-1 cells were shown to process the MT1-MMP zymogen to
two propeptide-deleted forms. Because only the 60-kDa species was
recognized by a mAb directed against the catalytic domain, the smaller
product represents an inactive product of the mature enzyme (Lehti
et al., 1998; Stanton et al., 1998). However,
because the enzymically inactive 45-kDa species was formed 1) in the
presence of a broad-spectrum MMP inhibitor, 2) after transfection with
a catalytically inactive form of MT1-MMP, or 3) in the presence of
1PDX, routes other than the autocatalytic degradation of mature MT1-MMP are operative under these conditions.
A Role for Multibasic Motifs in MT1-MMP Processing
Based on earlier studies with stromelysin-3 (Pei and Weiss, 1995),
the 108RRKR domain in full-length MT1-MMP was
predicted to play a critical role in the activation process by acting
as a recognition motif for proprotein convertases. Indeed, in the
HT-1080 cell line, two groups have identified the N terminus of the
60-kDa mature form of MT1-MMP as Y112, which lies
immediately downstream of the 108RRKR sequence
(Strongin et al., 1995; Lehti et al., 1998).
However, whereas the processing of the 108RRKR
AAAA mutant of soluble MT1-MMP was blocked completely in
transiently transfected COS cells, the A4 mutant of the full-length enzyme continued to undergo processing. Thus, despite an unequivocal role for the tetrabasic motif in regulating the maturation of the
soluble mutant form of the enzyme, the processing of the full-length proteinase proceeded in a divergent manner.
Given that proprotein convertase can recognize alternative motifs
within a framework (Arg/Lys)-(X)n-(Arg-Lys) (Zhou
et al., 1999) wherein n = 0, 2, 4, or 6 residues, we
noted that MT1-MMP displays an overlapping tandem of potential cleavage
sites 22 amino acid residues upstream of the
108RRKR site at 86KXXRRXR
(i.e., 86KXXR, 89RRXR, and
89RR). To eliminate these cleavage sites, an
89R A mutation was inserted into either
wild-type MT1-MMP or the A4 mutant. Although the
89R A mutant of full-length MT1-MMP was
processed normally, the partial processing of the A4 mutant was
completely inhibited when the putative alternative sites within the
86KXXRRXR motif were eliminated. In agreement
with these findings, the inability of the proMT1-MMP double mutant to
undergo processing correlated with the enzyme's inability to activate
progelatinase A. Although we have been unable to recover processed
MT1-MMP from transiently transfected cells in sufficient quantities for
microsequencing, together these data support a model wherein wild-type
MT1-MMP is processed primarily, if not solely, at the
108RRKR
site to generate the active
proteinase. Given that the secondary site within
86KXXRRXR was apparently used only when the
primary 108RRKR motif was mutated, a
physiological role for the alternative site has not been demonstrated
in transfected cells. Nonetheless, primary and secondary cleavage sites
for proprotein convertases have been posited in other target molecules,
including Notch, bone morphogenic protein-4, and insulin growth
factor-1 (Duguay et al., 1997; Cui et al., 1998;
Logeat et al., 1998).
Proprotein Convertases as Processing Enzymes for MT1-MMP
The ability of basic motif mutations within the MT1-MMP prodomain
to interfere with processing is consistent with a role for any
proteinase that cleaves target molecules on the C-terminal side of the
RRKR sequence. Indeed, others have proposed roles for plasmin, trypsin,
and urokinase in MT1-MMP processing at or near the C-terminal end of
the RRKR motif (Will et al., 1996; Okumura et
al., 1997; Kazes et al., 1998). However, proteinase inhibitors directed against these enzymes did not affect MT1-MMP maturation or activity. Instead, MT1-MMP processing was blocked by the
mutant 1 proteinase inhibitor
1PDX, which specifically inactivates furin and
PC6 (Cui et al., 1998; Jean et al., 1998) (although higher concentrations of 1PDX may
inhibit PACE4; Benjannet et al., 1997). Significantly,
1PDX inhibited both the processing of MT1-MMP
to its mature form and the enzyme's proteolytic activity against
either progelatinase A or a subjacent matrix of denatured collagen.
Although we note that the inactive 45-kDa form of MT1-MMP was
occasionally formed under these conditions, MT1-MMP proteolytic activity was nonetheless completely inhibited.
These findings are in contrast to those of Zucker and colleagues, who
reported proMT1-MMP to act as a fully functional enzyme (Cao et
al., 1996, 1998; Zucker et al., 1998). However, their conclusions were predicated, in part, on the following assumptions: 1)
that COS-1 cells do not process the MT1-MMP zymogen; 2) that 108RRKR mutations would completely prevent
MT1-MMP processing; and 3) that MT1-MMP processing would be blocked by
the furin inhibitor 1PIPITT. Based on our
findings that COS-1 cells can process wild-type MT1-MMP as well as its
108RRKR mutant, coupled with the recent
demonstration that
1PIPITT is unable to
efficiently inhibit proprotein convertase-dependent pathways
(Vollenweider et al., 1996; Cui et al., 1998), we
propose that the removal of the MT1-MMP prodomain is a prerequisite for the efficient display of proteolytic activity. Interestingly, although
partial blockade of proMT1-MMP processing was reported recently with a
chloromethylketone inhibitor (Maquoi et al., 1998; Kurschat
et al., 1999), this agent is not specific for proprotein convertases and also exerts cytotoxic effects (Hallenberger et al., 1992; Okumura et al., 1997; Jean et
al., 1998).
Inhibition of MT1-MMP processing by 1PDX alone
does not allow one to definitively identify the target proprotein
convertase(s). However, furin-deficient cell lines have proven to be a
useful adjunct for discriminating between furin-dependent and
furin-independent processing pathways (Pei and Weiss, 1995; Duguay
et al., 1997; Inocencio et al., 1997; Logeat
et al., 1998). As expected, neither of the
well-characterized furin-deficient cell lines, LoVo or RPE.40,
processed soluble MT1-MMP to its mature form. LoVo cells have been
reported to express PACE4 as well as PC7, and this furin-deficient cell
line can process other RXKR-encrypted targets including human immunodeficiency virus-1, E-cadherin and gp-160 (Gu et al.,
1995; Decroly et al., 1997; Zarkik et al., 1997;
Posthaus et al., 1998). However, neither PACE4 nor PC7
appeared to play a major role in the processing of MT1-MMP in LoVo
cells. In turn, a role for furin as the major processing enzyme for
MT1-MMP was further buttressed by the ability of furin-reconstituted
LoVo cells to efficiently process MT1-MMP and the ability of
extracellular soluble furin to cleave proMT1-MMP displayed on the cell surface.
The processing of proMT1-MMP by furin is consistent with the ability of
this proprotein convertase to recognize RX(K/R)R, RXXR, KXXR, or RR
motifs (Duguay et al., 1997; Nakayama, 1997; Cui et
al., 1998; Zhou et al., 1999). Unexpectedly, however,
MT1-MMP was processed after cleavage immediately downstream of the
108RRKR motif in the furin-deficient RPE.40 cell
line by a predominately 1PDX-sensitive
process. These findings demonstrate not only that furin-independent
maturation pathways exist but also that soluble and membrane-anchored
MT1-MMP were processed in distinct fashions. The
1PDX-inhibitable proprotein convertase
responsible for wild-type MT1-MMP processing has not been identified.
However, in contrast to LoVo cells, RPE.40 cells do express PC6 (as
both the transmembrane-anchored and soluble forms, PC6B and P6CA,
respectively; Duguay et al., 1997). Interestingly, PC6, like
furin, can process RXKR-, RXXR-, and KXXR-containing molecules (Zarkik
et al., 1997; Cui et al., 1998). Furthermore,
because PC6 cannot be detected in LoVo cells (Vollenweider et
al., 1996; Logeat et al., 1998), differences in the
MT1-MMP processing pathways displayed between LoVo and RPE.40 cells are
likely attributed to each cell line's distinct repertoire of
processing enzymes. Whatever the relative roles of furin and PC6 in
MT1-MMP processing, we note that 1PDX did not
completely inhibit MT1-MMP maturation in RPE.40 or LoVo cells. Additional studies are needed to determine the role of PC7, an 1PDX-resistant proprotein convertase
(Benjannet et al., 1997; Jean et al., 1998), as
well as proprotein convertase-independent systems in MT1-MMP processing.
The ability of furin-deficient RPE.40 cells to process
membrane-anchored, but not soluble, MT1-MMP by an
1PDX-sensitive process illustrates the
complexities associated with attempts to extrapolate results obtained
with transmembrane-deleted mutants to membrane-anchored wild-type
proteins. Along these lines, recent studies have suggested that the
cytosolic tail of MT1-MMP may contain signals critical for membrane
trafficking (Nakahara et al., 1997; Fernandez-Larrea et al., 1999; Urena et al., 1999). Nonetheless,
cytosolic tail-deletion mutants as well as transmembrane domain
"swaps" failed to reveal roles for either domain. These results are
consistent with studies demonstrating the ability of cells transfected
with cytosolic domain-deleted MT1-MMP to express heightened invasive
activity (Hiraoka et al., 1998; Hotary et al.,
2000). We conclude that membrane association alone may increase the
residence time of proMT1-MMP at cell surfaces where the likelihood of
its cleavage by proprotein convertases is enhanced. Although the
cellular sites at which MT1-MMP processing takes place have not been
defined, both furin and PC6 can be expressed as membrane-anchored
enzymes that potentially could cleave MT1-MMP intracellularly or at the cell surface (Malloy et al., 1994).
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SUMMARY |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
SUMMARY
REFERENCES
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Previous efforts to identify MMP activation schemes have focused
largely on the role of the plasminogen activator-plasminogen system
(Nagase and Woessner, 1999). However, a link between these systems is
based primarily on in vitro studies, whereas in vivo studies in
plasminogen activator- or plasminogen-deleted animals clearly support
the existence of plasmin-independent activation cascades (Hiraoka
et al., 1998; Lund et al., 1999; Hotary et
al., 2000). Significantly, RXKR motifs are found in at least six
other human MMPs (i.e., MT2-, MT3-, MT4-, and MT5-MMP, stromelysin-3, and MMP-23) (Nagase et al., 1999; Pei, 1999). Furthermore,
RXXR and KXXR motifs can be identified in the prodomains of multiple MMPs (Figure 9), and similar processing
cascades may apply to the structurally related
metalloprotease-disintegrin family (Schlondorff and Blobel,
1999). Along these lines, it is noteworthy that preliminary reports
indicate that progelatinase A can be processed directly to its active
form after furin-dependent cleavage at 69RQPR
(Cao et al., 1999). Because proprotein convertases can
process target molecules in both intracellular and extracellular
compartments (Malloy et al., 1994; Zhou et al.,
1999), the proprotein convertase-metalloproteinase axis may play a
major role in regulating complex arrays of proteolytic activities in
vivo.
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ACKNOWLEDGMENTS |
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We thank Drs. B. Donohoe, E. Allen, and K. Hotary for help with the confocal laser microscopy and J. Lowe (Howard Hughes Medical Institute, University of Michigan), M. Seiki, and Y. Itoh (both University of Tokyo) for helpful discussions. The work was supported by the National Institutes of Health (5R01 CA71699) and Novartis.
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FOOTNOTES |
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* Corresponding author. E-mail address: sjweiss{at}umich.edu.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
SUMMARY
REFERENCES
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1-antitrypsin Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway.
J. Biol. Chem.
272, 26210-26218 to the cell surface.
Mol. Cell
3, 423-433[Medline].1-antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agent.
Proc. Natl. Acad. Sci. USA
95, 7293-7298 and membrane type matrix metalloprotease (M.T1-M.M.P).
J. Cell Sci.
112, 773-784[Abstract].This article has been cited by other articles:
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S. McMahon, F. Grondin, P. P. McDonald, D. E. Richard, and C. M. Dubois Hypoxia-enhanced Expression of the Proprotein Convertase Furin Is Mediated by Hypoxia-inducible Factor-1: IMPACT ON THE BIOACTIVATION OF PROPROTEINS J. Biol. Chem., February 25, 2005; 280(8): 6561 - 6569. [Abstract] [Full Text] [PDF]
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P. Ottino, J. He, T. W. Axelrad, and H. E. P. Bazan PAF-Induced Furin and MT1-MMP Expression Is Independent of MMP-2 Activation in Corneal Myofibroblasts Invest. Ophthalmol. Vis. Sci., February 1, 2005; 46(2): 487 - 496. [Abstract] [Full Text] [PDF]
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 B. Birsoy, L. Berg, P. H. Williams, J. C. Smith, C. C. Wylie, J. L. Christian, and J. Heasman XPACE4 is a localized pro-protein convertase required for mesoderm induction and the cleavage of specific TGF{beta} proteins in Xenopus development Development, February 1, 2005; 132(3): 591 - 602. [Abstract] [Full Text] [PDF]
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M. Mazzone, M. Baldassarre, G. Beznoussenko, G. Giacchetti, J. Cao, S. Zucker, A. Luini, and R. Buccione Intracellular processing and activation of membrane type 1 matrix metalloprotease depends on its partitioning into lipid domains J. Cell Sci., December 15, 2004; 117(26): 6275 - 6287. [Abstract] [Full Text] [PDF]
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 T.-H. Chun, F. Sabeh, I. Ota, H. Murphy, K. T. McDonagh, K. Holmbeck, H. Birkedal-Hansen, E. D. Allen, and S. J. Weiss MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix J. Cell Biol., November 22, 2004; 167(4): 757 - 767. [Abstract] [Full Text] [PDF]
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H. G. Munshi, Y. I. Wu, S. Mukhopadhyay, A. J. Ottaviano, A. Sassano, J. E. Koblinski, L. C. Platanias, and M. S. Stack Differential Regulation of Membrane Type 1-Matrix Metalloproteinase Activity by ERK 1/2- and p38 MAPK-modulated Tissue Inhibitor of Metalloproteinases 2 Expression Controls Transforming Growth Factor-{beta}1-induced Pericellular Collagenolysis J. Biol. Chem., September 10, 2004; 279(37): 39042 - 39050. [Abstract] [Full Text] [PDF]
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J.-M. Longpre and R. Leduc Identification of Prodomain Determinants Involved in ADAMTS-1 Biosynthesis J. Biol. Chem., August 6, 2004; 279(32): 33237 - 33245. [Abstract] [Full Text] [PDF]
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 R. L. de Cicco, J. C. Watson, D. E. Bassi, S. Litwin, and A. J. Klein-Szanto Simultaneous Expression of Furin and Vascular Endothelial Growth Factor in Human Oral Tongue Squamous Cell Carcinoma Progression Clin. Cancer Res., July 1, 2004; 10(13): 4480 - 4488. [Abstract] [Full Text] [PDF]
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 P. Stawowy, C. Margeta, H. Kallisch, N. G Seidah, M. Chretien, E. Fleck, and K. Graf Regulation of matrix metalloproteinase MT1-MMP/MMP-2 in cardiac fibroblasts by TGF-{beta}1 involves furin-convertase Cardiovasc Res, July 1, 2004; 63(1): 87 - 97. [Abstract] [Full Text] [PDF]
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P. Wang, M. Tortorella, K. England, A.-M. Malfait, G. Thomas, E. C. Arner, and D. Pei Proprotein Convertase Furin Interacts with and Cleaves Pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi Network J. Biol. Chem., April 9, 2004; 279(15): 15434 - 15440. [Abstract] [Full Text] [PDF]
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Y. I. Wu, H. G. Munshi, R. Sen, S. J. Snipas, G. S. Salvesen, R. Fridman, and M. S. Stack Glycosylation Broadens the Substrate Profile of Membrane Type 1 Matrix Metalloproteinase J. Biol. Chem., February 27, 2004; 279(9): 8278 - 8289. [Abstract] [Full Text] [PDF]
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P. Stawowy, H. Kallisch, J. P. Veinot, A. Kilimnik, W. Prichett, S. Goetze, N. G. Seidah, M. Chretien, E. Fleck, and K. Graf Endoproteolytic Activation of {alpha}v Integrin by Proprotein Convertase PC5 Is Required for Vascular Smooth Muscle Cell Adhesion to Vitronectin and Integrin-Dependent Signaling Circulation, February 17, 2004; 109(6): 770 - 776. [Abstract] [Full Text] [PDF]
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H. Nakamura, N. Suenaga, K. Taniwaki, H. Matsuki, K. Yonezawa, M. Fujii, Y. Okada, and M. Seiki Constitutive and Induced CD44 Shedding by ADAM-Like Proteases and Membrane-Type 1 Matrix Metalloproteinase Cancer Res., February 1, 2004; 64(3): 876 - 882. [Abstract] [Full Text] [PDF]
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T. Takino, H. Miyamori, Y. Watanabe, K. Yoshioka, M. Seiki, and H. Sato Membrane Type 1 Matrix Metalloproteinase Regulates Collagen-Dependent Mitogen-Activated Protein/Extracellular Signal-Related Kinase Activation and Cell Migration Cancer Res., February 1, 2004; 64(3): 1044 - 1049. [Abstract] [Full Text] [PDF]
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E. M. Majerus, X. Zheng, E. A. Tuley, and J. E. Sadler Cleavage of the ADAMTS13 Propeptide Is Not Required for Protease Activity J. Biol. Chem., November 21, 2003; 278(47): 46643 - 46648. [Abstract] [Full Text] [PDF]
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C. Guo and L. Piacentini Type I Collagen-induced MMP-2 Activation Coincides with Up-regulation of Membrane Type 1-Matrix Metalloproteinase and TIMP-2 in Cardiac Fibroblasts J. Biol. Chem., November 21, 2003; 278(47): 46699 - 46708. [Abstract] [Full Text] [PDF]
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A. Jiang and D. Pei Distinct Roles of Catalytic and Pexin-like Domains in Membrane-type Matrix Metalloproteinase (MMP)-mediated Pro-MMP-2 Activation and Collagenolysis J. Biol. Chem., October 3, 2003; 278(40): 38765 - 38771. [Abstract] [Full Text] [PDF]
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A. Remacle, G. Murphy, and C. Roghi Membrane type I-matrix metalloproteinase (MT1-MMP) is internalised by two different pathways and is recycled to the cell surface J. Cell Sci., October 1, 2003; 116(19): 3905 - 3916. [Abstract] [Full Text] [PDF]
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A. Borroto, S. Ruiz-Paz, T. V. de la Torre, M. Borrell-Pages, A. Merlos-Suarez, A. Pandiella, C. P. Blobel, J. Baselga, and J. Arribas Impaired Trafficking and Activation of Tumor Necrosis Factor-{alpha}-converting Enzyme in Cell Mutants Defective in Protein Ectodomain Shedding J. Biol. Chem., July 3, 2003; 278(28): 25933 - 25939. [Abstract] [Full Text] [PDF]
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M. Leighton and K. E. Kadler Paired Basic/Furin-like Proprotein Convertase Cleavage of Pro-BMP-1 in the trans-Golgi Network J. Biol. Chem., May 9, 2003; 278(20): 18478 - 18484. [Abstract] [Full Text] [PDF]
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G. Mayer, G. Boileau, and M. Bendayan Furin interacts with proMT1-MMP and integrin {alpha}V at specialized domains of renal cell plasma membrane J. Cell Sci., May 1, 2003; 116(9): 1763 - 1773. [Abstract] [Full Text] [PDF]
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D. V. Rozanov and A. Y. Strongin Membrane Type-1 Matrix Metalloproteinase Functions as a Proprotein Self-convertase. EXPRESSION OF THE LATENT ZYMOGEN IN PICHIA PASTORIS, AUTOLYTIC ACTIVATION, AND THE PEPTIDE SEQUENCE OF THE CLEAVAGE FORMS J. Biol. Chem., February 28, 2003; 278(10): 8257 - 8260. [Abstract] [Full Text] [PDF]
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 D. E. Bassi, H. Mahloogi, R. Lopez De Cicco, and A. Klein-Szanto Increased Furin Activity Enhances the Malignant Phenotype of Human Head and Neck Cancer Cells Am. J. Pathol., February 1, 2003; 162(2): 439 - 447. [Abstract] [Full Text] [PDF]
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H. G. Munshi, Y. I. Wu, E. V. Ariztia, and M. S. Stack Calcium Regulation of Matrix Metalloproteinase-mediated Migration in Oral Squamous Cell Carcinoma Cells J. Biol. Chem., October 25, 2002; 277(44): 41480 - 41488. [Abstract] [Full Text] [PDF]
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E. M. Tam, Y. I. Wu, G. S. Butler, M. S. Stack, and C. M. Overall Collagen Binding Properties of the Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Hemopexin C Domain. THE ECTODOMAIN OF THE 44-kDa AUTOCATALYTIC PRODUCT OF MT1-MMP INHIBITS CELL INVASION BY DISRUPTING NATIVE TYPE I COLLAGEN CLEAVAGE J. Biol. Chem., October 4, 2002; 277(41): 39005 - 39014. [Abstract] [Full Text] [PDF]
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T. Kang, Y.-G. Zhao, D. Pei, J. F. Sucic, and Q.-X. A. Sang Intracellular Activation of Human Adamalysin 19/Disintegrin and Metalloproteinase 19 by Furin Occurs via One of the Two Consecutive Recognition Sites J. Biol. Chem., July 5, 2002; 277(28): 25583 - 25591. [Abstract] [Full Text] [PDF]
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J. Mercapide, R. Lopez De Cicco, D. E. Bassi, J. S. Castresana, G. Thomas, and A. J. P. Klein-Szanto Inhibition of Furin-mediated Processing Results in Suppression of Astrocytoma Cell Growth and Invasiveness Clin. Cancer Res., June 1, 2002; 8(6): 1740 - 1746. [Abstract] [Full Text] [PDF]
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A.-M. Khatib, G. Siegfried, M. Chretien, P. Metrakos, and N. G. Seidah Proprotein Convertases in Tumor Progression and Malignancy : Novel Targets in Cancer Therapy Am. J. Pathol., June 1, 2002; 160(6): 1921 - 1935. [Abstract] [Full Text] [PDF]
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 H. Mahloogi, D. E. Bassi, and A. J.P. Klein-Szanto Malignant conversion of non-tumorigenic murine skin keratinocytes overexpressing PACE4 Carcinogenesis, April 1, 2002; 23(4): 565 - 572. [Abstract] [Full Text] [PDF]
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G. Croissandeau, A. Basak, N. G. Seidah, M. Chretien, and M. Mbikay Proprotein convertases are important mediators of the adipocyte differentiation of mouse 3T3-L1 cells J. Cell Sci., March 15, 2002; 115(6): 1203 - 1211. [Abstract] [Full Text] [PDF]
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E. I. Deryugina, B. I. Ratnikov, T. I. Postnova, D. V. Rozanov, and A. Y. Strongin Processing of Integrin alpha v Subunit by Membrane Type 1 Matrix Metalloproteinase Stimulates Migration of Breast Carcinoma Cells on Vitronectin and Enhances Tyrosine Phosphorylation of Focal Adhesion Kinase J. Biol. Chem., March 15, 2002; 277(12): 9749 - 9756. [Abstract] [Full Text] [PDF]
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K. Lehti, J. Lohi, M. M. Juntunen, D. Pei, and J. Keski-Oja Oligomerization through Hemopexin and Cytoplasmic Domains Regulates the Activity and Turnover of Membrane-type 1 Matrix Metalloproteinase J. Biol. Chem., March 1, 2002; 277(10): 8440 - 8448. [Abstract] [Full Text] [PDF]
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T. Kang, H. Nagase, and D. Pei Activation of Membrane-type Matrix Metalloproteinase 3 Zymogen by the Proprotein Convertase Furin in the trans-Golgi Network Cancer Res., February 1, 2002; 62(3): 675 - 681. [Abstract] [Full Text] [PDF]
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 P. Stawowy, F. Blaschke, A. Kilimnik, S. Goetze, H. Kallisch, M. Chretien, M. Marcinkiewicz, E. Fleck, and K. Graf Proprotein Convertase PC5 Regulation by PDGF-BB Involves PI3-Kinase/p70s6-Kinase Activation in Vascular Smooth Muscle Cells Hypertension, February 1, 2002; 39(2): 399 - 404. [Abstract] [Full Text] [PDF]
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 K. B. Hotary, I. Yana, F. Sabeh, X.-Y. Li, K. Holmbeck, H. Birkedal-Hansen, E. D. Allen, N. Hiraoka, and S. J. Weiss Matrix Metalloproteinases (MMPs) Regulate Fibrin-invasive Activity via MT1-MMP-dependent and -independent Processes J. Exp. Med., January 28, 2002; 195(3): 295 - 308. [Abstract] [Full Text] [PDF]
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 K. Drbal, P. Angelisova, I. Hilgert, J. Cerny, P. Novak, and V. Horejsi A proteolytically truncated form of free CD18, the common chain of leukocyte integrins, as a novel marker of activated myeloid cells Blood, September 1, 2001; 98(5): 1561 - 1566. [Abstract] [Full Text] [PDF]
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S. Aznavoorian, B. A. Moore, L. D. Alexander-Lister, S. L. Hallit, L. J. Windsor, and J. A. Engler Membrane Type I-Matrix Metalloproteinase-Mediated Degradation of Type I Collagen by Oral Squamous Cell Carcinoma Cells Cancer Res., August 1, 2001; 61(16): 6264 - 6275. [Abstract] [Full Text] [PDF]
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M. Kajita, Y. Itoh, T. Chiba, H. Mori, A. Okada, H. Kinoh, and M. Seiki Membrane-Type 1 Matrix Metalloproteinase Cleaves Cd44 and Promotes Cell Migration J. Cell Biol., May 28, 2001; 153(5): 893 - 904. [Abstract] [Full Text] [PDF]
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D. V. Rozanov, E. I. Deryugina, B. I. Ratnikov, E. Z. Monosov, G. N. Marchenko, J. P. Quigley, and A. Y. Strongin Mutation Analysis of Membrane Type-1 Matrix Metalloproteinase (MT1-MMP). THE ROLE OF THE CYTOPLASMIC TAIL CYS574, THE ACTIVE SITE GLU240, AND FURIN CLEAVAGE MOTIFS IN OLIGOMERIZATION, PROCESSING, AND SELF-PROTEOLYSIS OF MT1-MMP EXPRESSED IN BREAST CARCINOMA CELLS J. Biol. Chem., July 6, 2001; 276(28): 25705 - 25714. [Abstract] [Full Text] [PDF]
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A.-M. Khatib, G. Siegfried, A. Prat, J. Luis, M. Chretien, P. Metrakos, and N. G. Seidah Inhibition of Proprotein Convertases Is Associated with Loss of Growth and Tumorigenicity of HT-29 Human Colon Carcinoma Cells. IMPORTANCE OF INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) RECEPTOR PROCESSING IN IGF-1-MEDIATED FUNCTIONS J. Biol. Chem., August 10, 2001; 276(33): 30686 - 30693. [Abstract] [Full Text] [PDF]
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X. Wang and D. Pei Shedding of Membrane Type Matrix Metalloproteinase 5 by a Furin-type Convertase. A POTENTIAL MECHANISM FOR DOWN-REGULATION J. Biol. Chem., September 14, 2001; 276(38): 35953 - 35960. [Abstract] [Full Text] [PDF]
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M. Fugere, P. C. Limperis, V. Beaulieu-Audy, F. Gagnon, P. Lavigne, K. Klarskov, R. Leduc, and R. Day Inhibitory Potency and Specificity of Subtilase-like Pro-protein Convertase (SPC) Prodomains J. Biol. Chem., March 1, 2002; 277(10): 7648 - 7656. [Abstract] [Full Text] [PDF]
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M. Pavlaki, J. Cao, M. Hymowitz, W.-T. Chen, W. Bahou, and S. Zucker A Conserved Sequence within the Propeptide Domain of Membrane Type 1 Matrix Metalloproteinase Is Critical for Function as an Intramolecular Chaperone J. Biol. Chem., January 18, 2002; 277(4): 2740 - 2749. [Abstract] [Full Text] [PDF]
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T. Uekita, Y. Itoh, I. Yana, H. Ohno, and M. Seiki Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity J. Cell Biol., December 24, 2001; 155(7): 1345 - 1356. [Abstract] [Full Text] [PDF]
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