From the Faculty of Dentistry and Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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ABSTRACT |
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Recombinant collagen-binding domain (rCBD)
comprising the three fibronectin type II-like modules of human
gelatinase A was found to compete the zymogen form of this matrix
metalloproteinase from the cell surface of normal human fibroblasts in
culture. Upon concanavalin A treatment of cells, the induced cellular
activation of gelatinase A was markedly elevated in the presence of the
rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays.
Fibroblasts attached to rCBD-coated microplate wells in a manner that
was inhibited by soluble rCBD, blocking antibodies to the
1-integrin subunit but not the
2-integrin subunit, and bacterial collagenase treatment.
Addition of soluble collagen rescued the attachment of
collagenase-treated cells to the rCBD. As a probe on ligand blots of
octyl-
-D-thioglucopyranoside-solubilized cell membrane
extracts, the rCBD bound 140- and 160-kDa protein bands. Their
identities were likely procollagen chains being both bacterial
collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen
1(I) and
2(I)
chains. A rCBD mutant protein (Lys263
Ala) with reduced
collagen affinity showed less cell attachment, whereas a
heparin-binding deficient mutant (Lys357
Ala),
heparinase treatment, or heparin addition did not alter attachment.
Thus, a cell-binding mechanism for gelatinase A is revealed that does
not involve the hemopexin COOH domain. Instead, an attachment complex
comprising gelatinase A-native type I
collagen-
1-integrin forms as a result of interactions
involving the collagen-binding domain of the enzyme. Moreover, this
distinct pool of cell collagen-bound proenzyme appears recalcitrant to
cellular activation.
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INTRODUCTION |
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The plasma membrane of various human cancer cells contains high
levels of collagenolytic and gelatinolytic proteinases (1, 2) with a
positive correlation shown between the expression of the matrix
metalloproteinase (MMP)1
gelatinase A and invasive potential (3). Moreover, certain tumor cell
lines, which do not express gelatinase A, can bind the enzyme to their
cell membranes by a membrane-associated receptor in trans
(2, 4). Activation of progelatinase A by cell membranes of concanavalin
A (ConA)-stimulated (5, 6) or
12-O-tetradecanoyl-phorbol-13-acetate-stimulated (7, 8)
normal cells requires a specific mode of enzyme-cell interaction that
utilizes the COOH-terminal domains of gelatinase A and the tissue
inhibitor of MMPs, TIMP-2 (8-10). Four membrane type (MT)-MMPs
possessing a hydrophobic transmembrane domain have been shown to
activate progelatinase A at the cell surface (11, 12) in an activation
complex comprising progelatinase A, TIMP-2, and MT-MMP (12, 13). Here,
the active site of MT-MMP functions as a receptor for the inhibitory
NH2 domain of TIMP-2, leaving the TIMP-2 COOH domain free
to interact with progelatinase A. Recent site-directed mutagenesis
studies have mapped the TIMP-2-binding site on gelatinase A to the
junction of the outer rim of -blades III and IV of the
hemopexin-like COOH-terminal domain (C
domain)2. However, alternative
interactions of the gelatinase A C domain with TIMP-4 (14) and cell
surface components such as the
v
3 integrin receptor (15), fibronectin (16), and heparin (16-18) have
also been identified.
The C domain of MMPs is involved in several important protein-protein interactions. In gelatinase B the C domain binds TIMP-1, whereas interstitial and neutrophil collagenases utilize the C domain for binding and cleavage of native type I collagen (19). However, the gelatinase A C domain does not bind collagen (16, 20). Instead, a different collagen-binding domain (CBD) is found in gelatinases A and B consisting of three fibronectin type II-like modules inserted in the catalytic domain (21, 22). In addition to binding denatured type I collagen (23-25), our characterization of recombinant human gelatinase A CBD (rCBD) showed that this domain accounts for all of the binding properties of the enzyme to native and denatured collagen types I, V, and X and elastin and also contains a heparin-binding site (17, 25).3 The importance of these functions is shown by CBD deletion, which reduces gelatinase A cleavage of denatured type I collagen by 90% (20) and abolishes elastin binding and cleavage (26).
The gelatinase A CBD may also serve to localize the enzyme to matrix
components in tissues (17, 20, 25). These properties may similarly
provide another mode of cell binding to membrane-associated matrix
proteins, including collagen and heparan sulfate proteoglycans, and
thus may play a role in gelatinase A activation (18) and its
physiological function on the cell surface. Here we report experiments
that establish that the fibronectin-like CBD localizes gelatinase A to
fibroblast cell surfaces by the formation of a gelatinase A-type I
collagen-1-integrin complex. Notably, this distinct pool
of cell-bound enzyme shows a lowered cellular activation potential
compared with soluble progelatinase A. This finding has important
implications for the role of cell membrane-bound stromal gelatinase A
on tumor cells.
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EXPERIMENTAL PROCEDURES |
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Recombinant Gelatinase A Domains and Antibodies-- rCBD (Val191-Gln364) and the rC domain (Gly417-Cys631) of human gelatinase A were expressed in Escherichia coli and purified by Zn2+-chelate and gelatin-Sepharose chromatography as appropriate (14, 25). Electrospray mass spectrometry of the recombinant proteins was performed on a SCIEX API 300 (Perkin-Elmer) mass spectrometer. The convention used in this paper to distinguish between the recombinant protein comprised of the gelatinase A triple fibronectin type II-like repeat and the domain present in the natural enzyme will be to refer to the recombinant collagen-binding domain as the rCBD and to the domain in the enzyme as the CBD (no r).
Rabbit polyclonal antibody (Cell Culture--
Human gingival fibroblasts, kindly provided by
Drs D. Brunette and H. Larjava (University of British Columbia), were
maintained in -minimal essential medium (
-MEM) (Life
Technologies, Inc.) containing 10% newborn calf serum (Life
Technologies, Inc.) and antibiotics at 37 °C. To minimize
proteolysis of membrane proteins during cell harvesting for cell
attachment assays, 0.2 mM EDTA with a low concentration of
trypsin (0.05%) in phosphate-buffered saline (PBS) (140 mM
NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4·7H2O, 1.5 mM
KH2PO4, pH 7.4) was used for 30-60 s only.
Competition Experiments--
Fibroblasts in 96-microwell tissue
culture plates were treated with soluble rCBD (1.0 × 104 to 1.0 × 10
8 M) or rC
domain (5.6 × 10
6 to 1.0 × 10
8
M) for 24-28 h during and/or after ConA treatment (20 µg/ml) (5) of quiescent cells in serum-free conditions. Conditioned medium and cell extracts were analyzed by zymography on 10%
polyacrylamide/40 µg/ml gelatin SDS-PAGE gels (27). To determine
whether progelatinase A could bind unstimulated cells by the CBD,
quiescent cells were thoroughly rinsed with PBS to remove unbound
secreted enzyme. Gelatinase A was then competed from cell surfaces by
incubation of the cell layers with 1.2 or 12 × 10
6
M rCBD in serum-free
-MEM at 22 °C for 5 min only.
This short time was selected to minimize contributions from newly
synthesized enzyme to the medium during the incubation. After medium
harvesting, the remaining cell-associated enzyme was assessed after
lysis of the cell layer with SDS-PAGE sample buffer.
Cell Attachment Assay--
Tissue culture surface treated
96-microwell plates were coated with 2-fold serially diluted rCBD
(50-0.25 µg/ml) in 100 µl PBS/well for 18 h at 4 °C. After
blocking with 10 mg/ml heat-denatured bovine serum albumin (BSA) for 30 min, 4 × 104 fibroblasts were added per well in
serum-free -MEM (to avoid cell attachment from serum proteins) and
incubated for 90 min at 37 °C. Cells were then thoroughly rinsed
with PBS and fixed with 4% formaldehyde in PBS. The attached cells
were stained with 0.1% crystal violet in 200 mM boric
acid, pH 6.0 (28). After extensive rinses, cellular stain was dissolved
in 10% acetic acid, and cell numbers were quantitated by measurement
of the optical density at 590 nm in a microplate reader. Positive
control wells were coated with fibronectin (Chemicon) or acid soluble
type I collagen prepared from rat tail collagen (25) or were nonblocked wells. Any cell attachment to BSA-blocked wells served to adjust for
nonspecific attachment. Experiments were performed in duplicate or
triplicate and repeated several times, but results were only compared
for experiments on the same plate.
Cell Morphology and Spreading Characterization--
For scanning
electron microscopy, cells were seeded and grown in serum-free -MEM
on rCBD-coated glass coverslips (1 cm2) blocked with BSA.
After 1 or 2 h, cells were rinsed and fixed with 2.5%
glutaraldehyde in PBS. Slides were stained with 1% osmium in PBS,
treated with 2% tannic acid, dried by critical point drying, and
sputter-coated with gold for analysis on a Stereoscan 260 (Cambridge
Instruments) scanning electron microscope. Phase contrast microscopy
was used to quantitate cell spreading at different time points after
seeding 5 × 103 cells on rCBD- or fibronectin-coated
wells. Cells were fixed in 4% formaldehyde for 30 or 60 min at
22 °C, and cell spreading, as judged by the appearance of lamellar
cytoplasm, was then quantitated.
Mechanisms of Cell Attachment--
Harvested cells were treated
with 0.075-7.5 units/100 µl highly pure bacterial collagenase
(clostridiopeptidase A, Type III, fraction A (EC 3.4.24.3), Sigma) or
0.01 and 0.1 units/ml highly pure heparinase (Flavobacterium
heparinum heparinase, Seikagaku Corporation) in -MEM with 10 mM Ca2+ acetate and 0.1% BSA for 15-30 min at
37 °C. Enzymes were then removed by repeated cell sedimentation
(120 × g, 5 min) and washes in serum-free
-MEM
prior to seeding in rCBD (25 µg/ml)-coated wells. Attachment of
bacterial collagenase-treated cells to native type I collagen bound to
rCBD-coated wells was also quantitated. In addition, cells were seeded
in the presence of blocking monoclonal antibody mAb13 (0.6-20 µg/ml)
to the
1-integrin subunit (kindly provided by Dr. K. Yamada, NIDR, National Institutes of Health) or ascites fluid antibody
(P1E6, Life Technologies, Inc.) to the
2-integrin
subunit diluted 1:10 to 1:100. Affinity purified
CBD and
His6 antibodies served as controls in the 90-min
incubations. The effect of 1 or 10 µg of heparin (Sigma) in 100 µl
of PBS added to rCBD-coated wells for 1 h prior to seeding was
also assessed.
Ligand Blot Analyses--
Confluent fibroblast cultures were
rinsed thoroughly with PBS and then treated with 50 mM
octyl--D-thioglucopyranoside (Sigma) in PBS for 30 min
at 15 °C (29). After clarification at 10,000 × g
for 15 min at 22 °C, detergent-solubilized cell membrane protein was
precipitated at 0 °C and then collected by centrifugation at
10,000 × g for 10 min at 0 °C. The protein pellet
was dissolved in PBS, separated under nonreducing or reducing (65 mM DTT) conditions by 7.5% SDS-PAGE, and transferred to
Immobilon-P polyvinylidene difluoride membrane (Millipore). The blots
were BSA-blocked and then incubated with 20 µg/ml rCBD in 150 mM NaCl, 10 mM Tris, pH 7.2, with 0.2% BSA for
1 h at 22 °C. After washes, rCBD bound to the blotted proteins
was detected using
CBD antibody and enhanced chemiluminescence
reagents (Amersham Pharmacia Biotech). The rCBD-binding proteins were
characterized by digestion with pepsin (0.1 mg/ml (Sigma) for 3 h
at 15 °C, pH 2.0) or highly pure bacterial collagenase (4 units/100
µl for 18 h at 37 °C, pH 7.0). An aliquot of the pepsin-treated sample was adjusted to pH 7.0 and incubated with bacterial collagenase for 18 h at 37 °C. The efficiency and
specificity of the enzyme digestions was verified using BSA, type I
collagen and rCBD as control substrates.
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RESULTS |
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Recombinant Protein Expression-- The rCBD mass was measured by electrospray mass spectrometry to be 21,218 Da, confirming NH2-terminal methionine processing of the recombinant protein (predicted mass 21, 212 Da), fidelity of expression, and homogeneity of the protein preparation. The typical yield of purified rCBD from 3.6 liters of culture was 120 mg.
The Collagen-binding Domain Mediates Binding of Gelatinase A to
Cells--
When rCBD was incubated with human fibroblasts for 24 h during and after ConA treatment (Fig.
1A) or for 24 h after
ConA treatment only (not shown), an increase in gelatinase A activation
was apparent in six separate experiments. At high rCBD concentrations,
essentially all the soluble gelatinase A was converted to the 59-kDa
(DTT) activated form (5). Although quantitation of enzyme levels from
zymograms is only semiquantitative, less than ~3% of the total
soluble gelatinase A remained as the 66-kDa (
DTT) zymogen form in the
presence of 100 µM rCBD (lane 100 +) compared
with ~28-34% in those cells not treated with rCBD (lanes 0 +). This trend was also apparent at 50 µM rCBD. In
contrast, recombinant gelatinase A C domain reduced cellular activation
of the enzyme as before (17) (not shown).
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The Collagen-binding Domain of Gelatinase A Mediates Cell Attachment-- The mechanistic aspects of gelatinase A cell binding via the CBD were further investigated by adaptation of cell attachment assays. Fibroblasts attached to rCBD-coated microwells in a concentration-dependent manner (Fig. 2A), but this was less efficient than cell attachment to fibronectin (Fig. 2B). Incubation of fibroblasts with soluble rCBD prior to seeding inhibited attachment to rCBD-coated wells in a concentration-dependent manner, confirming binding specificity (Fig. 2C). Attachment was not observed in wells coated with 10 mg/ml BSA, whereas cell attachment to tissue culture-treated plastic alone or to type I collagen-coated wells was similar to that on fibronectin under saturating conditions. As assessed by phase contrast microscopy significantly fewer fibroblasts displayed cytoplasmic spreading on rCBD coated at 10 µg/ml (23%) compared with fibronectin (50%) after 30 min. Greater differences in cell spreading were apparent between rCBD and fibronectin using 25 µg/ml coated protein with 23 and 90%, respectively, of the cells spreading after 30 min. Although the kinetics of cell attachment and spreading differed at these early time points, spreading of cells on both substrates plateaued at 80-90% of the attached cells by 60 min. Scanning electron microscopy confirmed both cell attachment to rCBD protein and these differences. After 1 and 2 h on fibronectin (Fig. 3, A and C, respectively), cells demonstrated typical cytoplasmic spreading (arrows) with a diameter of ~100 µm. In contrast, cells on rCBD were smaller (diameter of ~50 µm) and more rounded after 1 h (Fig. 3B) with limited spreading and extension of only delicate filopodia (arrowheads) after 2 h (Fig. 3D). Thus, this novel use of cell attachment assays confirmed the potential for gelatinase A binding to cells via the CBD of the enzyme.
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1-Integrins Are Involved in Cell Attachment to
rCBD--
A role for
1-integrins in CBD-mediated
gelatinase A cell binding was demonstrated using mAb13, an
anti-
1-integrin blocking monoclonal antibody. At 2.5 µg/ml antibody, more than 50% of the cell attachment to rCBD-coated
wells was inhibited (Fig. 4). This inhibition increased to 90% at antibody concentrations >5 µg/ml. In
comparison,
2-integrin blocking antibody and affinity
purified
CBD and
His6 control antibodies showed no
significant blocking effects at these concentrations.
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The Role of Pericellular Collagen in Cell Attachment to rCBD-- In addition to any direct interaction with other cell membrane proteins, the ligand blots indicated that binding of gelatinase A CBD to native cellular collagen might represent one mode of gelatinase A cell binding. To test this, rCBD-coated wells were incubated with 10 µg of soluble type I collagen in 100 µl of PBS/well to saturate rCBD collagen-binding sites prior to cell seeding. On the rCBD-collagen complexes, cell attachment levels approached that for wells coated with 1.0 µg/well collagen alone (Fig. 6). Cell attachment diminished with decreasing amounts of collagen bound to the rCBD.
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Cell Attachment to rCBD Is Not Heparan
Sulfate-dependent--
To determine the potential for rCBD
to interact with cell membrane heparan-sulfate proteoglycans through
the low affinity heparin-binding site on the rCBD3 (25),
Lys357 Ala, a mutant of rCBD that shows complete loss
of heparin binding,3 did not
display any differences in mediating cell attachment compared with the
wild type rCBD (Fig. 8A). In
contrast, a rCBD mutant (Lys263
Ala), characterized as
having reduced type I collagen binding affinity,3 showed
reduced cell attachment properties compared with the wild type rCBD
(Fig. 8A). In other experiments, cells were treated with
heparinase prior to plating, but even at concentrations as high as 0.1 units/ml there was no alteration in cell attachment to the rCBD (Fig.
8B). Lastly, heparin was added to rCBD-coated wells prior to
cell seeding to block heparin-binding sites, but this too did not
reduce attachment levels from controls (not shown). Collectively, these
results show that pericellular collagen was a rate-limiting component
of cell attachment to rCBD-coated wells and that heparan-sulfate
proteoglycans were not involved. Therefore, this reveals the potential
for gelatinase A binding to cells via interactions involving the CBD of
the enzyme and native cellular collagen that is cell associated by
1-integrins.
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DISCUSSION |
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By studying fibroblast cell attachment to the recombinant CBD of
human gelatinase A we have developed a novel approach to mechanistically explore cell-binding mechanisms of gelatinase A. Our
data indicate that the interaction between rCBD and cells in the
attachment assays is representative of gelatinase A utilizing this
domain to bind cell surfaces. Notably, the potential for gelatinase A
interaction with cells via the CBD of the enzyme was shown by the
competitive release of progelatinase A from unstimulated fibroblasts by
the rCBD. The importance of the gelatinase A C domain-TIMP-2 C domain
interaction for activation by MT-MMPs is also thereby demonstrated
because cell surface localization of progelatinase A through the CBD
was not sufficient for activation. This confirms previous reports using
CBD and C domain deletion mutants of the enzyme (20). Although the rCBD
binds heparin (25), we found no evidence of rCBD binding to cell
membrane heparan sulfate proteoglycans. Rather, our studies overall
indicate that the gelatinase A CBD mediates cell surface localization
of the enzyme to fibroblasts by binding pericellular collagen that in
turn is anchored to cell membrane 1-integrins, among
which
1
1,
2
1, and
3
1
are type I collagen receptors (31).
Direct binding of rCBD to 1-integrins as an
integrin-associated protein or via the integrin ligand-binding site is
a possibility not completely ruled out by our studies. However, no RGD
sequence occurs in the gelatinase A CBD. Interestingly, the gelatinase B CBD contains a RGD sequence that aligns with RSDG at positions 339-342 in gelatinase A. In support of a
1-integrin-collagen-gelatinase A CBD bridge model,
detergent solubilized fibroblast cell surface proteins that were bound
by rCBD on ligand blots were fully degraded by bacterial collagenase.
Furthermore, these proteins were also pepsin-resistant, a
characteristic of the native triple helical collagen domain. This
points to native collagen or procollagen mediating the cell binding
interactions with rCBD. Cell attachment to the rCBD-collagen complexes
showed that rCBD occupancy of the major type I collagen-binding site on
the collagen telopeptides (25) did not sterically block
1-integrin attachment to the collagen. This indicates
that the integrin receptors and rCBD recognize different binding sites
on collagen.
Other evidence supporting this mode of cell binding was that bacterial collagenase treatment of fibroblasts greatly reduced cell attachment to rCBD, and this was rescued in a concentration-dependent manner by adding native type I collagen to the coated rCBD films. That collagen was a rate-limiting component in attachment of untreated cells was also shown by binding type I collagen to coated rCBD-coated wells prior to cell seeding. This produced a concentration-dependent increase in cell attachment ultimately approaching that of cells attached to collagen alone.
Our demonstration of cell binding by the gelatinase A CBD reveals a
distinct mode of gelatinase A cell localization additional to that
involving the hemopexin-like C domain (5-7, 10, 13). We have
previously proposed that gelatinase A cellular activation involves
TIMP-2 bridging the C domain of progelatinase A and MT-MMP with
activation occurring by a second MT-MMP (17). Indeed, activation does
not occur with C domain deletion mutants of gelatinase A (10) or in the
absence of TIMP-2 (13) and can be inhibited by adding rC domain to
ConA-treated cells (17) or membranes (13). However, the mechanism of
release of active gelatinase A is enigmatic (17) given the
Kd values of TIMP-2 binding the C domain of
gelatinase A (14) and that of the inhibitory N domain of TIMP-2 binding
the active site of MT-MMP. Possibly, release of active gelatinase A
occurs upon MT-MMP degradation from the active 60-kDa form to a
truncated membrane-bound 42-kDa form of MT-MMP that was recently
reported (32). Despite convincing evidence for the trimolecular complex
of gelatinase A-TIMP-2-MT-MMP, alternative cell-binding mechanisms for
gelatinase A are indicated from other studies. Because TIMP-2 as well
as the progelatinase A-TIMP-2 complex can bind to cell surfaces, a
specific TIMP-2 receptor, possibly distinct from active MT-MMPs, may
also mediate enzyme binding (33). Indeed, signal transduction events
and growth factor effects can be elicited upon TIMP-2 cell binding (33), and gelatinase A can bind to cells not expressing MT1-MMP (34).
Binding of the gelatinase A C domain, which has homology to
vitronectin, to the v
3 integrin
vitronectin receptor can also occur (15). Vitronectin binding to
v
3 integrins also enhances gelatinase A
expression and cell penetration of basement membranes (35), emphasizing
the important role of integrins in gelatinase A function. Thus,
gelatinase A may localize to cell surfaces by a number of distinct
mechanisms including the CBD, the C domain via the TIMP-2-MT-MMP
complex, a distinct TIMP-2 receptor, and the C domain via the
v
3 integrin receptor.
As shown in Fig. 1, when rCBD was added to ConA-treated cells this
produced an elevated activation of the progelatinase A in the medium,
but not in the cell layer, over that seen by ConA alone as first
described by Overall and Sodek (5). Because the total amount of enzyme
recovered in the cell lysates, which also includes proenzyme in the
secretory pathway, was approximately 10-fold less than that found in
the medium, the total cellular response to the rCBD was one
characterized by a marked elevation in ConA-induced gelatinase A
activation. The explanation we favor for this new finding is presented
in Fig. 9. The displacement of
progelatinase A by the rCBD in cells treated with ConA would promote enzyme activation before release to the medium because of the
proximity of the released enzyme with the cell membrane and MT-MMPs.
This is likely to be the mechanism because the active enzyme
accumulated in the medium rather than in the cell layer, which retained
relatively unaltered levels of latent and active gelatinase A. The
progelatinase A on the cell-bound collagen would be replenished from
newly synthesized enzyme bound at the time of secretion. Thus, the
relative proportions of latent to active gelatinase A in the
collagen-bound pool would not necessarily alter significantly upon rCBD
addition. Together with the ongoing activation of soluble progelatinase
A, the presence of rCBD would also compete for binding of newly
activated soluble active gelatinase A to cell-bound collagen. This
would result in the accumulation of active gelatinase A in the medium
relative to the zymogen form of the enzyme over time. Thus, these data
strongly indicate that the pool of gelatinase A that is cell-bound via
1-linked collagen resists entry into the MT-MMP
activation pathway.
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We propose that CBD-mediated cell binding of progelatinase A may provide a means of maintaining a pool of latent enzyme at the cell membrane. Cell binding by the CBD also has the potential to target progelatinase A from one cell to another in trans, a mechanism thought to be important for increasing the proteolytic potential of tumor cells. However, our data indicate that in these cells MT-MMPs would not necessarily readily activate enzyme so targeted unless subsequently released from the collagen. Of note, MT1-MMP can cleave native type I collagen (36, 37). Therefore, on MT-MMP induction, release of CBD-bound progelatinase A from the MT-MMP degraded cell-bound collagen may provide the means for entry of this pool of progelatinase A into the C domain-TIMP-2-MT-MMP activation pathway. Activated gelatinase A would thereby be localized at sites of ongoing cell matrix degradation and gelatinolysis.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge invaluable advice in cell attachment assays from Dr. H. Larjava and C. Sperantia and in scanning electron microscopy analysis by A. Wong.
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FOOTNOTES |
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* This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Medical Research Council Fellowship. Present
address: Department of Periodontics, University of Texas Health Science
Center at San Antonio, San Antonio, TX 78284-7894.
§ Recipient of a Medical Research Council of Canada Clinician Scientist Award. To whom correspondence should be addressed: Faculty of Dentistry, University of British Columbia, 2199 Wesbrook Mall, Vancouver, V6T 1Z3 BC, Canada. Tel.: 604-822-2958; Fax: 604-822-8279; E-mail: overall{at}unixg.ubc.ca.
The abbreviations used are:
MMP, matrix
metalloproteinase; C domain, MMP COOH-terminal hemopexin-like domain; ConA, concanavalin A; -MEM,
-minimal essential medium; MT-MMP, membrane type MMP; TIMP, tissue inhibitor of metalloproteinases; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; CBD, collagen-binding domain; rCBD, recombinant CBD; BSA, bovine serum
albumin; DTT, dithiothreitol.
2 C. M. Overall, A. King, D. Sam, A. Ong, T. T. Y. Lau, U. M. Wallon, Y. A. DeClerck, and J. J. Atherstone, submitted for publication.
3 B. Steffensen, R. Maurus, E. Rydberg, and C. M. Overall, submitted for publication.
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REFERENCES |
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