Download The Involvement of the Fibronectin Type II-like Modules

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 273, No. 32, Issue of Aug 7, pp. 20622–20628, 1998
Printed in U.S.A.
The Involvement of the Fibronectin Type II-like Modules of Human
Gelatinase A in Cell Surface Localization and Activation*
(Received for publication, February 3, 1998, and in revised form, May 26, 1998)
Bjorn Steffensen‡, Heather F. Bigg, and Christopher M. Overall§
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
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 b1-integrin subunit but not the a2integrin 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-b-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 112and 126-kDa bands that co-migrated with collagen a1(I)
and a2(I) chains. A rCBD mutant protein (Lys263 3 Ala)
with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant
(Lys357 3 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 collagenb1-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.
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 in-
* 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. This 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: [email protected].
1
The abbreviations used are: MMP, matrix metalloproteinase; C
vasive 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-phorbol13-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 b-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 avb3 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 hepadomain, MMP COOH-terminal hemopexin-like domain; ConA, concanavalin A; a-MEM, a-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.
20622
This paper is available on line at http://www.jbc.org
Gelatinase A Cell Surface Binding by Fibronectin Modules
ran 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-b1integrin 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.
EXPERIMENTAL PROCEDURES
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 Zn21-chelate and gelatinSepharose 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 collagenbinding domain as the rCBD and to the domain in the enzyme as the
CBD (no r).
Rabbit polyclonal antibody (aCBD) was raised against rCBD injected
with sarcosyl-extracted rCBD inclusion bodies and was then affinity
purified over rCBD-AffiGel 10 (Bio-Rad) columns. Anti-peptide antibody (aHis6) to the NH2-terminal His6 fusion tag on the recombinant
proteins was affinity purified as before (16).
Cell Culture—Human gingival fibroblasts, kindly provided by Drs D.
Brunette and H. Larjava (University of British Columbia), were maintained in a-minimal essential medium (a-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 Na2HPO4z7H2O, 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 3 1024 to 1.0 3 1028 M)
or rC domain (5.6 3 1026 to 1.0 3 1028 M) for 24 –28 h during and/or
after ConA treatment (20 mg/ml) (5) of quiescent cells in serum-free
conditions. Conditioned medium and cell extracts were analyzed by
zymography on 10% polyacrylamide/40 mg/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 3 1026
M rCBD in serum-free a-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 mg/ml) in
100 ml PBS/well for 18 h at 4 °C. After blocking with 10 mg/ml heatdenatured bovine serum albumin (BSA) for 30 min, 4 3 104 fibroblasts
were added per well in serum-free a-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 a-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 micro-
20623
scope. Phase contrast microscopy was used to quantitate cell spreading
at different time points after seeding 5 3 103 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 ml 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 a-MEM with 10 mM Ca21 acetate and
0.1% BSA for 15–30 min at 37 °C. Enzymes were then removed by
repeated cell sedimentation (120 3 g, 5 min) and washes in serum-free
a-MEM prior to seeding in rCBD (25 mg/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 mg/ml)
to the b1-integrin subunit (kindly provided by Dr. K. Yamada, NIDR,
National Institutes of Health) or ascites fluid antibody (P1E6, Life
Technologies, Inc.) to the a2-integrin subunit diluted 1:10 to 1:100.
Affinity purified aCBD and aHis6 antibodies served as controls in the
90-min incubations. The effect of 1 or 10 mg of heparin (Sigma) in 100 ml
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-b-D-thioglucopyranoside (Sigma) in PBS for 30 min at 15 °C (29). After clarification
at 10,000 3 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 3 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 mg/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 aCBD 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 ml 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.
RESULTS
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 (2DTT) 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 (2DTT) zymogen
form in the presence of 100 mM rCBD (lane 100 1) compared
with ;28 –34% in those cells not treated with rCBD (lanes 0 1).
This trend was also apparent at 50 mM rCBD. In contrast,
recombinant gelatinase A C domain reduced cellular activation
of the enzyme as before (17) (not shown).
Cell lysates containing gelatinase A that was bound to cells
via the C domain of the enzyme or was intracellular in the cell
secretory pathway were also prepared after rCBD treatment.
Unlike the effect of rCBD on gelatinase A levels in the medium
(Fig. 1A), addition of rCBD to cells during and/or after ConA
treatment did not alter the ratios of latent (66 kDa) to active
(59 kDa) gelatinase A in the lysates (Fig. 1B). As estimated
20624
Gelatinase A Cell Surface Binding by Fibronectin Modules
FIG. 2. Attachment of human fibroblasts to rCBD and fibronectin. Human fibroblasts were seeded onto microwell plates coated with
serially diluted (0.25–50 mg/ml) rCBD (panel A) or fibronectin (panel B).
In panel C, harvested cells were first treated with 50 – 0.25 mg/ml
soluble rCBD in a-MEM for 30 min at 37 °C prior to seeding in wells
coated with 3.0 mg/ml (150 nM) rCBD (representing the amount of
coated protein producing ;50% maximal cell attachment). After 90 min
at 37 °C, the number of attached fibroblasts was quantitated as described under “Experimental Procedures.” Data points are the means of
duplicate measurements and are representative of three separate
experiments.
FIG. 1. rCBD competitively displaces progelatinase A from human fibroblasts and enhances ConA-induced enzyme activation. Confluent fibroblasts were incubated in serum-free a-MEM with
or without ConA (20 mg/ml) in the presence of serially diluted rCBD as
indicated (mM). The medium was then changed, and the cells were
incubated for a further 24 h in the presence of the same amounts of
rCBD as before. Medium samples (panel A) were analyzed on 10%
SDS-PAGE/40 mg/ml gelatin zymograms. Essentially identical results
were obtained when rCBD was only added after ConA treatment for
24 h. Cell lysates from this single addition of rCBD were analyzed on
zymograms (panel B). In panel A, the first lane (0 2) shows that in the
absence of both ConA (2) and rCBD (0), essentially 100% of the gelatinase A present in the medium is in the zymogen form with an apparent molecular mass of 66 kDa (2DTT). Upon ConA treatment of cells for
24 h in the absence of added rCBD protein (lanes 0 1), activation of
gelatinase A occurred. Laser densitometry showed that ;66 –72% of the
enzyme was converted to the fully active 59-kDa form (2DTT) as
indicated. A minor amount of the 62-kDa (2DTT) activation intermediate was detected. In cells treated with ConA (1) and at the highest
rCBD concentration shown (100 mM) (100 1), essentially all of the
gelatinase A was present in the 59-kDa active form. In contrast, the
adjacent control lane (far right lane) retains a strong latent gelatinase
band. Panel C, cell layers were rinsed three times with PBS (PBS, lanes
1, 2, and 3) to remove all unbound secreted enzyme from the medium
(lane M). Cells were then pulsed with 12 or 1.2 mM rCBD in PBS for 5
min at 22 °C. Gelatinase A, competitively released by the rCBD, was
analyzed by zymography. No gelatinase A was released without rCBD
(1rCBD, lane 0). Gelatinase A was then extracted from the corresponding cells (Cells) with SDS-PAGE sample buffer. Panel D, fibroblasts
were seeded in uncoated wells or wells coated with 25 mg/ml rCBD. At
confluence, the cells were rinsed with PBS and then incubated with
serum-free a-MEM with or without ConA (ConA, 1 or 2) for 18 h before
zymographic analysis of the medium.
from enzyme levels per microliter, the total enzyme recovered
in the lysates of ConA-activated cells was ;10-fold less than
that in the medium. In other experiments, zymography also
demonstrated that cell-bound progelatinase A (the 66-kDa zymogen form) was competitively displaced from unstimulated
cells that had not been ConA-treated. This was found even
after a short 5-min pulse of the rCBD intended to minimize
accumulations of newly secreted progelatinase A during the
experiment (Fig. 1C). Extraction of the cell layer with SDSPAGE sample buffer revealed that additional gelatinase A
remained associated with the cells that was either not fully
released by the short exposure to the rCBD or was bound by the
C domain or was intracellular. That the increased gelatinase A
activation upon ConA addition combined with rCBD treatment
was not because of a direct cellular response to binding rCBD
was shown in cultures incubated in the absence of ConA where
rCBD addition for 24 h did not induce gelatinase A activation
(not shown). Moreover, neither gelatinase A expression nor
activation was altered in cells that were attached to rCBD-coated
plates (see “The Collagen-binding Domain of Gelatinase A Mediates Cell Attachment”) without ConA treatment (Fig. 1D).
Thus, these data show that in addition to interactions involving the C domain, progelatinase A can bind to cells via another
domain of the enzyme, the CBD. Because only latent and not
active gelatinase A was displaced in unstimulated cultures by
the rCBD, these competition experiments also show that cell
binding via the CBD of progelatinase A is not sufficient for
enzyme activation. Indeed, because gelatinase A activation
upon ConA treatment increases in the presence of excess rCBD,
we conclude that cellular progelatinase A bound by the CBD
has a lower cellular activation potential than the soluble enzyme in the medium. Hence, displacement of CBD-bound
progelatinase A by the rCBD in ConA-treated cells may facilitate entry of the latent enzyme into the cellular activation
pathway.
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 seedinginhibitedattachmenttorCBD-coatedwellsinaconcentrationdependent 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 mg/ml (23%) compared with fibronectin (50%) after 30 min. Greater differences
in cell spreading were apparent between rCBD and fibronectin
using 25 mg/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,
Gelatinase A Cell Surface Binding by Fibronectin Modules
FIG. 3. Morphological differences between cells cultured on
rCBD and fibronectin. 1 3 103 human fibroblasts were seeded onto
glass coverslips coated with 25 mg/ml rCBD or fibronectin and blocked
with BSA. After 1 and 2 h at 37 °C, the cells were fixed with glutaraldehyde and processed for scanning electron microscopy. Bars, 25 mm.
respectively), cells demonstrated typical cytoplasmic spreading
(arrows) with a diameter of ;100 mm. In contrast, cells on
rCBD were smaller (diameter of ;50 mm) 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.
b1-Integrins Are Involved in Cell Attachment to rCBD—A
role for b1-integrins in CBD-mediated gelatinase A cell binding
was demonstrated using mAb13, an anti-b1-integrin blocking
monoclonal antibody. At 2.5 mg/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
mg/ml. In comparison, a2-integrin blocking antibody and affinity purified aCBD and aHis6 control antibodies showed no
significant blocking effects at these concentrations.
Ligand blotting was performed to identify cell proteins that
may interact with the rCBD. On polyvinylidene difluoride blots
of octyl-b-D-thioglucopyranoside solubilized cell membrane proteins, rCBD bound two distinct protein bands having apparent
masses of 140 and 160 kDa under reducing conditions (Fig. 5)
in the approximate positions of a- and b-integrin subunits or
procollagen chains. However, both bands were degraded by
bacterial collagenase. The 140- and 160-kDa bands were also
partially pepsin-sensitive, being degraded to pepsin-resistant,
but collagenase-sensitive, 112- and 126-kDa proteins. These
co-migrated with collagen a1(I) and a2(I) chains that were also
bound by the rCBD (Fig. 5). Thus, these data exclude the
identity of the 140- and 160-kDa protein bands as integrin
chains. Rather, the data provide strong evidence that the rCBD
can interact with procollagen chains in cell membrane protein
extracts. Nonetheless, other proteins, including those that do
not renature on these blots or that require subunit interactions, might also be involved in the CBD interaction.
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 mg of soluble type I collagen in
100 ml of PBS/well to saturate rCBD collagen-binding sites
20625
FIG. 4. Cell attachment to rCBD is inhibited by anti-b1-integrin antibodies. To 96-well plates coated with 25 mg/ml rCBD, fibroblasts were seeded in the presence of blocking monoclonal antibodies to
the b1-integrin subunit (mAb13) and to the a2-integrin subunit (P1E6),
and as controls, affinity purified aCBD and aHis6 antibodies or medium
alone was used. The concentration of the P1E6 monoclonal antibody
was estimated from the ascites fluid protein concentration. Cell attachment was quantitated after 90 min. Means of duplicate measurements
are shown (n 5 3).
FIG. 5. Ligand blotting. Detergent-solubilized cell proteins were
digested with pepsin or bacterial collagenase followed by SDS-PAGE on
7.5% gels and subsequent transfer to polyvinylidene difluoride membranes. Blots were probed with 20 mg/ml rCBD for 1 h at 22 °C. Control
membranes were not probed (Control). rCBD bound to the blotted
proteins was detected with affinity-purified aCBD antibody and enhanced chemiluminescence reagents. St, type I collagen standard; CB,
Coomassie Blue-stained detergent solubilized protein. The amount (in
kDa) of marker proteins and the collagen a-chain, pro a-chain, and
b-components are shown.
prior to cell seeding. On the rCBD-collagen complexes, cell
attachment levels approached that for wells coated with 1.0
mg/well collagen alone (Fig. 6). Cell attachment diminished
with decreasing amounts of collagen bound to the rCBD.
In other experiments, cells pretreated with bacterial collagenase showed a concentration-dependent decrease in cell attachment to rCBD-coated wells (Fig. 7A). In control experiments, 0.75 unit of collagenase completely digested 100 mg of
purified native type I collagen after a 15-min incubation at
37 °C and did not cleave rCBD or BSA (not shown). The digestions were not cytotoxic as evident from unaltered attachment
of the treated cells to fibronectin or uncoated tissue culture
wells (not shown). That the reduced cell attachment was because of pericellular collagen removal and not to integrin degradation was supported by antibody blocking experiments. Incubation of bacterial collagenase-treated cells (0.75 units/100
ml) with b1-integrin blocking antibody further reduced cell attachment to rCBD (not shown) or to the coated rCBD collagen
complexes by ;75% (Fig. 7B). In positive control experiments,
binding of collagenase-treated cells to wells coated with colla-
20626
Gelatinase A Cell Surface Binding by Fibronectin Modules
FIG. 6. Cell attachment to rCBD-type I collagen complex. 96well plates were coated with either rCBD (1.5 or 25 mg/ml) or native
type I collagen (1, 3, or 10 mg/100 ml PBS coated in each well). After
blocking with BSA, rCBD-coated wells were then incubated with soluble native type I collagen (1, 3, or 10 mg/100 ml PBS/well). Tissue culture
treated plastic (P) or wells blocked with BSA (B) served as positive and
negative controls (Cont), respectively. Human fibroblasts were seeded,
and cell attachment was analyzed after 90 min. Data points are the
means of duplicate wells (n 5 3).
FIG. 7. Effect of bacterial collagenase treatment on cell attachment to rCBD. Panel A, fibroblasts were treated with highly pure
bacterial collagenase (0.075, 0.75, or 7.5 units/100 ml) in serum-free
a-MEM for 15 min at 37 °C. Collagenase-treated cells (4 3 104) were
then seeded in wells coated either with 25 mg/ml rCBD alone or subsequently bound with native type I collagen (1, 3, or 10 mg/100 ml PBS in
each well). Panel B, collagenase (0.75 units/100 ml)-treated fibroblasts
were plated for 90 min in the presence of anti-b1-integrin antibody/
serum-free a-MEM in wells coated with either 25 mg/ml rCBD subsequently complexed with native type I collagen (10 mg/100 ml PBS in each
well) or native type I collagen alone (0.5 mg/well). Means (n 5 3) and
S.D. bars are shown.
gen alone was also blocked with the b1-integrin blocking antibody (Fig. 7B). However, it should be noted that cell attachment had already been greatly diminished by treatment with
bacterial collagenase, and so this result does not necessarily
show a direct interaction between b1-integrins and the rCBD,
but it is a possibility that cannot be totally excluded. Alternatively, the further minor contribution to the rCBD cell interaction by b1-integrins may have been through molecules not
degraded by bacterial collagenase that were bound to this integrin class and the rCBD. As further evidence for the role of
the interaction of the rCBD with b1-integrin-bound collagen,
cell attachment to bacterial collagenase-treated cells could be
rescued in a concentration-dependent manner by addition of
soluble native type I collagen to the rCBD-coated wells before
cell seeding (Fig. 7A). Rescue was not complete, due either to
release of endocytosed collagenase (30) or to nonrescued cell
FIG. 8. The relative roles of collagen and heparin in cell attachment to the CBD. Panel A, wild type rCBD and two mutant rCBD
proteins characterized by reduced binding to collagen, Lys263 3 Ala, or
no binding to heparin, Lys357 3 Ala, were coated in 96-well plates.
Fibroblasts were plated, and cell attachment was quantitated after 90
min. Although no differences were observed at saturating conditions,
the half-maximal cell attachment concentration of coated protein for
Lys263 3 Ala was ;3.5 mg/ml compared with ;1.7 mg/ml for the Lys357
3 Ala mutant protein. Data points are the means of duplicate wells and
representative of four experiments. Bars, range. Panel B, fibroblasts
were treated with heparinase prior to seeding in rCBD-coated wells,
and cell attachment was quantitated. Data points are the means (n 5 3)
and are representative of two experiments.
interactions involving other collagen types.
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 3 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 3 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 ratelimiting 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 b1-integrins.
DISCUSSION
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 in-
Gelatinase A Cell Surface Binding by Fibronectin Modules
FIG. 9. Model of the influence of cell surface collagen on progelatinase activation at the cell membrane after ConA stimulation. Panel A, in unstimulated cells, secreted progelatinase A accumulates extracellularly (1) or binds (2) to cellular collagen (3), which is
bound to the cell membrane (4) via b1-integrins (5). The TIMP-2 (6) C
domain (shaded square) interacts with the progelatinase A C domain to
form progelatinase A-TIMP-2 complexes (7). Synthesis and secretion of
progelatinase A is indicated by the arrows originating from within the
cell. Collagen binding by the progelatinase A is mediated through the
CBD of the enzyme in an equilibrium with the much larger pool of
soluble progelatinase A. Panel B, ConA treatment of cells induces the
expression of active MT-MMP (8), which is anchored to the cell membrane by a transmembrane domain and cytoplasmic extension (9). The
TIMP-2-progelatinase A complex (7) binds to the active site of the
MT-MMP via the NH2-terminal “inhibitory” domain of TIMP-2 (shaded
half-circle). A second active MT-MMP molecule (10) then cleaves (asterisk) the prodomain of the gelatinase A to generate the 62-kDa
(2DTT) activation intermediate. Full gelatinase A activation then proceeds autocatalytically by another active gelatinase A molecule (not
shown) to generate active gelatinase A that is bound to the MT-MMP on
the cell surface (7). The 59-kDa (2DTT) active gelatinase A (11) is then
released from the MT-MMP, possibly by MT-MMP degradation as discussed in the text. Active proteinases are indicated by a line in the open
circle representing the active cleft in the catalytic domain after removal
of the prodomain (shaded circle). Ongoing synthesis and secretion of
progelatinase A replenishes the pools of progelatinase A extracellularly
and that bound to integrin-linked collagen on the cell surface. Panel C,
in unstimulated cells, the addition of recombinant CBD protein (13)
competes for cell surface collagen binding with the natural CBD of the
collagen-bound progelatinase A (2). As experimentally shown in Fig.
1C, a short incubation time with rCBD displaces progelatinase A (14)
only from cell surfaces. Panel D, addition of recombinant CBD (13) with
ConA competes off collagen-bound progelatinase A (2) in proximity to
the MT-MMP-TIMP-2 activation complexes on the cell membrane (8,
10). This increases the amount of progelatinase A activation and release
of active enzyme (11). The presence of recombinant CBD also competes
for any binding of newly activated gelatinase A to the cell-bound colla-
20627
dicate 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 b1-integrins,
among which a1b1, a2b1, and a3b1 are type I collagen receptors
(31).
Direct binding of rCBD to b1-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 b1-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 b1-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 concentrationdependent 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 avb3 integrin
vitronectin receptor can also occur (15). Vitronectin binding to
avb3 integrins also enhances gelatinase A expression and cell
gen. Together with the ongoing activation of soluble progelatinase A (1),
this results in the accumulation of active gelatinase A extracellularly
relative to the zymogen form of the enzyme over time, as shown experimentally in the medium in Fig. 1A.
20628
Gelatinase A Cell Surface Binding by Fibronectin Modules
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 TIMP2-MT-MMP complex, a distinct TIMP-2 receptor, and the C
domain via the avb3 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 b1-linked collagen resists entry into the MT-MMP activation pathway.
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 cellbound 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.
Acknowledgments—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.
REFERENCES
1. Zucker, S., Wieman, J. M., Lysik, R. M., Wilkie, D. P., Ramamurthy, N., and
Lane, B. (1987) Biochim. Biophys. Acta 924, 225–237
2. Emonard, H. P., Remacle, A. G., Noel, A. C., Grimaud, J.-A., Stetler-Stevenson,
W. G., and Foidart, J.-M. (1992) Cancer Res. 52, 5845–5848
3. Stetler-Stevenson, W. G., Aznavoorian, S., and Liotta, L. A. (1993) Annu. Rev.
Cell Biol. 9, 541–573
4. Tryggvason, K., Hoyhtya, M., and Pyke, C. (1993) Breast Cancer Res. Treat. 24,
209 –218
5. Overall, C. M., and Sodek, J. (1990) J. Biol. Chem. 265, 21141–21151
6. Ward, R. V., Atkinson, S. J., Slocombe, P. M., Docherty, A. J. P., Reynolds, J. J.,
and Murphy, G. (1991) Biochim. Biophys. Acta 1079, 242–246
7. Brown, P. D., Levy, A. T., Margulies, I. M. K., Liotta, L. A., and StetlerStevenson, W. G. (1990) Cancer Res. 50, 6184 – 6191
8. Fridman, R., Fuerst, T. R., Bird, R. E., Hoyhtya, M., Oelkuct, M., Kraus, S.,
Komarek, D., Liotta, L. A., Berman, M. L., and Stetler-Stevenson, W. G.
(1992) J. Biol. Chem. 267, 15398 –15405
9. Strongin, A. Y., Marmer, B. L., Grant, G. A., and Goldberg, G. I. (1993) J. Biol.
Chem. 268, 14033–14039
10. Murphy, G., Willenbrock, F., Ward, R. V., Cockett, M. I., Eaton, D., and
Docherty, A. J. P. (1992) Biochem. J. 283, 637– 641
11. Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and
Seiki, M. (1994) Nature 370, 61– 65
12. Kolkenbrock, H., Hecker-Kia, A., Orgel, D., Ulbrich, N., and Will, H. (1997)
Biol. Chem. Hoppe-Seyler 378, 71–76
13. Strongin, A. Y., Collier, I., Bannikov, G., Marmer, B. L., Grant, G. A., and
Goldberg, G. I. (1995) J. Biol. Chem. 270, 5331–5338
14. Bigg, H. F., Shi, Y. E., Liu, Y. E., Steffensen, B., and Overall, C. M. (1997)
J. Biol. Chem. 272, 15496 –15500
15. Brooks, P. C., Stromblad, S., Sanders, L. C., von Schalscha, T. L., Aimes, R. T.,
Stetler-Stevenson, W. G., and Cherish, D. (1996) Cell 85, 683– 693
16. Wallon, U. M., and Overall, C. M. (1997) J. Biol. Chem. 272, 7473–7481
17. Overall, C. M., Wallon, U. M., Steffensen, B., De Clerck, Y., Tschesche, H., and
Abbey, R. S. (1998) in Inhibitors of Metalloproteinases in Development and
Disease (Edwards, D., Hawkes, S., and Khokha, R., eds) Gordon and
Breach, Amsterdam, Holland, in press
18. Crabbe, T., Joannou, C., and Docherty, A. J. P. (1993) Eur. J. Biochem. 218,
431– 438
19. Windsor, L. J., Birkedal-Hansen, H., Birkedal-Hansen, B., and Engler, J. A.
(1991) Biochemistry 30, 641– 647
20. Murphy, G., Nguyen, Q., Cockett, M. I., Atkinson, S. J., Allan, J. A., Knight,
C. G., Willenbrock, F., and Docherty, A. J. P. (1994) J. Biol. Chem. 269,
6632– 6636
21. Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer,
J. L., Kronberger, A., He, C., Bauer, E. A., and Goldberg, G. I. (1988) J. Biol.
Chem. 263, 6579 – 6587
22. Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A., and
Goldberg, G. I. (1989) J. Biol. Chem. 264, 17213–17221
23. Banyai, L., and Patthy, L. (1991) FEBS Lett. 282, 23–25
24. Collier, I. E., Krasnov, P. A., Strongin, A. Y., Birkedal-Hansen, H., and Goldberg, G. I. (1992) J. Biol. Chem. 267, 6776 – 6781
25. Steffensen, B., Wallon, U. M., and Overall, C. M. (1995) J. Biol. Chem. 270,
11555–11566
26. Shipley, J. M., Doyle, G. A. R., Fliszar, C. J., Ye, Q.-Z., Johnson, L. L., Shapiro,
S. D., Welgus, H. G., and Senior, R. M. (1996) J. Biol. Chem. 271,
4335– 4341
27. Overall, C. M., and Limeback, H. (1988) Biochem. J. 256, 965–972
28. Keung, W., Silber, E., and Eppenberger, U. (1989) Anal. Biochem. 182, 16 –19
29. Pytela, R., Pierschbacher, M. D., Argraves, S., Suzuki, S., and Ruoslahti, E.
(1987) Methods Enzymol. 144, 475– 489
30. Sodek, J., and Heersche, J. N. (1981) Calcif. Tissue Int. 33, 255–260
31. Hemler, M. E. (1988) Immunol. Today 9, 109 –113
32. Lohi, J., Lehti, K., Westermarck, J., Kahari, V., and Keski-Oja, J. (1996) Eur.
J. Biochem. 239, 239 –247
33. Corcoran, M. L., and Stetler-Stevenson, W. G. (1995) J. Biol. Chem. 270,
13453–13459
34. Sato, H., Takino, T., Kinoshita, T., Imai, K., Okada, Y., Stetler-Stevenson,
W. G., and Seiki, M. (1996) FEBS Lett. 385, 238 –240
35. Seftor, R. E. B., Seftor, E. A., Stetler-Stevenson, W. G., and Hendrix, M. J. C.
(1993) Cancer Res. 53, 3411–3415
36. Pei, D., and Weiss, S. J. (1996) J. Biol. Chem. 271, 9135–9140
37. Ohuchi, E., Imai, K., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1997) J. Biol.
Chem. 272, 2446 –2451