(Received for publication, November 21, 1994)
From the
Several recent investigations have demonstrated that matrix
metalloproteinase-2 (MMP-2) binds to the cell surface and undergoes
zymogen activation via a plasma membrane-associated activity. The
purpose of this study was to determine if association of MMP-2 with the
plasma membrane also modulates the catalytic efficiency of the active
enzyme. Using density gradient centrifugation, we isolated the plasma
membrane fractions of two ovarian adenocarcinoma cell lines, DOV 13 and
OVCA 432, previously described either to express MMP-2 or to express no
gelatinolytic metalloproteinases, respectively. While DOV 13 cells
contained plasma membrane-associated MMP-2 and OVCA 432 did not, both
cell types were able to bind exogenous MMP-2. Furthermore, plasma
membrane fractions from these cells significantly enhanced the rate of
cleavage of [C]gelatin I substrate by both
MMP-2
tissue inhibitor of metalloproteinases-2 (TIMP-2) complex
(2.5-8-fold) and TIMP-2-free MMP-2 (5.9-fold). This stimulatory
activity was dose-dependent, soluble in Triton X-100, and abolished by
trypsin treatment of the membranes, but was stable to heat treatment.
Plasma membrane stimulation of MMP-2 resulted in a 3.8-4.6-fold
increase in the catalytic efficiency of gelatinolysis. These data
suggest that, in addition to promoting zymogen activation, cell surface
binding of MMP-2 may regulate enzyme activity by increasing the rate of
substrate cleavage. Via this mechanism, tumor cell types that do not
express MMPs (such as OVCA 432) nevertheless may be able to utilize
exogenous MMP-2 to mediate proteolysis associated with invasion and
metastasis.
Proteolytic degradation of the extracellular matrix is a
postulated mechanism by which tumor cells initiate tissue invasion and
metastasis(1) . Overexpression of plasminogen activators (PAs) ()has long been associated with malignancy and, more
recently, elevated secretion of matrix metalloproteinases (MMPs) by
tumor cells also has been demonstrated (2, 3, 4) . The combined actions of the broad
specificity serine proteinase plasmin (formed upon conversion of the
zymogen plasminogen by PAs) and MMPs on the glycoprotein, proteoglycan,
and collagenous components of basement membranes enable tumor cells to
penetrate these tissue barriers. Furthermore, increasing evidence
demonstrates that cell surface localization of proteinases is a common
cellular strategy for regulating pericellular proteolysis, as
exemplified by urinary-type PA (u-PA), the activity of which is
localized predominantly on the cell surface via binding to a specific
receptor, u-PA
receptor(5, 6, 7, 8, 9) .
Although recent evidence suggests that MMPs may also be associated
with the cell surface, the mechanism of these interactions as well as
the potential relevance to tumor metastasis remains to be elucidated.
Previous studies employing subcellular fractionation and electron
microscopic immunolocalization have demonstrated MMP-2 and MMP-9-like
type IV collagenases as well as interstitial collagenase (MMP-1)
associated with the plasma membranes of human lung and pancreatic
cancer cells(10, 11, 12) . Furthermore, a
class of specific, saturable cell surface binding sites for MMP-2 (K = 2 nM) has been
described on breast cancer cells(13) , although no membrane
protein that functions as a metalloproteinase receptor has been
identified. In addition, a plasma membrane-associated component that
activates the zymogen of MMP-2 (proMMP-2) has been
described(14, 15, 16, 17) . This
activator is detergent-soluble and trypsin- and heat-sensitive, and its
function requires the carboxyl-terminal domain of
MMP-2(14, 15, 16, 17) . A newly
discovered ``membrane-type'' MMP (MT-MMP), an MMP family
member with a transmembrane domain, catalyzes the activation of
proMMP-2 when overexpressed in HT1080 cells (18) and may be
related to the previously described membrane activator. These data
indicate that a distinct cell surface binding molecule as well as a
membrane-tethered activator may be present on the cell surface. Taken
together, these findings suggest that cell surface localization may
function as a mechanism by which the activity of MMPs, particularly
MMP-2, is regulated.
The current study is designed to assess the
effect of membrane association on the catalytic activity of active
MMP-2. To address this question, we have prepared the plasma membrane
fractions of two ovarian adenocarcinoma cell lines, DOV 13 and OVCA
432, which either express MMP-2 or express no gelatinolytic
metalloproteinases, respectively (19) . These membrane
fractions were utilized to determine the effects of plasma membrane
association on the rate of [C]gelatin I
hydrolysis by active MMP-2. Our data demonstrate that the catalytic
activity of MMP-2 is enhanced by plasma membrane association,
suggesting a mechanism by which the activity of MMPs may be regulated.
Figure 1:
MMP-2 binding to ovarian
carcinoma cell membranes. DOV 13 cells (panelA, lanes 1 and 2, and panelB) and
OVCA 432 cells (panel A, lanes 3 and 4, and panelC) were fractionated as described under
``Experimental Procedures,'' and aliquots of the membrane
preparations were analyzed using gelatin zymography. PanelA, aliquots of the crude plasma membrane fraction of both
cell types were incubated with 0.16 µM proMMP-2TIMP-2 complex (lanes2 and 4) or with buffer only (lanes1 and 3) in Tris-Ca-sucrose for 1 h at 37 °C. Subsequently, the
membrane samples were centrifuged at 50,000
g for 30
min, the supernatant was discarded, and the membranes were resuspended
and washed twice. Following resuspension to the original volume,
10-µl aliquots of the MMP-2-treated and untreated membranes were
electrophoresed. Panel B, the Percoll gradient fractions
1-6 (lanes 1-6) of DOV 13 cells were subjected to
gelatin zymography (2 µg of membrane protein/lane). Panel
C, the Percoll gradient fractions 1-6 (lanes1-6) of OVCA 432 cells were subjected to gelatin
zymography (2 µg of membrane protein/lane). The migration positions
of proMMP-2 (72 kDa) and proMMP-9 (92 kDa) standards are indicated. The
gelatinase activities observed were inhibited by o-phenanthroline in parallel experiments (data not shown),
with the exception of the faint gelatinase activity migrating above
proMMP-2 in crude OVCA 432 membranes (panel A, lane
3).
Figure 2:
A
plasma membrane-associated component enhances gelatinolysis by MMP-2.
The rate of cleavage of [C]gelatin type I (714
nM) by MMP-2
TIMP-2 (5.38 nM) was determined in
the absence (designated No on the x axis) and
presence of density gradient membrane fractions (designated 1-6) from DOV 13 (panel A) and OVCA 432 (panel B) cells or their Triton X-100 extracts (1 µg of
membrane protein added) as described under ``Experimental
Procedures.'' As controls, the membrane fractions were incubated
with substrate in the absence of exogenous MMP-2 TIMP-2. Data represent
the means of five to seven experiments, and error bars signify standard
deviation. The symbols are as follows: opencircles,
membrane fraction + enzyme; filled circles, membrane
extract + enzyme; open squares, membrane fraction alone; filled squares, membrane extract alone. The 5`-nucleotidase
activity (µM phosphate/µg protein/h) of the membrane
fractions are indicated. The fraction density values were: DOV 13,
fraction 1 = 1.038 g/ml; fraction 2 = 1.042: fraction 3
= 1.045; fraction 4 = 1.048; fraction 5 = 1.050;
fraction 6 = 1.052; OVCA 432, fraction 1 = 1.038 g/ml;
fraction 2 = 1.043; fraction 3 = 1.044; fraction 4
= 1.047; fraction 5 = 1.049; fraction 6 =
1.052.
To determine the ability of membrane preparations to
bind exogenous MMP-2, purified proMMP-2TIMP-2 complex was
incubated with the membranes and the unbound ligand was removed by
washing. Increased MMP-2 activity was observed in association with both
the DOV13 and OVCA 432 membranes incubated with proMMP-2
TIMP-2 (Fig. 1A), indicating that these cells contain a
membrane-associated MMP-2 binding activity that may be related to the
MMP-2 receptor detected previously on breast cancer cells(13) .
In addition, these data demonstrate that cells such as OVCA 432, which
do not express any gelatinolytic MMPs, nonetheless have the capacity to
utilize exogenous MMP-2, suggesting that cell surface MMP-2 binding may
be an important determinant of tumor cell invasion and metastasis.
To characterize further the plasma membrane-associated stimulatory activity, dose-response experiments were performed. Using DOV 13 plasma membranes and detergent extracts, a dose-dependent increase in MMP-2 gelatinase activity was observed in the presence of increasing amounts of membrane material (Fig. 3). Maximal stimulation required the addition of 1000 or 250 ng of protein for the membranes and detergent extracts, respectively. Pretreatment of the DOV 13 plasma membrane samples with trypsin abolished the stimulatory activity (Fig. 3, inset, column3), whereas heat treatment (100 °C, 10 min) had no effect (Fig. 3, inset, column4). In contrast, the previously described plasma membrane-associated proMMP-2 activator was observed to be both trypsin- and heat-sensitive(14, 16) . These data suggest that the stimulatory activity is a thermostable membrane protein distinct from the activator protein. The mechanism for the increase in stimulatory activity observed in the detergent extracts relative to the membrane fractions is not clear. This positive effect of solubilization does not appear to be related to the presence of inside-out vesicles in the membrane preparation, since solubilization of trypsin-treated membranes (as shown in Fig. 3, inset) did not partially restore the stimulatory activity abolished by trypsinization (data not shown), as would be predicted if a portion of the binding protein molecules were protected inside vesicles.
Figure 3:
Characterization of the MMP-2 stimulatory
activity. The dose dependence of the plasma membrane-associated MMP-2
stimulatory activity from DOV 13 cells (density gradient fraction 4)
was determined by measuring the rate of cleavage of
[C]gelatin type I (214 nM) by
MMP-2
TIMP-2 (5.38 nM) in the presence of increasing
amounts of plasma membranes (open circles) or plasma membrane
detergent extracts (closed circles). Base-line gelatin
cleavage in the absence of added membrane material is indicated by a solid horizontal line. Data represent the mean of five
experiments. Inset, the effect of trypsin and heat treatment
on the MMP-2 stimulatory activity. The rate of cleavage of
[
C]gelatin type I (714 nM) by
MMP-2
TIMP-2 (5.38 nM) was determined in the absence of
modulator (column1), in the presence of 1 µg of
DOV 13 cell plasma membrane fraction 4 (column 2), in the
presence of an equivalent volume of the same membranes trypsin-treated
as described under ``Experimental Procedures'' (column3), and in the presence of 1 µg of the same fraction
heated to 100 °C for 10 min (column4). Data
represent the mean of three experiments.
The effects of DOV 13 and OVCA 432
membranes on the kinetics of gelatin hydrolysis by MMP-2 were
determined. The reactions obeyed Michaelis-Menten kinetics in the
presence and absence of membranes (Fig. 4). When the kinetic
data was analyzed using a nonlinear regression fit to the
Michaelis-Menten equation, the stimulatory mechanism was similar for
membrane extracts from both cell types, with the observed stimulation
resulting predominantly from an increase in k (3-3.5-fold) (Table 1). The small changes in K
do not appear to be significant. These data
suggest that association with a plasma membrane protein causes a
molecular alteration in MMP-2, such as a conformational shift, that
promotes catalysis but does not alter the binding affinity for
substrate. Overall, the catalytic efficiency (k
/K
) of MMP-2 increased
4.6- and 3.8-fold when incubated with DOV 13 and OVCA 432 plasma
membranes, respectively. To the authors' knowledge, this report
is the first description of modulation of MMP activity by a protein
that functions as a nonessential activator.
Figure 4:
Kinetic analysis of MMP-2 catalysis in the
absence and presence of membrane stimulation. Michaelis-Menten plot of
the initial rates of [C]gelatin type I cleavage
of MMP-2
TIMP-2 (5.38 nM) determined in the presence of
varying amounts of [
C]gelatin type I with no
modulator (open circles) or with added DOV 13 (filled
circles) or OVCA 432 (open triangles) membrane detergent
extract (fraction 4; 100 ng of membrane protein). Data represent the
mean of six experiments. Kinetic constants were derived from the data
as described in Table 1.
To summarize, we have observed that endogenous MMP-2 is found in association with the plasma membranes of ovarian adenocarcinoma cells. These membranes, even when derived from cells not expressing MMPs, also have the ability to bind exogenous MMP-2. Furthermore, as an apparent consequence of this binding interaction, MMP-2 activity is stimulated by a direct effect on the active metalloproteinase, resulting in an increase in the catalytic efficiency of gelatin type I hydrolysis. The thermostable stimulatory activity appears to be a plasma membrane-associated protein. These data demonstrate that, together with the inhibitory TIMPs and the newly described membrane activator (MT-MMP), a cell surface MMP-2-binding protein also appears to participate actively in the regulation of MMP-2 activity. The presence of this activity on tumor cells suggests a potentially important role in the regulation of proteolysis associated with invasion and metastasis.