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Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Emilia Mira, Department of Immunology and Oncology, Centro Nacional de Biotecnología, Spanish Research Council CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain. E-mail: emira{at}cnb.uam.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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vß5 integrin,
a vitronectin receptor. The levels of v- and
ß5-subunits expressed in MCF-7 cells depend on the IGF-I
concentration, which triggers an increase in both of these subunits.
Based on these results, we suggest that IGF-I-induced MCF-7 cell
migration is mediated by the MMP-9 activity on the cell surface and by
vß5 integrin. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tumor dissemination has been correlated with matrix metalloproteinases (MMP) due to their proteolytic activity on extracellular matrix proteins (6). More recently, MMP have been implicated in the release of the soluble form of several growth factors (7), and interestingly, several IGF-binding proteins have been described as MMP substrates (8, 9, 10). Together, these data enlarge the relevance of this proteinase family on sustaining an invasive phenotype. Among its members, MMP-9 exhibits a widespread expression in a variety of human cancers, suggesting its involvement in human tumor dissemination (11). Direct evidence of its role in tumor progression is derived from transfection experiments in which the MMP-9 gene in nonmetastatic cells endowed them with the ability to metastasize (12).
Besides the requirement of proteolysis, adhesion to the extracellular
matrix is necessary for tumor invasion (13). Integrin molecules belong
to the largest family of extracellular matrix (ECM) adhesion receptors
and have been implicated in several processes, including cell
migration, invasion, and metastasis (14, 15). The link between
integrins and MMP comes from experimental evidence such as the
colocalization of the integrin vß3 and
MMP-2 (16) and the positive regulation of MMP-1 expression by the
integrin 2ß1 (17). Furthermore, integrin
function is a requirement for IGF-I-mediated chemotaxis; in MCF-7
cells, migration through vitronectin and type IV collagen is dependent
on vß5 and
2ß1, respectively, and in smooth muscle
cells, ligand occupancy of vß3 modulates
the migration process (2, 3). The dissemination of several malignant
tumor cells is also dependent on the cooperation between
vß5 and IGF-I (4).
Although IGF-I-triggered migration of MCF-7 has been previously
reported (3), the mechanism mediating the MCF-7 migratory response to
IGF-I through the ECM component vitronectin has not yet been
elucidated. Here we have identified MMP-9 and its proteolytic activity
on the MCF-7 cell surface; we describe the IGF-I-induced changes in
MMP-9 and the vitronectin receptor vß5 and
their correlation with the migration response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-IR3), anti-MMP-9 (Ab-1), and anti-MMP-2 (Ab-3) mAb were purchased
from Oncogene (Cambridge, MA). The polyclonal anti-MMP-9
antibody (Ab), obtained by immunization with a synthetic peptide (amino
acids 422–434), was purchased from Research Genetics, Inc. (Huntsville, AL). Anti-v (K267) is a
polyclonal Ab obtained by immunization with a synthetic peptide (amino
acids 372–385), and anti-v (6D1) mAb was obtained by
immunizing with purified human placenta integrin
vß5; both were produced in our laboratory.
The anti-ß5 polyclonal Ab and
anti-vß5 (p1F6) mAb were purchased from
Boehringer Ingelheim (Heidelberg, Germany), the anti-ß1 (DF5) mAb was
obtained from Chemicon (Temecula, CA), and the anti-2 mAb (p1E6) was
obtained from Life Technologies (Paisley, Scotland, UK).
Migration assays
MCF-7 cells (105) were trypsinized and resuspended
in serum-free DMEM with 0.01% BSA, then seeded in the upper chamber of
vitronectin-coated transwells (5 µg/ml in PBS, 18 h at room
temperature). The lower chambers were loaded with DMEM-BSA, with or
without IGF-I. In some assays, cells were preincubated with Abs or
inhibitors for 30 min at room temperature before seeding in the
transwell. Cells were incubated for 18 h at 37 C in 5%
CO2. The chamber was disassembled, and cells on the upper
surface were removed. Filters were stained, and cell numbers were
calculated as previously described (3).
MMP activity measurement
Cell surface MMP activity was measured using a modification of
previously described methods (20, 21). Briefly, 5 x
104 cells resuspended in complete medium were seeded in
96-well microtiter plates (Nunc, Roskilde, Denmark). Cells were washed
three times with HBSS (Life Technologies), and the
fluorogenic peptide
Dnp-Pro-ß-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)-NH2
(Bachem, Bubendorf, Switzerland) was added in HBSS
containing 0.01% NaN3. Plates were incubated at 37 C, and
at the indicated time the increment in fluorescence was measured at an
excitation wavelength of 485 nm and an emission wavelength of 530 nm
with a Cytofluor microplate reader (Millipore Corp.,
Bedford, MA). When IGF-I was assayed, cells were starved for 6 h
in serum-free medium and incubated with or without IGF-I for the time
indicated. Medium was then removed, and after washing with HBSS,
proteolytic activity was measured. When inhibitors were tested, they
were preincubated for 30 min before the addition of the fluorogenic
substrate. Fluorescence was normalized by cell staining with crystal
violet (Sigma Chemical Co.). Zymograms were performed as
previously described (20) in 10% SDS-polyacrylamide gels containing
gelatin (1 mg/ml).
Fluorescence-activated cell sorting (FACS) analysis
MCF-7 cells were detached, washed with ice-cold PBS, and
resuspended at 106 cells/ml in PBS with 0.5% BSA and
0.01% NaN3. Anti-MMP-9, anti-MMP-2, or
anti-vß5 mAb were added, followed by a
fluorescein isothiocyanate-labeled goat antimouse IgG (Southern Biotechnology Associates, Birmingham, AL). Cell-associated
fluorescence was visualized in an EPICS XL flow cytometer
(Beckman Coulter, Inc., Hialeah, FL).
PCR analysis
First strand complementary DNA (cDNA) was prepared from
MCF-7-isolated messenger RNA and HT1080 total RNA. Oligonucleotide
primers for PCR amplification of gelatinase B were synthesized as
previously described (22), and each cycle consisted of denaturation at
94 C, annealing at 55 C, and elongation at 72 C for 30 sec in each step
after 1 min at 94 C in the first cycle. The primer sequences for PCR
amplification of GAPDH were as follows: sense primer,
5'-TCCTGCACCACCAAC TGCTTA-3'; and antisense primer,
5'-ACCACCCTGTTGCTGTAGCC-3'. Each cycle consisted of denaturation at 94
C and annealing at 57 C for 30 sec in each step and elongation at 72 C
for 1 min after 1 min at 94 C in the first cycle.
Western blot analysis
Conditioned medium samples were electrophoresed on 10%
SDS-polyacrylamide gels under reducing conditions, electroblotted, and
blocked with 5% (wt/vol) nonfat milk in PBS-Tween-20 (0.1%, vol/vol).
The filter was incubated with an IgG fraction of the rabbit anti-MMP-9
serum, followed by a peroxidase-conjugated goat antirabbit Ig
(DAKO Corp. A/S, Glostrup, Denmark). Reactions were
developed using the ECL system (Amersham, Aylesbury, UK).
Cell lysates were obtained by incubating monolayer cells in lysis
buffer containing 1% Triton X-100, 50 mM Tris (pH 7.5),
and 300 mM NaCl at 4 C for 15 min. After detaching cells by
scraping, insoluble material was removed by centrifugation. To detect
integrin subunits, samples were electrophoresed on 8%
SDS-polyacrylamide gels under nonreducing conditions, then processed as
described above.
Immunoprecipitation of biotin-labeled integrin
Cells were grown to 80% confluence and labeled with
Sulfo-NHS-LC-Biotin (Pierce Chemical Co., Rockford, IL)
following the manufacturer’s instructions. Cell lysates were obtained
as previously described (23) and were immunoprecipitated with mAb
anti-v or anti-vß5 (4 C,
overnight), followed by agarose-conjugated goat antimouse IgG. The
immunoprecipitated proteins were separated on 10% SDS-polyacrylamide
gels under reducing conditions, and blots were developed using
peroxidase-labeled streptavidin (Amersham) followed by
enhanced chemiluminescence.
Adhesion assay
Vitronectin-coated 96-well microtiter plates (1 µg/ml in PBS,
overnight at 4 C) were blocked with 5% BSA in PBS for 3 h at 37
C. MCF-7 cells (3 x 105) in DMEM-BSA were
preincubated with anti-vß5 and
anti-2 mAb for 30 min and seeded. After a 2-h
incubation, plates were washed three times with DMEM-BSA, and adherent
cells were stained with
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(Sigma Chemical Co.) (24).
Statistical analysis
All experiments were repeated two or three times. The mean and
SD of triplicate points from one or two experiments are
shown. Zymographic data and the corresponding densitometry quantitation
are presented from a representative experiment. The significant
differences between group values were analyzed using the Kruskal-Wallis
test, and the Mann-Whitney test was used to compare paired values.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). IGF-I-mediated MCF-7 cell migration
is inhibited by anti-IGF-I mAb (data not shown) as well as by
anti-IGF-1R mAb, as previously described (3). This migration response
to IGF-I is independent of de novo protein synthesis, as
cycloheximide has no effect on this assay (data not shown).
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). We thus conclude that
IGF-I-triggered MCF-7 cell migration is specific and dose dependent, is
mediated through the IGF-1R, and involves an MMP.
MMP-9 activity is anchored to the MCF-7 cell surface
MMP activity is usually analyzed in cell culture-conditioned
medium, as most MMP family members are secreted proteases (6). To
characterize the MMP involved in MCF-7 migration, we therefore analyzed
MCF-7 cell-conditioned medium by gelatin zymography, using
HT-1080-conditioned medium as a control (25). MCF-7-conditioned medium
shows gelatinolytic activity (Fig. 2A, upper panel, lane 3) coincident with the MMP-9 induced in
the HT-1080 medium after PMA treatment (Fig. 2A, upper
panel, lanes 1–2). Although the MMP-9 detected is the 92-kDa
latent enzyme form (25), it is activated by the
denaturation-renaturation process in the gel, allowing analysis of its
activity (20). To confirm the identity of this gelatinolytic activity
as MMP-9, Western blot analysis was performed with a polyclonal
anti-MMP-9 Ab. A specific band was detected in MCF-7-conditioned medium
that coincides with the mol wt found in PMA-induced HT-1080
cell-conditioned medium and absent in the medium from untreated HT-1080
cells (Fig. 2A, lower panel).
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). This activity is
independent of the presence of vitronectin (data not shown). BB-94
inhibited this proteolytic activity (Fig. 2C), with 80% inhibition
after 1 h of incubation in the presence of the substrate. Less
than 20% inhibition of the proteolytic activity was observed when
aprotinin was included in the assay (Fig. 2C).
To further identify this cell-bound MMP activity as that of MMP-9, FACS
analysis was performed using MMP-9-specific mAb and MMP-2 as a negative
control. Incubation of MCF-7 with the anti-MMP-9 Ab promoted a
cell-associated shift in fluorescence intensity compared with that
observed using anti-MMP-2 or the second Ab alone (Fig. 2D). Finally,
using MMP-9-specific primers, we detected messenger RNA encoding MMP-9
in MCF-7 cells (Fig. 2E, lane 3), with PMA-treated HT-1080 cells as a
control (Fig. 2E, lane 1). We conclude that MMP-9 is expressed by MCF-7
cells, is located on the cell surface, and is found in the conditioned
medium, and that its activity is at least in part responsible for MCF-7
cell-associated protease activity.
IGF-I triggers changes in MMP-9 activity in MCF-7 cells
MMP-9 is present in MCF-7 cell-conditioned medium, and its
activity is associated with the cell surface in the absence of IGF-I
treatment. As MCF-7 cells migrate through vitronectin only in the
presence of IGF-I, we analyzed the possible effect of IGF-I treatment
on MMP-9 activity, measuring both cell surface-associated proteolytic
activity and MMP-9 in the conditioned medium. A dose-dependent decrease
(r2 = 0.71; P < 0.05) in cell
surface-associated MMP-9 activity was observed when starved cells were
treated with IGF-I for 18 h (Fig. 3A), whereas a 2.5-fold increase in the
latent soluble form of MMP-9 was detected in the conditioned medium
(Fig. 3B). This treatment does not activate the latent MMP-9 form, as
we observed no lower mol wt bands corresponding to activated forms
(29). MMP-9 enhancement in the conditioned medium is vitronectin
independent, as also found for the MMP-9 membrane-associated activity
(data not shown). The MMP-9 in the conditioned medium appears to be
independent of MCF-7 migration, as this zymogen form is not activated
by IGF-I treatment.
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). To further analyze the possible correlation between
proteolytic activity and invasion in MCF-7 cells, we performed a
time-course experiment with cells untreated or IGF-I treated, and
measured cell surface proteolytic activity. Although a slight increase
in this activity is detected in unstimulated cells, IGF-I addition (1
ng/ml) produced a significant increase in proteolytic activity, with a
maximum value after 3–4 h of preincubation (Fig. 4). After 18 h of incubation, there
was a decrease in cell membrane-associated proteolytic activity,
similar to that observed in the dose-response experiment (Fig. 3A).
This early increase in proteolytic activity in response to IGF-I,
therefore, correlates with MCF-7 cell invasion.
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vß5 integrinvß5 vitronectin
receptor in this cell line (3). An
anti-vß5 mAb specifically blocks MCF-7
cell adhesion to vitronectin (data not shown) and inhibits migration,
as previously shown (3). To explore the effect of IGF-I treatment on
vß5, MCF-7 cells were incubated with
IGF-I, and the cell lysates were analyzed in Western blot. When IGF-I
was used at 1 ng/ml, neither v- nor
ß5-subunit levels changed with respect to those in
untreated cells; an increase in both subunits was found, however, when
cells were incubated with IGF-I at higher doses (100 and 1000 ng/ml;
Fig. 5A). The ß1 integrin
subunit remained unchanged at any IGF-I concentration employed (Fig. 5A). To ascertain whether the integrin vß5
levels in total extracts can be correlated with their presence on the
MCF-7 cell surface, we performed immunoprecipitation experiments with
biotinylated extracts. Starved cells were untreated or treated with
IGF-I (1 ng/ml) overnight and subsequently biotinylated. Biotinylated
extracts were immunoprecipitated with anti-v,
anti-vß5, or an irrelevant isotype-matched
Ab as a control. Although the band corresponding to v
was quite faint, both v- and ß5-subunits
were immunoprecipitated by anti-v and
anti-vß5 (Fig. 5B). The band intensity was
similar in both extracts (Fig. 5B), confirming the result observed in
Western blot analysis of total extracts. No changes in
vß5 staining were detected by FACS
analysis in MCF-7 cells under the same incubation conditions (data not
shown). In this system, therefore, the IGF-I concentration leading to
maximal migration (1 ng/ml) does not alter
vß5 levels; higher IGF-I doses, associated
with a lack of MCF-7 migration, produce an increase in
vß5 integrin levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most MMPs described to date are soluble enzymes secreted as proenzymes,
processed to generate their active forms (31), but cell
surface-associated MMP activity has recently been described (32, 33).
Membrane-type MMP have been identified on fibroblast cells of human
carcinomas (28, 33), and in addition, the soluble MMP gelatinase A
exhibits cell surface association in invasive cells (16). We have
described the presence of MMP-9 in the conditioned medium of MCF-7
cells, as analyzed by Western blot and gelatin zymography. We have also
detected MMP-9 on the MCF-7 cell surface; using an MMP-specific
substrate, we observed activity in intact MCF-7 cells, and as expected
for an MMP, this activity was inhibited by BB-94, but not by aprotinin.
Furthermore, an anti-MMP-9 mAb specifically stains MCF-7 cells. These
results led us to conclude that MMP-9 is expressed by MCF-7 cells and
is located on the cell surface as well as in the conditioned medium.
Although both MMP-2 and MMP-9 gelatinases have been found on the cell
surface (16, 32, 34, 35, 36), this is, to our knowledge, the first
description of MMP-9 activity on the cell membrane. At least two events
must take place in or near the cell surface to allow the appearance of
MMP-9 activity, the binding of the newly synthesized proform, followed
by its activation. High affinity binding of pro-MMP-9 to the collagen
type IV 2(IV) chain has been described in MCF10A cells (34). This
binding does not, however, result in zymogen activation, which, in
turn, has been associated with components of the urokinase-plasmin
system (35, 36). In our experimental system, we found the inactive
proform in the conditioned medium and detectable MMP-9 activity only at
the cell membrane level. Our data, therefore, support the idea that
although inactive MMP-9 binds to ECM components, an activation step is
required at the cell membrane. The mechanism of this activation in
MCF-7 requires further study, although the fact that aprotinin does not
block MCF-7 invasion suggests that the plasmin system is not
involved.
Our data further support the hypothesis of protease focalization on the cell membrane as a possible mechanism to increase proteolytic activity, producing more efficient invasion, as previously suggested (26, 30, 37). Long term IGF-I treatment of MCF-7 cells produces a dose-dependent decrease in MMP-9 activity on the cell surface and an increment in MMP-9 in the conditioned medium. When cycloheximide was included in the invasion assay, the results were unchanged; de novo protein synthesis is therefore not required for MCF-7 cell migration in response to IGF-I. From these observations, we hypothesize that IGF-I promotes a redistribution of an MMP-9 pool between the cell surface and the medium. Only the MMP-9 proform has been detected in the MCF-7-conditioned medium; therefore, MMP-9 in the MCF-7-conditioned medium does not appear to correlate with the invasive phenotype. A cell surface-associated protease activity would thus seem to be more relevant than a soluble form in the enhancement of a migration response. The maximal migration rate was observed with 1 ng/ml IGF-I, a dose leading to a significant increase in MCF-7 cell surface-associated MMP-9 activity. The decrease detected after 18-h incubation could be explained by the autoproteolytic activity of the protease, leading to its inactivation. In the absence of exogenous IGF-I, the increase in proteolytic activity is gradual, probably due to the autocrine IGF-I produced (38), and the concomitant inactivation must be delayed.
Cell movement can be observed as a continuous interplay between
adhesion and de-adhesion events (39, 40). Adhesion receptors such as
integrins are needed to promote cellular invasion (41). Indeed, our
results indicate that IGF-I-triggered MCF-7 chemotaxis requires cell
adhesion to a specific ECM substrate through integrins, as blocking of
vß5 integrin binding with an antagonist Ab
results in the inhibition of the IGF-I-induced invasive response on
vitronectin. These data are similar to those reported previously (3).
Although cell attachment to the proper ECM substrate is required for
migration, it is not sufficient in itself, as MCF-7 does not invade
through vitronectin in the absence of this chemoattractant. It could
thus be hypothesized that some cooperation should exist between
integrins and IGF-I, either by modification of the integrin levels or
by affecting the integrin-mediated signaling pathways.
IGF-I treatment of MCF-7 cells at the dose that induces maximum
invasion does not promote any change in the
vß5 integrin levels. These data concur
with results reported previously for several cell lines in which cell
migration is regulated by mechanisms independent of changes in the
integrin levels (4, 42, 43). On the other hand, IGF-I at high doses
produces an increase in vß5 integrin
levels that parallels with decreased migration. An excess of cell
adhesion to the underlying ECM could therefore be negative for cell
migration, as described for CHO cells, in which integrin accumulation
in the cell membrane diminishes cell migration speed (40).
Adhesion to the ECM and proteolysis of ECM components are necessary for
tumor invasion (16, 17, 44). A functional link between adhesion and
proteolysis has been shown in melanoma cells, in which MMP-2 is induced
and cellular invasion increased after vitronectin ligation to the
integrin vß3 (45). In our system, adhesion
and proteolysis are effected by the integrin
vß5 and MMP-9, respectively. Precise
regulation of these two components must take place to achieve the
invasive phenotype. MMP-9 must be localized and active on the cell
surface, where membrane association may affect its activity, as has
also been suggested for MMP-2 (45). The levels of integrin and its
activated state in response to IGF-I treatment must modify cell
spreading, adhesion, and detachment, which could, in turn, affect MMP
activity, leading to an invasive phenotype.
In conclusion, in addition to the role of integrins in cell migration, we highlight the importance of cell surface-anchored MMP-9 activity in mediating IGF-I chemoattractant activity. This finding emphasizes cell surface proteases as suitable targets in the prevention of cell dissemination, particularly in the case of tumor metastasis.
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Acknowledgments |
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v Ab; I.
López-Vidriera for help with flow cytometry; Dr. F. Ortego for
help with statistical analyses; and C. Mark for editorial
assistance. |
Footnotes |
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Received May 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 integrin
is necessary for smooth muscle cells to migrate in response to
insulin-like growth factor I. Proc Natl Acad Sci USA 93:2482–2487vß5 to promote tumor cell
dissemination in vivo. J Clin Invest 99:1390–1398[Medline]
v-integrins in tumour progression and metastasis. Semin
Cancer Biol 7:129–138[CrossRef][Medline]
vß3. Cell 85:683–693[CrossRef][Medline]
2ß1 is a positive regulator of collagenase
(MMP-1) and collagen 1 (I) gene expression. J Biol Chem 270:13548–135525ß1 and 4ß1
integrins regulates metalloproteinase gene expression in fibroblasts
adhering to fibronectin. J Cell Biol 129:867–879This article has been cited by other articles:
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Z. Lu, G. Jiang, P. Blume-Jensen, and T. Hunter Epidermal Growth Factor-Induced Tumor Cell Invasion and Metastasis Initiated by Dephosphorylation and Downregulation of Focal Adhesion Kinase Mol. Cell. Biol., June 15, 2001; 21(12): 4016 - 4031. [Abstract] [Full Text]
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), with
aprotinin (10 µg/ml; 
), or with BB94 (10 µM; •)
for 30 min before substrate addition. The fluorescence observed for
cells in the absence of proteinase inhibitors was considered 100% for
each time point indicated. The mean ± SD of
triplicate wells are represented. The asterisks indicate
a significant difference (P < 0.05) between
untreated and inhibitor-treated cells for each time point (by
Mann-Whitney test). D, MCF-7 cells were gently detached and incubated
with the Abs indicated followed by an FITC-labeled antimouse IgG Ab,
and the fluorescence intensity shift was analyzed by FACS. E, cDNA from
HT-1080 treated with PMA (lane 1) and MCF-7 treated with 1 ng/ml IGF-I
(lane 3) were employed in PCR amplification for 32 cycles with MMP-9-
and glyceraldehyde-3-phosphate dehydrogenase-specific oligonucleotides.
As a control, cDNA was replaced by H2O (lane 2).
