1
Laboratory of Tumor and Developmental Biology, University of
Liège, C.H.U. Sart-Tilman, B23,
Liège, Belgium
2
Unité INSERM U.514, Laboratoire Pol Bouin, IFR
53, C.H.U. Maison Blanche, Reims, France
3
Unité INSERM U.385, Faculty of Medicine, Nice,
France
4
Institut de Biologie et de Chimie des
Protéines, CNRS, U.P.R. 412, Lyon,
France
*
These authors contributed equally to this research
Author for correspondence (e-mail:
cgilles{at}ulg.ac.be
)
Accepted May 9, 2001
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SUMMARY |
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Key words: MT1-MMP, Laminin-5, Migration, MCF10A, Epithelial cells
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INTRODUCTION |
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The remodelling of the extracellular matrix (ECM) surrounding epithelial
cells is accordingly a mechanism frequently associated with cell migration.
Matrix metalloproteases (MMPs), a family of proteases that degrades specific
components of the ECM, have largely been implicated in the ECM degradation
associated with many processes that involve epithelial cell migration
(Matrisian, 1992; Werb,
1997
; Shapiro,
1998
; Murphy and Gavrilovic,
1999
, Nagase and Woessner,
1999
). Most MMPs are secreted
as inactive proenzymes and their activation requires the proteolytic removal
of the N-terminal pro-fragment (Nagase and Woessner,
1999
). By contrast, MT1-MMP
(membrane type-MMP) contains a transmembrane domain, which ensures the
anchorage of the protein at the cell membrane (Sato et al.,
1994
; Sato and Seiki,
1996
). MT1-MMP has been
described as a major activator of MMP-2 (Sato et al.,
1994
; Sato and Seiki,
1996
) but MT1-MMP also
possesses the ability to degrade ECM components, including gelatin,
-elastin, fibronectin, vitronectin, native fibrillar type I, II and III
collagen, and laminin-5 (Ln-5; Imai et al.,
1996
; Pei and Weiss,
1996
; Sato and Seiki,
1996
; Ohuchi et al.,
1997
; Koshikawa et al.,
2000
). The activity of MMPs,
including MT1-MMP, is regulated by specific tissue inhibitors of MMPs (TIMPs).
MMP-2 is unique in its binding of TIMP-2, and a trimolecular complex formed by
MT1-MMP, TIMP-2 and MMP-2 has been described (Strongin et al.,
1995
). The expression of
MT1-MMP has been detected in processes that involve epithelial cell migration
and ECM remodelling, and has been correlated to high invasive abilities in
tumour cell lines (Gilles et al.,
1996
; Sato and Seiki,
1996
; Gilles et al.,
1997
; Pulyaeva et al.,
1997
; Polette et al.,
1998
; Quaranta,
2000
). However, the mechanisms
by which MMPs and particularly MT1-MMP facilitate migration are still poorly
understood. The anchoring of MT1-MMP at the plasma membrane is thought to
contribute to a pericellular ECM degradation, either by its own degradative
potential or through its ability to activate MMP-2 at the cell surface
(Nakahara et al., 1997
; Chen
and Wang, 1999
; Quaranta,
2000
). A crucial issue is
therefore the determination of the expression and spatial distribution of
MT1-MMP in epithelial cells during cell migration. Indeed, a pericellular
degradation now appears as a key event, generating an adequate substrate for
cell migration in the immediate vicinity of migrating cells (Nakahara et al.,
1997
; Chen and Wang,
1999
; Murphy and Gavrilovic,
1999
; Hotary et al.,
2000
; Quaranta,
2000
).
Accordingly, rat laminin-5 (Ln-5) has recently been identified as a
substrate for both MT1-MMP and MMP-2 (Giannelli et al.,
1997; Koshikawa et al.,
2000
). Ln-5 is a heterotrimer
found in basement membranes (BM), which consists in the association of
3, ß3 and
2 subunits (Rousselle et al.,
1991
). It is a multifunctional
protein that, in apparent contrast, is involved in the static adhesion of
epithelial cells to the basement membrane (BM; Baker et al.,
1996
; Jones et al.,
1998
; Borradori and
Sonnenberg, 1999
; Nievers et
al., 1999
) and also in the
promotion of epithelial cell migration (Miyazaki et al.,
1993
; Zhang and Kramer,
1996
; Giannelli et al.,
1997
; Grassi et al.,
1999
; Goldfinger et al.,
1999
; Koshikawa et al.,
2000
). The proteolytic
degradation of Ln-5
3 and
2 subunits generates different
heterotrimeric forms of Ln-5 (Rousselle et al.,
1991
; Marinkovich et al.,
1992
; Vailly et al.,
1994
; Matsui et al.,
1995
), and appears to be
responsible for the contrasting activities (i.e. adhesion or migration)
attributed to Ln-5 (Giannelli et al.,
1997
; Goldfinger et al.,
1998
; Giannelli et al.,
1999
; Goldfinger et al.,
1999
; Koshikawa et al.,
2000
). Recent data have shown
that the degradation of
2 subunit of exogenously provided rat Ln-5 by
MMP-2 and/or MT1-MMP promotes cell migration in vitro (Giannelli et al.,
1997
; Giannelli et al.,
1999
; Koshikawa et al.,
2000
). Furthermore, evidence
that the degradation of Ln-5
2 chain could promote cell migration in
vivo has been given by the detection of specific fragments of
2 in
remodelling but not in quiescent mouse tissues and in rodent skin carcinoma
(Giannelli et al., 1997
;
Giannelli et al., 1999
).
In this study, we have used an in vitro migration assay coupled to
videomicroscopy analyses to study the expression and cellular distribution of
MT1-MMP in association with cell migration of human epithelial mammary MCF10A
cells. We show a stronger expression of MT1-MMP mRNA and protein in migratory
cells, and a distribution of MT1-MMP in lamellipodia and at the basal surface
of these migratory cells. Furthermore, we have found that Ln-5 was deposited
around migratory cells and also detected degraded fragments of Ln-5 2
subunit in migratory cultures of MCF10A cells. We have also used MMPs
inhibitors (BB94 and TIMP-2), MT1-MMP antisense oligonucleotides and Ln-5
blocking antibodies, which emphasized a functional contribution of MT1-MMP and
Ln-5 in cell migration.
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MATERIALS AND METHODS |
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In vitro migration assay
5x104 cells were seeded in growth medium inside a 6 mm
glass ring placed in the middle of a glass coverslip (22 mm in diameter).
Twenty-four hours after plating, the glass ring was removed and the cells were
covered with growth medium. In this model, the cells migrate as an outgrowth
from the confluent area initially delimited by the ring.
For epidermal growth factor (EGF)-induced migration, cells were plated inside the ring in complete growth medium for 24 hours. After the removal of the ring, cells were washed for 1 hour twice in serum-free medium and then covered with serum-free medium supplemented, or not, with EGF at 20 ng/ml.
For TIMP-2 and BB94 inhibition experiments, 72 hours after the ring removal TIMP-2 (at 1 µg/ml, Calbiochem, La Jolla, CA) and BB-94 (5x10-6 M, kindly provided by British Biotech, Oxford, UK) were added to the growth medium for 1 hour, before the quantification of cell migration.
For MT1-MMP antisense experiments, a T1-MMP antisense oligonucleotides kit was used (Biognostik, Gottingen, Germany). The control oligonucleotides were scrambled oligonucleotides of the MT1-MMP antisense oligonucleotides. The oligonucleotides were added in the growth medium at 4 µM (as recommended by the manufacturer) after the removal of the ring. Using FITC-labelled oligonucleotides, we determined that 144 hours were necessary to ensure an uptake of the oligonucleotides by at least 20% of the cells. The effect of control and MT1-MMP antisense oligonucleotides on cell migration were therefore measured 6 days after the removal of the ring and the addition of the oligonucleotides.
For Ln-5 antibody inhibition experiments, mouse Ln-5 blocking antibodies (clone P3H9-2, Bioproducts, Heidelberg, Germany) were added to the culture medium (at 25 µg/ml) 72 hours after the removal of the ring and incubated for 4 hours before quantification of the cell migration speed. Controls were incubated with mouse IgG.
Quantification of cell migration speed and cell trajectories
To quantify the expansion of the outgrowth, migratory monolayers were
placed on the stage of an inverted microscope (Nikon TMS-F, Tokyo, Japan),
connected to a video CCD camera (Cohu 4700, San Diego, CA) and a video monitor
(PVM 1371, Sony, Japan), so as to measure the outgrowth area.
To analyse and quantify the migratory speeds and trajectories of the cells,
the monolayers were first incubated with a fluorescent nuclear dye (Hoechst
33258, Molecular Probes, Eugene, OR). They were then placed in the
environmental chamber (37°C, 5% CO2) of a Zeiss IM35 inverted
microscope (Zeiss, Oberkochen, Germany) equipped with an epifluorescence
illumination source (excitation filter at 360 nm; emission filter at 510 nm)
and a low level SIT camera (Lhesa 4036) controlled by a microcomputer
(SparcClassic Workstation). An image was collected every 10 minutes for 30
minutes. Twenty cell nuclei selected in different zones of the culture were
then labelled manually on the computer for each time point. Cell migration was
characterized and quantified using a previously described software program
(Zahm et al., 1997) that
measures the nuclei trajectories, as well as the cell migration speed. As the
cell nuclei were labelled manually by the experimenter, the relative position
of the label on a nucleus pictured at different time points varied slightly.
Consequently, a movement of the cells nuclei could be detected (corresponding
to a speed of about 10 µm/hour) in the areas distant from the outgrowth
periphery. This movement was however random and was not considered as
migration. Hence, the corresponding areas were identified as stationary. By
contrast, the movement of the nuclei at the periphery of the outgrowth
corresponding to cell migration was clearly oriented towards the outside of
the outgrowth.
RT-PCR analyses
RNA extraction was performed from total migratory cultures using the RNA
miniprep kits as recommended by the manufacturer (Qiagen, Hilden, Germany).
RT-PCR was performed using 10 ng of total RNA. An internal control RNA
template containing the sequences of the different primers used to amplify
different MMPs was introduced in each sample for the standardization and
quantification of each RT-PCR reaction.
RT-PCR was performed using the GeneAmp Thermostable RNA PCR Kit (Perkin Elmer, Foster City, CA), and with pairs of primers for six MMPs and for 28S control amplification (Eurogentec, Seraing, Belgium). Forward and reverse primers for human MT1-MMP, MMP-2, MMP-9, MMP-1, MMP-3 and MMP-11 and 28S were designed as follows: MT1-MMP primers (forward 5'-CCATTGGGCATCCAGAAGAGAGC-3'; reverse 5'-GGATACCCAATGCCCATTGGCCA-3'), MMP-2 primers (forward 5'-GGCTGGTCAGTGGCTTGGGGTA-3'; reverse 5'-AGATCTTCTTCTTCAAGGACCGGTT-3'), MMP-1 primers (forward 5'-GAGCAAACACATCTGAGGTACAGGA-3'; reverse 5'-TTGTCCCGATGATCTCCCCTGACA-3'), MMP-3 primers (forward 5'-GATCTCTTCATTTTGGCCATCTCTTC-3'; reverse 5'-CTCCAGTATTTGTCCTCTACAAAGAA-3'), MMP-11 primers (forward 5'-ATTTGGTTCTTCCAAGGTGCTCAGT-3'; reverse 5'-CCTCGGAAGAAGTAGATCTTGTTCT-3') and 28S primers (forward 5'-GTTCACCCACTAATAGGGAACGTGA-3'; reverse 5'-GGATTCTGACTTAGAGGCGTTCAGT-3'). Reverse transcription was performed at 70°C for 15 minutes. Amplification cycles were as follows: 15 seconds at 94°C, 15 seconds at 68°C, 10 seconds at 72°C. Twenty-five cycles were allowed for MT1-MMP amplification, up to 35 cycles for MMP-2, MMP-1, MMP-3 and MMP-11 amplification, and 18 cycles for 28S amplification. Products were separated on acrylamide gels, stained with Gelstar (FMC, Bioproducts) and quantified by fluorimetric scanning (LAS-1000, Fuji). The ratio of each endogenous signal to its specific internal control was calculated and normalized to its the ratio to the 28S. These values were multiplied by the number of copies of internal controls added to the RT-PCR reactions. Results were therefore expressed as a number of copies per 10 ng of RNA, allowing the comparison of the expression of different MMPs that have been amplified using different PCR parameters.
Immunofluorescence
Monolayers were fixed with 4% paraformaldehyde in phosphate-buffered saline
(PBS) for 10 minutes at 37°C then treated with 0.1% Triton X-100 for
MT1-MMP and integrin labelling. For Ln-5 labelling, the cells were fixed with
methanol for 10 minutes at -20°C. The coverslips were then saturated for
30 minutes with 3% BSA in PBS.
For MT1-MMP immunostaining, monolayers were successively (after intermediate washes in PBS) incubated for 1 hour with a monoclonal antibody to MT1-MMP (clone 113-5B7 Chemicon, Temecula, CA), a biotinylated-sheep anti-mouse antibody (Amersham, Aylesbury, UK) and a Texas Red-conjugated streptavidin (Amersham).
For 6 integrin staining, cells were successively incubated with anti
6 integrin rat antibody (clone GoH3, Immunotech, Marseille, France), a
biotinylated goat anti-rat antibody (Sigma) and a Texas Red-conjugated
streptavidin (Amersham). For
3 integrin labelling, cells were incubated
subsequently with an anti
3 monoclonal antibody (clone P1B5, Dako) and
with a TRITC-conjugated rabbit anti-mouse antibody.
For Ln-5 labelling, the cells were successively incubated with an anti Ln-5
monoclonal antibody (clone GB3, described in Verrando et al.,
1987), a biotinylated-sheep
anti-mouse antibody (Amersham) and FITC-conjugated streptavidin
(Amersham).
After incubation with the different antibodies, nuclei were labelled with 4',6-diamidino-2-phenylindole (DAPI; 1 µg/ml) for 20 minutes. The coverslips were then mounted with Aquapolymount antifading solution (Agar, UK) onto glass slides and the slides were observed under a Zeiss fluorescence microscope or with a MRC 600 confocal laser scanning microscope (BioRad, Richmond, CA).
In situ hybridization
The cultures were fixed for 10 minutes in 4% paraformaldehyde in PBS,
dehydrated in ethanol 50% and 70%, rehydrated and treated with 0.2 N HCl for
20 minutes at room temperature. They were then washed in 2xSSC,
acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine for 10 minutes
and hybridized overnight with [35S]-labelled MT1-MMP antisense RNA
transcripts. This probe was prepared from the MT1-MMP cDNA insert that had
been cloned into pBluescript. The samples were then treated with RNase (20
µg/ml) for 1 hour at 37°C to remove the unhybridized probes, washed
under stringent conditions and detected autoradiographically by exposure to
D19 emulsion (Kodak, Rochester, NY) for 15 days. The control slides were
treated under the same conditions but were hybridized with
[35S]-labelled sense probes. Quantification of the in situ
hybridizations was performed using the Discovery system automated image
analyser (Becton-Dickinson, Mountain View, CA) that allowed the determination
of the mean area of the in situ hybridization grains per cell. The mean area
of the in situ hybridization grains per cell was measured automatically on 10
fields (500 cells) at high magnification (x500). In order to compare
MT1-MMP mRNA expression in stationary versus migratory cells, we performed
these measurements in the two rows of cells at the periphery of the outgrowth
and in the following rows of the cells (between the fourth and the tenth rows
of cells) on three independent experiments.
Degradation of purified human Ln-5 by recombinant MT1-MMP
Human Ln-5 was purified from human squamous carcinoma SCC25 cell
conditioned medium as described previously (Rousselle et al.,
1991). Purified Ln-5 (1 µg)
was incubated with the recombinant catalytic domain of human MT1-MMP (100-500
ng, Chemicon) in 50 mM Tris pH 7.5, 5 mM CaCl2, 150 mM NaCl for 16
hours at 37°C. The samples were then analysed by western blotting for the
expression of the
2 chain of Ln-5.
Western blotting analyses
Analyses of MT1-MMP and Ln-5 2 chain expression were performed on
protein extracts performed 72 hours after the removal of the ring on migratory
cultures of MCF10A cells (cultivated in complete growth medium or in
serum-free medium supplemented with EGF) or on stationary cultures of MCF10A
cells (cultivated in EGF/FCS-free medium) for 72 hours after the removal of
the ring.
Extracts were prepared by scraping the cells in RIPA buffer (50mM Tris (pH 7.4), 150 mM NaCl, 1% Igepal (v/v), 1% sodium deoxycholate (w/v), 5 mM iodoacetamide, 0.1% SDS (w/v)) containing protease inhibitors (1mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin and 10 µg/ml aprotinin).
Samples (8 µg for Ln-5 2 chain analyses and 20 µg for MT1-MMP
analyses) were mixed with 1/5 sample buffer (0.31 M Tris (pH6.8), 10% SDS
(w/v), 25% glycerol (v/v), 12.5% ß-mercaptoethanol (v/v) and 0.125%
bromophenol blue (w/v)) and boiled for 5 minutes. They were then separated on
7.5% and 12% SDS-PAGE gels for Ln-5 and MT1-MMP analyses respectively and
transferred to a PVDF filter (NEN, Boston, MA). The membranes were then
blocked with 5% milk (w/v), 0.1% Tween 20 (w/v) in PBS for 2 hours before
exposure to the primary antibody overnight at 4°C: a rabbit antibody
(clone L
2-1) generated against the C-terminal region of Ln-5
2
subunit or a monoclonal antibody directed against the hemopexin-like domain of
MT1-MMP (clone 2D7 kindly provided by Dr Rio, IGBMC, Illkirch, France). The
filters were then incubated either with a horseradish peroxydase-conjugated
swine anti-rabbit or goat anti-mouse antibody (Dako). Signals were detected
with an enhanced chemoluminescence (ECL) kit (NEN, Boston, MA).
Statistical analyses
Experiments were performed in triplicate at least three times each. Data
are expressed as means±s.d. Student's t test was used to
compare the migration speeds of the cells under various experimental
conditions. P<0.05 was considered to be significant.
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RESULTS |
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|
MT1-MMP is overexpressed in migratory MCF10A cell
In order to study more precisely the relationship between MT1-MMP and cell
migration, we performed in situ hybridization and immunofluorescence analyses
on migratory cultures of MCF10A cells. A stronger expression of MT1-MMP was
clearly observed both at the mRNA (Fig.
3A) and protein level (Fig.
3B) in cells at the periphery of the outgrowth, which has been
shown to be a subpopulation of migratory cells. Densitometric quantification
of the in situ hybridizations revealed a 2- to 2.4-fold increase in the
density of grains in the rows of cells at the periphery of the outgrowth when
compared to the density in the eight subsequent rows. Moreover,
immunofluorescence combined with phase contrast analyses clearly revealed that
MT1-MMP is present in lamellipodia identified as a phase-dark rim at the
leading edge of the cells (Fig.
3C). Confocal microscopy confirmed this particular distribution of
MT1-MMP in lamellipodia at the leading edge of migratory cells but also
revealed its presence at the basal surface of these migratory cells in contact
with the substrate (Fig.
3D).
|
Furthermore, using MMP inhibitors BB94 and TIMP-2, we showed a decrease of MCF10A cell migration (Fig. 4). The addition of MT1-MMP antisense oligonucleotides (with a maximal uptake of the oligonucleotides by about 20% of the cells) resulted in a decrease of MT1-MMP expression, as evaluated by immunofluorescence, and also diminished cell migration (Fig. 4). These data suggest a functional contribution of MT1-MMP overexpression and activity to MCF10A cell migration.
|
Ln-5 and MCF10A cell migration
AS rat Ln-5 has recently been described as a potential substrate for
MT1-MMP (Koshikawa et al.,
2000), the presence and
deposition of endogenously produced human Ln-5 was investigated in migratory
cultures of MCF10A cells. By immunofluorescence, Ln-5 appeared to be
overexpressed by migratory cells at the periphery of the outgrowth
(Fig. 5A, part a). The
deposition of Ln-5 was also emphasized by the presence of traces of Ln-5
behind cells which had migrated out of the outgrowth
(Fig. 5A, part b). Ln-5
blocking antibodies were also found to decrease MCF10A cell migration,
suggesting a contribution of the interactions between MCF10A cells and Ln-5 in
their migratory behaviour (Fig.
5B).
|
The distribution of 6 and
3 integrins, which constitute the
6ß4 and
3ß1 receptors known as two major receptors for
Ln-5, was also examined by immunofluorescence as a function of MCF10A cell
migration. These integrins were not only present in MCF10A migratory cultures
but also reorganized in function of cell migration. They were indeed mainly
found in lamellipodia of migratory cells, whereas in stationary cells,
3 was detected rather in places cell-cell contact and
6 at the
basal surface (Fig. 5C). These
results show that Ln-5 and its major receptors are expressed in our model but
also display a specific distribution during cell migration.
Ln-5 degradation and MCF10A cell migration
In order to examine the potential contribution of MT1-MMP on the
degradation of Ln-5 2 chain degradation during epithelial cell
migration, the pattern of endogenously produced
2 chain of Ln-5 was
examined by western blotting analyses in migratory cultures of MCF10A cells.
Four Ln-5
2 fragments could clearly be identified in total extracts of
migratory MCF10A cultures (Fig.
6A). The two largest fragments corresponded in size to those
detected in Ln-5 purified from squamous carcinoma SCC25 cell-conditioned
medium, and have previously been described as the unprocessed human Ln-5
2 subunit (155 kDa) and a processed fragment of human
2 (105
kDa; Rousselle et al., 1991
).
Furthermore, the incubation of purified Ln-5 with the recombinant catalytic
domain of human MT1-MMP generated fragments corresponding in size to those
identified in our migratory cultures of MCF10A cells
(Fig. 6A).
|
In order to relate the presence of the degraded fragments of Ln-5 2
chain to cell migration, we compared the Ln-5
2 pattern in migratory
cultures (incubated in complete medium) with the one obtained in cells from
cultures incubated in EGF/serum-free medium previously characterized as
stationary cultures (Gilles et al.,
1999
). In order to minimize
the potential contribution of serum-derived proteases in the processing of
Ln-5
2 chain, we also examined the
2 chain pattern in cells
cultivated in serum-free medium supplemented with EGF, which has previously
been shown to promote cell migration in our model (Gilles et al.,
1999
). It was verified that,
as observed for migratory cultures of MCF10A cells incubated in complete
medium, both MT1-MMP and Ln-5 expression was increased specifically in cells
at the periphery of the outgrowth in EGF-induced migratory cultures (data not
shown). Using these different culture conditions, we could show increased
amounts of Ln-5
2 and its three degraded fragments in migratory
cultures (incubated in serum- or in EGF-containing medium) in comparison with
those detected in EGF/serum-free stationary cultures
(Fig. 6B). As shown by MT1-MMP
western blotting analyses, this increase in Ln-5 fragments in migratory
cultures of MCF10A cells clearly correlated with increased amount of MT1-MMP
(Fig. 6C). These results also
revealed that MT1-MMP was mostly present as the 60 kDa form, known to be the
active form of the enzyme. As MMP-2 has also been shown to cleave rat Ln-5
2, we looked at MMP-2 expression by zymography analyses and did not
find any detectable levels of MMP-2 in cell extracts of MCF10A cells
cultivated with or without EGF in the migration assay (data not shown). These
data therefore suggest that MT1-MMP could contribute to the degradation of
endogenously produced Ln-5
2 during cell migration.
![]() |
DISCUSSION |
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Overexpression of MT1-MMP in epithelial cells associates with their
migratory status
Using an in vitro migration assay, we have shown a stronger expression of
MT1-MMP in cells located at the outgrowth periphery that, as determined by
time lapse videomicroscopy, represents a subpopulation of migratory cells. In
close relation to our observations obtained with the MCF10A cells, a
downregulation of MT1-MMP associated with increased cell-cell contacts has
been reported in a mouse mammary epithelial cell line (Tanaka et al.,
1997). In vivo, MT1-MMP has
also been reported in epithelial cells involved in processes that require cell
migration, such as nephrogenesis (Tanney et al.,
1998
; Kanwar et al.,
1999
), placentation (Nawrocki
et al., 1996
; Bjorn et al.,
1997
; Tanaka et al.,
1998
) or tumour invasion (Sato
and Seiki, 1996
; Ellerbroek
and Stack, 1999
). Our in vitro
model coupled to the videomicroscopy analyses nevertheless allowed us to
demonstrate a specific induction of MT1-MMP both at the protein and mRNA level
within the same cell line, in association with the expression of migratory
properties. Furthermore, our results showing that BB94, TIMP-2 and MT1-MMP
antisense oligonucleotides diminished MCF10A cell migration clearly emphasized
a functional contribution of MT1-MMP overexpression and activity in MCF10A
cell migration. It can therefore be suggested that MT1-MMP expression in
epithelial cells can be temporally and spatially regulated during cell
migration, and that it functionally contributes to this process.
MT1-MMP is distributed in lamellipodia and at the basal surface of
migratory cells in contact with the substrate
More than demonstrating an increase of MT1-MMP expression in relation with
cell migration, our results also showed a particular subcellular organization
of MT1-MMP in migratory epithelial cells. Immunofluorescence analyses indeed
clearly showed that MT1-MMP is mostly located in lamellipodia. Confocal
microscopy confirmed these data but also revealed a punctiform labelling of
MT1-MMP at the basal surface of migratory cells. Accordingly, Nakahara et al.
have shown that RPMI-7951 melanoma cells transfected with MT1-MMP organized
the protein in invadopodia at the basal surface of the cells and in
lamellipodia at the leading edge of the cells, and displayed an enhanced
ability to degrade FITC-labelled gelatin films (Nakahara et al.,
1997). By contrast, they
reported that ConA-induced overexpression of MT1-MMP, which did not generate
the subcellular localisation in invadopodia, did not enhance gelatin
degradation. It has also been shown that the degradation of the gelatin film
was mostly accomplished by the enzyme present in the invadopodia rather than
by the enzyme present in lamellipodia (Chen and Wang,
1999
). Belien et al. (Belien et
al., 1999
) have also shown a
distribution of MT1-MMP in the lamellipodia of MT1-MMP transfected rat glioma
cells. Taken together with our results, these data suggest that the
subcellular organization of MT1-MMP plays a major role in its degradative
ability and emphasize the importance of a pericellular proteolysis in cell
migration. Accordingly, Hotary et al. (Hotary et al.,
2000
) have shown that MT-MMPs,
but not soluble MMPs, participate to cell invasion and morphogenesis of MDCK
cells in collagen gels. A high level of expression of MT1-MMP and a
subcellular organization at the leading edge of migratory cells and at their
basal surface would thus contribute to a pericellular proteolysis involved in
cell migration.
Laminin-5 deposition associates with MCF10A cell migration
We found that Ln-5, a potential substrate for MT1-MMP, is preferentially
deposited by MT1-MMP-overexpressing migratory MCF10A cells at the periphery of
the outgrowth. In agreement with our findings, several reports have shown that
Ln-5 is expressed and deposited by migratory epithelial cells during would
healing both in vivo (Larjava et al.,
1993; Kainulainen et al.,
1998
) and in vitro (Zhang and
Kramer, 1996
; Lotz et al.,
1997
; Qin and Kurpakus,
1998
). Ln-5 has also been
shown to be deposited adjacent to carcinoma cell clusters and has been related
to the invasiveness of several types of tumours (Pyke et al.,
1995
; Sordat et al.,
1998
; Kosmehl et al.,
1999
; Maatta et al.,
1999
; Skyldberg et al.,
1999
; Lohi et al.,
2000
). Other evidence that the
interaction between Ln-5 and MCF10A cells could be involved in their migration
comes from our present results and those of Goldfinger et al. (Goldfinger et
al., 1999
) showing that the
6 and
3 integrin are not only present in MCF10A cells but also
reorganized in relation with cell migration and that Ln-5 blocking antibodies
decreased MCF10A cell migration. Accordingly,
6ß4 and
3ß1 are known as the main receptors for Ln-5 and have been
implicated in adhesion but also in migration of epithelial cell lines on Ln-5
(Zhang and Kramer, 1996
;
Goldfinger et al., 1999
).
Furthermore, the overexpression and cellular redistribution of
6ß4
has been described in invasive tumours (Rabinovitz and Mercurio,
1996
) and in wound healing
(Kainulainen et al., 1998
). It
can thus be suggested that an increased deposition of Ln-5 by MCF10A cells at
the periphery of the outgrowth and a reorganization of the Ln-5 integrin
receptors in such cells are involved in their migratory properties.
Laminin-5 2 chain degradation associates with MCF10A cell
migration
Supporting the concept that MT1-MMP-mediated pericellular proteolysis could
be involved in epithelial cell migration, we identified by western blotting
increased amounts of fragments of the 2 chain in migratory cultures of
MCF10A cells versus stationary cultures. The incubation of the recombinant
catalytic domain of human MT1-MMP with purified human Ln-5 generated degraded
fragments corresponding in size to those observed in our migratory cultures of
MCF10A cells, suggesting a contribution of MT1-MMP in the generation of the
Ln-5
2 fragments associated with MCF10A cell migration. In apparent
contrast to our data, Goldfinger et al. (Goldfinger et al.,
1999
) did not find the two
smallest fragments of Ln-5 in the ECM of wounded MCF10A cell cultures that
were allowed to heal for 8 hours. This discrepancy could be explained by the
fact that, in our model, cells were allowed to migrate for 72 hours. This
could indeed lead to an enrichment of the ECM with degraded Ln-5
2
chain if, as suggested by our data, migratory cells newly synthesize Ln-5,
overexpress MT1-MMP that subsequently cleaves the
2 chain of Ln-5.
However, these authors found that plasmin-mediated modifications of the
3 subunit of Ln-5 regulated cell migration (Goldfinger et al.,
1999
). Taken together with our
results, these data suggest that modifications of both
2 and
3
chains of Ln-5 can regulate MCF10A epithelial cell migration. Supporting our
findings that Ln-5
2 degradation associates with the overexpression of
MT1-MMP and cell migration, the cleavage of the
2 chain of rat Ln-5 has
been shown to be mediated by MMP-2 and/or MT1-MMP in a dose-dependent manner
(Giannelli et al., 1997
;
Koshikawa et al., 2000
), and
not by other proteases such as plasmin or MMP-9 (Giannelli et al.,
1997
). The degraded fragments
identified by Koshikawa et al. (Koshikawa et al.,
2000
) after the cleavage of
rat Ln-5
2 chain by MT1-MMP differed in number and in size from those
we observed after MT1-MMP-mediated degradation of human Ln-5
2 chain.
Also in agreement with our data, rat Ln-5 cleaved by MMP-2, but not intact rat
Ln-5, has bee reported to promote the migration of epithelial cells (Giannelli
et al., 1997
). It has also been
shown using a transwell assay that human tumour cells constitutively
expressing MT1-MMP display a higher migrating ability towards exogenously
provided rat Ln-5 than MT1-MMP negative cells (Koshikawa et al.,
2000
). We report an enhanced
production of both Ln-5 and MT1-MMP and a cleavage of Ln-5
2 chain
specifically associated with the expression of a migratory status by MCF10A
epithelial cells.
In conclusion, our data demonstrate that the acquisition of a migratory
phenotype by MCF10A epithelial cells is accompanied by an overexpression of
MT1-MMP and a localization of the protein in the lamellipodia and at the basal
surface of the cells in contact with the ECM substrate. This could participate
to a pericellular degradation of the 2 chain of Ln-5, which is more
specifically deposited by the migratory cells themselves, thereby providing a
modified substrate that promotes cell migration.
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ACKNOWLEDGMENTS |
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