1 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ,
UK
2 Department of Oncology, Cambridge Institute for Medical Research, Hills Road,
Cambridge CB2 2XY, UK
* Author for correspondence (e-mail: gm290{at}cam.ac.uk)
Accepted 12 June 2003
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Summary |
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Key words: Caveolae, Clathrin-coated pit, Endocytosis, MT1-MMP, TIMP-2
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Introduction |
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Membrane-type 1-matrix metalloproteinase (MT1-MMP) has been widely studied
since its identification and is invoked in the remodelling of the ECM in
physiological (wound healing, bone growth and remodelling) as well as
pathological processes (arthritis, tumour growth). This enzyme has been
invoked in the regulated turnover of various ECM components such as type I,
II, III and IV collagens, fibronectin, vitronectin, laminin, fibrin and
proteoglycan (d'Ortho et al.,
1997; Ohuchi et al.,
1997
) and participates in the activation of secreted MMPs such as
pro-MMP-2 (Sato et al., 1994
)
and pro-MMP-13 (Knaüper et al.,
1996
), which, in turn, will cleave multiple matrix substrates (for
a review, see Seiki, 2002
).
MT1-MMP has also been reported to process various cell membrane components -
for example, cell-surface protein-glutamine
-glutamyltransferase (tTG)
(Belkin et al., 2001
),
pro-
v integrin subunit (Deryugina
et al., 2002a
) and CD44H, a major receptor for hyaluronan
(Kajita et al., 2001
).
MT1-MMP expression has been reported to correlate with the malignancy of
multiple tumour types including lung, gastric, colon, breast, cervical
carcinomas, gliomas and melanomas (for a review, see
Yana and Seiki, 2002) and is
thought to be an important mediator of cell migration and invasion
(Hotary et al., 2000
;
Kajita et al., 2001
). MT1-MMP
has been reported to concentrate at the leading edge of various migrating
cells, and its focusing at specific sites of the cell surface is thought to be
determined by interaction with other membrane proteins at focal adhesions
(Ellerbroek et al., 2001
).
CD44H (Mori et al., 2002
) as
well as collagen (Tam et al.,
2002
) have also been specifically implicated. More recently,
MT1-MMP expression has been correlated with tumour growth and angiogenesis
through upregulation of vascular endothelial growth factor (VEGF) expression
(Deryugina et al., 2002b
;
Sounni et al., 2002
).
MT1-MMP proteolytic activity is highly regulated, and the absence of
MT1-MMP activity contributes to abnormal development
(Holmbeck et al., 1999;
Zhou et al., 2000
) and is a
key determinant for tumour progression and cancer metastasis (for a review,
see Yana and Seiki, 2002
).
Tissue inhibitors of metalloproteinases (TIMPs)-2, -3 and -4 (for a review,
see Baker et al., 2002
) as well
as the GPI anchored glycoprotein RECK (reversion-inducing cysteine-rich
protein with Kazal motifs) (Oh et al.,
2001
) have been found to inhibit MT1-MMP enzymatic activity.
Homophilic oligomerisation of MT1-MMP via its hemopexin domain alone
(Itoh et al., 2001
) or in
cooperation with the cytoplasmic and transmembrane domains
(Lehti et al., 2002
) promotes
pro-MMP2 activation. Dimerisation of MT1-MMP molecules via a disulfide bridge
between cysteine residues present in the cytoplasmic tails has also been
reported at the cell surface of MT1-MMP-transfected MCF7 cells
(Rozanov et al., 2001
).
Autocatalytic processing is also facilitated by such clustering and appears to
act as a level of downregulation (Stanton
et al., 1998
; Itoh et al.,
2001
; Lehti et al.,
2002
).
Recently, several laboratories have proposed that a major short-term level
of MT1-MMP regulation at the cell surface is by intracellular trafficking
(Jiang et al., 2001;
Uekita et al., 2001
). MT1-MMP
exocytosis is poorly understood; however, key features of this process include
the export of latent pro-MT1-MMP from the endoplasmic reticulum to the Golgi
apparatus, the proteolytic removal of the propeptide by furin or a related
preprotein convertase (Sato et al.,
1996
; Yana and Weiss,
2000
) probably in the trans Golgi network (TGN), the sorting of
the activated enzyme from the TGN and its delivery to the cell surface. More
recently, Jiang et al. (Jiang et al.,
2001
) and Uekita et al.
(Uekita et al., 2001
) have
reported the internalisation of MT1-MMP from the cell surface as a means of
controlling the net amount of active enzyme present at the plasma membrane.
This mechanism would obviously represent a rapid response mechanism which
could also be used by the cell for relocalising active MT1-MMP at the leading
edge during migration (Nabeshima et al.,
2000
; Kajita et al.,
2001
).
Endocytosis is a major mechanism by which cells regulate the level of
cell-surface proteins. Endocytosis occurs by clathrin-dependent, as well as
clathrin-independent, mechanisms (Nichols
and Lippincott-Schwartz, 2001). Clathrin-dependent endocytosis is
the most well-defined process to date and is responsible for the rapid uptake
of hormones, growth factors and transport molecules such as epidermal growth
factors (EGF) and transferrin. The interaction of molecules involved in this
process has been investigated both in vivo and in vitro, resulting in the
characterisation of several important proteins including clathrin, adaptors
and dynamin (Schmid, 1997
).
Besides clathrin-dependent endocytosis, different forms of
nonclathrin-mediated internalisation have been also identified but are much
less well characterised (Nichols and
Lippincott-Schwartz, 2001
). Caveolae are one example of a
clathrin-independent and cholesterol-sensitive uptake pathway. Caveolae are
flask shaped, non-coated plasma membrane invaginations present in many but not
all cell types. They are abundant in fibroblasts and endothelial cells and
have been recognised as centres for signalling activity at the cell surface.
Despite the growing number of ligands, receptors and lipids using caveolae as
a pathway for endocytosis, very little is known about the molecular mechanisms
that control uptake by this pathway and where exactly this pathway leads into
the cell.
The endocytosis studies conducted thus far have revealed that MT1-MMP is
internalised by using a dynamin-dependent pathway in MDCK cells
(Jiang et al., 2001).
Additionally a clathrin-mediated uptake has been reported in CHO-K1 cells by
virtue of interaction of the membrane proximal di-leucine motif localised
within the enzyme cytoplasmic domain with the AP-2 adaptor complex
(Uekita et al., 2001
). Two
further studies implicate a role for caveolae. MT1-MMP has been found in
detergent-insoluble, glycolipid-rich membrane microdomains
(Annabi et al., 2001
) and
co-sediments with caveolin-1 in U-87 glioblastoma cells, HT1080 and
MT1-MMP-transfected COS-7 cells (Annabi et
al., 2001
). In addition, MT1-MMP has been found to colocalise with
caveolin-1 at the cell surface of concanavalin A-treated microsvascular
endothelial HMEC-1 cells (Puyraimond et
al., 2001
).
Therefore, the present study aimed to determine whether MT1-MMP uses more
than one pathway for its internalisation from the cell surface. To this end,
we used the EpsE95/295 (EGF receptor pathway substrate clone
15) dominant negative mutant which specifically blocks clathrin-mediated
endocytosis, allowing the study of clathrin-independent MT1-MMP
internalization. In addition, we addressed the extent to which different forms
of MT1-MMP and its TIMP-2-associated complex may be endocytosed and whether
the enzyme is subsequently destined for degradation or recycled to the cell
surface.
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Materials and Methods |
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Antibodies
Anti-EEA1 (early endosome autoantigen-1) mouse monoclonal antibody (mAb
clone 14); anti-Eps15 (EGF receptor pathway substrate clone number 15) mAb
(clone 17) and anti-caveolin-1 (C13630) rabbit polyclonal antibody (pAb) were
purchased from BD Transduction Laboratories (Lexington, KY). Anti-TIMP-2
(67.4H11) mAb was a gift from K. Iwata (Fuji Chemical Industries, Toyama,
Japan). Anti-biotin mouse mAb was obtained from Jackson ImmunoResearch
Laboratories (West Grove, PA). Anti-Rab4-CT rabbit pAb was form Stressgen
Biotechnologies Corporation (BC, Canada). Anti-LAMP-1 (lysosome-associated
membrane protein-1) mouse mAb was purchased from the Developmental Studies
Hybridoma Bank (Iowa City, IA). Affinity-purified rabbit anti-clathrin heavy
chain immunoglobulins (IgGs) were kindly provided by Margaret S. Robinson
(Cambridge Institute for Medical Research, Cambridge, UK). Anti-MT1-MMP sheep
pAb IgGs (N175/6) (d'Ortho et al.,
1998) were affinity purified using MT1-MMP ectodomain immobilised
on a HiTrap NHS-activated HP (Amersham Pharmacia Biotech UK, Little Chalfont,
UK). Bound IgGs were eluted in 100 mM glycine pH 2.5 containing 10% dioxane
and the pH of the eluate was neutralised immediately by adding 0.1 volume of 1
M Tris-HCl pH 8.0. Anti-TIMP-2 sheep pAb (H225/9) was described previously
(Ward et al., 1991
). All
secondary antibodies were purchased from Jackson ImmunoResearch Laboratories
and used according to the manufacturer's instructions.
Cell culture conditions, transfections and DNA constructs
All cell culture reagents were purchased from Invitrogen (Paisley, UK)
unless indicated. HT1080 human fibrosarcoma and HeLa cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol)
fetal calf serum (Hyclone Laboratories, UT), 2 mM L-glutamine, 100 units/ml
penicillin and 100 µg/ml streptomycin at 37°C in an atmosphere of 5%
CO2. HT1080 stably transfected with wild-type MT1-MMP cDNA
(bbHT1080 clone #2) were prepared and grown as described in Stanton et al.
(Stanton et al., 1998). T47D
and MCF7 cell lines were kindly donated by A. Noel (Laboratoire de Biologie
des Tumeurs et du Développement, Liège, Belgium) and cultured as
previously described.
Transfections were performed using FuGENE-6 reagent, according to manufacturer's instructions (Roche Diagnostics, Lewes, UK).
Wild-type dyn2aa (dynwt) and K44A (dynK44A) dynamin
cDNAs in pEGFP vector were a gift from Mark McNiven (Mayo Clinic and
Foundation, Rochester) (Cao et al.,
2000). Vectors containing the inserts encoding
EGFP-EpsE
95/295 and EGFP-Eps15wt were a gift from
Alexandre Benmerah (Pasteur Institute, Paris)
(Benmerah et al., 1999
).
Full-length MT1-MMP cDNA without 5' and 3' untranslated regions
was inserted between the HindIII and EcoRI site of pCDNA3.1
Zeo+ mammalian expression vector (Invitrogen). Cytotail truncated
MT1-MMP construct (MT1C) was prepared from full-length MT1-MMP by
inserting a stop codon after Phe561 by site-directed
mutagenesis.
Cell-surface biotinylation, recycling assay and
immunoprecipation
Cell-surface protein biotinylation and recycling assay were performed
according to Neuhaus and Soldati (Neuhaus
and Soldati, 2000) with the following modifications. Wild-type
HT1080 and bbHT1080 cells grown to 80-90% confluence in a six-well plate were
washed twice with ice-cold SBS (Soerensen Buffer, 14.7 mM
KH2PO4 and 2 mM Na2HPO4 with 120
mM Sorbitol pH 7.8) and incubated for a further 10 minutes in ice-cold SBS.
Biotinylation was performed by adding 2.5 ml of freshly prepared ice-cold SBS
containing 0.3 mg/ml EZ-Link NHS-SS-biotin (sulfosuccinimidyl-2-(biotinamido)
ethyl-1,3-dithiopropionate, Perbio Science UK) per well for 15 minutes. After
two washes with ice-cold SBS, the unreacted biotin was quenched for 10 minutes
by incubation with ice-cold SBS containing 100 mM glycine. Quenched cells were
then washed twice with ice-cold SBS and incubated at 37°C in CIM/ITS to
allow the internalisation of biotinylated plasma membrane proteins. After 15
minutes, the uptake was stopped by washing the cells with ice-cold SBS and
placing them on an ice bath for 10 minutes. Biotin present on cell-surface
components was cleaved off by incubating the cells with 150 mM of membrane
impermeant reducing agent MESNA (2-mercaptoethane sulfonic acid) in ice-cold
SBS, pH 8.2 for 25 minutes. Cells were washed three times with ice-cold SBS,
and then lysed in 0.2 ml/well of RIPA buffer (20 mM Tris-HCl pH 7.4, 150 mM
NaCl, 0.1% SDS, 1% Triton X-100, 1% Deoxycholate, 1% NP-40) containing
proteinase inhibitor cocktail set III (Calbiochem-Novabiochem Corporation, San
Diego, CA).
For recycling assay, MESNA treated cells were washed with ice-cold SBS and incubated in CIM/ITS at 37°C to allow recycling of the endocytosed biotinylated proteins. Recycling was then stopped after 15, 30 and 45 minutes by washing the cells with ice-cold SBS and placing them on an ice bath for 10 minutes. Biotin re-exposed at the cell surface was cleaved off by a second MESNA treatment (see above). Finally, the cells were washed three times with ice-cold SBS, and lysed in RIPA buffer as previously described.
RIPA protein extracts were centrifuged at 16,000 g for 10 minutes at 4°C. Supernatants were precleared with 25 µl of protein G-agarose beads for 1 hour at 4°C. After centrifugation, biotinylated proteins were immunoprecipated for 1 hour at 4°C using 25 µl protein G-agarose beads saturated with an anti-biotin mouse mAb. Immobilised complexes were collected, washed five times with 1 ml of RIPA buffer, eluted from protein G with 30 µl of 2.5x Laemmli sample buffer. Samples (20 µl) were separated by SDS-PAGE and subjected to immunoblot analysis.
Endocytosis assays by indirect immunofluorescence microscopy
analysis
HT1080 cells seeded onto 13 mm sterile round glass coverslips and grown to
60-70% confluence were washed twice with PBS. After 30 minutes incubation at
37°C in a serum-free CO2 independent medium (CIM) containing 1%
Insulin-Transferrin-Selenium (ITS) supplement, cells were washed with ice-cold
CIM/ITS and incubated for a further 15 minutes at 4°C. Intact cells were
then incubated for 1 hour at 4°C with 10 µg/ml affinity-purified
anti-MT1-MMP sheep pAb (N175/6) or 10 µg/ml anti-TIMP-2 mouse mAb (67.4H11)
in CIM/ITS. Cells were extensively washed with ice cold serum-free CIM/ITS to
remove unbound antibody and surface-bound material was internalised by
incubating the cells at 37°C in CIM/ITS for indicated time points. Texas
red-labelled transferrin (25 ug/ml, Molecular Probes, Eugene, OR) was
internalised in serum-free CIM/ITS for 20 minutes at 37°C. Cells were then
fixed and permeabilised (see below) and internalised primary antibody was
detected using the appropriate fluorescently labelled secondary antibody and
samples were then processed as described under Indirect Immunofluorescence
Microscopy.
Indirect immunofluorescence microscopy
Cells were seeded on 13 mm round glass coverslips and grown at 37°C
until reaching 70-80% confluence. Cells were washed in phosphate-buffered
saline (PBS; 140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1
mM Na2HPO4), fixed either with methanol at -20°C for
5 minutes or with 4% paraformaldehyde in PBS for 25 minutes at room
temperature. In the latter case, cells were permeabilised for 4 minutes in
0.1% Triton X-100 (Surfact-Amps X-100 grade, Pierce, Rockford, IL) in PBS.
Staining was carried out as described by Roghi and Allan
(Roghi and Allan, 1999). After
extensive washing in PBS, coverslips were briefly rinsed in H2O and
then mounted on glass slides in Mowiol containing 25 mg/ml
1,4-diazobicyclo-[2.2.2]-octane to reduce photo-bleaching. Pictures of
fluorescently labelled cells were collected using a cooled, slow scan CCD
camera (Micromax 1401E, Roper Scientific, Harlow, UK) attached to a Nikon
Eclipse E800 microscope (Nikon UK, Kingston upon Thames, UK) using a 60x
Plan-Apo (NA 1.4) oil objective. Images were also acquired using a
Photometrics Coolsnap HQ CCD camera (Roper Scientific, Harlow, UK) attached to
a Zeiss Axioplan 2 imaging microscope equipped with a motorised stage and
fitted with 63x and 100x plan apochromat objectives (Carl Zeiss,
Welwyn Garden City, UK). Image acquisition was performed using Metamorph
software (Universal Imaging Corporation, Downingtown, PA). Images were
transferred to Photoshop (Adobe Systems, San Jose, CA) imaging software for
digital processing.
Drug treatments
Cells were incubated for 1 hour at 37°C in CIM/ITS containing 30
µg/ml nystatin or 50 µg/ml concanavalin A (ConA) or for 30 minutes at
37°C in CIM/ITS containing 4 mM methyl-ß-cyclodextrin before
cell-surface biotinylation or antibody uptake assay. bbHT1080 cells were
incubated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 16 hours.
Microtubule-disrupting drug nocodazole (10 µg/ml in DMSO) was added to the
cells for 1 hour. As controls, carriers were added to the cells at a final
concentration of 0.1% and did not have any effect on the internalisation (data
not shown).
Detergent-free isolation of caveolae-enriched membrane fraction
Caveolae-enriched membranes were prepared from HT1080 cells using carbonate
extraction according to the protocol described by Song et al.
(Song et al., 1996). Fractions
(1 ml) were collected from the top of the gradient and transferred into new
ultracentrifuge tubes containing 8 ml of MBS [25 mM MES (2[N-morpholino]
ethanesulfonic acid), pH 6.5, 150 mM NaCl]. After 90 minutes at 208,429
gmax (Ti70.1 rotor, Beckman Coulter, High Wycombe,
UK) at 4°C, pellets were resuspended with 150 µl of Laemmli sample
buffer and 15 µl aliquots were separated by SDS-PAGE and subjected to
immunoblot analysis as described below.
SDS-PAGE and immunoblot analysis
All reagents for electrophoresis were purchased from Bio-Rad (High Wycombe,
UK). Proteins were electrophoretically transfered for 1 hour at 15 Volts onto
Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech) using a Mini
Trans-Blot Electrophoretic transfer cell (Bio-Rad). After blocking with 5%
(wt/vol) nonfat dry milk in PBS (2x 20 minutes at room temperature),
blots were probed with 1.5 µg/ml affinity purified anti-MT1-MMP IgGs or 50
ng/ml anti-caveolin-1 pAb in 2.5% milk in PBS for 16 hours at 4°C. After
several washings (PBS containing 0.05% (vol/vol) Tween 20), membranes were
incubated for 1 hour at room temperature with appropriate HRP-conjugated
secondary antibody diluted in 2.5% milk in PBS. After extensive washes,
specific immunocomplexes were detected using an ECL western blotting detection
kit (Pierce).
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Results |
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Internalised MT1-MMP accumulates in a perinuclear located vesicular
compartment in bbHT1080 cells
On the basis of our previous observation, we decided to assess MT1-MMP
internalisation in bbHT1080 using an antibody uptake assay to visualize
MT1-MMP localisation after its internalisation. bbHT1080 cells were incubated
with affinity-purified anti-MT1-MMP antibody for 1 hour at 4°C. Unbound
antibody was washed off and cells were warmed to 37°C for varying times.
Cells were fixed and IgG-bound MT1-MMP complexes were detected after
permeabilisation using a fluorescently labelled secondary antibody. Using this
protocol, we observed that IgG-bound MT1-MMP complexes originally present at
the cell surface (Fig. 2A) were
quickly internalised after switching the cells from 4°C to 37°C
(Fig. 2B-E). Vesicles
containing IgG-bound MT1-MMP complexes accumulated in the cell cytoplasm and
concentrated with time in the perinuclear region of the cell
(Fig. 2C-E). Addition of the
microtubule-depolymerising drug nocodazole during the uptake assay did not
inhibit internalisation of IgG-bound MT1-MMP complexes but induced the
redistribution of perinuclear IgG-bound MT1-MMP complexes to the cell
periphery (Fig. 2F), suggesting
the accumulation of internalised MT1-MMP in the endocytic pathway. MT1-MMP
internalisation was also observed using a Fab fraction prepared from the
affinity-purified anti-MT1-MMP IgGs. This shows that MT1-MMP internalisation
did not result from an antibody-mediated clustering effect (data not shown).
In addition, no intracellular vesicular staining was observed when
internalisation experiments were carried out in MCF7 and T47D cells, which do
not express MT1-MMP (data not shown).
|
MT1-MMP is found in the endocytic compartment
To characterise the vesicular structures containing IgG-bound MT1-MMP
complexes, we performed double-immunofluorescence microscopy experiments.
Nonstimulated bbHT1080 cells, which were allowed to internalise IgG-bound
MT1-MMP complexes for 30, 45 and 60 minutes, were fixed, permeabilised and
incubated with primary antibodies directed against membrane markers for
various endocytic compartments. The early endosomal antigen EEA1 interacts
with Rab5 and phosphatidylinositol-3-phosphate, facilitating early endosome
fusion (Christoforidis et al.,
1999). After 30 minutes internalisation, IgG-bound MT1-MMP
complexes were found to extensively colocalise with
EEA1-(Fig. 3A-C, arrowheads)
and, to a certain extent, with Eps15-containing early endosomes
(Fig. 3D-F, arrowheads). We did
not observe colocalisation with Rab4, a marker for recycling endosomes
(Sönnichsen et al., 2000
)
or with LAMP-1, a marker for late endosomes/lysosomes
(Hunziker and Geuze, 1996
) at
this particular time point (data not shown). When IgG-bound MT1-MMP complexes
were internalised for 45 minutes, IgG-bound MT1-MMP complexes were still
largely present in EEA1-positive endosomes (data not shown) but in addition,
they were also found in rab4-positive recycling endosomes
(Fig. 3G-I, arrowheads).
Colocalisation with LAMP-1 could not be detected at this time point but could,
however, be observed when IgG-bound MT1-MMP complexes were internalised for 60
minutes (Fig. 3J-L,
arrowheads). We did not observe colocalisation between MT1-MMP and TGN43, a
specific marker for the TGN, suggesting that internalized MT1-MMP is probably
not recycled to the TGN.
|
Taken together, this set of experiments clearly shows that, in bbHT1080,
cell-surface IgG-bound MT1-MMP complexes are internalised from the cell
surface and distributed with time in various compartments of the endocytic
pathway. Our results correlate well with observations reported previously by
other groups (Jiang et al.,
2001; Uekita et al.,
2001
), enabling us to pursue our studies with bbHT1080 cells.
Exocytosis of internalised active MT1-MMP
The presence of IgG-bound MT1-MMP in Rab4-positives structures
(Fig. 3G-I) suggests that
internalised MT1-MMP may recycle back to the cell surface. To test this
hypothesis, wild-type nontransfected HT1080 cells were biotinylated at 0°C
(Fig. 1A, biotinylation, and
Fig. 4, lane 2). After 15
minutes internalisation at 37°C (Fig.
1A, uptake), cells were cooled again to 0°C and MT1-MMP
remaining at the cell surface was de-biotinylated with MESNA
(Fig. 1A, cleavage 1, and
Fig. 4, lane 3). In parallel,
cells with internalised biotinylated MT1-MMP were warmed again to 37°C for
15, 30 and 45 minutes (Fig. 1A,
chase) to allow recycling of internalised biotinylated proteins to the cell
surface, cooled to 0°C and treated
(Fig. 1A, cleavage 2, and
Fig. 4, lanes 5, 7 and 9) or
not (Fig. 4, lanes 4, 6 and 8)
with MESNA. In this case, the difference between MESNA-treated and
MESNA-nontreated samples corresponds to the fraction of biotinylated MT1-MMP
that has been re-exported to the cell surface during the second warming to
37°C. This experiment clearly shows for the first time that internalised
MT1-MMP is recycled to the surface of the cell.
|
Selective inhibition of the clathrin-dependent pathway does not
completely inhibit active MT1-MMP internalisation
Previous reports have shown that active MT1-MMP internalisation is
completely blocked in cells expressing the dynK44A
dominant-negative dynamin mutant (Jiang et
al., 2001; Uekita et al.,
2001
). Because dynK44A overexpression was shown to
block both clathrin-dependent (Pearse and
Robinson, 1990
; De Camilli et
al., 1995
) and clathrin-independent endocytosis
(Oh et al., 1998
;
Schnitzer, 2001
), the
previously reported experiments were unable to precisely identify the
pathway(s) involved in active MT1-MMP internalisation. In bbHT1080, expression
of EGFP-dynK44A completely inhibited MT1-MMP
(Fig. 5A,B) and transferrin
receptor (Fig. 5C,D)
internalisation. Interestingly, when the cells were transiently transfected
with the E
95/295 dominant-negative Eps15 mutant tagged with EGFP
(EGFP-EpsE
95/295), which has been previously described to
specifically block clathrin-dependent endocytosis without disrupting
clathrin-independent endocytosis (Benmerah
et al., 1999
), we observed a significant uptake of active MT1-MMP
from the cell surface (Fig.
5E,F), whereas Texas red-labelled transferrin internalisation was
completely inhibited (Fig.
5G,H). No effects were observed when EGFPEps15wt was
expressed before MT1-MMP internalisation (data not shown). In addition,
IgG-bound MT1-MMP complexes were observed at the cell surface of
EGFP-EpsE
95/295-transfected cells
(Fig. 5F) compared with
untransfected cells, suggesting that internalisation of MT1-MMP also required
a fully functional clathrin-dependent pathway. Interestingly, the expected
plasma membrane staining observed in
EGFPEpsE
95/295-expressing cells was not found in
EGFP-dynK44A-expressing cells
(Fig. 5B), indicating that
expression of dynK44A has actually a more profound effect on
MT1-MMP expression at the cell surface than previously described
(Jiang et al., 2001
).
|
Taken together, our observations showed that active MT1-MMP was still internalised when the clathrin-dependent pathway was selectively disrupted, suggesting that both clathrin-dependent and -independent pathways are probably involved in the endocytosis of active MT1-MMP.
Active MT1-MMP form is internalised through a cholesterol-dependent
pathway
We next focused our interest on the analysis of the previously identified
clathrin-independent pathway involved in active MT1-MMP internalisation.
Previous reports have shown the presence of MT1-MMP in caveolae
(Puyraimond et al., 2001;
Annabi et al., 2001
), and to
assess whether or not active MT1-MMP could be endocytosed through a
caveolae-mediated pathway, we examined IgG-bound MT1-MMP internalisation in
cells with perturbed caveolae. bbHT1080 cells were treated with nystatin, a
cholesterol-binding drug that inhibits endocytosis from caveolae and
caveolae-like domains by impairing invagination, and thereby internalisation
(Schnitzer et al., 1994
;
Deckert et al., 1996
), without
interfering with clathrin-coated pit formation. In cells treated with nystatin
(Fig. 6B) or with
methyl-ß-cyclodextrin (Fig.
6D), we observed a more profound cell-surface staining compared
with untreated cells (Fig.
6A,C), suggesting that cholesterol-sensitive structures are
probably involved in the internalisation of MT1-MMP from the cell surface.
Internalised IgG-bound MT1-MMP complexes can be seen in nystatin-treated cells
(Fig. 6B, arrowheads), as well
as in methyl-ß-cyclodextrin-treated cells
(Fig. 6D, arrowheads),
suggesting that some internalisation still occurred in these cells most
probably via the clathrin-mediated pathway. Texas-red-labelled transferrin
internalisation was not affected in nystatin-treated cells (data not shown) as
reported previously by others (Torgersen
et al., 2001
).
|
The involvement of both pathways was also assessed using our biotinylation
approach using concanavalin A- or nystatin-treated bbHT1080 cells. In
nontreated cells (Fig. 6E,
lanes 2 and 3), we observed, as previously described in this paper, only the
internalisation of 60 kDa active MT1-MMP
(Fig. 6E, lane 3). ConA has
previously been reported to block both caveolaeand clathrin-coated
pits-mediated pathways (for a review, see
Lefkowitz, 1998) and, as
expected, completely inhibited internalisation of active MT1-MMP from the cell
surface (Fig. 6E, lane 7).
Interestingly, when bbHT1080 cells were treated with nystatin
(Fig. 6E, lane 5) we observed
that 60 kDa active MT1-MMP was still internalised but the amount of
internalised enzyme was clearly reduced compared with untreated cells
(Fig. 6E, lane 3). This
approach provides further evidence that MT1-MMP internalisation requires a
combination of clathrin-dependent and -independent pathways.
To support this result, unstimulated bbHT1080 cells with internalised IgG-bound active MT1-MMP complexes were permeabilised and stained with antibodies directed against caveolin-1 and clathrin heavy chain. We observed that MT1-MMP was found in caveolin-1-positive vesicular structures (Fig. 7C, arrowheads), as well as in clathrin-positive structures (Fig. 7F, arrowheads), again suggesting the involvement of the caveolae system and clathrin-coated pits in MT1-MMP internalisation.
|
Finally, to have a more biochemical confirmation that caveolae might be
involved in MT1-MMP internalisation, we isolated caveolae-enriched membrane
domains using a detergent-free procedure
(Song et al., 1996) from
nonstimulated HT1080 cells. Isolated crude membranes were subjected to a
carbonate extraction, followed by a subcellular fractionation using a
discontinuous sucrose-density gradient. Analysis of the gradient fractions by
immunoblotting (Fig. 7G)
revealed that active MT1-MMP was present in the caveolin-1-positive fractions
(Fig. 7G, lanes 4 and 5), as
well as in other fractions (Fig.
7G, lanes 3, 6 and 7-9), suggesting its distribution within
multiple membrane compartments. Interestingly, the proteolytically inactivated
45 kDa MT1-MMP form was only found in the caveolin-1-enriched membrane
fraction (Fig. 7G, lanes 4 and
5).
Taken altogether, our results suggest that in HT1080, MT1-MMP is internalised by a clathrin-independent pathway, most probably caveolae, alongside the clathrin-mediated endocytosis.
TIMP-2 is co-internalised with MT1-MMP
Numerous reports have described the binding of TIMP-2 to the MT1-MMP
catalytic domain and the role of the complex as the physiological plasma
membrane receptor for proMMP-2 activation
(Strongin et al., 1995;
Butler et al., 1998
). Recently,
Maquoi et al. (Maquoi et al.,
2000
) have reported that, in PMA-treated cells, TIMP-2 was first
internalised before being partially degraded. We therefore examined whether or
not TIMP-2 internalisation could be mediated by MT1-MMP. HeLa cells, which
express low levels of MT1-MMP and a substantial amount of TIMP-2, were
transiently transfected with full-length MT1-MMP or cytotail truncated
MT1
C cDNAs. Transfected cells were then subjected to an antibody uptake
assay using affinity-purified anti-MT1-MMP IgGs together with anti-TIMP-2
antibody. After 30 minutes at 37°C, cells were fixed and permeabilised and
IgG-bound active MT1-MMP and IgG-bound TIMP-2 complexes were revealed using
fluorescently labelled secondary antibodies. Using this strategy, we observed
a very strong colocalisation between internalised TIMP-2 and internalised
active MT1-MMP (Fig. 8B) in
MT1-MMP-transfected HeLa cells. Interestingly, most, if not all, intracellular
TIMP-2-positive vesicles were MT1-MMP positive, suggesting that TIMP-2
internalisation is a MT1-MMP-mediated event in which TIMP-2 and MT1-MMP could
be internalised as a bimolecular complex. In addition, a few MT1-MMP-positive
vesicles were TIMP-2-negative, indicating the presence of TIMP-2-free MT1-MMP
in the cell cytoplasm (Fig. 8B,
arrowhead, for an example). It is still not known whether this TIMP-2 free
MT1-MMP resulted from the endocytosis of uncomplexed MT1-MMP or the
endocytosis of the bimolecular complex, followed by the dissociation of the
complex due to the low pH of the endocytic compartment. To confirm that TIMP-2
endocytosis is indeed MT1-MMP mediated, HeLa cells were transiently
transfected with MT1-MMP deleted from its cytoplasmic tail (MT1
C).
MT1
C can traffic to the cell surface but was then poorly endocytosed
and mainly retained at the cell surface
(Jiang et al., 2001
;
Uekita et al., 2001
). In
MT1
C-transfected HeLa cells, IgG-bound MT1-MMP and IgG-bound TIMP2
complexes were mainly detected at the cell surface
(Fig. 8D). Taken together, our
data suggest that TIMP-2 internalisation is exclusively and specifically
mediated by MT1-MMP.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Active MT1-MMP is internalised by two different pathways
Previous work has shown the clathrin-mediated and dynamin-dependent
internalisation of MT1-MMP internalisation from the cell surface
(Uekita et al., 2001;
Jiang et al., 2001
). Our
results confirm and extend these recent findings by showing clearly that
MT1-MMP is internalised by a clathrin-independent mechanism, which we suspect
to be caveaole, in addition to a the clathrin-mediated endocytic pathway. The
most direct evidence is the continued internalisation of MT1-MMP in bbHT1080
cells expressing EGFP-EpsE
95/295 in which clathrin-coated
pit endocytosis is selectively blocked. Moreover, the nondetection, using
immunofluorescence techniques, of IgG-bound complexes in
EGFP-dynK44A transfected bbHT1080 cells suggests that most, if not
all, MT1-MMP internalisation is dynamin-dependent and a third internalisation
pathway is not likely be involved in MT1-MMP internalisation.
Clathrin-dependent and -independent pathways seem to coexist in
nonstimulated HT1080, and they are probably nonredundant internalisation
pathways for MT1-MMP. It will be interesting to assess whether or not these
two pathways coexist in a migrating fibroblast. Obviously, the definitive role
of each pathway is as yet unknown and remains to be identified, thus their
potential function can only be hypothesised. It may be that both pathways
internalise different subpopulations of MT1-MMP present at the cell surface.
Another difference between caveolae and clathrin-mediated internalisations is
speed and localisation of internalisation. In a resting cell, the speed of
internalisation, as well as the localisation, may not be that important for
the cell. However, in a migratory situation, speed may be an issue. It has
been shown previously that internalisation by caveolae is two to four times
slower that clathrin-mediated endocytosis
(Fishman, 1982;
Tran et al., 1987
).
Clathrin-mediated and caveolae-dependent pathways may be involved in the
differential localisation of internalisation at the cell surface. In migrating
endothelial cells, caveolae have been found to relocalise at the trailing edge
of the migrating cell (Isshiki et al.,
2002
). MT1-MMP is internalised at the adherent edge of cells
plated on gelatin (Uekita et al.,
2001
). Recent studies have shown a functional interaction between
MT1-MMP and integrins. However, the mechanisms by which ECM and integrins
might regulate MT1-MMP functionality remain unexplored. Recently,
Gálvez et al. (Gálvez et
al., 2002
) have shown that ß1 integrin interacts with MT1-MMP
and appears to be involved in the modulation of MT1-MMP internalisation
together with ECM components. Integrin ß1 has been found to
immunoprecipitate with caveolin-1, a defining protein component of caveolae,
and uPAR in uPAR-transfected 293 cells
(Wei et al., 1999
) and in
detergent-lysed A431cells (Wary et al.,
1996
). In addition in WI-38 human lung fibroblasts, a fraction of
ß1 coprecipitates with caveolin-1 and promotes Fyn-dependent Shc
phosphorylation in response to integrin ligation, leading to mitogen-activated
protein kinase (MAPK) activation and cellular growth
(Wary et al., 1998
).
Caveolin-1 is also important to ß1 integrin-dependent fibronectin
adhesion and focal adhesion kinase activation
(Wei et al., 1999
). It is
possible that MT1-MMP internalisation from the adherent edge could be caveolae
and/or clathrin mediated. The absence of internalization of cytotail depleted
MT1-MMP from the adherent edge implies the absence of determinant signal
sequence(s) which direct MT1-MMP to caveolar structures in these cells.
Caveolae have been described as chemical switchboards enriched in molecules
that play pivotal roles in intracellular signal transduction. MT1-MMP is
present in caveolae located at the cell surface, as well as in internalised
caveolae that have been pinched off from the cell surface. Signal transduction
can certainly happen in both situations. Caveolae-located MT1-MMP could be
involved in extracellular signal-related kinase (ERK)-dependent activation of
transcription via its cytoplasmic tail
(Gingras et al., 2001).
Overexpression of caveolin-1 has been reported to inhibit caveolae formation
and, in MT1-MMP-expressing COS-7 cells, suppresses the increased migration on
gelatin promoted by MT1-MMP expression
(Annabi et al., 2001
). The
absence of caveolae in these cells could reflect the absence of signalling
between cell-surface MT1-MMP and the inside of the cell, and thus explain why
these cells are unable to achieve MT1-MMP-induced migration.
Postendocytic sorting of MT1-MMP
Internalised MT1-MMP is also recycled to the cell surface. The presence of
MT1-MMP in a rab4-positive endocytic compartment suggests that the recycling
is probably effected from this intracellular compartment. However, it is
possible that recycling may also occur from internalized caveolae. Studies on
the internalisation of the folate receptor
(Smart et al., 1994), cholera
toxin and alkaline phosphatase (Parton et
al., 1994
) have indicated that membrane recycling occurs during
caveolae-mediated endocytosis.
In our resting HT1080 cells, we suspect that internalised MT1-MMP is
randomly re-inserted with internalised membranes at the cell surface. However,
reinsertion may be redirected to the sites of protusion when migration is
induced by motogenic stimuli (Bretscher and
Aguado-Velasco, 1998). Cell migration is driven by the protrusive
activity at the leading edge of the cell, where continuous remodelling of
actin and adhesive contacts is required. It has been hypothesised that
membrane internalised from the cell surface is recycled to the front of
migrating cells to contribute to the extension of the cell border
(Bretscher and Aguado-Velasco,
1998
). Given the rapid rate of membrane internalization
(Hao and Maxfield, 2000
),
large amounts of recycling membrane would be made available for polarized
delivery by such a mechanism.
MT1-MMP recycling could also be involved in the dissociation of the MT1-MMP/TIMP-2 complex. The cointernalisation experiment of MT1-MMP and TIMP-2 has revealed the presence of both molecules in the same vesicular structure, probably early endosomes. It is not known whether or not both molecules are still interacting together, but the low pH of this endosomal compartment may provoke the dissociation of the complex and both proteins may recycle independently and to different regions of the cell surface. In addition, it would be interesting to investigate whether or not pro-MMP-2 is found in the same compartment as TIMP-2 and MT1-MMP after internalisation and thus could be activated in an intracellular endocytic membrane compartment and released as an active MMP-2 in the extracellular milieu.
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Acknowledgments |
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