Báo cáo Y học: Matrilysin (matrix metalloprotease-7) cleaves membrane-bound annexin II and enhances binding of tissue-type plasminogen activator to cancer cell surfaces docx

Matrilysin (matrix metalloprotease-7) cleaves
membrane-bound annexin II and enhances binding of
tissue-type plasminogen activator to cancer cell surfaces
Jun Tsunezumi
1,2
, Kazuhiro Yamamoto
1
, Shouichi Higashi
1,2
and Kaoru Miyazaki
1,2
1 Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan
2 Graduate School of Integrated Sciences, Yokohama City University, Japan
Matrix metalloproteinases (MMPs) form a group of
more than 20 zinc-dependent enzymes that are
involved in the processing of several components of
the extracellular matrix (ECM). They play roles in
many physiological processes, such as bone remodeling
and organogenesis, and have additional roles in the
reorganization of tissues during pathological
conditions such as inflammation and invasion and
metastasis of cancer cells [1,2]. Many recent studies
have provided evidence that the biological activities of
various cell surface molecules are proteolytically
modulated by several MMPs, including membrane-
type MMPs, gelatinase A (MMP-2), gelatinase B
(MMP-9), stromelysin (MMP-3), and matrilysin
(MMP-7) [3–6]. These metalloproteinases are likely to
regulate cellular functions by activating, inactivating or
releasing membrane proteins. Such regulation of cell
surface proteins, as well as MMP-catalyzed degra-

dation of the ECM, a natural barrier against tumor
invasion, is important for tumor metastasis.
Matrilysin, the smallest of the MMPs, has broad
substrate specificity and has been demonstrated to
Keywords
annexin II; cancer cells; matrilysin; matrix
metalloproteinase; plasminogen activator
Correspondence
K. Miyazaki, Division of Cell Biology, Kihara
Institute for Biological Research, Yokohama
City University, 641-12 Maioka-cho, Totsuka-
ku, Yokohama, Kanagawa 244-0813, Japan
Fax: +81 45 820 1901
Tel: +81 45 820 1905
E-mail:
(Received 9 May 2008, revised 22 July
2008, accepted 30 July 2008)
doi:10.1111/j.1742-4658.2008.06620.x
Matrilysin (matrix metalloproteinase-7) plays important roles in tumor
progression. It was previously found that matrilysin binds to the surface of
colon cancer cells to promote their metastatic potential. In this study, we
identified annexin II as a novel membrane-bound substrate of matrilysin.
Treatment of human colon cancer cell lines with active matrilysin released
a 35 kDa annexin II form, which lacked its N-terminal region, into the
culture supernatant. The release of the 35 kDa annexin II by matrilysin
was significantly enhanced in the presence of serotonin or heparin. Matri-
lysin hydrolyzed annexin II at the Lys9–Leu10 bond, thus dividing the
protein into an N-terminal nonapeptide and the C-terminal 35 kDa frag-
ment. Annexin II is known to serve as a cell surface receptor for tissue-type
plasminogen activator (tPA). Although the matrilysin treatment liberated

the 35 kDa fragment of annexin II from the cell surface, it significantly
increased tPA binding to the cell membrane. A synthetic N-terminal non-
apeptide of annexin II bound to tPA more efficiently than intact annexin II.
This peptide formed a heterodimer with intact annexin II in test tubes and
on cancer cell surfaces. These and other results suggested that the nonapep-
tide generated by matrilysin treatment might be anchored to the cell mem-
brane, possibly by binding to intact annexin II, and interact with tPA via
its C-terminal lysine. It is supposed that the cleavage of cell surface annex-
in II by matrilysin contributes to tumor invasion and metastasis by enhanc-
ing tPA-mediated pericellular proteolysis by cancer cells.
Abbreviations
ECM, extracellular matrix; MMP, matrix metalloproteinase; PVDF, poly(vinylidene difluoride); siRNA, small interfering RNA; TAPI-1, N-(R)-[2-
(hydroxyaminocarbonyl)-methyl]-4-methylpentanoyl-
L-naphthylalanyl-L-alanine-2-aminoethyl amide; tPA, tissue-type plasminogen activator.
4810 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS
degrade or process a variety of matrix and nonmatrix
molecules [7]. Unlike most MMPs, which are expressed
by stromal cells, matrilysin is principally expressed by
epithelial cells [8]. This enzyme seems to be one of the
most important MMPs in human colon cancers,
because the expression of matrilysin is highly corre-
lated with malignancy and metastatic potential of the
cancers, especially in their liver metastasis [9]. It has
recently been reported that active matrilysin specifi-
cally binds to the surface of colon cancer cells and
induces notable cell aggregation due to processing of
the cell membrane protein(s). Furthermore, these
aggregated cells showed greatly enhanced metastatic
potential in the nude mouse model [10,11]. Therefore,
it seems important to identify cell surface proteins that

are specifically cleaved by matrilysin, to elucidate the
mechanism of the matrilysin-induced phenotypic
changes of cancer cells, such as enhancement of homo-
typic cell adhesion and metastatic potential.
Annexin II belongs to a family of calcium-dependent
phospholipid-binding proteins that are expressed in
diverse tissues and cell types [12]. Annexin II was ini-
tially identified as an intracellular molecule without a
signal peptide, but later studies revealed extracellular
localization of annexin II in many kinds of tissues and
cells [13]. The mechanism of secretion of cytoplasmic
annexin II is mostly unknown, but a stress-induced
protein secretion pathway has been suggested in vascu-
lar endothelial cells [14]. Many studies have shown
that extracellular annexin II is involved in the regula-
tion of a variety of cellular processes, including pericel-
lular proteolysis, cell–cell or cell–ECM adhesion, and
regulation of membrane architecture [13–16]. One of
the important functions of extracellular annexin II is
its involvement in the tissue-type plasminogen activa-
tor (tPA)–plasminogen system on cell surfaces [17].
The N-terminal sequence of annexin II is required for
its binding with tPA [18].
In this study, we identified annexin II as a novel
membrane-associated substrate for matrilysin, and
investigated the biological consequence of annexin II
cleavage by matrilysin. Our results suggest that the
specific cleavage of annexin II by matrilysin enhances
the binding of tPA to cancer cell surfaces, leading to
activation of the tPA-mediated pericellular proteolytic

cascade on cancer cells.
Results
Cleavage of annexin II by matrilysin
It was previously found that active matrilysin specifi-
cally binds to surfaces of colon cancer cells and
induces prominent cell aggregation [10,11]. In the pres-
ent study, we first analyzed membrane proteins that
are cleaved by matrilysin. A membrane fraction of
WiDr human colon carcinoma cells was prepared by
the phase separation method with Triton X-114. When
the membrane fraction was treated with matrilysin,
several proteins, including a major protein of approxi-
mately 35 kDa, were released from the membrane
fraction (Fig. 1A). The N-terminal amino acid
sequence of the 35 kDa protein was determined to be
B
MAT
−+
116
97
200
(kDa)
A
97
66
45
31
21
N-
-C

338 10 9 1
9
1
0

STVHEILCK
LSLEGD STPPSAYGSVKAYT……
Fig. 1. Cleavage of membrane proteins by matrilysin (MAT) and
identification of annexin II. (A) Membrane fraction of WiDr cells
was prepared by Triton X-114 phase separation as described in
Experimental procedures. The membrane fraction obtained from
one confluent culture in a 90-mm dish (approximately 3 · 10
8
cells)
was incubated without ()) or with (+) 100 n
M matrilysin at 37 °C
for 3 h. The incubated membrane proteins were again subjected to
phase separation with Triton X-114, and proteins released into the
aqueous phase were separated by SDS/PAGE, transferred to
a PVDF membrane, and visualized by staining with Coomassie
Brilliant Blue R250. Closed arrowhead, a major 35-kDa band
identified as an annexin II fragment; open arrowheads, other major
differential bands in the matrilysin-treated sample. Ordinate,
molecular sizes in kDa of marker proteins. Other experimental con-
ditions are described in Experimental procedures. (B) Domain struc-
ture of annexin II and the site where it is cleaved by matrilysin.
N-terminal sequence analysis of the 35 kDa protein band revealed
that annexin II had been cleaved between Lys9 and Leu10.
J. Tsunezumi et al. Cleavage of annexin II by matrilysin
FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4811

LSLEGDHSTPPSAY by automated protein sequenc-
ing, and this sequence was identical to the amino acid
sequence from residues 10 to 23 of annexin II
(Fig. 1B).
To determine whether annexin II is directly cleaved
by matrilysin, we used both a recombinant human
annexin II and a natural annexin II purified from
CaR-1 human colon carcinoma cells. Matrilysin effec-
tively cleaved the 36 kDa recombinant annexin II and
converted it to the 35 kDa form (Fig. 2). This cleavage
was inhibited by an MMP inhibitor, N-(R)-[2-(hydrox-
yaminocarbonyl)-methyl]-4-methylpentanoyl-l-naphthyl-
alanyl-l-alanine-2-aminoethyl amide (TAPI-1), but not
by a mixture of inhibitors for serine, aspartic and
cysteine proteinases. The N-terminal amino acid
sequence of the 35 kDa, cleaved annexin II was iden-
tical to that of the membrane-derived annexin II
fragment (LSLEGDHSTPPSAY). These results indi-
cate that matrilysin cleaves the peptidyl bond between
Lys9 and Leu10 of annexin II (Fig. 1B).
When the annexin II purified from CaR-1 cells was
analyzed by immunoblotting, it showed two distinct
bands at approximately 36 and 72 kDa under non-
reducing conditions, but a single 36 kDa band under
reducing conditions (Fig. 3A). The 72 kDa protein was
thought to be a homodimer of annexin II cross-linked
with a disulfide bond. Next, the natural annexin II was
incubated with matrilysin and four other MMPs, and
then analyzed by immunoblotting under nonreducing
conditions (Fig. 3B). Matrilysin and MMP-2 almost

completely converted the 36 kDa annexin II to the
35 kDa cleaved form. In addition, these MMPs also
extinguished the 72 kDa band, suggesting that the
72 kDa annexin II dimer had been cross-linked by a
disulfide bond between the cysteine residues of two
monomer molecules at amino acid position 8 (Fig. 1B).
MMP-9 appeared to cleave annexin II weakly.
Matrilysin-catalyzed cleavage of annexin II on the
cell surface
Although annexin II does not have a signal sequence,
it is found on cell surfaces of many types of cultured
cells [19–21]. Indeed, flow cytometric analysis revealed
the existence of annexin II on cell surfaces of WiDr
cells (data not shown). To examine whether matrilysin
cleaves annexin II on cell surfaces, three kinds of
human colon cancer cell lines (CaR-1, WiDr and
DLD-1) and a human breast cancer cell line
(MDA-MB) were individually incubated with purified
matrilysin in culture conditions. After the treatment,
annexin II fragments released into the culture media
−
−−
++ +
++
(MAT)
(Inh.)
Native
Cleaved
(36)
(35)

(TAPI) (Mix.)
Fig. 2. Cleavage of purified annexin II by matrilysin (MAT). Recom-
binant annexin II (1 lgÆmL
)1
) was incubated at 37 °C for 3 h with
(+) or without ())50n
M matrilysin in the presence or absence of
the MMP inhibitor TAPI (4 l
M) or a proteinase inhibitor mixture
[Mix.; 0.2 m
M 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.16 lM ap-
rotinin, 0.025 m
M bestatin, 7.5 lM E-64, 0.01 mM leupeptin, and
5 l
M pepstatin]. The digests were analyzed by immunoblotting with
an antibody against annexin II under reducing conditions. Other
experimental conditions are described in Experimental procedures.
Arrowheads indicate native and cleaved annexin II bands at 36 and
35 kDa, respectively.
A
B
Fig. 3. Immunoblotting of purified natural annexin II and its cleav-
age by five kinds of MMP. (A) Immunoblotting of natural annexin II
purified from CaR-1 cells under nonreducing ()) and reducing (+)
conditions. 2ME, 2-mercaptoethanol. The bands at 72 and 36 kDa
correspond to dimeric and monomeric forms of annexin II, respec-
tively. (B). The natural annexin II (2 lgÆmL
)1
protein) was incubated
in 50 lL of a reaction mixture without (None) or with 5 n

M each of
MMP-1, MMP-2, MMP-3, MMP-9, or matrilysin (MAT) (MMP-7) at
37 °C for 16 h. The sample was subjected to nonreducing SDS/
PAGE followed by immunoblotting analysis with annexin II anti-
body. Other experimental conditions are described in Experimental
procedures. Arrowheads, annexin II bands.
Cleavage of annexin II by matrilysin J. Tsunezumi et al.
4812 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS
were analyzed by immunoblotting. Although the super-
natants from the control cultures did not contain
annexin II at detectable levels, all supernatants from
the matrilysin-treated cultures contained the 35 kDa
annexin II fragment (Fig. 4A). The amount of released
annexin II was highest in CaR-1 cells. The whole
lysate of WiDr cells contained only the 36 kDa native
annexin II. These results, as well as the result shown in
Fig. 1A, demonstrate that matrilysin cleaves annexin II
on the cell surface and releases the 35 kDa, C-terminal
fragment of annexin II into the culture medium.
The loss of cell surface annexin II after matrilysin
treatment was confirmed using CaR-1 cells by two dif-
ferent methods. Cell ELISA indicated that the immu-
noreactivity for cell surface annexin II was decreased
by about 40% after matrilysin treatment (Fig. 4B).
Immunofluorescence staining for annexin II also indi-
cated partial loss of the immunosignals for annexin II
on the cell surface after matrilysin treatment (Fig. 4C).
As MMP-2 cleaved purified annexin II in a test
tube, as shown in Fig. 3B, we also tested whether this
MMP cleaved annexin II on the cell surface and

released its soluble form. When CaR-1 cells were trea-
ted with active MMP-2, however, no annexin II frag-
ment was detectable in the culture supernatant,
suggesting that the cleavage of annexin II on the cell
surface is specific for matrilysin (data not shown).
It is known that annexin II binds to glycosaminogly-
cans such as heparan sulfate proteoglycans and sialo-
glycoproteins and phospholipids on the cell surface,
and many of the interactions are mediated by calcium
ions [22,23]. Serotonin (5-hydroxytryptamine) interacts
with N-acetylneuraminic acid, which is often contained
in glycolipids and glycoproteins on the cell surface
[24]. We examined the synergistic effects of matrilysin
with serotonin, heparin and EDTA on the release of
annexin II from the cell surface. WiDr cells and CaR-1
cells were treated with serotonin, heparin or EDTA in
the presence or absence of matrilysin, and the released
annexin II was analyzed by immunoblotting (Fig. 5).
When WiDr cells were treated with each of these
reagents, the intact (or full-length) annexin II was
released into the culture supernatant at a higher level
than the 35 kDa annexin II released by the matrilysin
treatment alone. When the cells were treated with sero-
tonin or heparin in the presence of matrilysin, release
MAT
–
+
–
+
–

+
–
+
CaR-1
DLD-1
WiDr
MDA-MB
WiDr
lysate
36
35
120
100
80
C ll ELISA ith C R 1ell ELISA with CaR-1
Annexin II (%)
60
40
20
None MAT
0
None MAT
A
B
C
Fig. 4. Matrilysin-catalyzed cleavage of annexin II on the cell sur-
face. (A) Three human colon carcinoma cell lines (CaR-1, WiDr, and
DLD-1) and human breast carcinoma cell line MDA-MB in mono-
layer cultures were incubated in 2 mL of serum-free medium with
(+) or without ())50n

M matrilysin (MAT) at 37 °C for 3 h. Proteins
released into the culture medium were concentrated by trichloro-
acetic acid precipitation and analyzed by immunoblotting under
reducing conditions with the antibody against annexin II. As a con-
trol, whole lysate of WiDr cells was run on the same gel. (B) CaR-1
cells were treated with (MAT) or without (None) matrilysin as
above, and the amount of annexin II remaining on the cell surface
was measured by cell ELISA. Each value represents the
mean ± SD of three independent results. (C) CaR-1 cells were
treated with (MAT) or without (None) matrilysin as above, and ann-
exin II remaining on the cell surface was visualized by immunofluo-
rescence staining. Detailed experimental conditions are described
in Experimental procedures.
Fig. 5. Release of membrane-bound annexin II by matrilysin and
three reagents. CaR-1 cells and WiDr cells in monolayer cultures
were incubated in the serum-free medium without ()) or with (+)
50 n
M matrilysin (MAT) in the presence or absence (None) of
0.2 m
M serotonin or 1 mgÆmL
)1
heparin at 37 °C for 3 h as
described in Fig. 4. Alternatively, the same cultures were incubated
with 5 m
M EDTA at 25 °C for 5 min. Proteins released into the
culture medium were analyzed by immunoblotting with the
antibody against annexin II as described in Fig. 4. Other experi-
mental conditions are described in Experimental procedures.
J. Tsunezumi et al. Cleavage of annexin II by matrilysin
FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4813

of cleaved annexin II was significantly increased as
compared with the level after the single treatment with
matrilysin. In contrast to the case of WiDr cells, intact
annexin II was slightly or never released when CaR-1
cells were treated with serotonin, heparin or EDTA
alone. However, when they were treated with both
matrilysin and serotonin or heparin, the amount of
cleaved annexin II released was significantly increased.
These results suggest that annexin II is bound to sialic
acid-containing molecules and heparan sulfate proteo-
glycans on the cell surface, and the strength of inter-
action differs between the two cell types. It seems
likely that serotonin and heparin promote matrilysin-
catalyzed annexin II cleavage by weakening the inter-
action of annexin II with the cell surface receptors.
Binding of tPA to surfaces of matrilysin-treated
cells
It has been shown that tPA binds to annexin II on
the surfaces of human endothelial cells [18,25]. In this
study, we investigated whether annexin II cleavage by
matrilysin affects tPA binding to the cell surface
(Fig. 6). First, we examined the effects of matrilysin
AB
C
D
Fig. 6. Effects of matrilysin and annexin II siRNA on binding of tPA to cancer cells. (A) CaR-1 cells were treated with (+) or without ())
50 n
M matrilysin (MAT) and/or 0.2 mM serotonin (Sero.) as shown in Fig. 5. After the treatment, the cells were washed and then further
incubated with 5 n
M tPA and 5 lM TAPI-1 at 37 °C for 1 h. The incubated cells were collected, washed and dissolved in the SDS/PAGE sam-

ple buffer and subjected to immunoblotting for tPA (top panel) and enolase as an internal loading control (center panel). Annexin II released
into the culture supernatant by the matrilysin/serotonin treatment is shown in the lower panel. (B) Detection of tPA bound to the cell surface
by cell ELISA. CaR-1 cells were pretreated without (None) or with 50 n
M matrilysin (MAT) on 96-well plates for 3 h, and incubated with tPA
and TAPI-1 as above. To quantify tPA bound to the cell surface, the cultures were subjected to cell ELISA according to the method
described in Experimental procedures. Each value represents the mean ± SD of triplicate assays. (C) Enzymatic activity of tPA bound to the
cell surface. CaR-1 cells were treated with the indicated concentrations of matrilysin and then with tPA as shown above. The catalytic activ-
ity of tPA bound to the cell surface was assayed using the fluorogenic peptide 3145v as a substrate. Each value represents the mean ± SD
of triplicate assays. Annexin II released into the culture supernatant by the matrilysin treatment is shown in the lower panel. (D) Effects of
annexin II siRNA on tPA binding to matrilysin-treated cells. CaR-1 cells were inoculated onto 24-well culture plates and treated with annex-
in II siRNA or a control RNA. Two days later, these cells were treated with matrilysin and then tPA as described above. The cells were
washed, lysed in the SDS/PAGE sample buffer and subjected to immunoblotting for tPA, annexin II (ANX-II) and enolase-1 as an internal
loading control. The cleaved annexin II (Sol. ANX-II) released into the culture medium is shown in the upper panel. Other experimental condi-
tions are described in Experimental procedures.
Cleavage of annexin II by matrilysin J. Tsunezumi et al.
4814 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS
and serotonin on tPA binding to CaR-1 cells
(Fig. 6A). Unexpectedly, the single treatment with
matrilysin significantly increased the binding of exoge-
nous tPA to CaR-1 cells, while releasing the cleaved
annexin II into the culture supernatant. Furthermore,
when CaR-1 cells were treated with both matrilysin
and serotonin, tPA binding to the cell surface was
greatly enhanced by the presence of serotonin. The
enhancement of tPA binding to the cell surface was
coincident with the release of cleaved annexin II. The
enhancement of tPA binding to the cell surface by
matrilysin was confirmed when the amount of tPA on
the cell surface was assayed by cell-based ELISA
(Fig. 6B). Moreover, the assay of tPA activity on the

cell surface clearly showed that matrilysin treatment
increased tPA activity in a dose-dependent manner
(Fig. 6C).
To show the direct link between tPA binding and
annexin II cleavage by matrilysin, we examined the
effect of suppression of annexin II expression on tPA
binding (Fig. 6D). Treatment of CaR-1 cells with a
small interfering RNA (siRNA) for annexin II signifi-
cantly and specifically suppressed expression of ann-
exin II protein. Suppression of annexin II expression
also suppressed binding of exogenous tPA to the
matrilysin-treated cells, as well as the release of cleaved
annexin II. All of these results strongly suggest that
matrilysin enhances binding of tPA to cancer cells by
cleaving annexin II on the cell surface. The bound tPA
maintained its enzymatic activity on the cancer cell
surface.
Mechanism for matrilysin-induced tPA binding to
the cell surface
Matrilysin cleaves annexin II on the cell surface into
the N-terminal peptide Ac-STVHEILCK and the
C-terminal large peptide of 35 kDa, releasing the latter
peptide from the cell surface. It was assumed that
Ac-STVHEILCK remained on the cell surface and
bound tPA. To test this possibility, we used a synthetic
Ac-STVHEILCK peptide.
First, the direct interaction between Ac-STVHEILCK
and tPA was examined using ELISA plates precoated
with the peptide. At an optimal dose, the peptide coat-
ing increased the amount of tPA bound to the plates to

about twice that bound to the control plates (Fig. 7A).
Next, tPA binding was examined in the presence or
absence of Ac-STVHEILCK on the plates precoated
with the purified, native annexin II or with the
annexin II cleaved by matrilysin. The tPA binding was
slightly but significantly more efficient on the cleaved
annexin II than on the native one, and on either plate
the addition of the soluble N-terminal peptide signifi-
cantly suppressed tPA binding to the plate (Fig. 7B).
These results suggested that tPA was able to bind
Ac-STVHEILCK. The competitive effect of the syn-
thetic peptide was also examined for tPA binding to
CaR-1 cells. tPA, with or without the peptide, was
applied to the cells pretreated with or without matrilysin
(Fig. 7C). Although Ac-STVHEILCK at 0.2 mm had
no effect on the nontreated cells, it significantly inhibited
tPA binding to the CaR-1 cells pretreated with
matrilysin. These results strongly suggested that the
matrilysin-enhanced tPA binding to cell membranes
depended, at least in part, on the N-terminal peptide
fragment Ac-STVHEILCK, which was generated by the
matrilysin-catalyzed cleavage of annexin II.
To obtain further evidence that the N-terminal
peptide binds tPA on the cell surface, CaR-1 cells,
without matrilysin treatment, were incubated with
the N-terminal peptide and then with tPA. Treat-
ment of CaR-1 cells with the peptide increased the
amount of tPA bound to the cell surface (Fig. 8A).
This implies that the N-terminal peptide binds both
an unidentified cell surface molecule and tPA on the

cell surface. Annexin II is known to form a hetero-
tetramer complex, which consists of two annexin II
molecules and two p11 (or S100A10) molecules [26].
Indeed, the CaR-1 cell-derived annexin II formed a
homodimer cross-linked with a disulfide bond, as
shown in Fig. 3. Therefore, it seemed possible that
the N-terminal peptide bound an annexin II mono-
mer on the cell surface. To test this possibility,
CaR-1 cells were first incubated with the N-terminal
peptide, and then with heparin to release it from the
cell surface, and the released annexin II was analyzed
by immunoblotting (Fig. 8B). In the absence of hep-
arin, annexin II was scarcely detected in the culture
supernatant of CaR-1 cells. When heparin was added
to the cells without the peptide treatment, a single
band of the 36 kDa annexin II was detected in the
culture supernatant. However, when heparin was
applied to the peptide-treated cells, the immunoblot-
ting of the culture supernatant showed two close
bands at 36 and 37 kDa under nonreducing condi-
tions, but a single band of 36 kDa under reducing
conditions. The 37 kDa band appeared to be the
heterodimer protein of the 36 kDa annexin II
monomer with Ac-STVHEILCK cross-linked with a
disulfide bond. Indeed, the 37 kDa annexin II hetero-
dimer was observed more clearly when purified ann-
exin II was incubated with Ac-STVHEILCK in a
test tube (Fig. 8C, lane 4). The production of the
37 kDa annexin II heterodimer appeared to be
enhanced when the treatment was done in the

J. Tsunezumi et al. Cleavage of annexin II by matrilysin
FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4815
presence of heparin (Fig. 8C, lane 5). In addition,
the 37 kDa annexin II heterodimer was faintly
detected even when the purified annexin II was
digested by matrilysin (Fig. 8C, lane 2), although it
did not increase in amount when the cleaved
annexin II was incubated with the uncleaved form
(lane 3). These results suggested that the 37 kDa
nonapeptide–intact annexin II complex might be
produced on the matrilysin-treated cancer cells.
However, we failed to recover the 37 kDa annexin II
complex from the matrilysin-treated cells (data
not shown). This is probably due to the low
concentration of the peptide in the matrilysin-treated
cells.
It has been reported that tPA binds to a lysine
residue via its kringle-2 domain [27]. This suggests
that tPA binds to the C-terminal lysine residue of
Ac-STVHEILCK, which is produced from annexin II
by matrilysin treatment. To test this possibility, we
performed a competition assay using e-aminocaproic
acid as a C-terminal lysine analog. e-Aminocaproic
acid at 10 mm strongly inhibited tPA binding to both
the matrilysin-treated cells and the untreated cells
(Fig. 8D). This competitive effect was much more
evident than that obtained with 0.2 mm
Ac-STVHEILCK (Fig. 7C). However, 1 mm e-amino-
caproic acid scarcely inhibited tPA binding (data not
shown). These results support the hypothesis that the

N-terminal Ac-STVHEILCK peptide may be linked
to an annexin II monomer on the cell surface, and
tPA efficiently binds to the C-terminal lysine of this
peptide. The relatively low blocking activity of
exogenous Ac-STVHEILCK suggests that the
membrane-bound peptide may have higher affinity
than the free peptide.
A
B
C
Fig. 7. Interaction of tPA with Ac-STVHEILCK. (A) Direct interaction
between Ac-STVHEILCK and tPA. Ninety-six-well microtiter plates
were coated with the indicated concentrations of Ac-STVHEILCK
(N-peptide) in NaCl/P
i
at 4 °C overnight. These wells were fixed
with 10% formaldehyde for 10 min and washed three times with
NaCl/P
i
containing 0.1% Tween-20. After blocking with 1.2% BSA
in NaCl/P
i
, each well was incubated with 5 nM tPA at 37 °C for
2.5 h. After fixing, the relative amount of tPA bound to each well
was assayed by ELISA as described in Experimental procedures.
(B) Binding of tPA to native and matrilysin-cleaved annexin II (ANX-
II) proteins. Purified natural annexin II (1 l
M) was digested with
50 n
M matrilysin at 37 °C for 3 h. The untreated (Native) and

digested (Cleaved) annexin II were individually coated on 96-well
microtiter plates overnight. Using these annexin II-coated wells, the
tPA binding assay in the presence (+) or absence ()) of 200 l
M
Ac-STVHEILCK was carried out as described above. (C) Competitive
inhibitory effect of Ac-STVHEILCK on tPA binding to CaR-1 cells.
CaR-1 cells were incubated with 50 n
M matrilysin on 96-well plates
at 37 °C for 3 h. The tPA binding to the CaR-1 cells in the presence
(+) or absence ()) of 200 l
M Ac-STVHEILCK was analyzed as
shown in Fig. 5B. In (A), (B) and (C), the data represent the
mean ± SD of triplicate assays.
Cleavage of annexin II by matrilysin J. Tsunezumi et al.
4816 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS
Discussion
The present study identified annexin II as a novel mem-
brane-bound substrate for matrilysin. Matrilysin
cleaved annexin II on the surfaces of human colon
cancer cells, releasing a major C-terminal sequence of
annexin II from the cell membrane. The matrilysin
treatment of cancer cells facilitated the binding of tPA
to the cell surface. We previously showed that active
matrilysin efficiently binds to cholesterol sulfate on the
cell membranes of colon cancer cells, retaining its enzy-
matic activity [11]. This MMP, together with cholesterol
sulfate, was localized in the lipid microdomain so-called
raft of cell membrane [11]. Annexin II is also localized
in the membrane domain raft [28]. Thus, it is highly
likely that the membrane-bound active matrilysin

A
C
D
B
Fig. 8. Interaction of tPA with Ac-STVHEILCK on the cell surface and its inhibition by e-aminocaproic acid. (A) Enhancement of tPA binding
to cells by Ac-STVHEILCK. CaR-1 cell were incubated with (+) or without ()) 400 l
M Ac-STVHEILCK (N-peptide) on 24-well plates containing
serum-free medium at 37 °C for 6 h and then with 5 n
M tPA for 1 h. The tPA bound to the cells, as well as enolase as an internal loading
control, was analyzed by immunoblotting as shown in Fig. 6A. (B) Formation of 37 kDa heterodimer on cells treated with Ac-STVHEILCK.
CaR-1 cells were incubated with (+) or without ()) 400 l
M Ac-STVHEILCK and further incubated in the serum-free medium supplemented
with (+) or without ())1mgÆmL
)1
heparin for 2 h. Annexin II released into the culture medium was analyzed by immunoblotting under non-
reducing ()2ME) and reducing (+2ME) conditions. The 37 kDa band indicated by the open arrowhead seems to be a complex of annexin II
with Ac-STVHEILCK. Closed arrowheads indicate annexin II monomer. (C) Formation of 37 kDa heterodimer in test tubes. Purified annexin II
(1 l
M) was incubated at 37 °C for 2 h without (lane 1) or with (lanes 4 and 5) 100 lM Ac-STVHEILCK in 50 mM Tris/HCl (pH 7.5) containing
150 m
M NaCl, 5 mM CaCl
2
and 0.01% Brji 35 in the absence (lanes 1 and 4) or presence (lane 5) of 100 lgÆmL
)1
heparin. In other tubes, the
purified annexin II (1 l
M) was incubated with 5 nM matrilysin at 37 °C for 2 h, and the reaction was terminated by adding 10 lM TAPI-1
(lane 2). The cleaved annexin II (1 l
M) was incubated with the uncleaved annexin II (1 lM)at37°C for 2 h (lane 3). These samples were ana-
lyzed by immunoblotting under nonreducing conditions. (D) Inhibition of tPA binding to cancer cells by e-aminocaproic acid. CaR-1 cells were

treated with (+) or without () ) matrilysin (MAT) on 24-well or 96-well culture plates and then incubated with 5 n
M tPA plus 5 lM TAPI-1 in
the presence (+) or absence ())of10m
M e-aminocaproic acid (eACA). The amounts of tPA on the cell surface were analyzed by immuno-
blotting (left panel) and cell ELISA (right panel). Each bar represents the mean ± SD of triplicate assays. Other experimental conditions are
described in Fig. 6 and Experimental procedures.
J. Tsunezumi et al. Cleavage of annexin II by matrilysin
FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4817
efficiently cleaves annexin II on cancer cell surfaces.
MMP-2 and MMP-9 cleaved purified annexin II, but
they appeared not to cleave annexin II on the cell sur-
face, indicating that the cleavage of the membrane-
bound annexin II is specific for matrilysin. The specific
cleavage of cell surface annexin II by matrilysin may
result from the specific binding of matrilysin to the
cancer cells. In our previous study, among three MMPs
tested (matrilysin, MMP-2 and MMP-3), only matrily-
sin was able to bind to the cancer cells [10] and choles-
terol sulfate [11].
Annexin II is expressed in epithelial cells of various
tissues, including the epidermis, pancreas and breast
[19–21], and vascular endothelial cells [25]. In these
kinds of cells, some annexin II molecules are found on
the cell surface. Annexin II is known to interact with
membrane phospholipids and glycosaminoglycans such
as heparin, heparan sulfate [22,23] and fucoidan as a
sulfated fucopolysaccharide, in a calcium-dependent or
calcium-independent manner [29,30]. Serotonin is
known to interact with glycolipids and glycoproteins
containing N-acetylneuraminic acid [24]. In this study,

heparin, serotonin and EDTA released different
amounts of intact or matrilysin-cleaved annexin II from
cell membranes, suggesting that annexin II binds to cell
membranes via multiple receptors. Our data also suggest
that the manner of annexinin II binding to cell mem-
branes varies considerably from one cell type to another.
Membrane-bound annexin II is thought to play impor-
tant roles in various biological processes, such as
fibrinolysis [31], cell–ECM adhesion [13], protease
binding to cell membranes [15], ligand-mediated cell
signaling [32], and virus infection [33]. One of the best
characterized functions of extracellular annexin II is its
action as a membrane-bound receptor for tPA on vascu-
lar endothelial cells [25,34]. Some previous studies have
demonstrated that Cys8 in the N-terminal region is
essential for tPA binding to the cell surface [18].
Unexpectedly, the present study showed that matri-
lysin treatment of colon cancer cells led to marked
enhancement of tPA binding to the cancer cell surface,
although the tPA receptor annexin II was cleaved and
released from the cell membrane. Our experiments with
the synthetic peptide Ac-STVHEILCK, which corre-
sponds to the N-terminal nine amino acid peptide of
annexin II generated by the matrilysin-catalyzed cleav-
age of annexin II, gave rise to the possibility that the
N-terminal peptide is responsible for the enhanced
binding of tPA to the cell surface. First, matrilysin-
induced tPA binding to cells correlated well with the
extent of annexin II cleavage by matrilysin, and the
suppression of annexin II expression by siRNA

decreased tPA binding (Fig. 6). Second, tPA bound to
the synthetic peptide coated on plastic plates in a dose-
dependent manner, and the tPA binding was more effi-
cient than that to the entire annexin II molecule
(Fig. 7A,B). Third, pretreatment of colon cancer cells
with the synthetic peptide significantly increased tPA
binding to the cells, whereas the peptide competitively
suppressed tPA binding to the matrilysin-treated cells
(Figs 7C and 8A). All these results support the hypoth-
esis that the N-terminal annexin II peptide produced
by matrilysin remains bound to cell membranes and
functions as a receptor for tPA. tPA is known to have
high affinity for lysine. tPA binds to lysyl–Sepharose
through its kringle-2 domain, and this interaction is
blocked by l-lysine or e-aminocaproic acid as a C-ter-
minal lysine analog [27]. The kringle-2 domain of tPA
directly interacts with e-aminocaproic acid [35]. Krin-
gle-2-mediated tPA binding to the C-terminal lysines
plays an important role in the degradation of fibrin
clots [36,37]. Partial degradation of fibrin by plasmin
generates C-terminal lysines, which function as new
binding sites for tPA, resulting in further activation of
plasminogen on the fibrin clot [38]. On the basis of
these facts, it seems very likely that tPA binds to the
C-terminal lysine of the N-terminal annexin II frag-
ment Ac-STVHEILCK remaining on cell membranes.
Although we cannot exclude the possibility that tPA
binds to the C-terminal lysines of other protein frag-
ments that are produced by the matrilysin activity, the
result of the siRNA experiment with annexin II shown

in Fig. 6D suggests that the annexin II fragment may
play a major role in matrilysin-induced tPA binding
to the tumor cell surface. On the basis of the amount
of annexin II fragment released by the treatment
with matrilysin plus heparin, we estimated that at
least 0.56–1.4 fmol of annexin II per 10
6
cells (3.4–
8.4 · 10
5
molecules per cell) exists on the surface
of CaR-1 cells (Fig. 5). We have determined that
matrilysin binds to the tumor cell surface with a K
d
of 7 nm (K. Yamamoto, J. Tsunezumi, S. Higashi and
K. Miyazaki, unpublished data). The binding capacity
of tumor cells for matrilysin was estimated to be
0.25–1.0 fmol per 10
6
cells (1.5–6.0 · 10
5
molecules per
cell). From the data shown in Fig. 4B, it is also
assumed that the matrilysin treatment produces at least
1.4–3.4 · 10
5
molecules per cell of the N-terminal ann-
exin II peptide, most likely on the tumor cell surface.
The N-terminal sequence of annexin II has previ-
ously been reported to interact with p11 (or S100A10),

which is often regarded as the annexin II light chain,
to form a heterotetramer complex [26]. In this study,
we detected the possible annexin II dimer of approxi-
mately 72 kDa, but we failed to detect p11 in the
annexin II complex with a specific antibody (data not
Cleavage of annexin II by matrilysin J. Tsunezumi et al.
4818 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS
shown). Our data indicated that exogenous N-terminal
annexin II peptide bound to the cancer cell surface, and
the bound peptide was recovered as a 37-kDa heterodi-
mer complex with the intact annexin II molecule from
the cell surface when the cells were treated with heparin.
This heterodimer complex was linked by a disulfide
bond, and was produced efficiently when the peptide
was incubated with the intact annexin II in test tubes.
These results strongly suggest that the N-terminal ann-
exin II peptide remains as the 37 kDa complex with the
intact annexin II on the surfaces of matrilysin-treated
cells. However, this possibility was not confirmed,
because we failed to detect this nonapeptide–annexin II
complex in the matrilysin-treated cancer cells (data not
shown). Thus, it is also possible that the N-terminal
annexin II peptide binds to cell membranes through p11
or other membrane molecules.
The plasminogen activator–plasmin system is well
known to play important roles not only in fibrinolysis
but also in ECM degradation during tissue remodeling
[39,40]. Like urokinase-type plasminogen activator,
tPA binds to some membrane proteins, including ann-
exin II [41]. The binding of tPA or urokinase-type

plasminogen activator to the membrane receptors
greatly increases plasminogen activator activity on the
membranes, i.e. the conversion of plasminogen to plas-
min [34,42]. In addition, it has become evident that the
receptor binding of the two plasminogen activators
induces cell growth signaling without the need for their
proteolytic activities [32,43]. In the present study, the
treatment of colon cancer cells with matrilysin resulted
in efficient cleavage of annexin II and enhanced bind-
ing of tPA to cell membranes. We confirmed that the
tPA bound to the matrilysin-treated cancer cells
showed plasminogen activator activity, but we could
not detect significant activation of MAP kinase signal-
ing in the tPA-treated cells (data not shown). Many
types of MMP, including matrilysin, MMP-3 and
MMP-2/9, are efficiently activated by trypsin-like
serine proteinases [44–46]. Plasmin is thought to be the
most important activator for these MMPs. It is
supposed that the tPA on the cell membrane activates
the proforms of these MMPs by producing plasmin
[47]. Thus, the cleavage of annexin II by matrilysin
may trigger the proteinase amplification cascade or
cycling in the pericellular space of cancer cells. These
proteolytic activities are likely to promote the invasive
growth of tumor cells and subsequent metastasis.
Past studies have suggested that matrilysin is
involved in the malignancy and metastasis of human
cancers [9]. We have previously reported that active
matrilysin binds to the surfaces of colon cancer cells
and induces notable cell aggregation, probably due to

cleavage of membrane protein(s) [13,14]. However, the
cleavage of annexin II by matrilysin appeared not to
induce cell–cell adhesion, because the suppression of
annexin II synthesis by RNA interference did not inhi-
bit cell aggregation (data not shown). Therefore, it is
likely that membrane proteins other than annexin II
are also degraded or processed by matrilysin. These
actions of matrilysin may also contribute to the malig-
nant growth of cancer cells. Understanding the patho-
logical significance of the cleavage of annexin II and
other membrane substrates by matrilysin seems to be
important in designing new targets for cancer therapies.
Experimental procedures
Antibodies and other reagents
The sources of reagents used were as follows: human
recombinant annexin II was from AmProx (Carlsbad, CA,
USA); human Glu-plasminogen and Lys-plasminogen were
from Hematologic Technologies (Essex Junction, VT,
USA); tPA and Protease Inhibitor Cocktail Set III were
from Calbiochem (San Diego, CA, USA); human recom-
binant MMP-9, human recombinant interstitial collage-
nase (MMP-1) and MMP-3 were from Chemicon
(Temecula, CA, USA); human recombinant matrilysin and
6-aminohexanoic acid (e-aminocaproic acid) were from
Wako Pure Chemical Industries (Osaka, Japan); the MMP
substrates 3145v (Pyr-Gly-Arg-MCA) and 3105v (Boc-Glu-
Lys-Lys-MCA) and the synthetic MMP inhibitor TAPI-1
were from Peptides Institute (Osaka, Japan); and serotonin
(5-hydroxytryptamine hydrochloride) was from Sigma
Aldrich (St Louis, MO, USA). Commercial antibodies

against human antigens used were: mouse monoclonal anti-
body against tPA from Abcam (Cambridge, MA, USA);
rabbit polyclonal antibody against enolase, mouse mono-
clonal antibody against annexin II and rabbit polyclonal
antibody against annexin II from Santa Cruz (Santa Cruz,
CA, USA); and goat polyclonal antibody against p11 from
R&D systems (Minneapolis, MN, USA). The mouse mono-
clonal antibody 11B4G against human matrilysin was a
kind gift from T. Tanaka (Nagahama Institute of Oriental
Yeast Co., Shiga, Japan). Human MMP-2 was prepared in
our laboratory as previously reported [10]. The N-terminal
annexin II peptide (Ac-STVHEILCK) was synthesized by
Hayashi Kasei (Osaka, Japan). All other chemicals used for
the experiments were of analytical grade or the highest
quality commercially available.
Cell lines and culture conditions
Four human colon cancer cell lines (Colo 201, DLD-1,
WiDr, and CaR-1) and the human breast cancer cell line
MDA-MB were obtained from Japanese Cancer Resources
J. Tsunezumi et al. Cleavage of annexin II by matrilysin
FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4819
Bank (Osaka, Japan). These cell lines were maintained in a
mixture of DMEM and Ham’s F-12 medium (DMEM/F12)
(Invitrogen, Carlsbad, CA, USA) supplemented with 10%
fetal bovine serum.
SDS/PAGE and immunoblotting analyses
SDS/PAGE was performed on 5–20% gradient polyacryl-
amide gels by the standard method. Separated proteins
were detected by staining with Coomassie Brilliant
Blue R250. For immunoblotting, proteins separated on gels

were transferred onto poly(vinylidene difluoride) (PVDF)
membranes (Millipore, Billerica, MA, USA) and visualized
by the alkaline phosphatase method or the enhanced chemi-
luminescence method (GE Healthcare, Amersham, UK)
with specific antibodies.
Suppression of annexin II expression by RNA
interference
A predesigned siRNA corresponding to the target sequence
for human annexin II (5¢-UGGAAAGCAUCAGGAAA
GAGGUUAA-3¢) and a control RNA were obtained from
iGENE (Tsukuba, Japan). Cells were inoculated on the day
before transfection at a cell density of approximately 30%
saturation in 24-well culture plates and treated with the
siRNA or the control RNA by using the HiperFect reagent
(Qiagen, Tokyo) according to the manufacturer’s protocol.
Two or three days later, the cells were used for experiments.
Phase separation of membrane-associated
molecules in Triton X-114 solution
To separate membrane-associated proteins from soluble
ones, we used phase separation of the Triton X-114 solu-
tion, as described previously [11]. WiDr cells (approxi-
mately 3 · 10
8
cells) were dissolved in 1.2 mL of 50 mm
Tris/HCl (pH 7.5) containing 150 mm NaCl and 5 mm
CaCl
2
(TBSC) supplemented with 0.1% Triton X-114 and
centrifuged at 95 g at 4 °C for 5 min. The resultant super-
natant was added to Triton X-114 to make a final concen-

tration of 2%, and incubated at 4 °C for 1 h. The
temperature of the extract was then increased to 37 ° C, and
the sample was incubated for a further 5 min. The resultant
detergent and aqueous phases were separated by centrifuga-
tion at 7500 g for 10 min at 25 °C. Membrane-associated
proteins were recovered in the lower, detergent phase.
Purification of natural annexin II
CaR-1 cells were dissolved in the TBSC buffer supple-
mented with 0.1% Triton X-100 and a proteinase inhibitor
mixture [0.2 mm 4-(2-aminoethyl)benzenesulfonyl fluoride,
0.16 lm aprotinin, 0.025 mm bestatin, 7.5 lm E-64,
0.01 mm leupeptin, and 5 lm pepstatin], sonicated for 10 s,
and then centrifuged at 95 g at 4 °C for 5 min. The cell
extract was applied to a heparin–Sepharose 6B column
(GE Healthcare). Annexin II was bound to the column and
eluted at 0.6–1.0 m NaCl. The annexin II fraction was dia-
lyzed against the TBSC plus Triton X-100 buffer and
applied to a Q-Sepharose column. A major part of annex-
in II was eluted from the column at 0.3 m NaCl. This frac-
tion, which contained two types of annexin II of 36 and
72 kDa, was further purified by heparin–Sepharose 6B col-
umn chromatography. The final annexin II preparation
showed a purity of 80–90% for the total annexin II as
judged by SDS/PAGE.
Cleavage of annexin II by MMPs and determina-
tion of N-terminal amino acid sequence
The proforms of matrilysin and other MMPs were activated
by incubation with 1 mm p-aminophenyl mercuric acetate.
Purified annexin II (7 lg of protein) was incubated with
50 nm matrilysin in 50 lL of a reaction buffer consisting of

TBSC and 0.01% Brij 35 at 37 °C for the indicated lengths
of time. The reaction was stopped by mixing with the SDS
sample buffer, and the reaction mixture was analyzed by
SDS/PAGE and immunoblotting. In some experiments,
membrane fractions instead of the purified annexin II were
used as the substrates. For determination of N-terminal
sequences, the digested proteins were separated by SDS/
PAGE, transferred to PVDF membranes, and stained with
Coomassie Brilliant Blue R-250. Stained protein bands were
cut from the membranes and analyzed with a Procise 49X
cLC protein sequencer (Applied Biosystems, Foster City,
CA, USA).
Cleavage of cell surface annexin II by matrilysin
Cancer cells were harvested by trypsinization, inoculated at
a density of 5 · 10
6
cells per 60 mm culture dish in the
growth medium, and incubated for 2 days. The cultures
were washed twice with the serum-free DMEM/F12 med-
ium and then incubated in 2 mL of the serum-free medium
containing 50 nm matrilysin at 37 °C for 3 h. Proteins
released into the culture medium were collected, precipi-
tated in 10% trichloroacetic acid, washed with ethanol, and
analyzed by immunoblotting.
Immunofluorescence staining of cell surface
annexin II
CaR-1 cells were inoculated onto four-well Lab-Tek chamber
slides (Nagel Nunc; Naperville, IL, USA) in the growth
medium for 2 days. The cultures were washed twice with the
serum-free medium, and incubated in the medium containing

50 nm matrilysin at 37 °C for 3 h. Adherent cells were fixed
Cleavage of annexin II by matrilysin J. Tsunezumi et al.
4820 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS
with 10% formaldehyde for 10 min, and washed three times
with NaCl/P
i
containing 1 mm CaCl
2
,1mm MgCl
2
and
6nm glucose. After blocking with 1% BSA in NaCl/P
i
, the
cells were incubated with a monoclonal antibody against
annexin II at 4 °C for 18 h. A fluorescein isothiocyanate-
conjugated second antibody (Vector Laboratories, Burlin-
game, CA, USA) was used for detection. The cell surface
annexin II was visualized under a fluorescence microscope
(Keyence, model BZ-8000, Osaka, Japan).
Analysis of membrane-bound annexin II after
matrilysin treatment
The relative amounts of annexin II on the cell surface were
determined by two different methods. For the cell ELISA
assay, CaR-1 cells were inoculated at a density of 1 · 10
5
cells per well of 96-well plates (Sumilon, Tokyo, Japan) in
the growth medium and incubated at 37 °C for 2 days. The
cultures were washed twice with the serum-free medium and
incubated in 0.2 mL of the serum-free medium supplemented

with 50 nm matrilysin at 37 °C for 3 h. After the incubation,
each culture was washed three times with NaCl/P
i
containing
1mm CaCl
2
,1mm MgCl
2
and 6 mm glucose, incubated
with 5 n m tPA and 5 lm TAPI-1 as an MMP inhibitor at
37 °C for 1 h, and washed three times. The cells were fixed
with 10% formaldehyde for 10 min, washed three times with
NaCl/P
i
containing 0.1% Tween-20, and blocked with 1.2%
BSA in NaCl/P
i
. Finally, each culture was sequentially incu-
bated with a monoclonal antibody against tPA and with a
biotinylated second antibody (Vector Laboratories) at 37 °C
for 1 h. The intensity of immunoreactive signals for tPA was
measured by the alkaline phosphatase method with p-nitro-
phenylphosphate as a substrate. Alternatively, tPA on the
cell surface was detected by measuring its enzyme activity.
In this assay, cells carrying the exogenous tPA were incu-
bated with 200 lm fluorogenic peptide 3145v as substrate at
37 °C for 40 min. Substrate hydrolysis was determined by
measuring fluorescence at 390 nm for excitation and at
460 nm for emission, using a Plate Chameleon spectrofluo-
rometer (Hidex, Turku, Finland).

Acknowledgements
We thank K. Moriyama and A. Kurosawa for techni-
cal support of protein sequencing and cytofluorometry,
respectively. We are grateful to S. Iiizumi for helpful
discussion. This work was supported by a Grant-in-
Aid from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
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