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

Matrilysin (matrix metalloprotease-7) cleaves membrane-bound annexin II and enhances binding of tissue-type plasminogen activator to cancer cell surfaces Jun Tsunezumi1,2, Kazuhiro Yamamoto1, Shouichi Higashi1,2 and Kaoru Miyazaki1,2

1 Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan 2 Graduate School of Integrated Sciences, Yokohama City University, Japan

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: miyazaki@yokohama-cu.ac.jp

(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.

enzymes than 20 zinc-dependent gelatinase A (MMP-2), (MMP-3), stromelysin

during of

including membrane- modulated by several MMPs, gelatinase B type MMPs, (MMP-9), 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.

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.

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Matrix metalloproteinases (MMPs) form a group of more 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 pathological tissues conditions such as inflammation and invasion and metastasis of cancer cells [1,2]. Many recent studies have provided evidence that the biological activities of surface molecules are proteolytically various cell Matrilysin, the smallest of the MMPs, has broad substrate specificity and has been demonstrated to

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Cleavage of annexin II by matrilysin

important MMPs

(Fig. 1A). The N-terminal amino 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 acid fraction sequence of the 35 kDa protein was determined to be

MAT

A

(kDa)

−

+

200

116

97 97

66

45

31

21

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 in human colon cancers, most 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 these cell membrane protein(s). Furthermore, 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 the matrilysin-induced phenotypic mechanism of changes of cancer cells, such as enhancement of homo- typic cell adhesion and metastatic potential.

1

9

10

B

338 -C

N-

STVHEILCK LSLEGD STPPSAYGSVKAYT……

9 10

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

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 · 108 cells) was incubated without ()) or with (+) 100 nM matrilysin at 37 (cid:2)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.

Cleavage of annexin II by matrilysin

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It was previously found that active matrilysin specifi- cally binds to surfaces of colon cancer cells and

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Cleavage of annexin II by matrilysin

A

from residues 10 to 23 of LSLEGDHSTPPSAY by automated protein sequenc- ing, and this sequence was identical to the amino acid sequence annexin II (Fig. 1B).

B

to that of

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 nM each of MMP-1, MMP-2, MMP-3, MMP-9, or matrilysin (MAT) (MMP-7) at 37 (cid:2)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.

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- the membrane-derived annexin II tical 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 it showed two distinct analyzed by immunoblotting, 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

(MAT)

(Inh.)

− −

+ −

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.

+ + (TAPI)

+ + (Mix.)

Matrilysin-catalyzed cleavage of annexin II on the cell surface

(36) Native Cleaved (35)

Fig. 2. Cleavage of purified annexin II by matrilysin (MAT). Recom- binant annexin II (1 lgÆmL)1) was incubated at 37 (cid:2)C for 3 h with (+) or without ()) 50 nM matrilysin in the presence or absence of (4 lM) or a proteinase inhibitor mixture the MMP inhibitor TAPI [Mix.; 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.16 lM ap- rotinin, 0.025 mM bestatin, 7.5 lM E-64, 0.01 mM leupeptin, and 5 lM 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.

and a human breast cancer cell

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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 lines (CaR-1, WiDr and human colon cancer cell DLD-1) line (MDA-MB) were individually incubated with purified matrilysin in culture conditions. After the treatment, annexin II fragments released into the culture media

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Cleavage of annexin II by matrilysin

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.

culture

A

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l

r D W

MAT

CaR-1 –+

DLD-1 –+

WiDr +–+

MDA-MB –

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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 supernatant, in the suggesting that the cleavage of annexin II on the cell surface is specific for matrilysin (data not shown).

B

120

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80

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60

40

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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 ()) 50 nM matrilysin (MAT) at 37 (cid:2)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 nM matrilysin (MAT) in the presence or absence (None) of 0.2 mM serotonin or 1 mgÆmL)1 heparin at 37 (cid:2)C for 3 h as described in Fig. 4. Alternatively, the same cultures were incubated with 5 mM EDTA at 25 (cid:2)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.

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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). these When WiDr cells were treated with each of reagents, (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

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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 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

A

B

D

C

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 nM 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 nM tPA and 5 lM TAPI-1 at 37 (cid:2)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 nM 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.

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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 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.

36 kDa protein the of

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lane 4). The production of tube (Fig. 8C, annexin II heterodimer 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 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 this possibility, surface. To test 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 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 the test appeared to be 37 kDa in the enhanced when the treatment was done

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A

B

the data represent

(B) and (C),

In (A),

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/Pi at 4 (cid:2)C overnight. These wells were fixed with 10% formaldehyde for 10 min and washed three times with NaCl/Pi containing 0.1% Tween-20. After blocking with 1.2% BSA in NaCl/Pi, each well was incubated with 5 nM tPA at 37 (cid:2)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 lM) was digested with 50 nM matrilysin at 37 (cid:2)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 lM 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 nM matrilysin on 96-well plates at 37 (cid:2)C for 3 h. The tPA binding to the CaR-1 cells in the presence (+) or absence ()) of 200 lM Ac-STVHEILCK was analyzed as shown in Fig. 5B. the mean ± SD of triplicate assays.

increase in amount when the

suggested that annexin II

from the matrilysin-treated cells is probably due shown). This to the

did not cleaved annexin II was incubated with the uncleaved form the 37 kDa (lane 3). These results complex might be nonapeptide–intact produced on the matrilysin-treated cancer cells. However, we failed to recover the 37 kDa annexin II (data complex low not concentration of the peptide in the matrilysin-treated cells.

C

obtained than with that

that 37 kDa annexin II heterodimer was

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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 0.2 mm evident 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 relatively low blocking activity of peptide. The the exogenous Ac-STVHEILCK suggests membrane-bound peptide may have higher affinity than the free peptide. lane 5). In addition, presence of heparin (Fig. 8C, faintly the detected even when the purified annexin II was lane 2), although it digested by matrilysin (Fig. 8C,

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Cleavage of annexin II by matrilysin

B

A

C

D

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 lM Ac-STVHEILCK (N-peptide) on 24-well plates containing serum-free medium at 37 (cid:2)C for 6 h and then with 5 nM 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 lM Ac-STVHEILCK and further incubated in the serum-free medium supplemented with (+) or without ()) 1 mgÆmL)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 lM) was incubated at 37 (cid:2)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 mM NaCl, 5 mM CaCl2 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 lM) was incubated with 5 nM matrilysin at 37 (cid:2)C for 2 h, and the reaction was terminated by adding 10 lM TAPI-1 (lane 2). The cleaved annexin II (1 lM) was incubated with the uncleaved annexin II (1 lM) at 37 (cid:2)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 nM tPA plus 5 lM TAPI-1 in the presence (+) or absence ()) of 10 mM 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.

Discussion

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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 the membrane-bound active matrilysin likely that 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

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than that

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].

fragment

Annexin II is expressed in epithelial cells of various including the epidermis, pancreas and breast tissues, [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 [31], cell–ECM adhesion [13], protease fibrinolysis 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].

it

the synthetic peptide coated on plastic plates in a dose- dependent manner, and the tPA binding was more effi- cient 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 released by the treatment with matrilysin plus heparin, we estimated that at least 0.56–1.4 fmol of annexin II per 106 cells (3.4– 8.4 · 105 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 Kd 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 106 cells (1.5–6.0 · 105 molecules per is also cell). From the data shown in Fig. 4B, assumed that the matrilysin treatment produces at least 1.4–3.4 · 105 molecules per cell of the N-terminal ann- exin II peptide, most likely on the tumor cell surface.

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expression by annexin II 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 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 siRNA decreased tPA binding (Fig. 6). Second, tPA bound to

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Cleavage of annexin II by matrilysin

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

(MMP-1)

Antibodies and other reagents 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 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- interstitial collage- binant MMP-9, human recombinant nase from Chemicon and MMP-3 were (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.

important activator these MMPs. for It

lines (Colo 201, DLD-1, Four human colon cancer cell WiDr, and CaR-1) and the human breast cancer cell line MDA-MB were obtained from Japanese Cancer Resources

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 including matrilysin, MMP-3 and types of MMP, MMP-2/9, are efficiently activated by trypsin-like serine proteinases [44–46]. Plasmin is thought to be the most 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 Cell lines and culture conditions

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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

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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.

0.01 mm leupeptin, and 5 lm pepstatin], sonicated for 10 s, and then centrifuged at 95 g at 4 (cid:2)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.

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.

SDS/PAGE and immunoblotting analyses

Cleavage of annexin II by MMPs and determina- tion of N-terminal amino acid sequence

A predesigned siRNA corresponding to the target sequence (5¢-UGGAAAGCAUCAGGAAA for human annexin II 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.

Suppression of annexin II expression by RNA interference

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 (cid:2)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).

Phase separation of membrane-associated molecules in Triton X-114 solution

Cancer cells were harvested by trypsinization, inoculated at a density of 5 · 106 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 (cid:2)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.

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 · 108 cells) were dissolved in 1.2 mL of 50 mm (pH 7.5) containing 150 mm NaCl and 5 mm Tris/HCl CaCl2 (TBSC) supplemented with 0.1% Triton X-114 and centrifuged at 95 g at 4 (cid:2)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 (cid:2)C for 1 h. The temperature of the extract was then increased to 37 (cid:2)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 (cid:2)C. Membrane-associated proteins were recovered in the lower, detergent phase.

Cleavage of cell surface annexin II by matrilysin

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 (cid:2)C for 3 h. Adherent cells were fixed

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, 7.5 lm E-64, 0.16 lm aprotinin,

0.025 mm bestatin,

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Immunofluorescence staining of cell surface annexin II Purification of natural annexin II

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Cleavage of annexin II by matrilysin

2 Egeblad M & Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2, 161–174.

with 10% formaldehyde for 10 min, and washed three times with NaCl/Pi containing 1 mm CaCl2, 1 mm MgCl2 and 6 nm glucose. After blocking with 1% BSA in NaCl/Pi, the cells were incubated with a monoclonal antibody against annexin II at 4 (cid:2)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).

3 Miyazaki K, Hasegawa M, Funahashi K & Umeda M (1993) A metalloproteinase inhibitor domain in Alzhei- mer amyloid protein precursor. Nature 362, 839–841. 4 Yu Q & Stamenkovic I (2000) Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angio- genesis. Genes Dev 14, 163–176.

5 Agnihotri R, Crawford HC, Haro H, Matrisian LM,

Havrda MC & Liaw L (2001) Osteopontin, a novel sub- strate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin). J Biol Chem 276, 28261–28267.

6 Li Q, Park PW, Wilson CL & Parks WC (2002) Matri- lysin shedding of syndecan-1 regulates chemokine mobi- lization and transepithelial efflux of neutrophils in acute lung injury. Cell 111, 635–646.

7 Ii M, Yamamoto H, Adachi Y, Maruyama Y &

Shinomura Y (2006) Role of matrix metalloproteinase-7 (matrilysin) in human cancer invasion, apoptosis, growth, and angiogenesis. Exp Biol Med 231, 20–27. 8 Miyazaki K, Hattori Y, Umenishi F, Yasumitsu H & Umeda M (1990) Purification and characterization of extracellular matrix-degrading metalloproteinase, matrin (pump-1), secreted from human rectal carcinoma cell line. Cancer Res 50, 7758–7764.

9 Hasegawa S, Koshikawa N, Momiyama N, Moriyama K, Ichikawa Y, Ishikawa T, Mitsuhashi M, Shimada H & Miyazaki K (1998) Matrilysin-specific antisense oligo- nucleotide inhibits liver metastasis of human colon cancer cells in a nude mouse model. Int J Cancer 76, 812–816.

10 Kioi M, Yamamoto K, Higashi S, Koshikawa N, Fujita K & Miyazaki K (2003) Matrilysin (MMP-7) induces homotypic adhesion of human colon cancer cells and enhances their metastatic potential in nude mouse model. Oncogene 22, 8662–8670.

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 · 105 cells per well of 96-well plates (Sumilon, Tokyo, Japan) in the growth medium and incubated at 37 (cid:2)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 (cid:2)C for 3 h. After the incubation, each culture was washed three times with NaCl/Pi containing 1 mm CaCl2, 1 mm MgCl2 and 6 mm glucose, incubated with 5 nm tPA and 5 lm TAPI-1 as an MMP inhibitor at 37 (cid:2)C for 1 h, and washed three times. The cells were fixed with 10% formaldehyde for 10 min, washed three times with NaCl/Pi containing 0.1% Tween-20, and blocked with 1.2% BSA in NaCl/Pi. Finally, each culture was sequentially incu- bated with a monoclonal antibody against tPA and with a biotinylated second antibody (Vector Laboratories) at 37 (cid:2)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 (cid:2)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).

Analysis of membrane-bound annexin II after matrilysin treatment

Acknowledgements

11 Yamamoto K, Higashi S, Kioi M, Tsunezumi J, Honke K & Miyazaki K (2006) Binding of active matrilysin to cell surface cholesterol sulfate is essential for its mem- brane-associated proteolytic action and induction of homotypic cell adhesion. J Biol Chem 281, 9170–9180.

12 Massey-Harroche D, Mayran N & Maroux S (1998)

Polarized localizations of annexins I, II, VI and XIII in epithelial cells of intestinal, hepatic and pancreatic tissues. J Cell Sci 111, 3007–3015.

13 Siever DA & Erickson HP (1997) Extracellular ann- exin II. Int J Biochem Cell Biol 29, 1219–1223. 14 Deora AB, Kreitzer G, Jacovina AT & Hajjar KA

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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