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 Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan 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, Totsukaku, Yokohama, Kanagawa 244-0813, Japan Fax: +81 45 820 1901 Tel: +81 45 820 1905 E-mail: miyazaki@yokohama-cu.ac.jp (Received 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 Matrilysin hydrolyzed annexin II at the Lys9–Leu10 bond, thus dividing the protein into an N-terminal nonapeptide and the C-terminal 35 kDa fragment 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 nonapeptide 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 nonapeptide generated by matrilysin treatment might be anchored to the cell membrane, 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 annexin II by matrilysin contributes to tumor invasion and metastasis by enhancing tPA-mediated pericellular proteolysis by cancer cells 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 membranetype 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 degradation 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 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 J Tsunezumi et al 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 correlated with malignancy and metastatic potential of the cancers, especially in their liver metastasis [9] It has recently been reported that active matrilysin specifically 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 homotypic 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 initially 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 vascular endothelial cells [14] Many studies have shown that extracellular annexin II is involved in the regulation of a variety of cellular processes, including pericellular 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 activator (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 specifically binds to surfaces of colon cancer cells and Cleavage of annexin II by matrilysin induces prominent cell aggregation [10,11] In the present 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 approximately 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 MAT A (kDa) − + 200 116 97 66 45 31 21 B N- 10 338 -C STVHEILCK LSLEGD STPPSAYGSVKAYT…… 10 Fig 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 · 108 cells) was incubated without ()) or with (+) 100 nM matrilysin at 37 °C for 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 conditions are described in Experimental procedures (B) Domain structure 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 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4811 Cleavage of annexin II by matrilysin J Tsunezumi et al LSLEGDHSTPPSAY by automated protein sequencing, 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 effectively 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-(hydroxyaminocarbonyl)-methyl]-4-methylpentanoyl-l-naphthylalanyl-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 identical to that of the membrane-derived annexin II fragment (LSLEGDHSTPPSAY) These results indicate 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 nonreducing 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.) − − + − + + (TAPI) B Fig Immunoblotting of purified natural annexin II and its cleavage 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, respectively (B) The natural annexin II (2 lgỈmL)1 protein) was incubated in 50 lL of a reaction mixture without (None) or with nM 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 antibody Other experimental conditions are described in Experimental procedures Arrowheads, annexin II bands disulfide bond between the cysteine residues of two monomer molecules at amino acid position (Fig 1B) MMP-9 appeared to cleave annexin II weakly + + (Mix.) (36) Native Cleaved (35) Fig Cleavage of purified annexin II by matrilysin (MAT) Recombinant annexin II (1 lgỈmL)1) was incubated at 37 °C for h with (+) or without ()) 50 nM matrilysin in the presence or absence of the MMP inhibitor TAPI (4 lM) or a proteinase inhibitor mixture [Mix.; 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 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 4812 A 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 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS J Tsunezumi et al Cleavage of annexin II by matrilysin A CaR-1 MAT + – DLD-1 + – WiDr + – MDA-MB + – WiDr lysate were analyzed by immunoblotting Although the supernatants 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 36 35 B 120 Cell ELISA with CaR-1 ll ith C R Annexin II (%) 100 80 60 40 20 C None None MAT MAT Fig Matrilysin-catalyzed cleavage of annexin II on the cell surface (A) Three human colon carcinoma cell lines (CaR-1, WiDr, and DLD-1) and human breast carcinoma cell line MDA-MB in monolayer cultures were incubated in mL of serum-free medium with (+) or without ()) 50 nM matrilysin (MAT) at 37 °C for h Proteins released into the culture medium were concentrated by trichloroacetic acid precipitation and analyzed by immunoblotting under reducing conditions with the antibody against annexin II As a control, 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 annexin II remaining on the cell surface was visualized by immunofluorescence staining Detailed experimental conditions are described in Experimental procedures The loss of cell surface annexin II after matrilysin treatment was confirmed using CaR-1 cells by two different methods Cell ELISA indicated that the immunoreactivity for cell surface annexin II was decreased by about 40% after matrilysin treatment (Fig 4B) Immunofluorescence staining for annexin II also indicated 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 treated with active MMP-2, however, no annexin II fragment 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 glycosaminoglycans such as heparan sulfate proteoglycans and sialoglycoproteins 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 serotonin or heparin in the presence of matrilysin, release Fig 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 mgỈmL)1 heparin at 37 °C for h as described in Fig Alternatively, the same cultures were incubated with mM EDTA at 25 °C for Proteins released into the culture medium were analyzed by immunoblotting with the antibody against annexin II as described in Fig Other experimental conditions are described in Experimental procedures FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4813 Cleavage of annexin II by matrilysin J Tsunezumi et al 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 proteoglycans on the cell surface, and the strength of interaction differs between the two cell types It seems likely that serotonin and heparin promote matrilysincatalyzed annexin II cleavage by weakening the interaction 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 A B C D Fig 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 After the treatment, the cells were washed and then further incubated with nM tPA and lM TAPI-1 at 37 °C for h The incubated cells were collected, washed and dissolved in the SDS/PAGE sample 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 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 activity 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 annexin 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 conditions are described in Experimental procedures 4814 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS J Tsunezumi et al and serotonin on tPA binding to CaR-1 cells (Fig 6A) Unexpectedly, the single treatment with matrilysin significantly increased the binding of exogenous 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 significantly and specifically suppressed expression of annexin 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 coating 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 Cleavage of annexin II by matrilysin the addition of the soluble N-terminal peptide significantly suppressed tPA binding to the plate (Fig 7B) These results suggested that tPA was able to bind Ac-STVHEILCK The competitive effect of the synthetic 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 Treatment 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 heterotetramer 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 Therefore, it seemed possible that the N-terminal peptide bound an annexin II monomer 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 heparin, 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 immunoblotting of the culture supernatant showed two close bands at 36 and 37 kDa under nonreducing conditions, 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 heterodimer was observed more clearly when purified annexin 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 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4815 Cleavage of annexin II by matrilysin J Tsunezumi et al Fig 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 °C overnight These wells were fixed with 10% formaldehyde for 10 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 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 (ANXII) proteins Purified natural annexin II (1 lM) was digested with 50 nM matrilysin at 37 °C for 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 °C for 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 In (A), (B) and (C), the data represent the mean ± SD of triplicate assays A B C 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 4816 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, mm e-aminocaproic 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 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS J Tsunezumi et al Cleavage of annexin II by matrilysin A B C D Fig 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 °C for h and then with nM tPA for 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 ()) mgỈmL)1 heparin for h Annexin II released into the culture medium was analyzed by immunoblotting under nonreducing ()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 °C for h without (lane 1) or with (lanes and 5) 100 lM Ac-STVHEILCK in 50 mM Tris/HCl (pH 7.5) containing 150 mM NaCl, mM CaCl2 and 0.01% Brji 35 in the absence (lanes and 4) or presence (lane 5) of 100 lgỈmL)1 heparin In other tubes, the purified annexin II (1 lM) was incubated with nM matrilysin at 37 °C for 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 °C for h (lane 3) These samples were analyzed 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 nM tPA plus 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 immunoblotting (left panel) and cell ELISA (right panel) Each bar represents the mean ± SD of triplicate assays Other experimental conditions are described in Fig and Experimental procedures Discussion The present study identified annexin II as a novel membrane-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 enzymatic 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 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4817 Cleavage of annexin II by matrilysin J Tsunezumi et al 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 surface, indicating that the cleavage of the membranebound 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 matrilysin was able to bind to the cancer cells [10] and cholesterol 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 membranes varies considerably from one cell type to another Membrane-bound annexin II is thought to play important 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 vascular 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 matrilysin 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 corresponds to the N-terminal nine amino acid peptide of annexin II generated by the matrilysin-catalyzed cleavage 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, matrilysininduced 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 4818 the synthetic peptide coated on plastic plates in a dosedependent manner, and the tPA binding was more efficient 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 hypothesis 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-terminal lysine analog [27] The kringle-2 domain of tPA directly interacts with e-aminocaproic acid [35] Kringle-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 fragment Ac-STVHEILCK remaining on cell membranes Although we cannot exclude the possibility that tPA binds to the C-terminal lysines of other protein fragments 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 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 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 cell) From the data shown in Fig 4B, it is also assumed that the matrilysin treatment produces at least 1.4–3.4 · 105 molecules per cell of the N-terminal annexin II peptide, most likely on the tumor cell surface The N-terminal sequence of annexin II has previously 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 approximately 72 kDa, but we failed to detect p11 in the annexin II complex with a specific antibody (data not FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS J Tsunezumi et al 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 heterodimer 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 annexin 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 annexin 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 plasmin [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 binding 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 signaling 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 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 inhibit 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 malignant growth of cancer cells Understanding the pathological 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 recombinant MMP-9, human recombinant interstitial collagenase (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-GluLys-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 antibody against tPA from Abcam (Cambridge, MA, USA); rabbit polyclonal antibody against enolase, mouse monoclonal 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 monoclonal 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 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4819 Cleavage of annexin II by matrilysin J Tsunezumi et al 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 polyacrylamide 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 chemiluminescence 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 solution, as described previously [11] WiDr cells (approximately · 108 cells) were dissolved in 1.2 mL of 50 mm Tris/HCl (pH 7.5) containing 150 mm NaCl and mm CaCl2 (TBSC) supplemented with 0.1% Triton X-114 and centrifuged at 95 g at °C for The resultant supernatant was added to Triton X-114 to make a final concentration of 2%, and incubated at °C for h The temperature of the extract was then increased to 37 °C, and the sample was incubated for a further The resultant detergent and aqueous phases were separated by centrifugation at 7500 g for 10 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 supplemented 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, 4820 0.01 mm leupeptin, and lm pepstatin], sonicated for 10 s, and then centrifuged at 95 g at °C for 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 dialyzed against the TBSC plus Triton X-100 buffer and applied to a Q-Sepharose column A major part of annexin II was eluted from the column at 0.3 m NaCl This fraction, which contained two types of annexin II of 36 and 72 kDa, was further purified by heparin–Sepharose 6B column 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 determination of N-terminal amino acid sequence The proforms of matrilysin and other MMPs were activated by incubation with 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 · 106 cells per 60 mm culture dish in the growth medium, and incubated for days The cultures were washed twice with the serum-free DMEM/F12 medium and then incubated in mL of the serum-free medium containing 50 nm matrilysin at 37 °C for h Proteins released into the culture medium were collected, precipitated 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 days The cultures were washed twice with the serum-free medium, and incubated in the medium containing 50 nm matrilysin at 37 °C for h Adherent cells were fixed FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS J Tsunezumi et al with 10% formaldehyde for 10 min, and washed three times with NaCl/Pi containing mm CaCl2, mm MgCl2 and nm glucose After blocking with 1% BSA in NaCl/Pi, the cells were incubated with a monoclonal antibody against annexin II at °C for 18 h A fluorescein isothiocyanateconjugated second antibody (Vector Laboratories, Burlingame, 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 · 105 cells per well of 96-well plates (Sumilon, Tokyo, Japan) in the growth medium and incubated at 37 °C for 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 h After the incubation, each culture was washed three times with NaCl/Pi containing mm CaCl2, mm MgCl2 and mm glucose, incubated with nm tPA and lm TAPI-1 as an MMP inhibitor at 37 °C for 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 incubated with a monoclonal antibody against tPA and with a biotinylated second antibody (Vector Laboratories) at 37 °C for h The intensity of immunoreactive signals for tPA was measured by the alkaline phosphatase method with p-nitrophenylphosphate 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 incubated with 200 lm fluorogenic peptide 3145v as substrate at 37 °C for 40 Substrate hydrolysis was determined by measuring fluorescence at 390 nm for excitation and at 460 nm for emission, using a Plate Chameleon spectrofluorometer (Hidex, Turku, Finland) Acknowledgements We thank K Moriyama and A Kurosawa for technical support of protein sequencing and cytofluorometry, respectively We are grateful to S Iiizumi for helpful 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metalloproteinase-1 and -9 activation by plasmin regulates a novel endothelial cell-mediated mechanism of collagen gel contraction and capillary tube regression in three-dimensional collagen matrices J Cell Sci 114, 917–930 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4823 ... Cleavage of annexin II by matrilysin J Tsunezumi et al 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... 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... specific binding of matrilysin to the cancer cells In our previous study, among three MMPs tested (matrilysin, MMP-2 and MMP-3), only matrilysin was able to bind to the cancer cells [10] and cholesterol