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Interaction between tumor cells and stromal fibroblasts plays essential roles in tumor progression. However, its detailed molecular mechanism remains unclear. To understand the mechanism, we investigated molecules mediating this interaction using the three-dimensional (3D) co-culture system of Panc-1 pancreatic carcinoma cells with normal fibroblasts. When the two kinds of cells were placed on the top of collagen gel, the tumor cells scattered into the fibroblast layer, apparently undergoing epithelial-mesenchymal transition. When fibroblasts were placed within collagen gel, Panc-1 cells actively invaded into the collagen gel, extending a microtubule-based long protrusion. Although transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF) individually stimulated the tumor cell invasion into collagen gel without fibroblasts, TGF-β signaling inhibitors (SB431542 and LY2157299) significantly enhanced the Panc-1 cell invasion in the 3D co-culture with fibroblasts. Experiments with HGF/Met signaling inhibitors or with the fibroblast conditioned medium revealed that HGF was a major invasion-promoting factor secreted from fibroblasts and SB431542 increased the HGF secretion by blocking the HGF-suppressing activity of cancer cell-derived TGF-β. These results indicate that HGF and TGF-β are critical regulators for both tumor-stroma interaction and tumor invasion. The results also suggest that TGF-β signaling inhibitors may promote tumor progression under some pathological conditions.

DOI： 10.1016/j.yexcr.2014.04.009

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Matrix metalloproteinase (MMP)-7 binds to cell surface cholesterol sulfate (CS) and acts as a membrane-associated protease. We have previously found that CS modulates the substrate preference of MMP-7, thereby regulating its pericellular proteolytic action. MMP-7 potentially associates with the cell surface via sulfatide (SM4) and cardiolipin (CL) when they are overexpressed on the cell surface. Here, we investigated the molecular interaction between these acidic lipids and MMP-7 or its substrates, and their effects on the activity of MMP-7. Studies using MMP-7 variants with low CS-binding ability suggested that these lipids interact with a similar site on MMP-7. The hydroxamate-based MMP inhibitor TAPI-1 markedly reduced the affinity of MMP-7 for CS and CL, whereas that for SM4 was not affected by TAPI-1. These three acidic lipids also had different effects on the hydrolytic activity of MMP-7 towards a small peptide substrate: SM4, CL and CS reduced the activity to 80%, 92%, and 20%, respectively. Nevertheless, SM4 and CS similarly accelerated the MMP-7-catalyzed degradation of fibronectin and laminin-332, whereas CL did not. The increased proteolysis of substrate was observed only when both substrate and enzyme had affinity for the lipid, suggesting that the lipids probably bring the reactants into closer proximity. Furthermore, MMP-7 bound to cell surface SM4 or CS cleaved specific cell surface proteins and released similar fragments, whereas the cleavage was not stimulated by cell surface CL-bound MMP-7. This study provides a novel mechanism by which acidic lipids differentially regulate pericellular proteolysis by MMP-7 through allosteric alteration of the substrate-binding site and their inherent affinities for MMP-7 substrates.

DOI： 10.1111/febs.12865

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Angiomodulin (AGM) is a member of insulin-like growth factor binding protein (IGFBP) superfamily and often called IGFBP-rP1 or IGFBP-7. AGM was originally identified as a tumor-derived cell adhesion factor, which was highly accumulated in blood vessels of human cancer tissues. AGM is also overexpressed in cancer-associated fibroblasts (CAFs) and activates fibroblasts. However, some studies have shown tumor-suppressing activity of AGM. To understand the roles of AGM in cancer progression, we here investigated the expression of AGM in benign and invasive breast cancers and its functions in cancer vasculature. Immunohistochemical analysis showed that AGM was highly expressed in cancer vasculature even in ductal carcinoma in situ (DCIS) as compared to normal vasculature, while its expression in CAFs was more prominent in invasive carcinomas than DCIS. In vitro analyses showed that AGM was strongly induced by vascular endothelial cell growth factor (VEGF) in vascular endothelial cells. Although AGM stimulated neither the growth nor migration of endothelial cells, it supported efficient adhesion of endothelial cells. Integrin alpha v beta 3 was identified as a novel major receptor for AGM in vascular endothelial cells. AGM retracted endothelial cells by inducing actin stress fibers and loosened their VE-cadherin-mediated intercellular junction. Consequently, AGM increased vascular permeability both in vitro and in vivo. Furthermore, AGM and integrin alpha v beta 3 were highly expressed and colocalized in cancer vasculature. These results suggest that AGM cooperates with VEGF to induce the aberrant functions of cancer vasculature as a ligand of integrin alpha v beta 3.

DOI： 10.1002/cam4.216

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Macrophages infiltrating tumor tissues (tumor-associated macrophages, TAM) affect the malignant behaviors of tumor cells. We previously reported that monocytes were differentiated into TAM-like cells secreting matrix metalloproteinase (MMP)-9 by co-culture with tumor cells, and that cell adhesion to extracellular matrix (ECM) proteins played a critical role in the differentiation. In this study, we found that the monocyte differentiation was promoted by laminin-332 (laminin-5), a major epithelial ECM component. We also demonstrated that the proteolytic processing of the gamma 2 chain of laminin-332 was essential for its activity but that the N-terminal short arm of the gamma 2 chain inhibited MMP-9 secretion. These results indicate that the activity of laminin-332 for monocyte differentiation is dynamically regulated by the proteolytic processing of the gamma 2 chain.

DOI： 10.1007/s10585-013-9627-0

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Laminin 2 (Lm2) chain, a subunit of laminin-332, is a typical molecular marker of invading cancer cells, and its expression correlates with poor prognosis of cancer patients. It was previously found that forced expression of Lm2 in cancer cells promotes their invasive growth in nude mice. However, the mechanism of the tumor-promoting activity of Lm2 remains unknown. Here we investigated the interaction between Lm2 and vascular endothelial cells. When treated with an N-terminal proteolytic fragment of 2 (2pf), HUVECs became markedly retracted or shrunken. The overexpression of Lm2 or treatment with 2pf stimulated T-24 bladder carcinoma cells to invade into the HUVEC monolayer and enhanced their transendothelial migration in vitro. Moreover, 2pf increased endothelial permeability in vitro and in vivo. As the possible mechanisms, 2pf activated ERK and p38 MAPK but inactivated Akt in HUVECs. Such effects of 2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor. In addition, 2pf induced delocalization of VE-cadherin and -catenin from the intercellular junction. As possible receptors, 2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs. Moreover, we localized the active site of 2pf to the N-terminal epidermal growth factor-like repeat. These data suggest that the interaction between 2pf and heparan sulfate proteoglycans induces cytoskeletal changes of endothelial cells, leading to the loss of endothelial barrier function and the enhanced transendothelial migration of cancer cells. These activities of Lm2 seem to support the aberrant growth of cancer cells.

DOI： 10.1111/cas.12323

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Laminin γ2 (Lmγ2) chain, a subunit of laminin-332, is a typical molecular marker of invading cancer cells, and its expression correlates with poor prognosis of cancer patients. It was previously found that forced expression of Lmγ2 in cancer cells promotes their invasive growth in nude mice. However, the mechanism of the tumor-promoting activity of Lmγ2 remains unknown. Here we investigated the interaction between Lmγ2 and vascular endothelial cells. When treated with an N-terminal proteolytic fragment of γ2 (γ2pf), HUVECs became markedly retracted or shrunken. The overexpression of Lmγ2 or treatment with γ2pf stimulated T-24 bladder carcinoma cells to invade into the HUVEC monolayer and enhanced their transendothelial migration in vitro. Moreover, γ2pf increased endothelial permeability in vitro and in vivo. As the possible mechanisms, γ2pf activated ERK and p38 MAPK but inactivated Akt in HUVECs. Such effects of γ2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor. In addition, γ2pf induced delocalization of VE-cadherin and β-catenin from the intercellular junction. As possible receptors, γ2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs. Moreover, we localized the active site of γ2pf to the N-terminal epidermal growth factor-like repeat. These data suggest that the interaction between γ2pf and heparan sulfate proteoglycans induces cytoskeletal changes of endothelial cells, leading to the loss of endothelial barrier function and the enhanced transendothelial migration of cancer cells. These activities of Lmγ2 seem to support the aberrant growth of cancer cells. © 2013 The Authors.

DOI： 10.1111/cas.12323

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Synthetic inhibitors of matrix metalloproteinases (MMPs), designed previously, as well as tissue inhibitors of metalloproteinases (TIMPs) lack enzyme selectivity, which has been a major obstacle for developing inhibitors into safe and effective MMP-targeted drugs. Here we designed a fusion protein named APP-IP-TIMP-2, in which the ten amino acid residue sequence of APP-derived MMP-2 selective inhibitory peptide (APP-IP) is added to the N terminus of TIMP-2. The APP-IP and TIMP-2 regions of the fusion protein are designed to interact with the active site and the hemopexin-like domain of MMP-2, respectively. The reactive site of the TIMP-2 region, which has broad specificity against MMPs, is blocked by the APP-IP adduct. The recombinant APP-IP-TIMP-2 showed strong inhibitory activity toward MMP-2 (K-i(app) = 0.68 pM), whereas its inhibitory activity toward MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, or MT1-MMP was six orders of magnitude or more weaker (IC50 > 1 mu M). The fusion protein inhibited the activation of pro-MMP-2 in the concanavalin A-stimulated HT1080 cells, degradation of type IV collagen by the cells, and the migration of stimulated cells. Compared with the decapeptide APP-IP (t(1/2) = 30 min), APP-IP-TIMP-2 (t(1/2) >> 96 h) showed a much longer half-life in cultured tumor cells. Therefore, the fusion protein may be a useful tool to evaluate contributions of proteolytic activity of MMP-2 in various pathophysiological processes. It may also be developed as an effective anti-tumor drug with restricted side effects.

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DOI： 10.1158/1538-7445.TIM2013-B81

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DOI： 10.15015/00000373

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Other Link： http://id.nii.ac.jp/1246/00000373/

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Epithelial-mesenchymal transition (EMT) is a crucial event in tumor invasion and metastasis. However, most of past EMT studies have been conducted in the conventional two-dimensional (2D) monolayer culture. Therefore, it remains unclear what invasive phenotypes are acquired by EMT-induced cancer cells. To address this point, we attempted to characterize EMT cells in more physiological, three-dimensional (3D) collagen gel culture. EMT was induced by treating three human carcinoma cell lines (A549, Panc-1 and MKN-1) with TGF-beta. The TGF-beta treatment stimulated these cells to overexpress the invasion markers laminin gamma 2 and MT1-MMP in 2D culture, in addition to the induction of well-known morphological change and EMT marker expression. EMT induction enhanced cell motility and adhesiveness to fibronectin and collagen in 2D culture. Although EMT cells showed comparable cell growth to control cells in 2D culture, their growth rates were extremely suppressed in soft agar and collagen gel cultures. Most characteristically, EMT-induced cancer cells commonly and markedly extended invasive protrusions in collagen gel. These protrusions were mainly supported by microtubules rather than actin cytoskeleton. Snail-introduced, stable EMT cells showed similar protrusions in 3D conditions without TGF-beta. Moreover, these protrusions were suppressed by colchicine or inhibitors of heat shock protein 90 (HSP-90) and protein phosphatase 2A. However, MMP inhibitors did not suppress the protrusion formation. These data suggest that EMT enhances tumor cell infiltration into interstitial stroma by extending microtubule-based protrusions and suppressing cell growth. The elevated cell adhesion to fibronectin and collagen and high cell motility also seem important for the tumor invasion.

DOI： 10.1371/journal.pone.0053209

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Angiomodulin (AGM/IGFBP-rP1), a glycoprotein of about 30 kDa, is overexpressed in tumor vasculature as well as some human cancer cell lines, but it has been suggested to be a tumor suppressor. To elucidate roles of angiomodulin (AGM) in tumor progression, we here examined distribution of AGM in three types of human cancer tissues by immunohistochemistry. The results showed that AGM was overexpressed in the stroma as well as the vasculature surrounding tumor cells in the human cancer tissues. AGM and a-smooth muscle actin (a-SMA) as an activated fibroblast marker were often colocalized in cancer-associated fibroblasts (CAFs). In vitro analysis indicated that transforming growth factor (TGF)-beta might be an important inducer of AGM in normal human fibroblasts. AGM strongly stimulated the expression of fibronectin and weakly that of alpha-SMA in normal fibroblasts. AGM significantly stimulated the proliferation and migration of fibroblasts. The AGM-induced expression of fibronectin and alpha-SMA was blocked by a TGF-beta signal inhibitor but neither the stimulation of cell growth nor migration. These results imply that AGM activates normal fibroblasts by TGF-beta-dependent and independent mechanisms. These findings also suggest that AGM and TGF-beta cooperatively or complementarily contribute to the stromal activation and connective tissue formation in human cancer tissues, contributing to tumor progression. (Cancer Sci 2012; 103: 691699)

DOI： 10.1111/j.1349-7006.2012.02203.x

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Unlike other synthetic or physiological inhibitors for matrix metalloproteinases (MMPs), the beta-amyloid precursor protein-derived inhibitory peptide (APP-IP) having an ISYGNDALMP sequence has a high selectivity toward MMP-2. Our previous study identified amino acid residues of MMP-2 essential for its selective inhibition by APP-IP and demonstrated that the N to C direction of the decapeptide inhibitor relative to the substrate-binding cleft of MMP-2 is opposite that of substrate. However, detailed interactions between the two molecules remained to be clarified. Here, we determined the crystal structure of the catalytic domain of MMP-2 in complex with APP-IP. We found that APP-IP in the complex is indeed embedded into the substrate-binding cleft of the catalytic domain in the N to C direction opposite that of substrate. With the crystal structure, it was first clarified that the aromatic side chain of Tyr(3) of the inhibitor is accommodated into the S1' pocket of the protease, and the carboxylate group of Asp(6) of APP-IP coordinates bidentately to the catalytic zinc of the enzyme. The Ala(7) to Pro(10) and Tyr(3) to Ile(1) strands of the inhibitor extend into the nonprime and the prime sides of the cleft, respectively. Therefore, the decapeptide inhibitor has long range contact with the substrate-binding cleft of the protease. This mode of interaction is probably essential for the high MMP-2 selectivity of the inhibitor because MMPs share a common architecture in the vicinity of the catalytic center, but whole structures of their substrate-binding clefts have sufficient variety for the inhibitor to distinguish MMP-2 from other MMPs.

DOI： 10.1074/jbc.M111.264176

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Laminins present in the basement membranes (BM) of blood vessels are involved in angiogenesis and other vascular functions that are critical for tumor growth and metastasis. Two major vascular laminins, the alpha 4 (laminin-411/421) and alpha 5 (laminin-511/521) types, have been well characterized. We recently found a third type of vascular laminin, laminin-3B11, consisting of the alpha 3B, beta 1 and gamma 1 chains, and revealed its biological activity. Laminin-3B11 potently stimulates vascular endothelial cells to extend lamellipodial protrusions. To understand the roles of laminin-3B11 in blood vessel functions and tumor growth, we examined localization of the laminin alpha 3B chain in normal mammary glands and breast cancers, in comparison with the alpha 4 and alpha 5 laminins. In the immunohistochemical analysis, the alpha 3B laminin was co-localized with the alpha 4 and alpha 5 laminins in the BM of venules and capillaries of normal breast tissues, but alpha 3B was scarcely detected in vessels near invasive breast carcinoma cells. In contrast, the alpha 4 laminin was overexpressed in capillaries of invasive carcinomas, where a large number of macrophages were found. The alpha 5 laminin appeared to be weakly downregulated in cancer tissues, especially in capillary vessels. Furthermore, our in vitro analysis indicated that TNF-alpha significantly suppressed the laminin alpha 3B expression in vascular endothelial cells, while it, as well as IL-1 beta and TGF-alpha, upregulated the alpha 4 expression. These results suggest that Lm3B11/3B21 may be required for normal mature vessels and interfere with tumor angiogenesis. (Cancer Sci 2011; 102: 1095-1100).

DOI： 10.1111/j.1349-7006.2011.01892.x

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The basement membrane (BM) proteins laminins, which consist of alpha, beta, and gamma chains, support tissue structures and cellular functions. To date only alpha 4 and alpha 5 types of laminins have been identified in the BMs of blood vessels. Our recent study suggested the presence of novel alpha 3B-containing laminins in vascular BMs. Here we identified and characterized the third member of vascular laminins, laminin-3B11 (Lm3B11). RT-PCR analysis showed that microvascular endothelial (MVE) cells and umbilical vein endothelial cells expressed the messages for the alpha 3B, beta 1, beta 2, and gamma 1 chains. In the culture of MVE cells, alpha 3B was associated with beta 1 and gamma 1, producing Lm3B11. Recombinant Lm3B11 was overexpressed by introducing the cDNAs of the three chains into HEK-293 cells and purified to homogeneity. Purified Lm3B11 exhibited relatively weak cell adhesion activity through both alpha 3 beta 1 and alpha 6 beta 1 integrins. Most characteristically, Lm3B11 strongly stimulated MVE cells to extend many lamellipodial protrusions. This pseudopodial branching was blocked by an inhibitor for Src or phosphatidylinositol 3-kinase. Consistently, Lm3B11 stimulated the phosphorylation of Src and Akt more strongly than other laminins, suggesting that the integrin-derived signaling is mediated by these factors. The unique activity of Lm3B11 appears to be favorable to the branching of capillaries and venules.

DOI： 10.1074/jbc.M110.146126

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Localization of secreted matrix metalloproteinases (MMPs) on the cell surface is required not only for processing of cell surface proteins, but also for controlled degradation of the extracellular matrix (ECM). Our previous study demonstrated that binding of MMP-7 (matrilysin) to cell surface cholesterol sulfate (CS) is essential for the cell membrane-associated proteolytic action of this MMP. In this study, we investigated the role of CS in the MMP-7-catalyzed degradation of protein components of ECM. We found that the degradation of laminin-332 (laminin-5) catalyzed by MMP-7 was accelerated dramatically in the presence of CS, whereas the sulfated lipid inhibited the degradation of casein catalyzed by the protease. The MMP-7 catalyzed degradation of fibronectin was partially inhibited in the presence of low concentrations of CS, whereas it was accelerated significantly at high concentrations of the lipid. Therefore, it is likely that CS alters the substrate preference of MMP-7. We also found that the proteins of which MMP-7-catalyzed degradation were accelerated by CS also had affinities for CS, suggesting that CS facilitates the proteolyses by cross-linking MMP-7 to its substrates. Moreover, MMP-7 tethered to cancer cell surface via CS degraded fibronectin and laminin-332 coated on a culture plate. The degradations of the adhesive proteins led to significant detachment of the cells from the plate. Taken together, our findings provide a novel mechanism in which cell surface CS promotes the proteolytic activities of MMP-7 toward selective substrates in the pericellular ECM, thereby contributing to cancer cell migration and metastasis.

DOI： 10.1074/jbc.M110.136994

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Matrilysin (MMP-7) plays important roles in tumor progression. Previous studies have suggested that MMP-7 binds to tumor cell surface and promotes their metastatic potential. In this study, we identified C-type lectin domain family 3 member A (CLEC3A) as a membrane-bound substrate of MMP-7. Although this protein is known to be expressed specifically in cartilage, its message was found in normal breast and breast cancer tissues as well as breast and colon cancer cell lines. Because few studies have been done on CLEC3A, we overexpressed its recombinant protein in human cancer cells. CLEC3A was found in the cell membrane, extracellular matrix (ECM), and culture medium of the CLEC3A-expressing cells. CLEC3A has a basic sequence in the NH(2)-terminal domain and showed a strong heparin-binding activity. MMP-7 cleaved the 20-kDa CLEC3A protein, dividing it to a 15-kDa COOH-terminal fragment and an NH(2)-terminal fragment with the basic sequence. The 15-kDa fragment no longer had heparin-binding activity. Treatment of file CLEC3A-expressing cells with MMP-7 released the 15-kDa CLEC3A into file culture supernatant, Furthermore, the 20-kDa CLEC3A promoted cell adhesion to laminin-332 and fibronectin substrates, but this activity was abrogated by the cleavage by MMP-7. These results suggest that CLEC3A binds to heparan sulfate proteoglycans oil cell surface, leading to the enhancement of cell adhesion to integrin ligands on ECM. It can be speculated that the cleavage of CLEC3A by MMP-7 weakens the stable adhesion of tumor cells to the matrix and promotes their migration in tumor microenvironments. J. Cell. Biochem. 106: 693-702, 2009. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/jcb.22062

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Matrix metalloproteinase-7 (MMP-7; matrilysin) induces homotypic adhesion of colon cancer cells by cleaving cell surface protein(s) and enhances their metastatic potential. Our previous study (Yamamoto, K., Higashi, S., Kioi, M., Tsunezumi, J., Honke, K., and Miyazaki, K. (2006) J. Biol. Chem. 281, 9170-9180) demonstrated that binding of MMP-7 to cell surface cholesterol sulfate (CS) is essential for the cell membrane-associated proteolytic action of the protease. To determine the region of MMP-7 essential for binding to CS, we constructed chimeric proteases consisting of various parts of MMP-7 and those of the catalytic domain of MMP-2; the latter protease does not have an affinity for CS. Studies of these chimeric proteases and other mutants of MMP-7 revealed that Ile(29), Arg(33), Arg(51), and Trp(55), in the internal sequence, and the C-terminal three residues corresponding to residues 171-173 of MMP-7 are essential for binding to CS. An MMP-7 mutant, which had the internal 4 residues at positions 29, 33, 51, and 55 of MMP-7 replaced with the corresponding residues of MMP-2 and the C-terminal 3 residues deleted, had essentially no affinity for CS. This mutant and wild-type MMP-7 showed similar proteolytic activity toward fibronectin, whereas the mutant lacked the ability to induce the colon cancer cell aggregation. In the three-dimensional structure of MMP-7, the residues essential for binding to CS are located on the molecular surface in the opposite side of the catalytic cleft of the protease. Therefore, it is assumed that the active site of MMP-7 bound to cell surface is directed outside. We speculate that the direction of the cell-bound MMP-7 makes it feasible for the protease to cleave its substrates on cell surface.

DOI： 10.1074/jbc.M806285200

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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.

DOI： 10.1111/j.1742-4658.2008.06620.x

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The extracellular domain of beta-amyloid precursor protein (APP) contains an inhibitor against matrix metalloproteinase-2 (MMP-2, gelatinase A). Our previous study (Higashi, S. and Miyazaki, K. (2003) J Biol Chem 278, 14020 - 14028) demonstrated that the inhibitor is localized within the ISYGN-DALMP sequence of APP, and a synthetic decapeptide containing this sequence (named APP-derived inhibitory peptide, APP-IP) selectively inhibits the activity of MMP-2. To determine the region of interaction that correlates with the selective inhibition, we constructed various MMP-2 mutants. An MMP-2 mutant, which had the hemopexin-like domain and three fibronectin-like type II domains of MMP-2 deleted, and native MMP-2 showed similar affinities for APP-IP, suggesting that only the catalytic domain of MMP-2 is essential for the interaction. Studies of chimeric proteases, consisting of various parts of the MMP-2 catalytic domain and those of MMP-7 (matrilysin) or MMP-9 (gelatinase B), further revealed that Ala88 and Gly94 in the non-prime side and Tyr145 and Thr146 in the prime side of the substrate- binding cleft of MMP-2 contribute separately to the selective inhibition. Replacement of the amino acid residue at position 94 of a chimeric MMP mutant affected its interaction with the C-terminal Pro(10) of APP-IP, whereas that of residues 145 148 affected the interaction with Tyr(3) of the inhibitor, suggesting that the N to C direction of APP-IP relative to the substrate- binding cleft of MMP is analogous to that of propeptide in proMMP, and opposite to that of substrate. When the APP-IP sequence was added to the N terminus of the catalytic domain of MMP-2, the activity of the protease was intramolecularly inhibited. We speculate that the direction of interaction makes the active site-bound APP-IP resistant to cleavage, thereby supporting the inhibitory action of the peptide inhibitor.

DOI： 10.1074/jbc.M709509200

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Matriptase/MT-SP1, a type II membrane serine protease widely expressed in normal epithelial cells and human carcinoma cells, is thought to be involved in cancer progression. To clarify this possibility, we overexpressed exogenous matriptase in the human stomach cancer cell line AZ521. In vitro, the matriptase transfectant (Mat-AZ521) and the control transfectant (Mock-AZ521) showed a similar growth rate, although the saturation cell density was significantly higher with the Mat-AZ521. When implanted into nude mice subcutaneously or intraperitoneally, Mat-AZ521 cells grew faster and produced much larger solid tumors than Mock-AZ521 cells. The overexpression of matriptase in AZ521 cells shortened the survival time of tumor-bearing mice. Histological analysis showed that both the number and the size of blood vessels in tumor tissues were significantly higher in the Mat-AZ521 tumors than the Mock-AZ521 ones. Moreover, it was found that purified matriptase activated one of the important matrix metalloproteinases, stromelysin (MMP-3). These results suggest the possibility that the matriptase-dependent activation of MMP-3, as well as the direct activity of matriptase, promotes tumor growth and angiogenesis by enhancing extracellular matrix degradation in tumor cell microenvironments.

DOI： 10.1111/j.1349-7006.2006.00328.x

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Regulation of cell surface molecules by matrix metalloproteinases (MMPs), as well as MMPs-catalyzed degradation of extracellular matrix, is important for tumor invasion and metastasis. Our previous study (Kioi, M., Yamamoto, K., Higashi, S., Koshikawa, N., Fujita, K., and Miyazaki, K. (2003) Oncogene 22, 8662-8670) demonstrated 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 a dramatically enhanced metastatic potential. To elucidate the mechanism of matrilysin-induced cell aggregation, we attempted to identify the matrilysin-binding substance on the cell surface. Here, we demonstrate that cholesterol sulfate on the cell surface is a major matrilysin-binding substance. We found that active matrilysin bound to the cell membrane and cholesterol sulfate incorporated into liposomes with similar affinities. Treatment of colon cancer cells with beta-cyclodextrin significantly reduced not only matrilysin binding to the cell surface but also matrilysin-dependent proteolysis and cell aggregation. Interestingly, replenishment of cholesterol sulfate, but not cholesterol, neutralized the effects of beta-cyclodextrin. Taken together, it is likely that binding of matrilysin to cholesterol sulfate facilitates the matrilysin-catalyzed modulation of cell surface proteins, thus inducing the cancer cell aggregation.

DOI： 10.1074/jbc.M510377200

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Insulin-like growth factor (IGF) binding protein-related protein-1 (IGFBP-rP1) modulates cellular adhesion and growth in an IGF/insulin-dependent or independent manner. It also shows tumor-suppressive activity in vivo. We recently found that a single-chain IGFB-rP1 is proteolytically cleaved to a two-chain form by a trypsin-like, endogenous serine proteinase, changing its biological activities. In this study, we attempted to identify the IGFBP-rP1-processing enzyme. Of nine human cell lines tested, seven cell lines secreted IGFBP-rP1 at high levels, and two of them, ovarian clear cell adenocarcinoma (OVISE) and gastric carcinoma (MKN-45), highly produced the cleaved TGFBP-rP1. Serine protemase inhibitors effectively blocked the IGFBP-rP1 cleavage in the OVISE cell culture. The conditioned medium of OVISE cells did not cleave purified IGFBP-rP1, but their membrane fraction had an IGFBP-rP1-cleaving activity. The membrane fraction contained an 80-kDa gelatinolytic enzyme, which was identified as the membrane-type serine proteinase matriptase (MT-SP1) by immunoblotting. When the membrane fraction was separated by SDS/PAGE, the IGFBP-rP1-cleaving activity comigrated with matriptase. A soluble form of matriptase purified in an inhibitor-free form efficiently cleaved IGFBP-rP1 at the same site as that found in a naturally cleaved IGFBP-rP1. Furthermore, small interfering RNAs for matriptase efficiently blocked both the matriptase expression and the cleavage of IGBP-rP1 in OVISE cells. These results demonstrate that IGFBP-rP1 is processed to the two-chain form by matriptase on the cell surface.

DOI： 10.1111/j.1742-4658.2005.05094.x

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The membrane-bound serine proteinase matriptase, which is often released from the plasma membrane of epithelial and carcinoma cells, has been implicated to play important roles in both physiological and pathological conditions. However, the regulatory mechanism of its activity is poorly understood. In the present study, we examined expression and activation state of soluble matriptase in 24 human cancer cell lines. Soluble matriptase was detected in the conditioned media from all of 5 colon and 4 breast carcinoma cell lines and 8 of 10 stomach carcinoma cell lines tested. Only two of five lung cancer cell lines released the matriptase protein into the culture media. Out of the five matriptase-negative cell lines, two cell lines expressed the matriptase mRNA. Among 24 cancer cell lines tested, 13 cell lines secreted trypsin in an active or latent form and all of them released matriptase. Most of the 24 cell lines released a latent, single-chain matriptase of 75 kDa as a major form, as well as low levels of complex forms of an activated two-chain enzyme with its specific inhibitor HAI-1. Thus, these soluble matriptases appeared to have little proteolytic activity. Treatment of stomach and colon cancer cell lines with epidermal growth factor stimulated the release of matripatase/HAI-1 complexes. In cancercell lines secreting active trypsin, however, matriptase was released mostly as an inhibitor-free, two-chain active form. Trypsin seemed to activate the membrane-bound, latent matriptase on the cell surface. These results suggest that matriptase and trypsin cooperatively function for extracellular proteolysis. J. Cell. Biochem. 95: 632-647, 2005. (c) 2005 Wiley-Liss, Inc.

DOI： 10.1002/jcb.20418

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Matrilysin (MMP-7) is thought to contribute to invasive growth and metastasis of colon carcinoma and many other human cancers. The present study demonstrates that treatment of human colon carcinoma cells with active matrilysin induces cell aggregation in vitro and promotes liver metastasis in nude mice. When two kinds of colon carcinoma cell lines were incubated with active matrilysin, this enzyme efficiently bound to the cell surface and induced loose cell aggregation, which led to E-cadherin-mediated tight cell aggregation. Synthetic MMP inhibitors inhibited both the membrane binding of matrilysin and matrilysin-induced cell aggregation, while TIMP-2 inhibited only the cell aggregation. Two other active MMPs, stromelysin and gelatinase A, neither bound to cell membrane nor induced cell aggregation. Tumor cells in loose cell aggregates could reaggregate even after they were freed from matrilysin and dispersed. When injected into the spleen of nude mice, the tumor cells in the stable aggregates produced much larger metastatic nodules in the livers than control cells and those in the loose aggregates. These results suggest that matrilysin may enhance metastatic potential of tumor cells by processing a cell surface protein(s) and thereby inducing loose and then tight aggregation of tumor cells.

DOI： 10.1038/sj.onc.1207181

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Insulin-like growth factor (IGF) binding protein-related protein-1 (IGFBP-rP1) was previously identified as tumor-derived adhesion factor (TAF) secreted from human bladder carcinoma cells. It exhibits growth-stimulatory activity in synergy with insulin or IGFs. In the present study, we found that IGFBP-rP1 was proteolytically cleaved to a two-chain form. The cleavage sequence suggested that a trypsin-like serine proteinase may be responsible for the processing. The cleavage of IGFBP-rP1 led to an almost complete loss of both insulin/IGF-1-binding activity and insulin/IGF-1-dependent growth-stimulatory activity. On the other hand, the cell attachment activity of IGFBP-rP1 was markedly increased by the proteolytic processing. Syndecan-1 was thought to be a cell surface receptor for both intact and cleaved IGFBP-rP1 forms. Although the proteolytic cleavage of IGFBP-rP1 decreased its heparin-binding activity, the cleaved form could bind syndecan-1 efficiently. Thus the proteolytic processing of IGFBP-rP1 seems to modulate its insulin/IGF-dependent and -independent biological functions. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2003.09.058

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

In various mammalian cell lines, beta-amyloid precursor protein (APP) is proteolytically processed to release its NH2-terminal extracellular domain as a soluble APP (sAPP) that contains the inhibitor domain against gelatinase A. To investigate roles of SAPP in the regulation of gelatinase A activity, we examined the correlation between the activation of progelatinase A and processing of APP. We found that stimulation of HT1080 fibrosarcoma cells with concanavalin A led to an activation of endogenous progelatinase A and to a novel processing of APP, which releases a COOH-terminally truncated form of sAPP (sAPPtrc) into the culture medium. Reverse zymographic analysis showed that sAPPtrc lacked an inhibitory activity against gelatinase A. Analyses of production of sAPPtrc in the presence of various metalloproteinase inhibitors showed that membrane type 1 matrix metalloproteinase (MT1-MMP), an activator of progelatinase A, is most likely responsible for the production of sAPPtrc. When the concanavalin A-stimulated HT 1080 cells were cultured in the condition that inhibited MT1-MMP activity, sAPP and APP were associated with the extracellular matrix deposited by the cells, whereas these gelatinase A inhibitors in the matrix were displaced by sAPPtrc after exertion of MT1-MMP activity. Taken together, these data support a model in which MT1-MMP-catalyzed release of sAPPtrc leads to reduction of the extracellular matrix-associated gelatinase A inhibitor, SAPP, thus making it feasible for gelatinase A to exert proteolytic activity only near its activator, MT1-MMP.

DOI： 10.1021/bi020643m

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Because beta-amyloid precursor protein (APP) has the abilities both to interact with extracellular matrix and to inhibit gelatinase A activity, this molecule is assumed to play a regulatory role in the gelatinase A-catalyzed degradation of extracellular matrix. To determine a region of APP essential for the inhibitory activity, we prepared various derivatives of APP. Functional analyses of proteolytic fragments of soluble APP (sAPP) and glutathione S-transferase fusion proteins, which contain various COOH-terminal parts of sAPP, showed that a site containing residues 579-601 of APP(770) is essential for the inhibitory activity. Moreover, a synthetic decapeptide containing the 579-601 sequence corresponding to residues 586-595 of APP(770) had a gelatinase A inhibitory activity slightly higher than that of sAPP. Studies of deletion of the NH2- and COOH-terminal residues and alanine replacement of internal residues of the decapeptide further revealed that Tyr(588), Asp(591), and Leu(593) of APP mainly stabilize the interaction between gelatinase A and the inhibitor. We also found that the residues of Ile(586), Met(594), and Pro(595) modestly contribute to the inhibitory activity. The APP-derived decapeptide efficiently inhibited the activity of gelatinase A (IC50 = 30 nm), whereas its inhibitory activity toward membrane type 1 matrix metalloproteinase was much weaker (IC50 = 2 muM). The decapeptide had poor inhibitory activity toward gelatinase B, matrilysin, and stromelysin (IC50 > 10 muM). The APP-derived inhibitor formed a complex with active gelatinase A but not with progelatinase A, and the complex formation was prevented completely by a hydroxamate-based synthetic inhibitor. Therefore, the decapeptide region of APP is likely an active site-directed inhibitor that has high selectivity toward gelatinase A.

DOI： 10.1074/jbc.M212264200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Factor VIIa (VIIa) is an unusual trypsin-type serine proteinase that appears to exist in an equilibrium between minor active and dominant zymogen-like inactive conformational states. The binding of tissue factor to VIIa is assumed to shift the equilibrium into the active state. The proteinase domain of VIIa contains a unique structure: a loop formed by a disulfide bond between Cys(310) and Cys(329), which is five residues longer than those of other trypsin types. To examine the functional role of the loop region, we prepared two mutants of VIIa, One of the mutants, named VII-11, had five extra corresponding residues 316-320 of VII deleted. The other mutant, VII-31, had all of the residues in its loop replaced! with those of trypsin, Functional analysis of the two mutants showed that VIIa-11 (K-d = 41 nM) and VIIa-31 (K-d = 160 nM) had lower affinities for soluble tissue factor as compared with the wild-type VIIa (K-d = 11 nM). The magnitude of tissue factor-mediated acceleration of amidolytic activities of VIIa-11 (7-fold) and that of VIIa-31 (2-fold) were also smaller than that of wild-type VIIa (30-fold). In the absence of tissue factor, VIIa-31 but not VIIa-11 showed enhanced activity; the catalytic efficiencies of VIIa-31 toward various chromogenic substrates were 2-18-fold greater than those off the wildtype VIIa. Susceptibility of the alpha -amino group of IIe-153 of VIIa-31 to carbamylation was almost the same as that of wild-type VIIa, suggesting that VIIa-31 as well as wildtype VIIa exist predominantly in the zymogen-like state. Therefore, the tested modifications in the loop region had adverse effects on affinity for tissue factor, disturbed the tissue factor-induced conformational transition, and changed the catalytic efficiency of VIIa, but they did not affect the equilibrium between active and zymogen-like conformational states.

DOI： 10.1074/jbc.M009206200

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DOI： 10.2302/kjm.50.supplement3_44

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Laminin-5 (alpha3 beta3 gamma2) is an important component of epithelial basement membranes. The 190-kDa alpha3 chain undergoes extracellular cleavage within the carboxyl (C) terminus consisting of five globular domains (G1 to G5), producing the mature laminin-5 with the 160-kDa alpha3 chain. To understand the physiological meaning of this processing, we isolated the C-terminal fragments of the alpha3 chain from the conditioned media of two kinds of human cell lines. The amino-terminal sequence of the fragments suggested that the cleavage occurs at Gln(1337)-Asp(1338) in the spacer region between the G3 and G4 domains. The G4-G5 fragment itself did not show significant activity, but it stimulated cell migration in the presence of a low concentration of the mature laminin-5, suggesting its regulatory role in cell migration. (C) 2000 Academic Press.

DOI： 10.1006/bbrc.2000.3851

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

It was recently found that overexpression of the trypsin gene in tumor cells stimulates their growth in culture and in nude mice. In the present study, expression of trypsin in various human cancer cell lines and tissues was studied by gelatin zymography and immunoblotting before and after enterokinase treatment and by immunohistochemistry. The analyses showed that many stomach, colon, and breast cancer cell lines secreted trypsinogens-1 and/or -2, as well as an unidentified serine proteinase of about 70 kDa, into culture medium. Lung cancer cell lines secreted 18- and 19-kDa unidentified trypsin-like proteins. Stomach cancer cell lines frequently secreted active trypsin, suggesting that they produced an endogenous activator of trypsinogen, most likely enterokinase. Active trypsin formed a complex with a soluble form of Alzheimer amyloid precursor protein (sAPP), a Kunitz-type trypsin inhibitor, which was secreted by all cell lines tested. This indicated that sAPP is a primary inhibitor of secreted trypsin, Immunohistochemical analysis showed that trypsin(ogen) was frequently expressed at high levels in stomach and colon cancers, but scarcely in breast cancers. In the stomach cancers, the trypsin immunoreactivity was higher in the malignant, non-cohesive type than in the cohesive type. These results support the hypothesis that tumor-derived trypsin is involved in the malignant growth of tumor cells, especially stomach cancer cells.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Tissue inhibitor of metalloproteinases-a (TIMP-2) is supposed to play a regulatory role in the cell-mediated activation of progelatinase A To investigate the mechanism of the regulation, we prepared and characterized a chemically modified TIMP-2, and examined its effects on the activation of progelatinase A. We found that treatment of TIMP-2 with cyanate ion led to loss of inhibitory activity toward matrilysin or gelatinase A Structural and functional analyses of the modified TIMP-2 showed that carbamylation of the alpha-amino group of the NH2-terminal Cys(1) of TIMP-2 led to complete loss of the inhibitory activity. When the reactive-site modified TIMP-2 was added to culture medium of concanavalin A-stimulated HT1080 cells, the conversion of endogenous progelatinase A to the intermediate form was partially inhibited, whereas that of the intermediate form to the mature one was strongly inhibited. The reactive site-modified TIMP-2 also prevented an accumulation of active gelatinase A on the cell surface. We speculate that occupation of the hemopexin-like domain of gelatinase A by the reactive site-modified TIMP-2 makes it unable for gelatinase A to be retained on the cell surface, thus preventing the autocatalytic conversion of the intermediate fern of gelatinase A to its mature form.

DOI： 10.1074/jbc.274.15.10497

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

To clarify the regulatory mechanism of pro-gelatinase A (proGelA) activation at a cellular level, expression of gelatinase A (GelA), three MT-RIMPs, and TIMP-2 was examined with 11 human cancer cell lines cultured in the presence and absence of stimulants. MT1-MMP mRNA was expressed in 8 cell lines, while MT2-MMP and MT3-MMP mRNAs were expressed in fewer cell lines, The cells with high proGelA activation strongly expressed MTI-MMP mRNA but not MTS-MMP and MT3-MMP mRNAs, suggesting that MT1-MMP was responsible for the proGelA activation in the cancer cells. Treatments with concanavalin A (Con A) and a phorbor ester (TPA) enhanced the MTI-MMP expression, but only Con A stimulated the proGelA activation in many cell lines. In HT1080 fibrosarcoma cells, however, TPA also stimulated the activation. The level of TIMP-8 secreted into culture medium inversely correlated with proGelA activation, For example, 2 squamous cell carcinoma lines (HSC-3 and HSC-4) and 3 HT1080 clones, which efficiently activated proGelA, secreted little TIMP-8 into medium, whereas other cell lines and other HT1080 clones, which hardly activated proGelA, secreted TIMP-8 at high levels, When HSC-3 cells were incubated with TIMP-8 protein or transfected with TIMP-8 cDNA, the proGelA activation was strongly inhibited, These results indicated that. extracellular TIMP-8 was an important negative regulator of proGelA activation. However, the level of extracellular TIMP-8 was not consistent with that of TIMP-2 mRNA in some cell lines. Other experimental results suggested that TIMP-2 might be rapidly metabolized after binding to MT1-MMP, and Con A treatment might stabilize the complex of TIMP-8 and MT1-MMP on cell membranes.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer-Verlag Tokyo  

Complex formation between serine protease factor VIIa (VIIa) and tissue factor (TF) dramatically enhances the catalytic activity of VIIa, leading to the initiation of extrinsic blood coagulation. The recently determined crystal structure of the complex formed between VIIa and the extracellular domain of TF has revealed in detail the interacting surfaces of the two molecules. Mutational and biochemical studies have further identified regions of VIIa and/or those of TF essential for the high affinity interaction. These studies indicate that the regions extending from the γ-carboxyglutamic acid domain through the first epidermal growth factor-like domain and protease domain of VIIa are involved separately in the interaction with the distinct sites of TF. The interaction of TF with the protease domain of VIIa apparently induces a conformational transition of the VIIa active site. Studies of the chemical modification of VIIa provided a model for the mechanism of TF-mediated acceleration of VIIa activity. In this model, the protease domain of VIIa exists in equilibrium between minor active and dominant zymogen-like inactive conformational states, and preferential binding of TF to the active state leads to a shift in equilibrium into the active state, thereby accelerating VIIa activity. Overall docking of TF with VIIa in the presence of phospholipids further supports a recognition of macromolecular substrates, such as factors IX and X. Therefore, single VIIa- TF interaction contains several distinct mechanisms for enhancing the coagulant activity of VIIa, which may be important for the initiation of coagulation specifically mediated by TF. Studies of the interaction between VIIa and TF provide not only a detailed understanding of the regulated initiation of blood coagulation but also the potential to design novel anticoagulants for the treatment of thrombotic diseases.

DOI： 10.1016/S0925-5710(98)00018-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Unlike other trypsin-type serine proteases, zymogen-to-enzyme transition of conformation of factor VII apparently requires not only conversion of the zymogen to active form factor VIIa (VIIa) but also interaction of VIIa with tissue factor (TF), To determine the region of interaction that correlates with maturation of the Wa active site, we modified intramolecular disulfide bonds in VIIa and examined the interaction of the modified VIIa with soluble TF (sTF). We found that partial reduction and S-carboxamidomethylation of disulfide bonds in VIIa led to losses of amidolytic activity and the binding ability to sTF. To determine the sites of modification that associate with the loss of functions, partially S-carboxamidomethylated Ma was separated on a column of immobilized sTF, Each of the sTF-bound and sTF-unbound fractions and native VIIa was then digested by trypsin, and the digest was analyzed by reversed-phase high performance liquid chromatography, We found that reduction and S-carboxamidomethylation of a disulfide bond between Cys-310 and Cys-329 in the protease domain of VIIa led to loss of the binding ability with sTF, and the modification of a disulfide bond between Cys-340 and Cys-368 of VIIa led to loss of the amidolytic activity. In the three-dimensional structures of trypsinogen and trypsin, the disulfide bonds corresponding to Cys-340-Cys-368 and Cys-310-Cys-329 of Ma are, respectively, in and adjacent to the activation domain, which has flexible conformation in trypsinogen but not in trypsin. Furthermore, the crystal structure of human VIIa TF complex indicates that the region next to Cys-310-Cys-329 is in contact with sTF, We speculate that a regional flexibility, reflected by the labile nature of disulfide bonds of Cys-310-Cys-329 and Cys-340-Cys-368 in the protease domain, contributes to the inability of VIIa to attain the active conformation. Interaction of TF with this flexible region may stabilize the structure in a conformation similar to that of the active state of VIIa.

DOI： 10.1074/jbc.272.41.25724

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The mechanism of the acceleration of the catalytic activity of factor VIIa (VIIa) in the presence of tissue factor (TF) was investigated, To explore the VIIa's site(s) that correlates with TF-mediated acceleration, zymogen VII, VIIa, and active site-modified VIIa were prepared, and dissociation constants (K-d) for their bindings to TF or soluble TF in solution were determined. We found that conversion of zymogen VII to VIIa led to an increase in affinity (Delta Delta G = 4.3-4.4 kJ/mol) for TFs., Dansyl-Glu-Gly-Arg chloromethyl ketone (DNS-EGRck) treatment of VIIa led to a further increase in the affinity (Delta Delta G = 7.3-12 kJ/mol), Neither removal of the Gla domain from VIIa nor truncation of the COOH-terminal membrane and cytoplasmic regions of TF affected the affinity enhanced after DNS-EGRck treatment of VIIa. Treatment of VIIa with (p-amidinophenyl)methanesulfonyl fluoride also enhanced its affinity for soluble TF, whereas treatment with 4-(2-aminoethyl)benzenesulfonyl fluoride, phenylmethylsulfonyl fluoride, or diisopropyl fluorophosphate had a slight effect on the affinity, On the other hand, DNS-EGRck and (p-amidinophenyl)methanesulfonyl fluoride treatments, but not diisopropyl fluorophosphate treatment, of VIIa led to protection of its alpha-amino group of Ile-153 from carbamylation, Protection of the alpha-amino group was consistent with formation of a critical salt bridge between Ile-153 and Asp-343 in the protease domain of VIIa, Therefore, TF may preferentially bind to the active conformational state of VIIa. When one assumes that free VIIa exists in equilibrium between minor active and dominant zymogen-like inactive conformational states, preferential binding of TF to the active state leads to a shift in equilibrium, We speculate that TF traps the active conformational state of VIIa and converts its zymogen-like state into an active state, thereby accelerating the VIIa activity.

DOI： 10.1074/jbc.271.43.26569

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Initiation of the extrinsic blood coagulation pathway is mediated by a complex formed between plasma-derived factor VII/VIIa and cell-derived tissue factor (TF). To identify the site(s) of interaction, zymogen VII and VIIa were enzymatically and chemically modified, and their affinities for TF were estimated by measuring their inhibitory effects on the amidolytic activity enhanced after formation of the VIIa-TF complex. We found that the VIIa-light chain (K-i = 3.5 x 10(-7) M) and its fragment consisting of the gamma-carboxyglutamic acid (Gla)-domain and the first epidermal growth factor (EGF)-like domain (Gla-EGF1 peptide; K-i = 1.0 x 10(-6) M) have an affinity for TF. Therefore, one of the binding sites of VII with TF is probably located in the Gla-EGF1 region. On the other hand, a dansyl-Glu-Gly-Arg chloromethyl ketone-treated Gla-domainless VIIa (K-i = 0.7 x 10(-7) M) showed a high affinity for TF: whereas the corresponding Gla-domainless VII similarly treated showed no binding potential, thereby indicating that binding site(s) other than in the Gla-EGF1 region are present in VIIa but not in VII. Acetylation or carbamylation of the alpha-amino group of the NH2-terminal Ile-153 of VIIa resulted in the loss of binding affinity for TF; such modifications convert VIIa into a zymogen-like inactive form by destroying the salt bridge between Ile-153 and Asp-343 in VIIa. The rate of carbamylation of VIIa was reduced in the presence of TF. Protection of the alpha-amino group of Ile-153 from carbamylation after complex formation was consistent with salt bridge formation between Ile-153 and Asp-343 in the VIIa-TF complex. Therefore, binding of TF with the heavy chain of VIIa may induce a conformational change that brings the alpha-amino group of Ile-153 close to the beta-carboxyl group of Asp-343 to make a stable salt bridge.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：W B SAUNDERS CO  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The extracellular domain of human tissue factor (TF, amino acids 1-217) was expressed in Saccharomyces cerevisiae, using the inducible yeast acid phosphatase promoter and the yeast invertase signal sequence to direct its secretion into the culture broth. Two active soluble forms sTFalpha (high molecular weight form) and sTFbeta (low molecular weight form) were purified, the yield being approximately 10 and 1 mg/liter of culture supernatant, respectively. sTFalpha had an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis and contained more than 200 residues of mannose/mol of protein. sTFbeta had an apparent molecular mass of 37 kDa and contained 22 residues of mannose/mol of protein. N-Glycosidase F treatments of both rTFs reduced the apparent molecular mass to 35 kDa. The amino-terminal sequences and amino acid compositions of sTFalpha and sTFbeta were consistent with those deduced from the cDNA sequence, thereby indicating that the difference in molecular mass is caused by heterogeneity of oligosaccharide structures. Of these recombinant TFs, sTFbeta enhanced factor VIIa-amidolytic activity 40-fold toward the chromogenic substrate and 147-fold toward the fluorogenic substrate, affecting mainly the k(cat) value. The enhancement was comparable with that of TF purified from human placenta. The TF-mediated enhancement of factor VIIa-amidolytic activity was inhibited by heparin-activated antithrombin III, forming a high molecular weight complex. As treatment of sTFbeta with denaturants such as guanidine hydrochloride or urea led to a biphasic loss of the activity, the extracellular domain of TF probably consists of two discrete domains. This expression system provides a significant amount of the extracellular domain of TF so that studies of interactions with factor VII are feasible.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We designed a simple and sensitive method to assay the activity of the factor VIIa-tissue factor complex, using as a substrate N(alpha)-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester (Z-Arg-ONb) (Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203-2209). The principle was to measure the amount of p-nitrobenzyl alcohol released during ester hydrolysis using reversed-phase high performance liquid chromatography. Z-Arg-ONb had a broad specificity for plasma serine proteases and factor IXa. Using this method, we examined the effect of tissue factor on the esterase activity of factor VIIa under various conditions. We found that tissue factor greatly potentiates the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb. Phospholipids were not required for the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb, even in the presence of tissue factor. The K(m) value of factor VIIa alone toward the ester substrate was six times higher than that of a VIIa-tissue factor complex (3.2 versus 0.54 mM), whereas the k(cat) value was 12 times lower than that of the VIIa-tissue factor complex (14.3 versus 173 s-1). Thus, tissue factor apparently affects the catalytic site of factor VIIa and enhances hydrolysis of the ester substrate. This enhancing effect of tissue factor disappeared on removal of the gamma-carboxyglutamic acid domain from factor VIIa, whereas the esterase activity in the absence of tissue factor was not affected by this modification. The gamma-carboxyglutamic acid domain is probably required as a potent determinant for interactions with tissue factor, even in the absence of phospholipids in the reaction mixture.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2011-3360

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DOI： 10.1074/jbc.M212264200

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DOI： 10.1021/bi020643m

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Language：English   Publisher：ACADEMIC PRESS INC  

Laminin-5 (alpha3 beta3 gamma2) is an important component of epithelial basement membranes. The 190-kDa alpha3 chain undergoes extracellular cleavage within the carboxyl (C) terminus consisting of five globular domains (G1 to G5), producing the mature laminin-5 with the 160-kDa alpha3 chain. To understand the physiological meaning of this processing, we isolated the C-terminal fragments of the alpha3 chain from the conditioned media of two kinds of human cell lines. The amino-terminal sequence of the fragments suggested that the cleavage occurs at Gln(1337)-Asp(1338) in the spacer region between the G3 and G4 domains. The G4-G5 fragment itself did not show significant activity, but it stimulated cell migration in the presence of a low concentration of the mature laminin-5, suggesting its regulatory role in cell migration. (C) 2000 Academic Press.

DOI： 10.1006/bbrc.2000.3851

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：ELSEVIER SCI IRELAND LTD  

Complex formation between serine protease factor VIIa (VIIa) and tissue factor (TF) dramatically enhances the catalytic activity of VIIa, leading to the initiation of extrinsic blood coagulation. The recently determined crystal structure of the complex formed between VIIa and the extracellular domain of TF has revealed in detail the interacting surfaces of the two molecules. Mutational and biochemical studies have further identified regions of VIIa and/or those of TF essential for the high affinity interaction. These studies indicate that the regions extending from the gamma-carboxyglutamic acid domain through the first epidermal growth factor-like domain and protease domain of VIIa are involved separately in the interaction with the distinct sites of TF. The interaction of TF with the protease domain of VIIa apparently induces a conformational transition of the VIIa active site. Studies of the chemical modification of VIIa provided a model for the mechanism of TF-mediated acceleration of VIIa activity. In this model, the protease domain of VIIa exists in equilibrium between minor active and dominant zymogen-like inactive conformational states, and preferential binding of TF to the active state leads to a shift in equilibrium into the active state, thereby accelerating VIIa activity. Overall docking of TF with VIIa in the presence of phospholipids further supports a recognition of macromolecular substrates, such as factors IX and X. Therefore, single VIIa-TF interaction contains several distinct mechanisms for enhancing the coagulant activity of VIIa, which may be important for the initiation of coagulation specifically mediated by TF. Studies of the interaction between VIIa and TF provide not only a detailed understanding of the regulated initiation of blood coagulation but also the potential to design novel anticoagulants for the treatment of thrombotic diseases. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0925-5710(98)00018-8

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Language：English   Publisher：JAPAN BIOCHEMICAL SOC  

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