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| 1.
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Takino, Takahisa ; Sato, Hiroshi ; Seiki, Motoharu ; 滝野, 隆久
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金沢大学国際基幹教育院GS教育系 / Institute of Liberal Arts and Science<br />A modified invasion assay using a three-dimensional collag
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en gel was developed that enables isolation of invasive living cells; it was named the invading cell trapping (iCT) assay. A small cell strainer consisting of a nylon mesh with 40-μm2 pores was used, and collagen gel layers formed across the membrane. Test cells were seeded in the lower gel layer and invasive cells were attracted upward and trapped in the upper gel. After incubation, the collagen gel layers in cell strainers were easily separated and living cells in the gel were counted and analyzed. An advantage of the iCT assay is that it can capture living invasive cells in the upper gel while leaving noninvasive ones in the lower layer. Further enrichment of the two cell populations can be achieved by repeating the assay. Thus, the iCT assay allows comparative analysis of invasive versus noninvasive cells.
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| 2.
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Takino, Takahisa ; Yoshimoto, Taisuke ; Nakada, Mitsutoshi ; Li, Zichen ; Domoto, Takahiro ; Kawashiri, Shuici ; Sato, Hiroshi
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Fibronectin matrix formation requires the increased cytoskeletal tension generated by cadherin adhesions, and is suppres
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sed by membrane-type 1 matrix metalloproteinase (MT1-MMP). In a co-culture of Rat1 fibroblasts and MT1-MMP-silenced HT1080 cells, fibronectin fibrils extended from Rat1 to cell-matrix adhesions in HT1080 cells, and N-cadherin adhesions were formed between Rat1 and HT1080 cells. In control HT1080 cells contacting with Rat1 fibroblasts, cell-matrix adhesions were formed in the side away from Rat1 fibroblasts, and fibronectin assembly and N-cadherin adhesions were not formed. The role of N-cadherin adhesions in fibronectin matrix formation was studied using MT1-MMP-silenced HT1080 cells. MT1-MMP knockdown promoted fibronectin matrix assembly and N-cadherin adhesions in HT1080 cells, which was abrogated by double knockdown with either integrin β1 or fibronectin. Conversely, inhibition of N-cadherin adhesions by its knockdown or treatment with its neutralizing antibody suppressed fibronectin matrix formation in MT1-MMP-silenced cells. These results demonstrate that fibronectin assembly initiated by MT1-MMP knockdown results in increase of N-cadherin adhesions, which are prerequisite for further fibronectin matrix formation. © 2014 Elsevier Inc. All rights reserved.
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| 3.
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Takino, Takahisa ; Miyamori, Hisashi ; Kawaguchi, Noriko ; Uekita, Takamasa ; Seiki, Motoharu ; Sato, Hiroshi
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金沢大学がん研究所<br />Membrane-type 1 matrix metalloproteinase (MT1-MMP) is known to be internalized from cell surface, however
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, the fate of internalized MT1-MMP is still unknown. Here we demonstrate that at least a part of internalized MT1-MMP is targeted for lysosomal proteolysis. Treatment with an inhibitor of lysosomal proteinases chloroquine suppressed degradation of internalized MT1-MMP and induced accumulation of MT1-MMP in CD63-positive lysosomes. Ectopic expression of CD63 accelerated degradation of MT1-MMP, which was blocked by chloroquine. MT1-MMP, and CD63 were shown to form a complex through hemopexin-like domain of MT1-MMP and N-terminal region of CD63, and thus accelerated degradation of MT1-MMP was not observed with mutants lacking these domains. CD63 mutant lacking lysosomal targeting motif was unable to promote MT1-MMP degradation. These results suggest that CD63 regulates MT1-MMP by targeting to lysosomes.
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| 4.
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Bayarsaikhan, Munkhuu ; Takino, Takahisa ; Gantulga, Davaakhuu ; Yoshioka, Katsuji ; Sato, Hiroshi
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金沢大学がん研究所がん分子細胞制御<br />金沢大学大学院医学系研究科<br />We previously reported that the level of c-Jun NH2-terminal kinase (JNK)/stres
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s-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, increases dramatically during nerve growth factor (NGF)-induced differentiation of PC12h cells. In the present study, we investigated the function of JSAP1 during PC12h cell differentiation by knocking down the level of JSAP1. The depletion of JSAP1 caused NGF-treated PC12h cells to form aggregates and impaired their differentiation. The aggregation was not observed in JSAP1-depleted cells that were untreated or treated with epidermal growth factor. Immunocytochemical studies indicated that N-cadherin, but not E-cadherin, was localized to sites of cell–cell contact in the aggregated cells. Furthermore, an inhibitory anti-N-cadherin antibody completely blocked the aggregation. Taken together, these results suggest that JSAP1 regulates cell–cell interactions in PC12h cells specifically in the NGF-induced signaling pathway, and does so by modulating N-cadherin.
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Takino, Takahisa ; Tsuge, Hisashi ; Ozawa, Terumasa ; Sato, Hiroshi
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金沢大学がん研究所<br />Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show he
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re that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21WAF1 and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin αvβ3 were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin. © 2010 Elsevier Inc. All rights reserved.
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| 6.
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Takino, Takahisa ; Tamura, Masahito ; Miyamori, Hisashi ; Araki, Masaru ; Matsumoto, Kazue ; Sato, Hiroshi ; Yamada, Kenneth M.
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CrkII belongs to a family of adaptor proteins that become tyrosine phosphorylated after various stimuli. We examined the role of CrkII tyrosine phosphorylation in fibronectin-induced cell migration. Overexpression of CrkII
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inhibited dephosphorylation of focal adhesion components such as p130 Crk-associated substrate (p130cas) and paxillin by protein tyrosine phosphatase 1B (PTP1B). Tyrosine-phosphorylated CrkII was dephosphorylated by PTP1B both in vitro and in vivo, showing for the first time that PTP1B directly dephosphorylates CrkII. A CrkII mutant in which tyrosine residue 221 was substituted by phenylalanine (CrkII-Y221F) could not be tyrosine phosphorylated, and it showed significantly increased binding to p130cas and paxillin. Enhanced binding of CrkII to p130cas has been reported to promote cell migration. Nonphosphorylated CrkII-Y221F promoted HT1080 cell migration on fibronectin, whereas wild-type CrkII did not at moderate expression levels. Moreover, co-expression of CrkII and PTP1B promoted HT1080 cell migration on fibronectin and retained tyrosine phosphorylation and binding of p130cas to CrkII, whereas paxillin tyrosine phosphorylation was reduced. These findings support the concepts that CrkII binding activity is regulated by tyrosine kinases and phosphatases, and that tyrosine phosphorylation of CrkII can downmodulate cell migration mediated by the focal adhesion kinase/p130cas pathway.
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| 7.
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Nishida, Yuki ; Miyamori, Hisashi ; Thompson, Erik W. ; Takino, Takahisa ; Endo, Yoshio ; Sato, Hiroshi
概要:
金沢大学がん研究所がん病態制御<br />The suggested model for pro-matrix metalloproteinase-2 (proMMP-2) activation by membrane type 1 MMP
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(MT1-MMP) implicates the complex between MT1-MMP and tissue inhibitor of MMP-2 (TIMP-2) as a receptor for proMMP-2. To dissect this model and assess the pathologic significance of MMP-2 activation, an artificial receptor for proMMP-2 was created by replacing the signal sequence of TIMP-2 with cytoplasmic/transmembrane domain of type II transmembrane mosaic serine protease (MSP-T2). Unlike TIMP-2, MSP-T2 served as a receptor for proMMP-2 without inhibiting MT1-MMP, and generated TIMP-2-free active MMP-2 even at a low level of MT1-MMP. Thus, MSP-T2 did not affect direct cleavage of the substrate testican-1 by MT1-MMP, whereas TIMP-2 inhibited it even at the level that stimulates proMMP-2 processing. Expression of MSP-T2 in HT1080 cells enhanced MMP-2 activation by endogenous MT1-MMP and caused intensive hydrolysis of collagen gel. Expression of MSP-T2 in U87 glioma cells, which express a trace level of endogenous MT1-MMP, induced MMP-2 activation and enhanced cell-associated protease activity, activation of extracellular signal-regulated kinase, and metastatic ability into chick embryonic liver and lung. MT1-MMP can exert both maximum MMP-2 activation and direct cleavage of substrates with MSP-T2, which cannot be achieved with TIMP-2. These results suggest that MMP-2 activation by MT1-MMP potentially amplifies protease activity, and combination with direct cleavage of substrate causes effective tissue degradation and enhances tumor invasion and metastasis, which highlights the complex role of TIMP-2. MSP-T2 is a unique tool to analyze physiologic and pathologic roles of MMP-2 and MT1-MMP in comparison with TIMP-2. ©2008 American Association for Cancer Research.全文公開200911
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| 8.
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Kitano, Ayako ; Shimasaki, Takeo ; Chikano, Yuri ; Nakada, Mitsutoshi ; Hirose, Mayumi ; Higashi, Tomomi ; Ishigaki, Yasuhito ; Endo, Yoshio ; Takino, Takahisa ; Sato, Hiroshi ; Sai, Yoshimichi ; Miyamoto, Ken-ichi ; Motoo, Yoshiharu ; Kawakami, Kazuyuki ; Minamoto, Toshinari
概要:
Background and Purpose: The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resis
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tance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer. Methods: Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined. Results: Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts. Conclusion: The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer. © 2013 Kitano et al.
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| 9.
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Yoshimoto, Taisuke ; Takino, Takahisa ; Li, Zichen ; Domoto, Takahiro ; Sato, Hiroshi
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Vinculin regulates a variety of cellular functions partly through stabilization of tumor suppressor PTEN. In order to study the role of vinculin in tumor progression other than PTEN stabilization, vinculin was knocked down in
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PTEN-deficient squamous cell carcinoma HSC-4 cells. Knockdown of vinculin induced phenotypical change by reducing cell-cell and cell-extracellular matrix adhesions, and enhanced MT1-MMP expression at transcription level and subsequent cell migration. Up-regulation of MT1-MMP transcription by vinculin knockdown was abrogated by ERK inhibition. These results suggest that vinculin negatively regulates malignant phenotype of tumor cells including MT1-MMP transcription through MEK/ERK pathway.
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| 10.
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Takino, Takahisa ; Luyang, Guo ; Domoto, Takahiro ; Sato, Hiroshi
概要:
The extracellular microenvironment plays a key role in regulation of cellular functions and growth control. We show here
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that membrane-type 1 matrix metalloproteinase (MT1-MMP) acts as a growth promoter in confluent culture. When MT1-MMP was silenced in HT1080 fibrosarcoma cells, cells created three dimensional (3D) fibronectin matrix in a confluent culture, and growth of cells embedded within it was retarded. Formation of 3D fibronectin matrix initiated by MT1-MMP silencing was impeded by knockdown of either FN or integrin β1, which resulted in restoration of cell growth. When cells in 3D fibronectin matrix were treated with integrin β1 inhibitory antibody, cells underwent S phase entry. These results suggest that MT1-MMP prevents growth suppression by 3D fibronectin matrix, which is mediated through integrin β1. © 2013 Elsevier Inc.
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