研究者詳細 - 江口　傑徳

論文 - 江口　傑徳

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エクソソーム招待査読

江口傑徳

岡山医学会雑誌 136 ( 1 ) 33 - 36 2024年1月

Extracellular Vesicles: New Classification and Tumor Immunosuppression. 招待査読国際誌

Mona Sheta, Eman A Taha, Yanyin Lu, Takanori Eguchi

Biology 12 ( 1 ) 2023年1月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles carrying various types of molecules. These EV cargoes are often used as pathophysiological biomarkers and delivered to recipient cells whose fates are often altered in local and distant tissues. Classical EVs are exosomes, microvesicles, and apoptotic bodies, while recent studies discovered autophagic EVs, stressed EVs, and matrix vesicles. Here, we classify classical and new EVs and non-EV nanoparticles. We also review EVs-mediated intercellular communication between cancer cells and various types of tumor-associated cells, such as cancer-associated fibroblasts, adipocytes, blood vessels, lymphatic vessels, and immune cells. Of note, cancer EVs play crucial roles in immunosuppression, immune evasion, and immunotherapy resistance. Thus, cancer EVs change hot tumors into cold ones. Moreover, cancer EVs affect nonimmune cells to promote cellular transformation, including epithelial-to-mesenchymal transition (EMT), chemoresistance, tumor matrix production, destruction of biological barriers, angiogenesis, lymphangiogenesis, and metastatic niche formation.

添付ファイル： 2023 01 Sheta et al, Eguchi, Biology EVs Review ★.pdf

DOI： 10.3390/biology12010110

Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches 招待査読

Joshua A Welsh, Deborah C.I. Goberdhan, Lorraine O'Driscoll, et al, MISEV Consortium, Takanori Eguchi, et al, Clotilde Thery, Kenneth W Witwer

Journal of Extracellular Vesicles 2023年

SCAND1 Reverses Epithelial-to-Mesenchymal Transition (EMT) and Suppresses Prostate Cancer Growth and Migration 査読国際誌

Takanori Eguchi, Eva Csizmadia, Hotaka Kawai, Mona Sheta, Kunihiro Yoshida, Thomas L. Prince, Barbara Wegiel, Stuart K. Calderwood

Cells 11 ( 24 ) 3993 2022年12月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Epithelial-mesenchymal transition (EMT) is a reversible cellular program that transiently places epithelial (E) cells into pseudo-mesenchymal (M) cell states. The malignant progression and resistance of many carcinomas depend on EMT activation, partial EMT, or hybrid E/M status in neoplastic cells. EMT is activated by tumor microenvironmental TGFβ signal and EMT-inducing transcription factors, such as ZEB1/2, in tumor cells. However, reverse EMT factors are less studied. We demonstrate that prostate epithelial transcription factor SCAND1 can reverse the cancer cell mesenchymal and hybrid E/M phenotypes to a more epithelial, less invasive status and inhibit their proliferation and migration in DU-145 prostate cancer cells. SCAND1 is a SCAN domain-containing protein and hetero-oligomerizes with SCAN-zinc finger transcription factors, such as MZF1, for accessing DNA and the transcriptional co-repression of target genes. We found that SCAND1 expression correlated with maintaining epithelial features, whereas the loss of SCAND1 was associated with mesenchymal phenotypes of tumor cells. SCAND1 and MZF1 were mutually inducible and coordinately included in chromatin with hetero-chromatin protein HP1γ. The overexpression of SCAND1 reversed hybrid E/M status into an epithelial phenotype with E-cadherin and β-catenin relocation. Consistently, the co-expression analysis in TCGA PanCancer Atlas revealed that SCAND1 and MZF1 expression was negatively correlated with EMT driver genes, including CTNNB1, ZEB1, ZEB2 and TGFBRs, in prostate adenocarcinoma specimens. In addition, SCAND1 overexpression suppressed tumor cell proliferation by reducing the MAP3K-MEK-ERK signaling pathway. Of note, in a mouse tumor xenograft model, SCAND1 overexpression significantly reduced Ki-67(+) and Vimentin(+) tumor cells and inhibited migration and lymph node metastasis of prostate cancer. Kaplan-Meier analysis showed high expression of SCAND1 and MZF1 to correlate with better prognoses in pancreatic cancer and head and neck cancers, although with poorer prognosis in kidney cancer. Overall, these data suggest that SCAND1 induces expression and coordinated heterochromatin-binding of MZF1 to reverse the hybrid E/M status into an epithelial phenotype and, inhibits tumor cell proliferation, migration, and metastasis, potentially by repressing the gene expression of EMT drivers and the MAP3K-MEK-ERK signaling pathway.

添付ファイル： cells-11-03993.pdf

DOI： 10.3390/cells11243993

Cancer Extracellular Vesicles, Tumoroid Models and Tumor Microenvironment 招待査読国際誌

Takanori Eguchi, Mona Sheta, Masanori Fujii, Stuart K. Calderwood

Seminars in Cancer Biology 86 ( Pt 1 ) 112 - 126 2022年1月

Triple knockdown of CDC37, HSP90-alpha and HSP90-beta diminishes extracellular vesicles-driven malignancy events and macrophage M2 polarization in oral cancer. 査読国際誌

Kisho Ono, Chiharu Sogawa, Hotaka Kawai, Manh Tien Tran, Eman A Taha, Yanyin Lu, May Wathone Oo, Yuka Okusha, Hirohiko Okamura, Soichiro Ibaragi, Masaharu Takigawa, Ken-Ichi Kozaki, Hitoshi Nagatsuka, Akira Sasaki, Kuniaki Okamoto, Stuart K Calderwood, Takanori Eguchi

Journal of extracellular vesicles 9 ( 1 ) 1769373 - 1769373 2020年5月

Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles. 査読国際誌

Eman A Taha, Chiharu Sogawa, Yuka Okusha, Hotaka Kawai, May Wathone Oo, Abdellatif Elseoudi, Yanyin Lu, Hitoshi Nagatsuka, Satoshi Kubota, Ayano Satoh, Kuniaki Okamoto, Takanori Eguchi

Cancers 12 ( 5 ) 1 - 29 2020年5月

Extracellular Vesicles Enriched with Moonlighting Metalloproteinase Are Highly Transmissive, Pro-Tumorigenic, and Trans-Activates Cellular Communication Network Factor (CCN2/CTGF): CRISPR against Cancer. 査読国際誌

Yuka Okusha, Takanori Eguchi, Manh T Tran, Chiharu Sogawa, Kaya Yoshida, Mami Itagaki, Eman A Taha, Kisho Ono, Eriko Aoyama, Hirohiko Okamura, Ken-Ichi Kozaki, Stuart K Calderwood, Masaharu Takigawa, Kuniaki Okamoto

Cancers 12 ( 4 ) 1 - 27 2020年4月

Cell Stress Induced Stressome Release Including Damaged Membrane Vesicles and Extracellular HSP90 by Prostate Cancer Cells. 査読国際誌

Takanori Eguchi, Chiharu Sogawa, Kisho Ono, Masaki Matsumoto, Manh Tien Tran, Yuka Okusha, Benjamin J Lang, Kuniaki Okamoto, Stuart K Calderwood

Cells 9 ( 3 ) 1 - 24 2020年3月

Roles of Extracellular HSPs as Biomarkers in Immune Surveillance and Immune Evasion. 査読国際誌

Eman A Taha, Kisho Ono, Takanori Eguchi

International journal of molecular sciences 20 ( 18 ) 1 - 32 2019年9月

HSP-enriched properties of extracellular vesicles involve survival of metastatic oral cancer cells. 査読国際誌

Kisho Ono, Takanori Eguchi, Chiharu Sogawa, Stuart K Calderwood, Junya Futagawa, Tomonari Kasai, Masaharu Seno, Kuniaki Okamoto, Akira Sasaki, Ken-Ichi Kozaki

Journal of cellular biochemistry 119 ( 9 ) 7350 - 7362 2018年9月

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment. 査読国際誌

Takanori Eguchi, Chiharu Sogawa, Yuka Okusha, Kenta Uchibe, Ryosuke Iinuma, Kisho Ono, Keisuke Nakano, Jun Murakami, Manabu Itoh, Kazuya Arai, Toshifumi Fujiwara, Yuri Namba, Yoshiki Murata, Kazumi Ohyama, Manami Shimomura, Hirohiko Okamura, Masaharu Takigawa, Tetsuya Nakatsura, Ken-Ichi Kozaki, Kuniaki Okamoto, Stuart K Calderwood

PloS one 13 ( 2 ) e0191109 - 2332 2018年

Intracellular MMP3 Promotes HSP Gene Expression in Collaboration With Chromobox Proteins. 査読国際誌

Takanori Eguchi, Stuart K Calderwood, Masaharu Takigawa, Satoshi Kubota, Ken-Ichi Kozaki

Journal of cellular biochemistry 118 ( 1 ) 43 - 51 2017年1月

OstemiR: a novel panel of microRNA biomarkers in osteoblastic and osteocytic differentiation from mesencymal stem cells. 査読国際誌

Takanori Eguchi, Ken Watanabe, Emilio Satoshi Hara, Mitsuaki Ono, Takuo Kuboki, Stuart K Calderwood

PloS one 8 ( 3 ) e58796 2013年

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

MicroRNAs (miRNAs) are small RNA molecules of 21-25 nucleotides that regulate cell behavior through inhibition of translation from mRNA to protein, promotion of mRNA degradation and control of gene transcription. In this study, we investigated the miRNA expression signatures of cell cultures undergoing osteoblastic and osteocytic differentiation from mesenchymal stem cells (MSC) using mouse MSC line KUSA-A1 and human MSCs. Ninety types of miRNA were quantified during osteoblastic/osteocytic differentiation in KUSA-A1 cells utilizing miRNA PCR arrays. Coincidently with mRNA induction of the osteoblastic and osteocytic markers, the expression levels of several dozen miRNAs including miR-30 family, let-7 family, miR-21, miR-16, miR-155, miR-322 and Snord85 were changed during the differentiation process. These miRNAs were predicted to recognize osteogenic differentiation-, stemness-, epinegetics-, and cell cycle-related mRNAs, and were thus designated OstemiR. Among those OstemiR, the miR-30 family was classified into miR-30b/c and miR-30a/d/e groups on the basis of expression patterns during osteogenesis as well as mature miRNA structures. In silico prediction and subsequent qRT-PCR in stable miR-30d transfectants clarified that context-dependent targeting of miR-30d on known regulators of bone formation including osteopontin/spp1, lifr, ccn2/ctgf, ccn1/cyr61, runx2, sox9 as well as novel key factors including lin28a, hnrnpa3, hspa5/grp78, eed and pcgf5. In addition, knockdown of human OstemiR miR-541 increased Osteopontin/SPP1 expression and calcification in hMSC osteoblastic differentiation, indicating that miR-541 is a negative regulator of osteoblastic differentiation. These observations indicate stage-specific roles of OstemiR especially miR-541 and the miR-30 family on novel targets in osteogenesis.

DOI： 10.1371/journal.pone.0058796

Novel transcription-factor-like function of human matrix metalloproteinase 3 regulating the CTGF/CCN2 gene. 査読国際誌

Takanori Eguchi, Satoshi Kubota, Kazumi Kawata, Yoshiki Mukudai, Junji Uehara, Toshihiro Ohgawara, Soichiro Ibaragi, Akira Sasaki, Takuo Kuboki, Masaharu Takigawa

Molecular and cellular biology 28 ( 7 ) 2391 - 413 2008年4月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC MICROBIOLOGY

Matrix metalloproteinase 3 (MMP3) is well known as a secretory endopeptidase that degrades extracellular matrices. Recent reports indicated the presence of MMPs in the nucleus (A. J. Kwon et al., FASEB J. 18:690-692, 2004); however, its function has not been well investigated. Here, we report a novel function of human nuclear MMP3 as a trans regulator of connective tissue growth factor (CCN2/CTGF). Initially, we cloned MMP3 cDNA as a DNA-binding factor for the CCN2/CTGF gene. An interaction between MMP3 and transcription enhancer dominant in chondrocytes (TRENDIC) in the CCN2/CTGF promoter was confirmed by a gel shift assay and chromatin immunoprecipitation. The CCN2/CTGF promoter was activated by overexpressed MMP3, whereas a TRENDIC mutant promoter lost the response. Also, the knocking down of MMP3 suppressed CCN2/CTGF expression. By cytochemical and histochemical analyses, MMP3 was detected in the nuclei of chondrocytic cells in culture and also in the nuclei of normal and osteoarthritic chondrocytes in vivo. The nuclear translocation of externally added recombinant MMP3 and six putative nuclear localization signals in MMP3 also were shown. Furthermore, we determined that heterochromatin protein gamma coordinately regulates CCN2/CTGF by interacting with MMP3. The involvement of this novel role of MMP3 in the development, tissue remodeling, and pathology of arthritic diseases through CCN2/CTGF regulation thus is suggested.

DOI： 10.1128/MCB.01288-07

Herbal medicine Ninjinyoeito inhibits RANKL-induced osteoclast differentiation and bone resorption activity by regulating NF-kB and MAPK pathway 査読国際誌

Kaung Htike, Kunihiro Yoshida, Takanori Eguchi, Katsuki Takebe, Xueming Li, Yaxin Qu, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

Journal of Oral Biosciences 66 ( 4 ) 49 - 57 2024年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier BV

OBJECTIVES: Osteoporosis is a systemic bone metabolism disorder characterized by decreased bone mass and strength. Osteoclasts (OCs) are giant multinucleated cells that regulate bone homeostasis by degrading bone matrix. Excessive OC differentiation and activity can lead to serious bone metabolic disorders including osteoporosis. Current treatments, including antiresorptive drugs, exert considerable adverse effects, including jaw osteonecrosis. Herbal medicines, such as Ninjinyoeito (NYT), may also offer efficacy, but with fewer adverse effects. In this study, we investigated NYT's effects on osteoclastogenesis. METHODS: Tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assays were performed to examine NYT's effects on OC differentiation and function. OC-related gene expression at mRNA and protein levels was investigated to confirm NYT's inhibitory action against osteoclastogenesis. We also demonstrated involvement of signaling pathways mediated by IκBα and mitogen-activated protein kinases (MAPK) [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38] and showed nuclear translocation of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and nuclear factor kappa B (NF-κB) p65 during osteoclastogenesis. RESULTS: TRAP staining and bone resorption assays confirmed that NYT significantly inhibited OC differentiation and function. Western blot and RT-PCR results showed that NYT ameliorated osteoclastogenesis by suppressing mRNA and protein level expression of OC-related genes. Moreover, blots and immunocytochemistry (ICC) data clarified that NYT abrogates signaling pathways mediated by IκBα and MAPK (ERK, JNK, p38), and demonstrated nuclear translocation of NFATc1 and NF-κB p65 during OC differentiation. CONCLUSIONS: These findings suggest NYT is an alternative therapeutic candidate for treating osteoporosis.

DOI： 10.1016/j.job.2024.09.007

Stress biology: Complexity and multifariousness in health and disease. 招待査読国際誌

Matthias P Mayer, Laura Blair, Gregory L Blatch, Thiago J Borges, Ahmed Chadli, Gabriela Chiosis, Aurélie de Thonel, Albena Dinkova-Kostova, Heath Ecroyd, Adrienne L Edkins, Takanori Eguchi, Monika Fleshner, Kevin P Foley, Sotirios Fragkostefanakis, Jason Gestwicki, Pierre Goloubinoff, Jennifer A Heritz, Christine M Heske, Jonathan D Hibshman, Jenny Joutsen, Wei Li, Michael Lynes, Marc L Mendillo, Nahid Mivechi, Fortunate Mokoena, Yuka Okusha, Veena Prahlad, Elizabeth Repasky, Sara Sannino, Federica Scalia, Reut Shalgi, Lea Sistonen, Emily Sontag, Patricija van Oosten-Hawle, Anniina Vihervaara, Anushka Wickramaratne, Shawn Xiang Yang Wang, Tawanda Zininga

Cell stress & chaperones 29 ( 1 ) 143 - 157 2024年2月

記述言語：英語掲載種別：研究論文（国際会議プロシーディングス）

Preserving and regulating cellular homeostasis in the light of changing environmental conditions or developmental processes is of pivotal importance for single cellular and multicellular organisms alike. To counteract an imbalance in cellular homeostasis transcriptional programs evolved, called the heat shock response, unfolded protein response, and integrated stress response, that act cell-autonomously in most cells but in multicellular organisms are subjected to cell-nonautonomous regulation. These transcriptional programs downregulate the expression of most genes but increase the expression of heat shock genes, including genes encoding molecular chaperones and proteases, proteins involved in the repair of stress-induced damage to macromolecules and cellular structures. Sixty-one years after the discovery of the heat shock response by Ferruccio Ritossa, many aspects of stress biology are still enigmatic. Recent progress in the understanding of stress responses and molecular chaperones was reported at the 12th International Symposium on Heat Shock Proteins in Biology, Medicine and the Environment in the Old Town Alexandria, VA, USA from 28th to 31st of October 2023.

DOI： 10.1016/j.cstres.2024.01.006

Rab11 suppresses head and neck carcinoma by regulating EGFR and EpCAM exosome secretion. 査読国際誌

Kunihiro Yoshida, Kaung Htike, Takanori Eguchi, Hotaka Kawai, Htoo Shwe Eain, Manh Tien Tran, Chiharu Sogawa, Koki Umemori, Tatsuo Ogawa, Hideka Kanemoto, Kisho Ono, Hitoshi Nagatsuka, Akira Sasaki, Soichiro Ibaragi, Kuniaki Okamoto

Journal of oral biosciences 66 ( 1 ) 205 - 216 2023年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

OBJECTIVES: Rab11(Rab11a and Rab11b) localizes primarily along recycling endosomes in cells and is involved in various intracellular trafficking processes, including membrane receptor recycling and secretion of exosomes or small extracellular vesicles (EVs). Although Rab11 is closely associated with the progression and metastasis of various cancer types, little is known about Rab11' role in head and neck squamous cell carcinoma (HNSCC). In this study, we investigated the roles of Rab11a and Rab11b in HNSCC. METHODS: The clinical significance of Rab11 expression in HNSCC was investigated using a public database and tissue microarray analysis. Stable cell lines with loss and gain of Rab11a or Rab11b were originally established to investigate their roles in the proliferative, migratory, and invasive capabilities of HNSCC cells. RESULTS: Database analysis revealed a significant association between Rab11b mRNA expression and a favorable patient survival rate in HNSCC. Tissue microarray analysis revealed that Rab11b expression was the highest in normal tissues and gradually decreased across the stages of HNSCC progression. Overexpression of Rab11a or Rab11b resulted in a decrease in epidermal growth factor receptor (EGFR), Epithelial cell adhesion molecule (EpCAM) exosome secretion, and the migratory and invasive potential of HNSCC cells. The knockdown of Rab11a or Rab11b increased EpCAM/CD9 exosome secretion in addition to the migratory and invasive potential of HNSCC cells. CONCLUSIONS: Rab11 suppresses HNSCC by regulating EGFR recycling and EpCAM exosome secretion in HNSCC cells. Our results indicate that Rab11b is a superior prognostic indicator of HNSCC and holds promise for developing novel therapeutic strategies.

添付ファイル： 2023 12 Yoshida K Rab11 HNSC Exosome.pdf

DOI： 10.1016/j.job.2023.11.007

EpEX, the soluble extracellular domain of EpCAM, resists cetuximab treatment of EGFR-high head and neck squamous cell carcinoma. 査読国際誌

Koki Umemori, Kisho Ono, Takanori Eguchi, Hotaka Kawai, Tomoya Nakamura, Tatsuo Ogawa, Kunihiro Yoshida, Hideka Kanemoto, Kohei Sato, Kyoichi Obata, Shoji Ryumon, Hirokazu Yutori, Naoki Katase, Tatsuo Okui, Hitoshi Nagatsuka, Soichiro Ibaragi

Oral oncology 142 106433 - 106433 2023年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

OBJECTIVES: Cetuximab (Cmab) is a molecularly targeted monoclonal antibody drug for head and neck squamous cell carcinoma (HNSC), although cetuximab resistance is a serious challenge. Epithelial cell adhesion molecule (EpCAM) is an established marker for many epithelial tumors, while the soluble EpCAM extracellular domain (EpEX) functions as a ligand for epidermal growth factor receptor (EGFR). We investigated the expression of EpCAM in HNSC, its involvement in Cmab action, and the mechanism by which soluble EpEX activated EGFR and played key roles in Cmab resistance. MATERIALS AND METHODS: We first examined EPCAM expression in HNSCs and its clinical significance by searching gene expression array databases. We then examined the effects of soluble EpEX and Cmab on intracellular signaling and Cmab efficacy in HNSC cell lines (HSC-3 and SAS). RESULTS: EPCAM expression was found to be enhanced in HNSC tumor tissues compared to normal tissues, and the enhancement was correlated with stage progression and prognosis. Soluble EpEX activated the EGFR-ERK signaling pathway and nuclear translocation of EpCAM intracellular domains (EpICDs) in HNSC cells. EpEX resisted the antitumor effect of Cmab in an EGFR expression-dependent manner. CONCLUSION: Soluble EpEX activates EGFR to increase Cmab resistance in HNSC cells. The EpEX-activated Cmab resistance in HNSC is potentially mediated by the EGFR-ERK signaling pathway and the EpCAM cleavage-induced nuclear translocation of EpICD. High expression and cleavage of EpCAM are potential biomarkers for predicting the clinical efficacy and resistance to Cmab.

添付ファイル： 2023 05 Umemori Ono EpEX.pdf

DOI： 10.1016/j.oraloncology.2023.106433

Stress-Inducible SCAND Factors Suppress the Stress Response and Are Biomarkers for Enhanced Prognosis in Cancers. 招待査読国際誌

Mona Sheta, Kunihiro Yoshida, Hideka Kanemoto, Stuart K Calderwood, Takanori Eguchi

International journal of molecular sciences 24 ( 6 ) 2023年3月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The cell stress response is an essential system present in every cell for responding and adapting to environmental stimulations. A major program for stress response is the heat shock factor (HSF)-heat shock protein (HSP) system that maintains proteostasis in cells and promotes cancer progression. However, less is known about how the cell stress response is regulated by alternative transcription factors. Here, we show that the SCAN domain (SCAND)-containing transcription factors (SCAN-TFs) are involved in repressing the stress response in cancer. SCAND1 and SCAND2 are SCAND-only proteins that can hetero-oligomerize with SCAN-zinc finger transcription factors, such as MZF1(ZSCAN6), for accessing DNA and transcriptionally co-repressing target genes. We found that heat stress induced the expression of SCAND1, SCAND2, and MZF1 bound to HSP90 gene promoter regions in prostate cancer cells. Moreover, heat stress switched the transcript variants' expression from long noncoding RNA (lncRNA-SCAND2P) to protein-coding mRNA of SCAND2, potentially by regulating alternative splicing. High expression of HSP90AA1 correlated with poorer prognoses in several cancer types, although SCAND1 and MZF1 blocked the heat shock responsiveness of HSP90AA1 in prostate cancer cells. Consistent with this, gene expression of SCAND2, SCAND1, and MZF1 was negatively correlated with HSP90 gene expression in prostate adenocarcinoma. By searching databases of patient-derived tumor samples, we found that MZF1 and SCAND2 RNA were more highly expressed in normal tissues than in tumor tissues in several cancer types. Of note, high RNA expression of SCAND2, SCAND1, and MZF1 correlated with enhanced prognoses of pancreatic cancer and head and neck cancers. Additionally, high expression of SCAND2 RNA was correlated with better prognoses of lung adenocarcinoma and sarcoma. These data suggest that the stress-inducible SCAN-TFs can function as a feedback system, suppressing excessive stress response and inhibiting cancers.

添付ファイル： 2023 03 Sheta IJMS Stress +supple.pdf

DOI： 10.3390/ijms24065168

Multiple Targeting of HSP Isoforms to Challenge Isoform Specificity and Compensatory Expression. 招待査読国際誌

Kisho Ono, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2693 141 - 161 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Heat shock proteins (HSPs) are molecular chaperones that assist in protein folding, trafficking, and metabolism. Intracellular chaperone functions of HSPs had been well-investigated, but extracellular and exosomal HSPs have been recently found. Exosomal HSPs are intercellularly transferred, while extracellular HSPs play cytokine-like roles called chaperokines. We have shown that exosomal HSPs play key roles in intercellular communication between tongue carcinoma and tumor-associated macrophages in the tumor microenvironment. Notably, HSP90 isoforms consist of HSP90alpha, HSP90beta, mitochondrial TRAP1, and GRP94 in the endoplasmic reticulum. Moreover, many pseudogenes of HSP90 can be transcribed into RNA. Besides, the function of HSP90 is defined by their cochaperones, such as CDC37 or AHA1. Therefore, isoform-specific small interfering RNA (siRNA) is necessary for precisely targeting each HSP90 isoform and cochaperone. Nevertheless, we often encountered compensatory expression of HSP90 isoforms in the knockdown studies. Here, we provide dual and triple knockdown methods to target multiple RNA for challenging isoform-specific roles and compensatory expression of intracellular, extracellular, and exosomal HSPs.

DOI： 10.1007/978-1-0716-3342-7_12

Proteomic Profiling of the Extracellular Vesicle Chaperone in Cancer. 招待査読国際誌

Kisho Ono, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2693 233 - 249 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Molecular chaperones are widely distributed intracellular proteins that play essential roles in maintaining proteome function by assisting in the folding of client proteins. Molecular chaperones, such as heat shock proteins (HSPs), are found intracellularly and extracellularly. Extracellular vesicles (EVs), such as exosomes, contain HSPs and horizontally transfer the functional chaperones into various recipient cells. Besides, mass spectrometry has enabled a comprehensive analysis of exosomal and EV proteins, which is useful in basic biomedical research to clinical biomarker search. We have performed deep proteome analysis of EVs, including exosomes, from metastatic tongue and prostate cancers and detected >700 protein types, including cytoplasmic, ER, mitochondrial, small, and large HSPs. Here, we provide protocols for isolating exosomes/EVs and deep proteome analysis to detect the EV chaperone.

DOI： 10.1007/978-1-0716-3342-7_18

Large-Scale Databases and Portals on Cancer Genome to Analyze Chaperone Genes Correlated to Patient Prognosis. 招待査読国際誌

Kisho Ono, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2693 293 - 306 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Molecular chaperones, such as heat shock proteins (HSPs), have attracted attention as molecules involved in malignant events in cancers and are potential therapeutic targets and biomarkers for tumor therapy. Furthermore, mutations in chaperones can significantly impact cancer risk and prognosis. Bioinformatics is a particularly useful method for developing biomarkers as a practical consideration for the immediate clinical application of data. Many large-scale databases and portals on cancer genome are nowadays publicly available, including the International Cancer Genome Consortium (ICGC); The Cancer Genome Atlas (TCGA), renamed as Genomic Data Commons (GDC); Catalogue of Somatic Mutations in Cancer (COSMIC); and Cancer Cell Line Encyclopedia (CCLE). Referring to these databases, advanced web portals are publicized, including cBioPortal, Human Protein Atlas (HPA), Kaplan-Meier (KM) plotter, Gene Expression Profiling Interactive Analysis 2 (GEPIA2), Genomics of Drug Sensitivity in Cancer (GDSC), and Dependency Map (DepMap). Here, we assemble these databases and portals to clarify what is available and useful for current cancer research and provide protocols to utilize the HPA, KM plotter, and GEPIA2 for studies on chaperone genes in cancer patients. Utilizing these portals will reveal the correlation between tumor subtype-specific high expression of chaperone genes and patient prognosis. Our protocols are useful to increase systematic awareness of chaperones and find new biomarkers for diagnosis and prognosis and new targets for anticancer drugs.

DOI： 10.1007/978-1-0716-3342-7_22

Monitoring of the Heat Shock Response with a Real-Time Luciferase Reporter. 招待査読国際誌

Andrew Ackerman, Toshiki Kijima, Takanori Eguchi, Thomas L Prince

Methods in molecular biology (Clifton, N.J.) 2693 1 - 11 2023年

記述言語：英語掲載種別：研究論文（学術雑誌）

The heat shock response (HSR) is a cellular mechanism for counteracting acute proteotoxic stress. In eukaryotes, transcriptional activation of the HSR is regulated by heat shock factor 1 (HSF1). Activation of HSF1 induces the expression of heat shock proteins (HSPs) that function as molecular chaperones to fold and maintain the three-dimensional structure of misfolded proteins. The regulation of the degree and duration of the HSR is controlled by multiple biochemical mechanisms that include posttranslational modification of HSF1 and numerous protein-protein interactions. In this chapter, we describe a method to evaluate the activation and deactivation of the HSR at the transcriptional level using a short half-life luciferase reporter assay. This assay can be used to further characterize the HSR or as a screen for small molecule inducers, amplifiers, or repressors.

DOI： 10.1007/978-1-0716-3342-7_1

Protocol for CRISPR/Cas Genome Editing for Investigating Cell Communication Network. 招待査読国際誌

Yuka Okusha, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2582 157 - 167 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The Cellular Communication Network Factor (CCN) family is composed of six members: CCN1/CYR61, CCN2/CTGF, CCN3/NOV, CCN4/WISP1, CCN5/WISP2, and CCN6/WISP3. The second member, CCN2/CTGF is a matricellular protein that promotes extracellular matrix (ECM) synthesis and controls angiogenesis. On the other hand, moonlighting/matrix metalloproteinase 3 (MMP3) is an ECM-degrading enzyme that also functions as an intracellular transcription factor. Importantly, extracellular MMP3 is uptaken into cells, translocating into nuclei, and transcriptionally activating CCN2/CTGF gene in cancer and chondrocytes. Thus, the MMP3-CTGF axis balances the matrix metabolism and turnover in the tissue and tumor microenvironments. We established an MMP3 knockout cell line using the CRISPR/Cas9 system, demonstrating the sequential regulatory events of the MMP3-CCN2 axis in the microenvironment. Notably, our protocol is useful for generation of CCN knockout cells as well. Here we serve a protocol of the CRISPR/Cas9-based gene targeting in cultured cells for investigating cellular communication network.

DOI： 10.1007/978-1-0716-2744-0_11

Transfection, Spinfection, Exofection, and Luciferase Assays for Analysis of CCN Genes Expression Mechanism. 招待査読国際誌

Takanori Eguchi, Yanyin Lu, Eman A Taha, Yuka Okusha

Methods in molecular biology (Clifton, N.J.) 2582 103 - 126 2023年

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Cell communication network factor 2 (CCN2), also known as connective tissue growth factor (CTGF), is protein inducible in response to TGFβ/Smad signal or the transcriptional activity of matrix metalloproteinase 3 (MMP3). We discovered that MMP3 in exosomes is transferable to recipient cells and then translocates into cell nuclei to transactivate the CCN2/CTGF gene. Exosomes and liposomes enable molecular transfection to recipient cells in vitro and in vivo. These small vesicles are surrounded by lipid membranes and carry proteins, RNA, DNA, and small chemicals. Here we define the exosome-based transfection as "exofection." In addition, spinfection increases the efficiencies of transfection, exofection, and viral infection, thus being compatible with various molecular transfer protocols. Here, we provide protocols, tips, and practical examples of transfection, spinfection, exofection, fluorescence microscopy, and luciferase assays to analyze the CCNs gene expression mechanisms.

DOI： 10.1007/978-1-0716-2744-0_9

Comprehensive Method for Exosome Isolation and Proteome Analysis for Detection of CCN Factors in/on Exosomes. 招待査読国際誌

Takanori Eguchi, Yuka Okusha, Yanyin Lu, Kisho Ono, Eman A Taha, Shiro Fukuoka

Methods in molecular biology (Clifton, N.J.) 2582 59 - 76 2023年

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Cellular Communication Network (CCN) proteins are secretory growth factors often associated with extracellular matrix (ECM) and extracellular vesicles (EVs) such as exosomes or matrix-coated vesicles. CCN factors and fragments loaded on/in EVs may play key roles in cell communication networks in cancer biology, bone and cartilage metabolism, wound healing, and tissue regeneration. CCN proteins and EVs/exosomes are found in body fluids, such as blood, urine, milk, and supernatants of the two-dimensionally (2D) cultured cells and three-dimensionally (3D) cultured tissues, such as spheroids or organoids. More than ten methods to isolate exosomes or EVs have been developed with different properties. Here, we introduce comprehensive protocols for polymer-based precipitation, affinity purification, ultracentrifugation methods combined with the ultrafiltration method for isolating CCN-loaded exosomes/EVs from 2D and 3D cultured tissues, and proteome analysis using mass spectrometry for comprehensive analysis of CCN proteins.

DOI： 10.1007/978-1-0716-2744-0_6

Western Blot Protocols for Analysis of CCN Proteins and Fragments in Exosomes, Vesicle-Free Fractions, and Cells. 査読国際共著国際誌

Kisho Ono, Yuka Okusha, Manh Tien Tran, Koki Umemori, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2582 39 - 57 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Cellular Communication Network (CCN) proteins are growth factors that play key roles in many pathophysiological events, including bone formation, wound healing, and cancer. CCN factors and fragments generated by metalloproteinases-dependent cleavage are often associated with extracellular matrix (ECM) or small extracellular vesicles (sEVs) such as exosomes or matrix-coated vesicles. We provide reliable methods and protocols for Western blotting to analyze CCN factors and fragments in cells, sEVs, and vesicle-free fractions.

DOI： 10.1007/978-1-0716-2744-0_5

Cdc37 as a Co-chaperone to Hsp90 招待査読国際誌

Thomas L Prince, Benjamin J Lang, Yuka Okusha, Takanori Eguchi, Stuart K Calderwood

Sub-cellular biochemistry 101 141 - 158 2023年

Multiplex Immunostaining Method to Distinguish HSP Isoforms in Cancer Tissue Specimens. 招待査読国際誌

Hotaka Kawai, Kisho Ono, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2693 281 - 291 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Heat shock proteins (HSPs) are often expressed in all nucleated cells, but their expression profiles differ. In particular, HSP90α and HSP90β have high sequence identity and have not been fully examined for their individual and compensatory functions as molecular chaperones, differences in client proteins, and extracellular distributions with exosomes. Immunohistochemical staining is a technique to visualize the presence and localization of target antigens using specific antibodies, of which the multiplex immunostaining method can reveal differences in protein expression in the same tumor tissue and the localization of proteins of interest within tumor tissue or single cells. The common multiplex immunostaining method uses multiple secondary antibodies of different reacting animal species to identify and detect different antigens, thus requiring different animals to be immunized with each primary antibody. Furthermore, the fluorescent-antibody method is the predominant multiplex staining method but has the critical disadvantage that permanent specimens cannot be prepared. Here, we outline a multiplex staining method for HSP90α and HSP90β based on the enzyme-antibody method that allows permanent specimens to be prepared without the restriction of immunized animal species.

DOI： 10.1007/978-1-0716-3342-7_21

Roles of Cancer Exosomes in Immunosuppression and Immune Evasion

Mona Sheta, Eman Ahmed Taha, Yanyin Lu, Takanori Eguchi

Preprints 2022110004 2022年11月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

Extracellular vesicles (EV), including exosomes and microvesicles, are released from various cells and alter recipient cell phenotypes and fates by their biomolecules. Here we review current knowledge about tumor EVs and how they prompt malignant cell communication with tumor-associated cells, such as cancer-associated fibroblasts, tumor endothelial cells, and immune cells. We delineate the major pathways and molecular players that influence each step of cancer initiation, progression, and resistance. Of note, cancer exosomes involve immunosuppression by tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Moreover, tumor exosomes can induce the apoptosis of killer T cells and immune checkpoint of dendritic cells and attenuate natural killer cells. An in-depth understanding of EV biology is essential to ensure the clinical development of exosome/EV-based therapeutic products, which will be of benefit to exosome manipulation in cancer management.

DOI： 10.20944/preprints202211.0004.v1

SCAND1 Reverts EMT and Suppresses Tumor Growth and Collective Migration

Takanori Eguchi, Eva Csizmadia, Thomas L. Prince, Barbara Wegiel, Stuart K. Calderwood

Preprints 2022年10月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

Epithelial-mesenchymal transition (EMT) is a reversible cellular program that transiently places epithelial (E) cells into pseudo-mesenchymal (M) cell states. The malignant progression and resistance of many types of carcinomas depends on EMT activation, partial EMT and hybrid E/M status in neoplastic cells. EMT is activated by tumor microenvironmental TGF&beta; signal and EMT-inducing transcription factors, such as ZEB1/2 in tumor cells. However, reverse EMT factors are less studied. We demonstrate that transcription factor SCAND1 can revert mesenchymal and hybrid E/M phenotype of cancer cells to a more epithelial, less invasive status and inhibit their proliferation and migration. SCAND1 is a SCAN domain-containing protein and hetero-oligomerizes with SCAN-zinc finger transcription factors, such as MZF1, for accessing DNA and transcriptional co-repression of target genes. We found that SCAND1-MZF1 co-expression and interaction correlated with maintaining epithelial features, whereas the simultaneous loss of SCAND1 and MZF1 correlated with mesenchymal features of tumor cells. Overexpression of SCAND1 over endogenous MZF1 in DU-145 prostate cancer cells reverted their hybrid E/M status into cobblestone morphology with increased epithelial adhesion by E-cadherin and &beta;-catenin relocation. Consistently, co-expression analysis in TCGA PanCancer Atlas revealed that both SCAND1 and MZF1 co-express and are negatively correlated with EMT driver genes, including CTNNB1, ZEB1, ZEB2 and TGFBR, in prostate tumor specimens. In addition, SCAND1 overexpression suppressed tumor cell proliferation by reducing the MAP3K-MEK-ERK signaling pathway. Of note, SCAND1-overexpressing DU-145 cells migrated slower than control cells with decreased lymph node metastasis of prostate cancer in a mouse tumor xenograft model. Kaplan-Meyer analysis showed high expression of MZF1 and SCAND1 to correlate with better prognoses in pancreatic cancer and head and neck cancers, although with poorer prognosis in kidney cancer. Overall, these data suggest that the combination of SCAND1-MZF1 complexes may revert the EMT mechanism in cancer to establish an epithelial phenotype. These effects seem to include co-repression of EMT-driver genes and suppression of tumor cell proliferation via inhibition of the MAP3K-MEK-ERK signaling pathway.

DOI： 10.20944/preprints202210.0475.v1

HSP90 drives the Rab11a-mediated vesicular transport of the cell surface receptors in osteoclasts. 査読国際誌

Manh Tien Tran, Yuka Okusha, Kaung Htike, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

Cell biochemistry and function 40 ( 8 ) 838 - 855 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

Rab11a, which ubiquitously localizes to early and recycling endosomes, is required for regulating the vesicular transport of cellular cargos. Interestingly, our previous study revealed that Rab11a served as a negative regulator of osteoclastogenesis by facilitating the lysosomal proteolysis of (1) colony-stimulating factor-1 (c-fms) receptor and (2) receptor activator of nuclear factor-κB (RANK) receptor, thereby resulting in inhibition of osteoclast (OC) differentiation, maturation, and bone-resorbing activity. However, the molecular mechanisms of how Rab11a negatively affected osteoclastogenesis were largely unknown. Heat shock protein (HSP90), including two isoforms HSP90α and HSP90β, necessitates the stability, maturation, and activity of a broad range of its clients, and is essentially required for a vast array of signal transduction pathways in nonstressful conditions. Furthermore, cumulative evidence suggests that HSP90 is a vital element of the vesicular transport network. Indeed, our recent study revealed that HSP90, a novel effector protein of Rab11b, modulated Rab11b-mediated osteoclastogenesis. In this study, we also found that Rab11a interacted with both HSP90α and HSP90β in OCs. Upon blockade of HSP90 ATPase activity by a specific inhibitor(17-allylamino-demethoxygeldanamycin), we showed that (1) the ATPase domain of HSP90 was a prerequisite for the interaction between HSP90 and Rab11a, and (2) the interaction of HSP90 to Rab11a sufficiently maintained the inhibitory effects of Rab11a on osteoclastogenesis. Altogether, our findings undoubtedly indicate a novel role of HSP90 in regulating Rab11a-mediated osteoclastogenesis.

DOI： 10.1002/cbf.3745

口腔癌間質細胞由来CCL2による骨髄由来細胞動員への関与(Resident stroma-secreted chemokine CCL2 governs myeloid-derived suppressor cells in the tumor microenvironment)

河合穂高, 冨田秀太, 江口傑徳, 大原利章, 小野喜章, 長塚仁

日本癌学会総会記事 81回 P - 3186 2022年9月

口腔癌間質由来のCCL2はCCR2陽性骨髄由来免疫抑制細胞の腫瘍間質への動員に関与する

河合穂高, メイ・ワトウ, 高畠清文, 冨田秀太, 小野喜章, 江口傑徳, 大原利章, 中野敬介, 長塚仁

日本がん免疫学会総会プログラム・抄録集 26回 91 - 91 2022年6月

Rab34 plays a critical role as a bi-directional regulator of osteoclastogenesis 査読国際誌

Yunxia Feng, Manh Tien Tran, co-first author, Yanyin Lu, Kaung Htike, Yuka Okusha, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

Cell Biochemistry and Function 40 ( 3 ) 263 - 277 2022年3月

Resident stroma-secreted chemokine CCL2 governs myeloid-derived suppressor cells in the tumor microenvironment. 査読国際誌

May Wathone Oo, Hotaka Kawai, Kiyofumi Takabatake, Shuta Tomida, Takanori Eguchi, Kisho Ono, Qiusheng Shan, Toshiaki Ohara, Saori Yoshida, Haruka Omori, Shintaro Sukegawa, Keisuke Nakano, Kuniaki Okamoto, Akira Sasaki, Hitoshi Nagatsuka

JCI insight 7 ( 1 ) 3186 2022年1月

Porphyromonas gingivalisはマクロファージの細胞外小胞を介して胎盤・胎児の成長発育を阻害する(Porphyromonas gingivalis impairs placental and fetal development through macrophage-derived extracellular vesicles)

棚井あいり, 福原瑶子, 江口傑徳, 河合穂高, 池亀美華, 岡村裕彦, 植田幸嗣

Journal of Oral Biosciences Supplement 2021 205 - 205 2021年10月

A novel role of HSP90 in regulating osteoclastogenesis by abrogating Rab11b-driven transport. 査読国際誌

Manh Tien Tran, Yuka Okusha, Yunxia Feng, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

Biochimica et biophysica acta. Molecular cell research 1868 ( 10 ) 119096 - 119096 2021年9月

Extracellular Hsp90 and protection of neuronal cells through Nrf2 査読国際誌

Stuart K Calderwood, Thiago J Borges, Takanori Eguchi, Benjamin J Lang, Ayesha Murshid, Yuka Okusha, Thomas L Prince

Biochemical Society Transactions 49 ( 5 ) 2299 - 2306 2021年8月

Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method. 査読国際誌

Yanyin Lu, Takanori Eguchi, Chiharu Sogawa, Eman A Taha, Manh Tien Tran, Toshiki Nara, Penggong Wei, Shiro Fukuoka, Takuya Miyawaki, Kuniaki Okamoto

Cells 10 ( 6 ) 1 - 23 2021年5月

Macrophage-Derived Small Exosomes: Efficient Transmission and Cytotoxicity to Cancer Cells

Yanyin Lu, Takanori Eguchi, Chiharu Sogawa, Eman A. Taha, Manh Tien Tran, Toshiki Nara, Penggong Wei, Shiro Fukuoka, Takuya Miyawaki, Kuniaki Okamoto

Preprints doi: 10.20944/preprints202104.0464.v1 2021年4月

Gel-Free 3D Tumoroids with Stem Cell Properties Modeling Drug Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer. 査読国際誌

Chiharu Sogawa, Takanori Eguchi, Yuri Namba, Yuka Okusha, Eriko Aoyama, Kazumi Ohyama, Kuniaki Okamoto

Cells 10 ( 2 ) 1 - 17 2021年2月

HSP Stimulation on Macrophages and Dendritic Cells Activates Innate Immune System 招待査読

Yanyin Lu, Takanori Eguchi

Heat Shock Proteins 22 53 - 67 2021年

Extracellular Vesicle-Associated Moonlighting Proteins: Heat Shock Proteins and Metalloproteinases 招待査読

Takanori Eguchi, Eman Ahmed Taha

Heat Shock Proteins 22 1 - 18 2021年

Heat Shock Proteins and Periodontitis – Cross-Reaction Between Bacterial and Human HSP in Periodontal Infection Linking with Cardiovascular Diseases 招待査読

Tadashi Yamamoto, Takanori Eguchi

Heat Shock Proteins 22 19 - 32 2021年

Liquid-based 3D Cultured Tumoroids Modeling Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer

Chiharu Sogawa, Takanori Eguchi, Yuri Namba, Eriko Aoyama, Kazumi Ohyama, Kuniaki Okamoto

Preprints doi: 10.20944/preprints202012.0582.v1 2020年12月

The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors. 査読国際誌

Manh Tien Tran, Yuka Okusha, Yunxia Feng, Masatoshi Morimatsu, Penggong Wei, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Hirohiko Okamura, Keiji Naruse, Takayuki Tsukuba, Kuniaki Okamoto

International journal of molecular sciences 21 ( 24 ) 9352 - 9352 2020年12月

侵襲性歯周炎の血液診断バイオマーカーとしての細胞外小胞由来マイクロRNAの探索

河本美奈, 山本直史, 河村麻理, 森彩乃, 山城圭介, 大森一弘, 小野喜章, 江口傑徳, 十川千春, 高柴正悟

岡山歯学会雑誌 39 ( 2 ) 35 - 36 2020年12月

Organoids and Liquid Biopsy in Oral Cancer Research. 招待査読国際誌

Takanori Eguchi

Journal of clinical medicine 9 ( 11 ) 1 - 5 2020年11月

Rab11A Functions as a Negative Regulator of Osteoclastogenesis through Dictating Lysosome-Induced Proteolysis of c-fms and RANK Surface Receptors. 査読国際誌

Yuka Okusha, Manh Tien Tran, Mami Itagaki, Chiharu Sogawa, Takanori Eguchi, Tatsuo Okui, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

Cells 9 ( 11 ) 2384 - 2384 2020年10月

Roles of HSP on Antigen Presentation 招待査読

Kazuyuki Furuta, Taka Eguchi

Heat Shock Proteins in Human Diseases 275 - 280 2020年7月

Outer membrane vesicles of Porphyromonas gingivalis attenuate insulin sensitivity by delivering gingipains to the liver. 査読国際誌

Mariko Seyama, Kaya Yoshida, Kayo Yoshida, Natsumi Fujiwara, Kisho Ono, Takanori Eguchi, Hotaka Kawai, Jiajie Guo, Yao Weng, Yuan Haoze, Kenta Uchibe, Mika Ikegame, Akira Sasaki, Hitoshi Nagatsuka, Kuniaki Okamoto, Hirohiko Okamura, Kazumi Ozaki

Biochimica et biophysica acta. Molecular basis of disease 1866 ( 6 ) 165731 - 165731 2020年6月

A Novel Model of Cancer Drug Resistance: Oncosomal Release of Cytotoxic and Antibody-Based Drugs. 査読国際誌

Takanori Eguchi, Eman Ahmed Taha, Stuart K Calderwood, Kisho Ono

Biology 9 ( 3 ) 1 - 22 2020年3月

Antiparkinson Drug Benztropine Suppresses Tumor Growth, Circulating Tumor Cells, and Metastasis by Acting on SLC6A3/DAT and Reducing STAT3. 査読国際誌

Chiharu Sogawa, Takanori Eguchi, Manh Tien Tran, Masayuki Ishige, Kilian Trin, Yuka Okusha, Eman Ahmed Taha, Yanyin Lu, Hotaka Kawai, Norio Sogawa, Masaharu Takigawa, Stuart K Calderwood, Kuniaki Okamoto, Ken-Ichi Kozaki

Cancers 12 ( 2 ) 1 - 22 2020年2月

Extracellular Oncosomes Rich in Moonlighting Metalloproteinase (MMP3) Are Transmissive, Pro-Tumorigenic, and Induces Cellular Communication Network Factor 2 (CCN2/CTGF): CRISPR against Cancer

Yuka Okusha, Takanori Eguchi, Manh Tien Tran, Chiharu Sogawa, Kaya Yoshida, Mami Itagaki, Eman Ahmed Taha, Kisho Ono, Eriko Aoyama, Hirohiko Okamura, Ken-ichi Kozaki, Stuart K. Calderwood, Masaharu Takigawa, Kuniaki Okamoto

Preprints doi: 10.20944/preprints202002.0281.v1 2020年2月

CDC37 and HSP90 are Essential for Stressome Release and Tumor Progression in Resistant Prostate Cancer

Takanori Eguchi, Chiharu Sogawa, Kisho Ono, Masaki Matsumoto, Manh Tien Tran, Yuka Okusha, Benjamin Lang, Kuniaki Okamoto, Stuart Calderwood

Preprints doi: 10.20944/preprints202002.0148.v1 2020年2月

Exosome Release of Drugs: Coupling with Epithelial-Mesenchymal Transition

Takanori Eguchi, Kisho Ono, Stuart Calderwood, Kuniaki Okamoto

Preprints doi: 10.20944/preprints201912.0386.v1 2019年12月

Exosome Release of Drugs: Coupling with Epithelial-Mesenchymal Transition

Takanori Eguchi, Kisho Ono, Stuart Calderwood, Kuniaki Okamoto

Preprints 2019年12月

担当区分：筆頭著者,　責任著者掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

Extracellular vesicles (EVs), such as exosomes or oncosomes are released with molecules unfavorable for survival from cells. In addition, accumulating evidence has shown that tumor cells often eject anti-cancer drugs such as chemotherapeutics and targeted drugs within EVs, a novel mechanism of drug resistance. The EV-releasing, drug resistance phenotype is often coupled with cellular dedifferentiation and transformation, cells undergoing epithelial-mesenchymal transition (EMT) and taking on a cancer stem cell phenotype. Recent studies have shown that the release of EVs is also involved in immunosuppression. The concept of the resistance-associated secretory phenotype (RASP) is reviewed herein.

DOI： 10.20944/preprints201912.0386.v1

オルガノイドを応用したドラッグリポジショニング開発

十川千春, 江口傑徳, Tran Tien Manh, 石毛真行, 河合穂高, 奥舎有加, 中野敬介, 十川紀夫, 小崎健一, 岡元邦彰

岡山歯学会雑誌 38 ( 2 ) 89 - 89 2019年12月

Regulatory Roles of HSP90-Rich Extracellular Vesicles

Takanori Eguchi, Kisho Ono, Kazumi Kawata, Kuniaki Okamoto, Stuart K. Calderwood

Heat Shock Proteins 3 - 17 2019年11月

A reporter system evaluates tumorigenesis, metastasis, β-catenin/MMP regulation, and druggability. 招待査読国際誌

Sogawa C, Eguchi T, C, i, auth, Corresponding author, Okusha Y, Ono K, Ohyama K, Iizuka M, Kawasaki R, Hamada Y, Takigawa M, Sogawa N, Okamoto K, Kozaki K

Tissue Engineering Part A 25 ( 19-20 ) 1413 - 1425 2019年10月

Tumor Angiogenic Inhibition Triggered Necrosis (TAITN) in Oral Cancer. 査読国際誌

Saori Yoshida, Hotaka Kawai, Takanori Eguchi, Shintaro Sukegawa, May Wathone Oo, Chang Anqi, Kiyofumi Takabatake, Keisuke Nakano, Kuniaki Okamoto, Hitoshi Nagatsuka

Cells 8 ( 7 ) 761 2019年7月

MZF1 and SCAND1 Reciprocally Regulate CDC37 Gene Expression in Prostate Cancer. 査読国際誌

Takanori Eguchi, Thomas L Prince, Manh Tien Tran, Chiharu Sogawa, Benjamin J Lang, Stuart K Calderwood

Cancers 11 ( 6 ) 792 2019年6月

Nicotine promotes lymph node metastasis and cetuximab resistance in head and neck squamous cell carcinoma. 査読国際誌

Rieko Shimizu, Soichiro Ibaragi, Takanori Eguchi, Daisuke Kuwajima, Shinichi Kodama, Takashi Nishioka, Tatsuo Okui, Kyoichi Obata, Kiyofumi Takabatake, Hotaka Kawai, Kisho Ono, Kuniaki Okamoto, Hitoshi Nagatsuka, Akira Sasaki

International journal of oncology 54 ( 1 ) 283 - 294 2019年1月

Carcinogenic epithelial-mesenchymal transition initiated by oral cancer exosomes is inhibited by anti-EGFR antibody cetuximab. 査読国際誌

Toshifumi Fujiwara, Takanori Eguchi, Chiharu Sogawa, Kisho Ono, Jun Murakami, Soichiro Ibaragi, Jun-Ichi Asaumi, Stuart K Calderwood, Kuniaki Okamoto, Ken-Ichi Kozaki

Oral oncology 86 251 - 257 2018年11月

Lipoprotein Cholesterols Are Stored in High-Resistant, Metastatic Cancer Cells and Released Upon Stress: Implication for a Mechanism Underlying Hypocholesterolemia in Cancer Patients

Taka Eguchi, Chiharu Sogawa, Kisho Ono, Mami Itagaki, Masaki Matsumoto

Preprints 2018年10月

担当区分：筆頭著者,　責任著者出版者・発行元：MDPI AG

Resistant cancer often shows a particular secretory trait such as heat shock proteins (HSPs) and extracellular vesicles (EVs), including exosomes and oncosomes surrounded by lipid bilayers. Lipoproteins are biochemical assemblies that transport hydrophobic lipid (a.k.a. fat) molecules in body fluid and are composed of a single-layer phospholipid and cholesterol outer shell, lipids molecules within the particles, and apolipoproteins embedded in the membrane. However, lipoprotein storage and secretion by cancer cells have not well-investigated yet. We found lipoproteins were stored and abundantly secreted by neuroendocrine, castration-resistant prostate cancer (NEPC / CRPC) cells but barely secreted by colon cancer cells and oral squamous cell carcinoma (OSCC) cells. In addition, large EVs (approx. 300 nm diameter) and potential oncosomes were released by CRPC and OSCC cells. Proteomics revealed that CRPC cells secreted EVs enriched with tetraspanins and extracellular matrices which were reduced upon heat shock stress and alternatively lipoproteins and HSPs were secreted upon stress. Heat shock stress triggered secretion of lipoprotein-EV complexes that contained apolipoprotein A, B, C and E. These data suggested that vesicular assembly composed of EVs and lipoproteins enriched with cholesterols and phospholipids may be stored in resistant cancer cells but released upon cell stress that is increased in cancer therapies.

DOI： 10.20944/preprints201810.0211.v1

侵襲性歯周炎患者の血漿エクソソーム由来microRNAの発現解析

高木美奈, 山本直史, 河村麻理, 高知信介, 山城圭介, 大森一弘, 江口傑徳, 十川千春, 高柴正悟

日本歯周病学会会誌 60 ( 秋季特別 ) 134 - 134 2018年10月

Anti-EGFR antibody cetuximab is secreted by oral squamous cell carcinoma and alters EGF-driven mesenchymal transition. 査読国際誌

Toshifumi Fujiwara, Takanori Eguchi, Chiharu Sogawa, Kisho Ono, Jun Murakami, Soichiro Ibaragi, Jun-Ichi Asaumi, Kuniaki Okamoto, Stuart K Calderwood, Ken-Ichi Kozaki

Biochemical and biophysical research communications 503 ( 3 ) 1267 - 1272 2018年9月

The intranuclear PEX domain of MMP involves proliferation, migration, and metastasis of aggressive adenocarcinoma cells. 査読国際誌

Yuka Okusha, Takanori Eguchi, Chiharu Sogawa, Tatsuo Okui, Keisuke Nakano, Kuniaki Okamoto, Ken-Ichi Kozaki

Journal of cellular biochemistry 119 ( 9 ) 7363 - 7376 2018年9月

エクソソーム研究の「がん」における新展開細胞外小胞のHSPに富む特性は、転移性口腔癌細胞の生存を担う(HSP-enriched properties of extracellular vesicles involve survival of metastatic oral cancer cells)

小野喜章, 江口傑徳, 十川千春, 佐々木朗

日本癌学会総会記事 77回 897 - 897 2018年9月

急速転移性癌細胞株由来細胞外小胞による破骨細胞分化および癌細胞浸潤の制御

板垣まみ, 十川千春, 奥舎有加, 江口傑徳, 小野喜章, 岡元邦彰

Journal of Oral Biosciences Supplement 2018 347 - 347 2018年9月

大腸癌の増殖,浸潤,転移を促進するPEXドメインの役割とその制御(The intranuclear PEX domain of MMP involves proliferation, migration, and metastasis of aggressive adenocarcinoma cells)

江口傑徳, 奥舎有加, 小野喜章, 十川千春, Calderwood Stuart K., 小崎健一

日本癌学会総会記事 77回 1332 - 1332 2018年9月

Genotoxic stress induces Sca-1-expressing metastatic mammary cancer cells. 査読国際誌

Jianlin Gong, Benjamin J Lang, Desheng Weng, Takanori Eguchi, Ayesha Murshid, Thiago J Borges, Sachin Doshi, Baizheng Song, Mary A Stevenson, Stuart K Calderwood

Molecular oncology 12 ( 8 ) 1249 - 1263 2018年8月

Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells. 査読国際誌

Yuri Namba, Chiharu Sogawa, Yuka Okusha, Hotaka Kawai, Mami Itagaki, Kisho Ono, Jun Murakami, Eriko Aoyama, Kazumi Ohyama, Jun-Ichi Asaumi, Masaharu Takigawa, Kuniaki Okamoto, Stuart K Calderwood, Ken-Ichi Kozaki, Takanori Eguchi

8 ( 376 ) 376 - 376 2018年

Monitoring of the Heat Shock Response with a Real-Time Luciferase Reporter

Toshiki Kijima, Takanori Eguchi, Len Neckers, Thomas L. Prince

Methods in Molecular Biology 35 - 45 2018年

異なる転移能を有する口腔扁平上皮癌細胞由来エクソソームに含まれるプロテオームの特性

小野喜章, 江口傑徳, 十川千春, 村上純, 藤原敏史, 笠井智成, 妹尾昌治, 佐々木朗, 小崎健一, 岡元邦彰

生命科学系学会合同年次大会 2017年度 [1LBA - 052] 2017年12月

A Tumor Suppressor Gene Product, Platelet-Derived Growth Factor Receptor-Like Protein Controls Chondrocyte Proliferation and Differentiation. 査読国際誌

Kazumi Kawata, Satoshi Kubota, Takanori Eguchi, Eriko Aoyama, Norifumi H Moritani, Morihiko Oka, Harumi Kawaki, Masaharu Takigawa

Journal of cellular biochemistry 118 ( 11 ) 4033 - 4044 2017年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY

The platelet-derived growth factor receptor-like (PDGFRL) gene is regarded as a tumor suppressor gene. However, nothing is known about the molecular function of PDGFRL. In this study, we initially clarified its function in chondrocytes. Among all cell lines examined, the PDGFRL mRNA level was the highest in chondrocytic HCS-2/8 cells. Interestingly, the proliferation of chondrocytic HCS-2/8 cells was promoted by PDGFRL overexpression, whereas that of the breast cancer-derived MDA-MB-231 cells was inhibited. Of note, in PDGFRL-overexpressing HCS-2/8 cells, the expression of chondrocyte differentiation marker genes, SOX9, ACAN, COL2A1, COL10A1, and ALP, was decreased. Moreover, we confirmed the expression of PDGFRL mRNA in normal cartilage tissue and chondrocytes. Eventually, the expression of PDGFRL mRNA in condrocytes except in the case of hypertrophic chondrocytes was demonstrated in vivo and in vitro. These findings suggest that PDGFRL plays the different roles, depending upon cell types. Particularly, in chondrocytes, PDGFRL may play a new and important role which is distinct from the function previously reported. J. Cell. Biochem. 118: 4033-4044, 2017. © 2017 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.26059

Catabolic effects of FGF-1 on chondrocytes and its possible role in osteoarthritis. 査読国際誌

Abdellatif El-Seoudi, Tarek Abd El Kader, Takashi Nishida, Takanori Eguchi, Eriko Aoyama, Masaharu Takigawa, Satoshi Kubota

Journal of cell communication and signaling 11 ( 3 ) 255 - 263 2017年9月

舌癌細胞株由来エクソソーム解析による頸部リンパ節転移マーカーの探索

小野喜章, 江口傑徳, 十川千春, 村上純, 笠井智成, 妹尾昌治, 佐々木朗, 小崎健一

日本癌学会総会記事 76回 J - 1062 2017年9月

Semaphorin 4D promotes bone invasion in head and neck squamous cell carcinoma. 査読国際誌

Hiroyuki Takada, Soichiro Ibaragi, Takanori Eguchi, Tatsuo Okui, Kyoichi Obata, Masanori Masui, Ayaka Morisawa, Kiyofumi Takabatake, Hotaka Kawai, Norie Yoshioka, Nur Mohammad, Monsur Hassan, Tsuyoshi Shimo, Guo-Fu Hu, Hitoshi Nagatsuka, Akira Sasaki

International journal of oncology 51 ( 2 ) 625 - 632 2017年8月

Promoter Analyses of CCN Genes

Takanori Eguchi, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 177 - 185 2017年

Cellular Reprogramming Using Defined Factors and MicroRNAs. 査読国際誌

Takanori Eguchi, Takuo Kuboki

Stem cells international 2016 7530942 - 7530942 2016年

担当区分：筆頭著者,　責任著者記述言語：英語出版者・発行元：Hindawi Publishing Corporation

Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES) cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS) cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1), Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs). This review reports histories, topics, and idea of cellular reprogramming.

DOI： 10.1155/2016/7530942

A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516. 査読国際誌

Kazuya Arai, Takanori Eguchi, M Mamunur Rahman, Ruriko Sakamoto, Norio Masuda, Tetsuya Nakatsura, Stuart K Calderwood, Ken-Ichi Kozaki, Manabu Itoh

PloS one 11 ( 9 ) e0162394 2016年

Targeting the hsp70 gene delays mammary tumor initiation and inhibits tumor cell metastasis 査読

J. Gong, D. Weng, T. Eguchi, A. Murshid, M. Y. Sherman, B. Song, S. K. Calderwood

ONCOGENE 34 ( 43 ) 5460 - 5471 2015年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

Elevated levels of the inducible heat-shock protein 70 (Hsp72) have been implicated in mammary tumorigenesis in histological investigations of human breast cancer. We therefore examined the role of Hsp72 in mice, using animals in which the hsp70 gene was inactivated. We used a spontaneous tumor system with mice expressing the polyomavirus middle T (PyMT) oncogene under control of the mouse mammary tumor virus (MMTV) long-terminal repeat (MMT mice). These mice developed spontaneous, metastatic mammary cancer. We then showed Hsp72 to be upregulated in a fraction of mammary cancer initiating cells (CIC) within the MMT tumor cell population. These cells were characterized by elevated surface levels of stem cell markers CD44 and Sca1 and by rapid metastasis. Inactivation of the hsp70 gene delayed the initiation of mammary tumors. This delay in tumor initiation imposed by loss of hsp70 was correlated with a decreased pool of CIC. Interestingly, hsp70 knockout significantly reduced invasion and metastasis by mammary tumor cells and implicated its product Hsp72 in cell migration and formation of secondary neoplasms. Impaired tumorigenesis and metastasis in hsp70-knockout MMT mice was associated with downregulation of the met gene and reduced activition of the oncogenic c-Met protein. These experiments therefore showed Hsp72 to be involved in the growth and progression of mammary carcinoma and highlighted this protein as a potential target for anticancer drug development.

DOI： 10.1038/onc.2015.1

Role and Regulation of Myeloid Zinc Finger Protein 1 in Cancer. 査読国際誌

Taka Eguchi, Thomas Prince, Barbara Wegiel, Stuart K Calderwood

Journal of cellular biochemistry 116 ( 10 ) 2146 - 54 2015年10月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

Myeloid zinc finger 1 (MZF1) belongs to the SCAN-Zinc Finger (SCAN-ZF) transcription factor family that has recently been implicated in a number of types of cancer. Although the initial studies concentrated on the role of MZF1 in myeloid differentiation and leukemia, the factor now appears to be involved in the etiology of major solid tumors such as lung, cervical, breast, and colorectal cancer. Here we discuss the regulation of MZF1 that mediated its recruitment and activation in cancer, concentrating on posttranslational modification by phosphorylation, and sumoylation, formation of promyelocytic leukemia nuclear bodies and its association with co-activators and co-repressors.

DOI： 10.1002/jcb.25203

HSF1 regulation of β-catenin in mammary cancer cells through control of HuR/elavL1 expression. 査読国際誌

Chou SD, Murshid A, Eguchi T, Gong J, Calderwood SK

Oncogene 34 ( 17 ) 2178 - 2188 2015年4月

OstemiR: A Novel Panel of MicroRNA Biomarkers in Osteoblastic and Osteocytic Differentiation from Mesencymal Stem Cells

Eguchi, Takanori, Hara, Emilio Satoshi, Ono, Mitsuaki, Watanabe, Ken, Kuboki, Takuo, Calderwood, Stuart

Faseb Journal 29 ( 3 ) e58796 2015年

miRNA-720 regulates the stem cell phenotype and differentiation of human dental pulp-derived mesenchymal stromal cells

Hara Emilio Satoshi, Ono Mitsuaki, Eguchi Takanori, Hai Thanh Pham, Tajima Shoji, Calderwood Stuart K, Matsumoto Takuya, Kuboki Takuo, IEEE

2014 International Symposium on Micro-Nanomechatronics and Human Science (Mhs) 1 - 2 2014年

Stress proteins in aging and life span. 査読国際誌

Ayesha Murshid, Takanori Eguchi, Stuart K Calderwood

International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group 29 ( 5 ) 442 - 7 2013年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：INFORMA HEALTHCARE

Heat shock proteins (HSP) are molecular chaperones and have been implicated in longevity and aging in many species. Their major functions include chaperoning misfolded or newly synthesised polypeptides, protecting cells from proteotoxic stress, and processing of immunogenic agents. These proteins are expressed constitutively and can be induced by stresses such as heat, oxidative stress and many more. The induction of HSP in aging could potentially maintain protein homeostasis and longevity by refolding the damaged proteins which accumulate during aging and are toxic to cells. HSP are shown to increase life span in model organisms such as Caenorhabditis elegans and decrease aging-related proteotoxicity. Thus, decrease in HSP in aging is associated with disruption of cellular homeostasis which causes diseases such as cancer, cell senescence and neurodegeneration. HSP levels are decreased with aging in most organs including neurons. Aging also causes attenuation or alteration of many signalling pathways as well as the expression of transcription factors such as heat shock factor (HSF). The alteration in regulation and synthesis of Forkhead box O3a (FoxO3a) family of transcription factors as well as major antioxidant enzymes (manganese superoxide dismutase, catalase) are also seen in aging. Among many signalling mechanisms involved in altering longevity and aging, the insulin/IGF-1 pathway and the Sir2 deacetylase are highly significant. This review enquires into the role of some of these pathways in longevity/aging along with HSP.

DOI： 10.3109/02656736.2013.798873

miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells. 査読国際誌

Emilio Satoshi Hara, Mitsuaki Ono, Takanori Eguchi, Satoshi Kubota, Hai Thanh Pham, Wataru Sonoyama, Shoji Tajima, Masaharu Takigawa, Stuart K Calderwood, Takuo Kuboki

PloS one 8 ( 12 ) e83545 2013年

記述言語：英語掲載種別：研究論文（学術雑誌）

Dental pulp cells (DPCs) are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs) have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP) cells from human DPCs and periodontal ligament cells (PDLCs), and performed a locked nucleic acid (LNA)-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP) cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs), which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.

DOI： 10.1371/journal.pone.0083545

Role of LRP1 in transport of CCN2 protein in chondrocytes. 査読国際誌

Kazumi Kawata, Satoshi Kubota, Takanori Eguchi, Eriko Aoyama, Norifumi H Moritani, Seiji Kondo, Takashi Nishida, Masaharu Takigawa

Journal of cell science 125 ( Pt 12 ) 2965 - 72 2012年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：COMPANY OF BIOLOGISTS LTD

Low-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-β-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.

DOI： 10.1242/jcs.101956

Role of low-density lipoprotein receptor related protein 1 (LRP1) in CCN2/connective tissue growth factor (CTGF) protein transport in chondrocytes 査読

Kawata., K, Kubota, S, Eguchi, T, Aoyama, E, Moritani, N, Kondo, S, Nishida, T, Takigawa, M

J Cell Sci 15 2965 - 2972 2012年

低密度リポタンパク受容体関連タンパク1(LRP1)による軟骨細胞でのCCNファミリー2/結合組織成長因子(CCN2/CTGF)タンパク質輸送

河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 近藤誠二, 滝川正春

日本結合組織学会学術大会・マトリックス研究会大会合同学術集会プログラム・抄録集 43回・58回 76 - 76 2011年5月

A coding RNA segment that enhances the ribosomal recruitment of chicken ccn1 mRNA. 査読国際誌

Yoshiki Mukudai, Satoshi Kubota, Takanori Eguchi, Kumi Sumiyoshi, Danilo Janune, Seiji Kondo, Satoru Shintani, Masaharu Takigawa

Journal of cellular biochemistry 111 ( 6 ) 1607 - 18 2010年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

CCN1, a member of the CCN family of proteins, plays important physiological or pathological roles in a variety of tissues. In the present study, we initially found a highly guanine-cytosine (GC)-rich region of approximately 200 bp near the 5'-end of the open reading frame, which was always truncated by amplification of the corresponding cDNA region through the conventional polymerase chain reaction. An RNA in vitro folding assay and selective ribonuclease digestion of the corresponding segment of the ccn1 mRNA confirmed the involvement of a stable secondary structure. Subsequent RNA electromobility-shift assays demonstrated the specific binding of some cytoplasmic factor(s) in chicken embryo fibroblasts to the RNA segment. Moreover, the corresponding cDNA fragment strongly enhanced the expression of the reporter gene in cis at the 5'-end, but did not do so at the 3'-end. According to the results of a ribosomal assembly test, the effect of the mRNA segment can predominantly be ascribed to the enhancement of transport and/or entry of the mRNA into the ribosome. Finally, the minimal GC-rich mRNA segment that was predicted and demonstrated to form a secondary structure was confirmed to be a functional regulatory element. Thus, we here uncover a novel dual-functionality of the mRNA segment in the ccn1 open reading frame, which segment acts as a cis-element that mediates posttranscriptional gene regulation, while retaining the information for the amino acid sequence of the resultant protein.

DOI： 10.1002/jcb.22894

軟骨細胞のCCN2蛋白質輸送における低比重リポ蛋白受容体関連蛋白質1(LRP1)の役割(Role of the low-density lipoprotein receptor related protein 1 (LRP1) in CCN2 protein transportation in chondrocytes)

河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 近藤誠二, 滝川正春

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 2T10 - 12 2010年12月

低密度リポタンパク受容体関連タンパク1(LRP1)による軟骨細胞でのタンパク質輸送

河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 近藤誠二, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 28回 188 - 188 2010年7月

Role of the low-density lipoprotein receptor-related protein-1 in regulation of chondrocyte differentiation. 査読国際誌

Kazumi Kawata, Satoshi Kubota, Takanori Eguchi, Norifumi H Moritani, Tsuyoshi Shimo, Seiji Kondo, Takashi Nishida, Shogo Minagi, Masaharu Takigawa

Journal of cellular physiology 222 ( 1 ) 138 - 48 2010年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

The low-density lipoprotein receptor-related protein 1 (LRP1) is known as an endocytic and signal transmission receptor. We formerly reported the gene expression and the localization of LRP1 in cartilage tissue and chondrocytes, but its roles in the differentiation of chondrocytes remained to be investigated. Here, in order to address this issue, we employed RNAi strategy to knockdown lrp1 in chondrocytic cells and obtained findings indicating a critical role therein. As a result of lrp1 knockdown, aggrecan and col2a1 mRNA levels were decreased. However, that of col10a1 or mmp13 mRNA was rather increased. Under this condition, we performed a promoter assay for Axin2, which is known to be induced by activation of the WNT/beta-catenin (betacat) signaling pathway. Thereby, we found that Axin2 promoter activity was enhanced in the lrp1 knockdown cells. Furthermore, when the WNT/beta-catenin pathway was activated in chondrocytic cells by WNT3a or SB216763, which inhibits the phosphorylation of GSK3beta, the mRNA levels of aggrecan and col2a1 were decreased, whereas that of mmp13 was increased. Additionally, the level of phosphorylated protein kinase C (PKC) zeta was also decreased in the lrp1 knockdown cells. When the phosphorylation of PKCzeta was selectively inhibited, aggrecan and col2a1 mRNA levels decreased, whereas the mmp13 mRNA level increased. These data demonstrate that LRP1 exerts remarkable effects to retain the mature phenotype of chondrocytes as a critical mediator of cell signaling. Our findings also indicate that the onset of hypertrophy during endochondral ossification appears to be particularly dependent on the WNT and PKC signaling initiated by LRP1.

DOI： 10.1002/jcp.21930

Novel Transcriptional Regulation of CCN2/CTGF by Nuclear Translocation of MMP3

Takanori Eguchi, Satoshi Kubota, Kazumi Kawata, Yoshiki Mukudai, Junji Uehara, Toshihiro Ohgawara, Soichiro Ibaragi, Akira Sasaki, Takuo Kuboki, Masaharu Takigawa

CCN PROTEINS IN HEALTH AND DISEASE: AN OVERVIEW OF THE FIFTH INTERNATIONAL WORKSHOP ON THE CCN FAMILY OF GENES 255 - + 2010年

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（国際会議プロシーディングス）出版者・発行元：SPRINGER-VERLAG BERLIN

CCN2/CTGF, previously known as Connective Tissue Growth Factor, is a crucial regulator of extra-cellular matrix (ECM), which promotes ECM synthesis and stabilization. As their family name clearly implies, matrix metalloproteases (MMPs) are also localized in the ECM, where they function as proteases, modulating cell signaling by cleaving proteins such as matrix proteins, growth factors and growth factor receptors. Strong expression of CCN2/CTGF in chondrocytic cells occurs through transcription enhancer dominant in chondrocytes (TRENDIC). Matrix metalloprotease-3 (MMP3) is a novel TRENDIC-binding transcription factor for CCN2/CTGF expression. First, MMP3 cDNA was cloned as a TRENDIC-binding factor by Southwestern screening. The interaction between MMP3 and TRENDIC was confirmed by a gel shift assay and chromatin immunoprecipitation. The CCN2/CTGF promoter was activated by transfected MMP3, whereas a TRENDIC mutant for the promoter lost the response. In addition, the knockdown of MMP3 suppressed CCN2/CTGF expression. Cytochemical and histochemical analyses demonstrated that MMP3 was detected in the nuclei of chondrocytic cells in culture and also in the nuclei of normal and osteoarthritic chondrocytes in vivo. The nuclear translocation of externally added recombinant MMP3 was observed in 30 min after the addition, and six putative nuclear localization signals were found in MMP3. These results indicated a novel trans-activation mechanism of CCN2/CTGF by the nuclear translocation of MMP3 through binding with TRENDIC in chondrocytes. Although MMPs historically had been recognized as a protease for extra-cellular proteins, this study indicated that it also stimulates ECM synthesis through CCN2/CTGF trans-activation. This novel regulatory role of the ECM may contribute to understanding the mechanism of not only the development, but also the pathogenesis of arthritis fibrosis and periodontitis.

DOI： 10.1007/978-90-481-3779-4_19

その他リンク： http://orcid.org/0000-0002-9600-4430

Nucleophosmin/B23: A Multifunctional Regulator that Determines the Fate of CCN2 mRNA

Satoshi Kubota, Yoshiki Mukudai, Harumi Kawaki, Seiji Kondo, Takanori Eguchi, Kumi Sumiyoshi, Toshihiro Ohgawara, Tsuyoshi Shimo, Masaharu Takigawa

CCN PROTEINS IN HEALTH AND DISEASE: AN OVERVIEW OF THE FIFTH INTERNATIONAL WORKSHOP ON THE CCN FAMILY OF GENES 41 - + 2010年

記述言語：英語掲載種別：研究論文（国際会議プロシーディングス）出版者・発行元：SPRINGER-VERLAG BERLIN

CCN2/CTGF is a multifunctional molecule that has been shown to play a central role in chondrocyte differentiation. During this process, the expression of ccn2 is tightly regulated to confer a maximal level at prehypertrophic - hypertrophic stages, in which the 3'-untranslated region (UTR) of the mRNA is critically involved in mediating its post-transcriptional regulation. In our previous studies, we found that a 40-kDa protein binding specifically to an RNA cis-element, 3'-100/50, in the 3'-UTR of the chicken ccn2 mRNA regulated the intracellular stability of the mRNA. The interaction of this 40-kDa protein with 3'-100/50 was enhanced in proliferating chondrocytes, in which ccn2 mRNA is rapidly degraded; whereas a prolonged half life of ccn2 mRNA is observed in hypertrophic chondrocytes, where the interaction of the 40 kDa-protein and 3'-100/50 is diminished. Collectively, the data suggested that this 40-kDa protein acts as a ccn2-specific mRNA destabilizer during chondrocyte differentiation.
In this present study we finally identified this 40-kDa protein as nucleophosmin (NPM)/B23. NPM is a nuclear-cytoplasmic shuttling protein that is characterized by its multiple functionality. This protein is known to be a histone chaperone, a regulator of ribosomal RNA transcription, as well as an RNA-binding post-transcriptional regulator of gene expression. In our hands, direct binding of NPM to 3'-100/50 was confirmed not only by RNA EMSA and UV crosslinking assays, but also by RNA immunoprecipitation analysis. By using recombinant chicken NPM, we could successfully reconstitute the post-transcriptional regulation of ccn2 by NPM in vitro and found that this regulation was more robust in chondrocytes than in fibroblasts. Furthermore, siRNA-mediated gene silencing of NPM in vivo clearly showed enhanced ccn2 gene expression and a prolonged half life of the ccn2 mRNA, confirming the functional property of NPM as a specific destabilizer of the ccn2 mRNA in living cells.
The 5'-100/50 element, a target of NPM, is evolutionally conserved among vertebrate species. Therefore, we consider NPM to be a critical post-transcriptional regulator of ccn2 acting via 3'-UTR during endochondral ossification and possibly, in other physiological and pathological states as well.

DOI： 10.1007/978-90-481-3779-4_4

その他リンク： http://orcid.org/0000-0002-9600-4430

低密度リポタンパク受容体関連タンパク-1(LRP1)の軟骨細胞分化における多面的作用機構

河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 森谷徳文, 近藤誠二, 西田崇, 皆木省吾, 滝川正春

日本生化学会大会プログラム・講演要旨集 82回 ( Vol.1 ) 4T4p - 9 2009年9月

軟骨細胞におけるCCN2遺伝子の転写後調節機構におけるNucleophosmin/B23の機能的意義

住吉久美, 久保田聡, 椋代義樹, 近藤誠二, 川木晴美, 江口傑徳, 大河原敏博, 山城隆, 滝川正春

日本生化学会大会プログラム・講演要旨集 82回 3T18a - 8 2009年9月

Nucleophosmin/B23によるChicken CCN2遺伝子の軟骨細胞特異的転写後調節

住吉久美, 久保田聡, 椋代義樹, 近藤誠二, 川木晴美, 江口傑徳, 山城隆, 滝川正春

Journal of Oral Biosciences 51 ( Suppl. ) 105 - 105 2009年8月

低密度リポタンパク受容体関連タンパク-1(LRP1)の軟骨細胞分化における機能とその作用機構

河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 森谷徳文, 近藤誠二, 西田崇, 皆木省吾, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 27回 179 - 179 2009年7月

Regulation of chondrocytic phenotype by micro RNA 18a: involvement of Ccn2/Ctgf as a major target gene. 査読国際誌

Toshihiro Ohgawara, Satoshi Kubota, Harumi Kawaki, Seiji Kondo, Takanori Eguchi, Naito Kurio, Eriko Aoyama, Akira Sasaki, Masaharu Takigawa

FEBS letters 583 ( 6 ) 1006 - 10 2009年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

We searched for miRNAs that were down-regulated in chondrocytic cells and predicted to target CCN2/connective tissue growth factor (CCN2/CTGF) that promotes endochondral ossification. Among them, expression of miR-18a was most strongly repressed in chondrocytic cells. Reporter gene analysis confirmed the functionality of an miR-18a target in the 3'-untranslated region of Ccn2 mRNA, which was predicted in silico. Indeed, introduction of miR-18a efficiently repressed the CCN2 production from chondrocytic cells. Finally, transfected miR-18a significantly repressed the mature chondrocytic phenotype. Our present study revealed a regulatory role for miR-18a in chondrocytic differentiation through CCN2.

DOI： 10.1016/j.febslet.2009.02.025

低密度リポタンパク受容体関連タンパク-1(LRP1)の軟骨細胞分化における多面的作用機構

河田かずみ, 河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 森谷徳文, 近藤誠二, 西田崇, 皆木省吾, 滝川正春

生化学 2009年

Posttranscriptional regulation of chicken ccn2 gene expression by nucleophosmin/B23 during chondrocyte differentiation. 査読国際誌

Yoshiki Mukudai, Satoshi Kubota, Harumi Kawaki, Seiji Kondo, Takanori Eguchi, Kumi Sumiyoshi, Toshihiro Ohgawara, Tsuyoshi Shimo, Masaharu Takigawa

Molecular and cellular biology 28 ( 19 ) 6134 - 47 2008年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC MICROBIOLOGY

CCN2/CTGF is a multifunctional factor that plays a crucial role in the growth and differentiation of chondrocytes. The chicken ccn2 gene is regulated not only at the transcriptional level but also by the interaction between a posttranscriptional element in the 3' untranslated region (3'-UTR) and a cofactor. In the present study, we identified a nucleophosmin (NPM) (also called B23) as this cofactor. Binding of NPM to the element was confirmed, and subsequent analysis revealed a significant correlation between the decrease in cytosolic NPM and the increased stability of the ccn2 mRNA during chondrocyte differentiation in vivo. Furthermore, recombinant chicken NPM enhanced the degradation of chimeric RNAs containing the posttranscriptional cis elements in a chicken embryonic fibroblast extract in vitro. It is noteworthy that the RNA destabilization effect by NPM was far more prominent in the cytosolic extract of chondrocytes than in that of fibroblasts, representing a chondrocyte-specific action of NPM. Stimulation by growth factors to promote differentiation changed the subcellular distribution of NPM in chondrocytes, which followed the expected patterns from the resultant change in the ccn2 mRNA stability. Therefore, the present study reveals a novel aspect of NPM as a key player in the posttranscriptional regulation of ccn2 mRNA during the differentiation of chondrocytes.

DOI： 10.1128/MCB.00495-08

マイクロRNA 18aによるCcn2/Ctgf遺伝子を介した軟骨細胞分化の制御機構の解明

大河原敏博, 久保田聡, 川木晴美, 近藤誠二, 江口傑徳, 佐々木朗, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 26回 168 - 168 2008年10月

軟骨細胞成熟における低密度リポ蛋白受容体関連タンパク-1(LRP1)の関与

河田かずみ, 河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 皆木省吾, 滝川正春

日本軟骨代謝学会プログラム・抄録集 21st 2008年

Gene expression and distribution of connective tissue growth factor (CCN2/CTGF) during secondary ossification center formation. 査読国際誌

Morihiko Oka, Satoshi Kubota, Seiji Kondo, Takanori Eguchi, Chisa Kuroda, Kazumi Kawata, Shogo Minagi, Masaharu Takigawa

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 55 ( 12 ) 1245 - 55 2007年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：HISTOCHEMICAL SOC INC

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metalloproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.

DOI： 10.1369/jhc.7A7263.2007

Different transcriptional strategies for ccn2/ctgf gene induction between human chondrocytic and breast cancer cell lines. 査読国際誌

Takanori Eguchi, Satoshi Kubota, Kazumi Kawata, Yoshiki Mukudai, Toshihiro Ohgawara, Kohei Miyazono, Kyouji Nakao, Seiji Kondo, Masaharu Takigawa

Biochimie 89 ( 3 ) 278 - 88 2007年3月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

Connective tissue growth factor (CTGF/CCN2) plays a critical role in endochondral bone formation; however, CCN2 also promotes angiogenesis and bone metastasis in breast cancer. Chondrocytic HCS-2/8 cells and breast cancer MDA231 cells produce over 6 times more CCN2 than any other cell type. In this study, we demonstrate that these cell lines employ different transcriptional strategies for ccn2 gene induction. Four tandem copies of the dominant transcriptional enhancer in chondrocytes (4 x TRENDIC) were chimerically connected to an SV40 promoter-luciferase construct and subsequently analyzed. The enhancement of the promoter activity by 4 x TRENDIC was greater in the HCS-2/8 cells (7-fold) than in the other 4 cell lines (3-4 fold). The TRENDIC-binding protein complex was detected at a higher signal in the HCS-2/8 cells than in the other cell lines. In addition, the HCS-2/8 nuclear factors strongly targeted not only TRENDIC, but also the previously reported basal control element and a novel enhancer element in the ccn2 promoter. In contrast, high-level ccn2 gene induction in MDA231 cells was largely dependent on Smad signaling through the Smad-binding element in the ccn2 promoter. Based on these results, we propose a model of differential transcription of the ccn2 gene between the chondrocytic cell line and the breast cancer cell line, and therefore imply that these cells utilize distinct transcriptional strategies to obtain the enhanced CCN2 production that is not observed in other types of cells.

DOI： 10.1016/j.biochi.2006.12.006

Pathogenic role of connective tissue growth factor (CTGF/CCN2) in osteolytic metastasis of breast cancer. 査読国際誌

Tsuyoshi Shimo, Satoshi Kubota, Norie Yoshioka, Soichiro Ibaragi, Sachiko Isowa, Takanori Eguchi, Akira Sasaki, Masaharu Takigawa

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 21 ( 7 ) 1045 - 59 2006年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BONE & MINERAL RES

UNLABELLED: The role of CTGF/CCN2 in osteolytic metastasis by breast cancer cells and its mechanism of action were studied. Osteolytic metastasis accompanied by CCN2 and PTHrP overproduction was efficiently inhibited by an anti-CCN2 antibody. Furthermore, we found that CCN2 was induced by PTHrP through PKA-, PKC-, and ERK-mediated pathways therein. INTRODUCTION: Connective tissue growth factor (CTGF/CCN2) is a mediator of local angiogenesis induced by breast cancer, but its role in osteolytic metastasis has not been evaluated. PTH-related peptide (PTHrP) is another critical factor in the development of the osteolytic metastasis. Using both in vivo and in vitro approaches, we studied whether/how neutralization of CCN2 prevented bone metastasis and how PTHrP signaling is related. MATERIALS AND METHODS: A mouse model of bone metastasis by human breast cancer cell line MDA231 was treated with a CCN2-neutralizing antibody, and osteolytic bone metastases were assessed on radiographs and immunohistochemistry. Ccn2 gene expression and transcription were examined by Northern blot and luciferase analysis. Immunoblot analysis and kinase inhibitors were used to identify the signaling pathways implicated. Anti-angiogenic/osteoclastogenic effects of ccn2 downregulation were also evaluated. RESULTS: Treatment of mice with a CCN2-neutralizing antibody greatly decreased osteolytic bone metastasis, microvasculature, and osteoclasts involved. The antibody also suppressed the growth of subcutaneous tumor in vivo and proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro. Downregulation of ccn2 also repressed osteoclastogenesis. CCN2 expression was specifically observed in cancer cells producing PTHrP and type I PTH/PTHrP receptor (PTH1R) invaded the bone marrow, and PTHrP strongly upregulated ccn2 in MDA231 cells in vitro. Activation of protein kinase C (PKC) and protein kinase A (PKA) was necessary and sufficient for the stimulation of ccn2 by PTHrP. Indeed, inhibition of the extracellular signal-regulated kinase (ERK1/2), PKC, or PKA by specific inhibitors counteracted the stimulation of ccn2 expression. Incubation of MDA231 cells with PTHrP induced the activation of ERK1/2. Consistent with these findings, inhibition of PKC prevented PTHrP-induced ERK1/2 activation, whereas 12-O-tetradecanoylphorbol13-acetate (TPA), a stimulator of PKC, upregulated it. CONCLUSIONS: CCN2 was critically involved in osteolytic metastasis and was induced by PKA- and PKC-dependent activation of ERK1/2 signaling by PTHrP. Thus, CCN2 may be a new molecular target for anti-osteolytic therapy to shut off the PTHrP-CCN2 signaling pathway.

DOI： 10.1359/JBMR.060416

Possible role of LRP1, a CCN2 receptor, in chondrocytes. 査読国際誌

Kazumi Kawata, Takanori Eguchi, Satoshi Kubota, Harumi Kawaki, Morihiko Oka, Shogo Minagi, Masaharu Takigawa

Biochemical and biophysical research communications 345 ( 2 ) 552 - 9 2006年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Low density lipoprotein receptor (LDLR)-related protein 1 (LRP1/CD91) is one of the receptors of CCN2 that conducts endochondral ossification and cartilage repair. LRP1 is a well-known endocytic receptor, but its distribution among chondrocytes remains to be elucidated. We herein demonstrate for the first time that the distribution of LRP1 in chondrocytes except for hypertrophic chondrocytes in vivo and in vitro. Interestingly, the LRP1 levels were higher in mature chondrocytic HCS-2/8 and osteoblastic SaOS-2 than in other cells, whereas the other LDLR family members involved in ossification were detected at lower levels in HCS-2/8. It was interesting to note that in HCS-2/8, LRP1 was observed not only on the cell surface and in the cytoplasm, but also in the nucleus. Exogenously added CCN2 was incorporated into HCS-2/8, which was partially co-localized with LRP1, and targeted to the recycling endosomes and nucleus as well as the lysosomes. These findings suggest specific roles of LRP1 in cartilage biology.

DOI： 10.1016/j.bbrc.2006.04.109

二次骨化中心形成過程に発現する結合組織成長因子CTGF/CCN2のパールカン陽性軟骨細胞への特異的集積

岡森彦, 久保田聡, 近藤誠二, 江口傑徳, 河田かずみ, 黒田知沙, 皆木省吾, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 23回 229 - 229 2005年6月

Collaborative action of M-CSF and CTGF/CCN2 in articular chondrocytes: possible regenerative roles in articular cartilage metabolism. 査読国際誌

Kyouji Nakao, Satoshi Kubota, Hideyuki Doi, Takanori Eguchi, Morihiko Oka, Takuo Fujisawa, Takashi Nishida, Masaharu Takigawa

Bone 36 ( 5 ) 884 - 92 2005年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE INC

It is known that expression of the macrophage colony-stimulating factor (M-CSF) gene is induced in articular chondrocytes upon inflammation. However, the functional role of M-CSF in cartilage has been unclear. In this study, we describe possible roles of M-CSF in the protection and maintenance of the articular cartilage based on the results of experiments using human chondrocytic cells and rat primary chondrocytes. Connective tissue growth factor (CTGF/CCN2) is known to be a potent molecule to regenerate damaged cartilage by promoting the growth and differentiation of articular chondrocytes. Here, we uncovered the fact that M-CSF induced the mRNA expression of the ctgf/ccn2 gene in those cells. Enhanced production of CTGF/CCN2 protein by M-CSF was also confirmed. Furthermore, M-CSF could autoactivate the m-csf gene, forming a positive feed-back network to amplify and prolong the observed effects. Finally, promotion of proteoglycan synthesis was observed by the addition of M-CSF. These findings taken together indicate novel roles of M-CSF in articular cartilage metabolism in collaboration with CTGF/CCN2, particularly during an inflammatory response. Such roles of M-CSF were further supported by the distribution of M-CSF producing chondrocytes in experimentally induced rat osteoarthritis cartilage in vivo.

DOI： 10.1016/j.bone.2004.10.015

Regulation of chicken ccn2 gene by interaction between RNA cis-element and putative trans-factor during differentiation of chondrocytes. 査読国際誌

Yoshiki Mukudai, Satoshi Kubota, Takanori Eguchi, Seiji Kondo, Kyouji Nakao, Masaharu Takigawa

The Journal of biological chemistry 280 ( 5 ) 3166 - 77 2005年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

CCN2/CTGF is a multifunctional growth factor. Our previous studies have revealed that CCN2 plays important roles in both growth and differentiation of chondrocytes and that the 3'-untranslated region (3'-UTR) of ccn2 mRNA contains a cis-repressive element of gene expression. In the present study, we found that the stability of chicken ccn2 mRNA is regulated in a differentiation stage-dependent manner in chondrocytes. We also found that stimulation by bone morphogenetic protein 2, platelet-derived growth factor, and CCN2 stabilized ccn2 mRNA in proliferating chondrocytes but that it destabilized the mRNA in prehypertrophic-hypertrophic chondrocytes. The results of a reporter gene assay revealed that the minimal repressive cis-element of the 3'-UTR of chicken ccn2 mRNA was located within the area between 100 and 150 bases from the polyadenylation tail. Moreover, the stability of ccn2 mRNA was correlated with the interaction between this cis-element and a putative 40-kDa trans-factor in nuclei and cytoplasm. In fact, the binding between them was prominent in proliferating chondrocytes and attenuated in (pre)hypertrophic chondrocytes. Stimulation by the growth factors repressed the binding in proliferating chondrocytes; however, it enhanced it in (pre)hypertrophic chondrocytes. Therefore, gene expression of ccn2 mRNA during endochondral ossification is properly regulated, at least in part, by changing the stability of the mRNA, which arises from the interaction between the RNA cis-element and putative trans-factor.

DOI： 10.1074/jbc.M411632200

Interaction of AP-1 and the ctgf gene: a possible driver of chondrocyte hypertrophy in growth cartilage. 査読

Norifumi H Moritani, Satoshi Kubota, Takanori Eguchi, Tomohiro Fukunaga, Takashi Yamashiro, Teruko Takano-Yamamoto, Hideki Tahara, Kazumi Ohyama, Toshio Sugahara, Masaharu Takigawa

Journal of bone and mineral metabolism 21 ( 4 ) 205 - 10 2003年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER-VERLAG TOKYO

The expression of the connective tissue growth factor ( ctgf) gene increases along with the differentiation of growth cartilage cells, and the highest expression is observed in the hypertrophic stage. Similarly, recent reports demonstrated c- fos expression in chondrocytes in the early hypertrophic zone of growth cartilage, and suggested that the c- fos gene may play a crucial role in the regulation of hypertrophic differentiation. A chondrocytic human cell line, HCS-2/8, is known to retain a variety of chondrocytic phenotypes. When such cells were kept overconfluent, they expressed increasing levels of c- fos transcripts along a time course phenotypically similar to that of hypertrophic differentiation. Moreover, by using a competitive electromobility-shift assay, we found that AP-1, a Fos/Jun heterodimer, in HCS-2/8 was capable of binding not only to a typical AP-1-binding DNA fragment but also to the enhancer fragment of the ctgf gene. Based on the findings above, we hypothesize that, prior to hypertrophic differentiation, AP-1-related oncogenes are activated and that their gene products subsequently activate ctgf gene expression, which might eventually induce hypertrophy.

DOI： 10.1007/s00774-003-0410-1

軟骨由来成長因子CTGF/Hcs24の細胞種特異的遺伝子発現抑制機構の解析

森谷徳文, 久保田聡, 江口傑徳, 近藤誠二, 菅原利夫, 滝川正春

生化学 74 ( 8 ) 913 - 913 2002年8月

A novel cis-element that enhances connective tissue growth factor gene expression in chondrocytic cells. 査読国際誌

Takanori Eguchi, Satoshi Kubota, Seiji Kondo, Takuo Kuboki, Hirofumi Yatani, Masaharu Takigawa

Biochemical and biophysical research communications 295 ( 2 ) 445 - 51 2002年7月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

To clarify the chondrocyte-specific regulatory mechanism of connective tissue growth factor (ctgf) gene expression, we analyzed the functionality and DNA-protein interaction of the CTGF promoter. Comparative luciferase assay of the CTGF promoter deletion mutants among HCS-2/8 chondrocytic cells and fibroblastic cells revealed that a 110-bp region in the promoter was crucial for the HCS-2/8-specific transcriptional enhancement. Subsequent competitive gel shift assay revealed that transcription factors in HCS-2/8 nuclei bound to a 60-bp portion in the corresponding region. Relative luciferase activity from a CTGF promoter with mutant TGF-beta response element (TbRE) was 16.9% lower than that from an intact promoter. On the other hand, relative luciferase activity from a CTGF promoter with 4bp point mutations at 30bp upstream of the TbRE was 47.7% lower than that from the intact one. The binding activity of HCS-2/8 nuclear factor(s) to the sequence over the 4-bp was remarkably higher than that of any nuclear extract from other types of cells. Therefore, we entitled the sequence 'TRENDIC', a transcription enhancer dominant in chondrocytes, which stands for a novel enhancer for chondrocyte-specific CTGF gene expression.

DOI： 10.1016/S0006-291X(02)00700-3

Connective tissue growth factor increased by hypoxia may initiate angiogenesis in collaboration with matrix metalloproteinases. 査読国際誌

Seiji Kondo, Satoshi Kubota, Tsuyoshi Shimo, Takashi Nishida, Gen Yosimichi, Takanori Eguchi, Toshio Sugahara, Masaharu Takigawa

Carcinogenesis 23 ( 5 ) 769 - 76 2002年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OXFORD UNIV PRESS

Connective tissue growth factor (CTGF) is known to be a potent angiogenic factor. Here we investigated how CTGF and matrix metalloproteinases (MMPs) are involved in the early stage of hypoxia-induced angiogenesis using human breast cancer cell line, MDA231, and vascular endothelial cells. Hypoxic stimulation (5% O(2)) of MDA231 cells increased their steady-state level of ctgf mRNA by approximately 2-fold within 1.5 h, and the levels remained at a plateau up to 6 h, and then decreased by 12 h as compared with the cells cultured under the normoxic condition. Membrane-type 1 MMP (MT1-MMP) mRNA levels was also increased within a few hours of the exposure to hypoxia. Indeed, ELISA revealed that the CTGF protein/cell in medium conditioned by MDA231 cells exposed to hypoxia was maximally greater at 24 h than in the medium from normoxic cultures and that the secretion rate (supernatant CTGF/cell layer CTGF) increased in a time-dependent manner from 24 to 72 h of hypoxic exposure. Hypoxic induction of CTGF was also confirmed by immunohistochemical analyses. Furthermore, zymogram analysis revealed that the production of active MMP-9 was also induced in MDA231 cells incubated under hypoxic conditions. Finally, we found that recombinant CTGF also increased the expression of a number of metalloproteinases that play a role in the vascular invasive processes and decreased the expression of tissue inhibitors of metalloproteinases by vascular endothelial cells. These findings suggest that hypoxia stimulates MDA231 cells to release CTGF as an angiogenic modulator, which initiates the invasive angiogenesis cascade by modulating the balance of extracellular matrix synthesis and degradation via MMPs secreted by endothelial cells in response to CTGF. This cascade may play critical roles in the hypoxia-induced neovascularization that accompanies tumor invasion in vivo.

遺伝子発現の抑圧的調節を仲介するマウスctgf3'-UTR segmentの性質

近藤誠二, 久保田聡, 江口傑徳, 服部高子, 中西徹, 菅原利夫, 滝川正春

日本口腔科学会雑誌 50 ( 6 ) 568 - 568 2001年11月

Cell-type-specific trans-activation of herpes simplex virus thymidine kinase promoter by the human T-cell leukemia virus type I Tax protein. 査読

Kubota S, Mukudai Y, Hattori T, Eguchi T, Kondo S, Takigawa M

DNA and cell biology 20 ( 9 ) 563 - 568 2001年9月

多機能成長因子CTGF/Hcs24遺伝子の転写後制御エレメント,CAESARの作用機序

久保田聡, 近藤誠二, 椋代義樹, 江口傑徳, 服部高子, 中西徹, 滝川正春

生化学 73 ( 8 ) 710 - 710 2001年8月

Regulatory Mechanism of Human Connective Tissue Growth Factor (CTGF/Hcs24) Gene Expression in a Human Chondrocytic Cell Line, HCS-2/8 査読

T. Eguchi, S. Kubota, S. Kondo, T. Shimo, T. Hattori, T. Nakanishi, T. Kuboki, H. Yatani, M. Takigawa

Journal of Biochemistry 130 ( 1 ) 79 - 87 2001年7月

Novel mode of processing and secretion of connective tissue growth factor/ecogenin（CTGF/Hcs24） in chondrocytic HCS-2/8 cells 査読

Kubota S, Eguchi T, Shimo T, Nishida T, Hattori T, Kondo S, Nakanishi T, Takigawa M

Bone 29 ( 2 ) 155 - 161 2001年

遺伝子発現の抑制的な調節をもたらすマウスのctgf3′-UTRセグメントの特性

近藤誠二, 久保田聡, 江口傑徳, 服部高子, 中西徹, 菅原利夫, 滝川正春

日本口腔科学会雑誌 278 ( 1 ) 119 - 124 2000年11月

Characterization of a Mouse ctgf 3′-UTR Segment That Mediates Repressive Regulation of Gene Expression 査読

Seiji Kondo, Satoshi Kubota, Takanori Eguchi, Takako Hattori, Tohru Nakanishi, Toshio Sugahara, Masaharu Takigawa

Biochemical and Biophysical Research Communications 278 ( 1 ) 119 - 124 2000年11月

Identification of an RNA element that confers post-transcriptional repression of connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/hcs24) gene: Similarities to retroviral RNA-protein interactions 査読

S Kubota, S Kondo, T Eguchi, T Hattori, T Nakanishi, RJ Pomerantz, M Takigawa

ONCOGENE 19 ( 41 ) 4773 - 4786 2000年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

The repressive effect of the 3'-untranslated region (3'-UTR) in human connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/hcs24) mRNA on gene expression had been demonstrated in our previous study. Here, we identified a minimal RNA element in the 3'-UTR, which acts as a cis-acting element of structure-anchored repression (CAESAR). Deletion analyses of the 3'-UTR led us to minimize the element of 84 bases at the junction of the coding region and the 3'-UTR. The minimized RNA segment is predicted, and actually capable of forming a stable secondary structure in vitro. Mutational analyses disclosed a significant relationship between the predicted structure and repressive effect. The utility of CAESAR as a post-transcriptional regulatory element was represented by the fact that steady-state mRNA levels were not affected by CAESAR linked in cis, while protein levels from such a chimeric gene were markedly reduced. Of note, the CAESAR sequence exerted no effect, when it was placed upstream of the promoter. Finally, RNA gel electromobility-shift analyses demonstrated a nuclear factor that interacts with the folded CAESAR. Taken together, it was uncovered that CAESAR of ctgf is a novel post-transcriptional structured RNA regulatory element, probably acting through direct interactions with a nuclear factor as observed in retroviral RNA elements with certain proteins.

軟骨由来の成長因子CTGF/Hcs24遺伝子の転写後制御エレメント,CAESARの構造機能連関

久保田聡, 近藤誠二, 江口傑徳, 服部高子, 中西徹, 滝川正春

生化学 72 ( 8 ) 972 - 972 2000年8月

軟骨細胞および線維芽細胞株におけるCTGF/Ecogenin遺伝子発現の3'-UTRによる調節

近藤誠二, 久保田聡, 江口傑徳, 服部高子, 中西徹, 菅原利夫, 滝川正春

岡山歯学会雑誌 19 ( 1 ) 205 - 206 2000年6月

軟骨由来の成長因子CTGF/Hcs24遺伝子に同定された新たな転写後制御エレメント,CAESAR

久保田聡, 近藤誠二, 江口傑徳, 服部高子, 中西徹, 滝川正春

日本骨代謝学会雑誌 18 ( 2 ) 119 - 119 2000年6月

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