Faculty Profiles - NAGATSUKA Hitoshi

Involvement of miR-140-3p in Wnt3a and TGFβ3 signaling pathways during osteoblast differentiation in MC3T3-E1 cells Reviewed International journal

Shigeko Fushimi, Tsutomu Nohno, Hitoshi Nagatsuka, Hironobu Katsuyama

Genes to Cells 23 ( 7 ) 517 - 527 2018.7

Language：English Publishing type：Research paper (scientific journal)

The Wnt/β-catenin signaling and TGFβ signaling pathways play a key role in osteoblast differentiation. The miRNAs play important roles in regulating gene expression at the post-transcriptional level through fine-tuning of protein-encoding gene expression. However, involvement of miRNAs is not established for Wnt3a and TGFβ signaling pathways in osteoblast differentiation. Here, we examined the role of miRNAs expressed differentially after Wnt3a expression during osteoblast differentiation. Over-expression of the Wnt3a gene increased ALP transcription, but decreased Col1, Runx2, and OCN transcription in osteoblastic MC3T3-E1 cells. Expression profiling and quantitative PCR for miRNAs showed that miR-140-3p decreased in Wnt3a-over-expressing osteoblastic cells. Wnt3a over-expression increased TGFβ3 expression, whereas transfection of the miR-140-3p mimic into MC3T3-E1 cells significantly inhibited TGFβ3 expression. Luciferase assay for the TGFβ3 transcript showed that TGFβ3 was a direct target of miR-140-3p. miR-140-3p mimic transfection resulted in significantly increased OCN transcription, but did not affect ALP, Col1, and Runx2 transcription in MC3T3-E1 cells. rTGFβ3 treatment decreased OCN transcription in MC3T3-E1 cells. These results suggest that the miR-140-3p is involved in osteoblast differentiation as a critical regulatory factor between Wnt3a and TGFβ3 signaling pathways.

DOI： 10.1111/gtc.12591

肺癌移植マウスを用いた高転移臓器における骨髄由来細胞の局在と役割の検討

河合穂高, 辻極秀次, 信長ひかり, 高畠清文, 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 22回 97 - 97 2018.7

Cell supplying to the experimentally induced absorbable suture thread foreign body granuloma from the bone marrow tissues

Y. Nakayasu, S. Aoki, M. Shoumura, N. Osuga, N. Okafuji, K. Nakano, H. Nagatsuka, H. Tsujigiwa, Toshiyuki Kawakami

International Journal of Dentistry and Oral Science 5 ( 6 ) 641 - 644 2018.6

Publishing type：Research paper (scientific journal)

Using the experimental system of GFP (Green florescence Protein) bone marrow transplanted rats, we implanted the Vicryl absorbable suture thread in subcutaneous tissue and examined the formation of foreign body granuloma histopathologically. We also compared the main cellular components, namely, the macrophage and foreign body giant cell immunohistochemically using GFP to confirm their origin. Histologically, the disintegrated suture thread was observed as a circular in void surrounded by the proliferation of macrophages and foreign body giant cells. Fibrous connective tissue including the fibroblasts was interposed among large masses. Even after 6 months, some clusters considered to be residues of macrophages that grew on the suture were still remained. Immunohistochemical examination of GFP showed that all proliferated macrophages and foreign body giant cells were GFP-positive. However, the relatively thin fibrous tissues formed on the outermost layer of the granulation tissue growth were mostly GFP-negative. Based on the results of the experiment, the macrophages and foreign body giant cells that are GFP-positive were derived from the mesenchymal tissue of transplanted bone marrow tissues.

DOI： 10.19070/2377-8075-18000126

The role of bone marrow-derived cells during ectopic bone formation of mouse femoral muscle in GFP mouse bone marrow transplantation model Reviewed International journal

Kiyofumi Takabatake, Hidetsugu Tsujigiwa, Yu Song, Hiroyuki Matsuda, Hotaka Kawai, Masae Fujii, Mei Hamada, Keisuke Nakano, Toshiyuki Kawakami, Hitoshi Nagatsuka

International Journal of Medical Sciences 15 ( 8 ) 748 - 757 2018.5

Language：English Publishing type：Research paper (scientific journal) Publisher：IVYSPRING INT PUBL

Multipotential ability of bone marrow-derived cells has been clarified, and their involvement in repair and maintenance of various tissues has been reported. However, the role of bone marrow-derived cells in osteogenesis remains unknown. In the present study, bone marrow-derived cells during ectopic bone formation of mouse femoral muscle were traced using a GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) mice were transplanted into C57BL/6 J wild type mice. After transplantation, insoluble bone matrix (IBM) was implanted into mouse muscle. Ectopic bone formation was histologically assessed at postoperative days 7, 14, and 28. Immunohistochemistry for GFP single staining and GFP-osteocalcin double staining was then performed. Bone marrow transplantation successfully replaced hematopoietic cells with GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts and osteocytes involved in ectopic bone formation were GFP-negative, whereas osteoclasts and hematopoietic cells involved in bone formation were GFP-positive. These results indicate that bone marrow-derived cells might not differentiate into osteoblasts. Thus, the main role of bone marrow-derived cells in ectopic osteogenesis may not be to induce bone regeneration by differentiation into osteoblasts, but rather to contribute to microenvironment formation for bone formation by differentiating tissue stem cells into osteoblasts.

DOI： 10.7150/ijms.24605

The prognostic implications of bone invasion in gingival squamous cell Carcinoma Reviewed International journal

Shoko Yoshida, Tsuyoshi Shimo, Yurika Murase, Kiyofumi Takabatake, Koji Kishimoto, Soichiro Ibaragi, Norie Yoshioka, Tatsuo Okui, Hitoshi Nagatsuka, Akira Sasaki

Anticancer Research 38 ( 2 ) 955 - 962 2018.2

Language：English Publishing type：Research paper (scientific journal) Publisher：INT INST ANTICANCER RESEARCH

Background/Aim: This study evaluated the associations between bone invasion of gingival squamous cell carcinoma (SCC) and clinicopathological manifestations, and aimed to determine whether bone invasion is an independent prognostic factor in gingival SCC. Patients and Methods: The study was a retrospective review of 78 patients with gingival SCC who underwent surgery with curative intent. The level of bone invasion was pathologically categorized as medullary, cortical or no bone invasion. Results: Cortical and medullary bone invasion was present in 29 and 22 patients, respectively. There was a significant association between medullary bone invasion and tumor size (p=0.017), pathological N classification (p0.001), differentiation (p=0.017) and lymphovascular invasion (p=0.007). Medullary bone invasion and lymphovascular invasion were independent predictors of reduced overall survival (p=0.015, 0.048); medullary bone invasion was also an independent predictor of reduced disease-specific survival (p=0.018). Conclusion: Pathologically-proven medullary bone invasion and lymphovascular invasion were found to be key prognostic factors in gingival SCC. The results suggest that it is necessary to consider adjuvant therapy in patients with medullary bone invasion.

DOI： 10.21873/anticanres.12309

Characterization and potential roles of bone marrow-derived stromal cells in cancer development and metastasis Reviewed International journal

Hotaka Kawai, Hidetsugu Tsujigiwa, Chong Huat Siar, Keisuke Nakano, Kiyofumi Takabatake, Masae Fujii, Mei Hamada, Ryo Tamamura, Hitoshi Nagatsuka

International Journal of Medical Sciences 15 ( 12 ) 1406 - 1414 2018

Language：English Publishing type：Research paper (scientific journal)

Background: The tumor microenvironment and its stromal cells play an important role in cancer development and metastasis. Bone marrow-derived cells (BMDCs), a rich source of hematopoietic and mesenchymal stem cells, putatively contribute to this tumoral stroma. However their characteristics and roles within the tumor microenvironment are unclear. In the present study, BMDCs in the tumor microenvironment were traced using the green fluorescent protein (GFP) bone marrow transplantation model. Methods: C57BL/6 mice were irradiated and rescued by bone marrow transplantation from GFP-transgenic mice. Lewis lung cancer cells were inoculated into the mice to generate subcutaneous allograft tumors or lung metastases. Confocal microscopy, immunohistochemistry for GFP, α-SMA, CD11b, CD31, CD34 and CD105, and double-fluorescent immunohistochemistry for GFP-CD11b, GFP-CD105 and GFP-CD31 were performed. Results: Round and dendritic-shaped GFP-positive mononuclear cells constituted a significant stromal subpopulation in primary tumor peripheral area (PA) and metastatic tumor area (MA) microenvironment, thus implicating an invasive and metastatic role for these cells. CD11b co-expression in GFP-positive cells suggests that round/dendritic cell subpopulations are possibly BM-derived macrophages. Identification of GFP-positive mononuclear infiltrates co-expressing CD31 suggests that these cells might be BM-derived angioblasts, whereas their non-reactivity for CD34, CD105 and α-SMA implies an altered vascular phenotype distinct from endothelial cells. Significant upregulation of GFP-positive, CD31-positive and GFP/CD31 double-positive cell densities positively correlated with PA and MA (P<0.05). Conclusion: Taken together, in vivo evidence of traceable GFP-positive BMDCs in primary and metastatic tumor microenvironment suggests that recruited BMDCs might partake in cancer invasion and metastasis, possess multilineage potency and promote angiogenesis.

DOI： 10.7150/IJMS.24370

Osteogenic capacity of mixed-acid and heat-treated titanium mesh prepared by a selective laser melting technique

Kayoko Yamamoto, Seiji Yamaguchi, Tomiharu Matsushita, Shigeo Mori, Azumi Hirata, Nahoko Kato-Kogoe, Hiroyuki Nakano, Yoichiro Nakajima, Yoshihiro Nishitani, Hitoshi Nagatsuka, Takaaki Ueno

RSC Advances 8 ( 46 ) 26069 - 26077 2018

Language：English Publishing type：Research paper (scientific journal) Publisher：ROYAL SOC CHEMISTRY

The practical use of additive manufacturing to create artificial bone as a material for repairing complex bone defects is currently attracting attention. In this study, we compared the osteogenic capacity of materials composited by the method developed by Kokubo et al. of treating 3D-printed titanium (Ti) mesh with a mixture of H2SO4 and HCl and heating (mixed-acid and heat treatment) with that of materials subjected to conventional chemical treatment. Ti plates treated with this method have been found to promote highly active bone formation on their surface when inserted into rabbit tibial bone defects. No previous study has compared this method with other surface treatment methods. In this study, we used histological and other observations to compare the bone formation process in bone defects when Ti meshes prepared by the selective laser melting technique (SLM) and treated either with mixed acids and heat or with conventional chemical Ti surface treatments were implanted in a rat calvarial bone defect model. We found that both micro-computed tomography and observations of undecalcified ground sections showed that the best bone formation was observed in rats implanted with mesh treated with mixed acids and heat. Our results suggest that mixed-acid and heat-treated Ti mesh prepared by SLM may have a high osteogenic capacity in bone defects.

DOI： 10.1039/c8ra04193h

Web of Science

DKK3 overexpression increases the malignant properties of head and neck squamous cell carcinoma cells Reviewed International journal

Naoki Katase, Shin Ichiro Nishimatsu, Akira Yamauchi, Masahiro Yamamura, Kumiko Terada, Masumi Itadani, Naoko Okada, Nur Mohammad Monsur Hassan, Hitoshi Nagatsuka, Tohru Ikeda, Tsutomu Nohno, Shuichi Fujita

Oncology Research 26 ( 1 ) 45 - 58 2018

Language：English Publishing type：Research paper (scientific journal)

DKK3, a member of the dickkopf Wnt signaling pathway inhibitor family, is believed to be a tumor suppressor because of its reduced expression in cancer cells. However, our previous studies have revealed that DKK3 expression is predominantly observed in head and neck/oral squamous cell carcinoma (HNSCC/OSCC). Interestingly, HNSCC/OSCC patients with DKK3 expression showed a high rate of metastasis and poorer survival, and siRNA-mediated knockdown of DKK3 in HNSCC-derived cancer cell lines resulted in reduced cellular migration and invasion. From these data, it was hypothesized that DKK3 might exert an oncogenic function specific to HNSCC. In the present research, the DKK3 overexpression model was established, and its influences were investigated, together with molecular mechanism studies. The DKK3 expression profile in cancer cell lines was investigated, including HNSCC/OSCC, esophageal, gastric, colorectal, pancreatic, prostatic, and lung cancers. DKK3 overexpression was performed in HNSCC-derived cells by transfection of expression plasmid. The effects of DKK3 overexpression were assessed on cellular proliferation, migration, invasion, and in vivo tumor growth. The molecular mechanism of DKK3 overexpression was investigated by Western blotting and microarray analysis. DKK3 overexpression significantly elevated cellular proliferation, migration, and invasion, as well as increased mRNA expression of cyclin D1 and c-myc. However, reporter assays did not show TCF/LEF activation, suggesting that the increased malignant property of cancer cells was not driven by the Wnt/β-catenin pathway. For the investigation of the pathways/molecules in DKK3-mediated signals, the Western blot analyses revealed that phosphorylation of Akt (S473) and c-Jun (Ser63) was elevated. The application of a PI3K kinase inhibitor, LY294002, on HSC-3 DKK3 cells significantly decreased tumor cell proliferation, migration, and invasion. From these results, we demonstrated that DKK3 might contribute to cellular proliferation, invasion, migration, and tumor cell survival in HNSCC cells through a mechanism other than the canonical Wnt signaling pathway, which might be attributed to PI3K-Akt signaling.

DOI： 10.3727/096504017X149268745963860965-0407

In vitro efficacy of CaCO<inf>3</inf> content in CaTiO<inf>3</inf> - CaCO<inf>3</inf> composites for bone growth Reviewed

Andrea Paola Rodríguez, María Alejandra Sánchez, Betiana Felice, Martín Lucas Zamora, Hidetsugu Tsujigiwa, Kiyofumi Takabatake, Hotaka Kawai, Keisuke Nakano, Hitoshi Nagatsuka

Journal of Hard Tissue Biology 27 ( 3 ) 250 - 256 2018

Publishing type：Research paper (scientific journal)

The effect of CaTiO3 compounded with different amounts of CaCO3 on osteoblastic KUSA/A1 cells was evaluated. CaTiO3-CaCO3 composites were obtained by alkoxide method, a simple, low-cost and reproducible technique used for large-scale production of material. The content of CaCO3 in our samples was controlled by varying the sintering time of the overall process. Composite morphology was assessed by scanning electron microscopy (SEM) showing particles with sizes ranging from100 to 500 nm. The presence of CaCO3 was revealed by XRD and thermogravimetric analyses, which suggested that samples treated at 650ºC for 30 min contained higher amounts of CaCO3 than samples treated for 2 and 10 h. Additionally, in vitro studies demonstrated that CaTiO3–CaCO3 composites sintered for 30 min induced augmented cell proliferation and mineralization in comparison to composites sintered for longer periods of time. Hence, our findings clearly suggest that the amount of CaCO3 within CaTiO3-CaCO3 composites exerts a critical effect on osteoblastic cells response. Enhanced bone regeneration could be achieved by increasing the content of CaCO3 within the composites, thus establishing CaTiO3-CaCO3 as a promising material for bone augmentation procedures in dental field.

DOI： 10.2485/jhtb.27.250

Web of Science