Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Satoshi Fukumoto Last modified date:2022.06.28

Professor / Division of Oral Health, Growth and Development / Department of Dental Science / Faculty of Dental Science

1. Clinical Study on the Restoration of Primary Teeth : Comparison with 1984 and 1994
This report investigated 1,466 primary-tooth restorations in 374 children, who attended the Nagasaki University Dental Clinic of Pediatric Dentistry in 1994. Data resulting from the investigation were compared with those obtained in 1984.
Results and Conclusions
1. Of all the restorations performed in 1984,37.5% were composite resin fillings; 28.8% were stainlesssteel crowns; 16.6% were metal inlays; 11.3% were amalgam fillings. On the other hand, of all the restorations performed in 1994,60.3% were composite resin fillings; 21.0% were metal inlays; 14.1% were stainless-steel crowns.
2. In 1984, of primary molars with decayed proximal surfaces,75.4% were restored with metal inlays,14.3% were restored with composite resin fillings and 10.0% were restored with amalgam fillings. In 1994, of the decayed proximal surfaces of primary molars,51.0% were restored with metal inlays and 47.5% were restored with composite resin fillings.
3. Both in 1984 and in 1994, composite resin fillings were used preferentially for primary anterior teeth in which caries extended only into the enamel or only into the dentin, and for primary molars in which caries extended only into the enamel. In 1994, composite resin fillings were used preferentially for primary anterior teeth in which caries extended into the pulp and for primary molars in which caries extended only into the dentin, too.
4. Compared with the results in 1984, in 1994, the percentages of the composite resin fillings increased both for primary anterior teeth and for primary molars. In 1994, no amalgam filling was used and glass-ionomer cement fillings were used as a new method..
2. 出力可変型キセノン照射器の光重合型レジンの物理的性質に及ぼす影響.
3. Masahiko Okada, Michi Ichiro Itoh, Masashi Haraguchi, Tetsuya Okajima, Masahiro Inoue, Hideto Oishi, Yoichi Matsuda, Tsutomu Iwamoto, Toshihiro Kawano, Satoshi Fukumoto, Hiroshi Miyazaki, Keiko Furukawa, Shinichi Aizawa, Koichi Furukawa, b-series Ganglioside deficiency exhibits no definite changes in the neurogenesis and the sensitivity to Fas-mediated apoptosis but impairs regeneration of the lesioned hypoglossal nerve, Journal of Biological Chemistry, 10.1074/jbc.C100395200, 277, 3, 1633-1636, 2002.01, The polymorphic carbohydrate structures of gangliosides play regulatory roles. In particular, b-series gangliosides, all of which contain α-2,8 sialic acids, have been considered to be critical in various biological events such as adhesion, toxin binding, neurite extension, cell growth, and apoptosis. To clarify the physiological functions of b-series gangliosides in vivo, we have established a gene knockout mouse of GD3 synthase. Although all b-series structures were deleted in the mutant mice, they showed an almost complete nervous tissue morphology with no apparent abnormal behavior. Moreover, no differences in Fas-mediated apoptotic reaction of lymphocytes between wild type and the mutant mice were detected. However, the mutant mice exhibited clearly reduced regeneration of axotomized hypoglossal nerves compared with the wild type, suggesting that b-series gangliosides are more important in the repair rather than in the differentiation of the nervous system and apoptotic process induced via Fas..
4. Kan Saito, Satoshi Fukumoto, Emiko Fukumoto, Michi-ichiro Itoh, Kenji Yuasa, Yuzo Kato, George Goto, Cleft palate- and lip-related gene expression analyzed by cDNA microarray hybridization, Pediatric Dental Journal, 12, 1, 35-42, 2002.03.
5. Masashi Nishio, Satoshi Fukumoto, Keiko Furukawa, Akiko Ichimura, Hiroshi Miyazaki, Susumu Kusunoki, Takeshi Urano, Koichi Furukawa, Overexpressed GM1 suppresses nerve growth factor (NGF) signals by modulating the intracellular localization of NGF receptors and membrane fluidity in PC12 cells, Journal of Biological Chemistry, 10.1074/jbc.M403816200, 279, 32, 33368-33378, 2004.08, Ganglioside GM1 has been considered to have a neurotrophic factor-like activity. To analyze the effects of endogenously generated GM1, the rat pheochromocytoma cell line PC12 was transfected with the GM1/GD1b/GA1 synthase gene and showed increased expression levels of GM1. To our surprise, GM1 +-transfectant cells (GM1+ cells) showed no neurite formation after stimulation with nerve growth factor (NGF). Autophosphorylation of NGF receptor TrkA and activation of ERK1/2 after NGF treatment were scarcely detected in GM1+ cells. Binding of 125I-NGF to PC12 cells was almost equivalent between GM1+ cells and controls. However, dimer formation of TrkA upon NGF treatment was markedly suppressed in GM1+ cells in both cross-linking analysis with Bis(sulfosuccinimidyl)suberate 3 and 125I-NGF binding assay. The sucrose density gradient fractionation of the cell lysate revealed that TrkA primarily located in the lipid raft fraction moved to the non-raft fraction in GM1+ cells. p75NTR and Ras also moved from the raft to non-raft fraction in GM1+ cells, whereas flotillin and GM1 persistently resided in the lipid raft. TrkA kinase activity was differentially regulated when GM1 was added to the kinase assay system in vitro, suggesting suppressive/enhancing effects of GM1 on NGF signals based on the concentration. Measurement of fluorescence recovery after photobleaching revealed that the membrane fluidity was reduced in GM1 + cells. These results suggested that overexpressed GM1 suppresses the differentiation signals mediated by NGF/TrkA by modulating the properties of the lipid raft and the intracellular localization of NGF receptors and relevant signaling molecules..
6. Kenji Yuasa, Satoshi Fukumoto, Yoko Kamasaki, Aya Yamada, Emiko Fukumoto, Kazuhiro Kanaoka, Kan Saito, Hidemitsu Harada, Eri Arikawa-Hirasawa, Yuko Miyagoe-Suzuki, Shinichi Takeda, Kuniaki Okamoto, Yuzo Kato, Taku Fujiwara, Laminin α2 is Essential for Odontoblast Differentiation Regulating Dentin Sialoprotein Expression, Journal of Biological Chemistry, 10.1074/jbc.M310013200, 279, 11, 10286-10292, 2004.03, Laminin α2 is subunit of laminin-2 (α2β1γ1), which is a major component of the muscle basement membrane. Although the laminin α2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin α2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin α2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin α2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins..
7. Kan Saito, Naoya Ohara, Hitoshi Hotokezaka, Satoshi Fukumoto, Kenji Yuasa, Mariko Naito, Taku Fujiwara, Koji Nakayama, Infection-induced Up-regulation of the Costimulatory Molecule 4-1BB in Osteoblastic Cells and Its Inhibitory Effect on M-CSF/-induced in Vitro Osteoclastogenesis, Journal of Biological Chemistry, 10.1074/jbc.M303791200, 279, 14, 13555-13563, 2004.04, Bacterial infection sometimes impairs bone metabolism. In this study, we infected the osteoblastic cell line MC3T3-E1 with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and identified genes that were up-regulated in the BCG-infected cells by the suppression subtractive hybridization method. A gene encoding 4-1BB (CD137), a member of the tumor necrosis factor-α receptor family, was found to be one of the up-regulated genes. Up-regulation of 4-1BB was also observed by infection with Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, and by treatment with lipopolysaccharides and heat-killed BCG. Bone marrow cells and the macrophage-like cell lines J774 and RAW264.7 were found to express 4-1BB ligand (4-1BBL). Recombinant 4-1BB (r4-1BB) that was immobilized on culture plates strongly inhibited macrophage colony stimulating factor (M-CSF)/receptor activator of nuclear factor-κB ligand (RANKL)-induced in vitro osteoclast formation from bone marrow cells. Anti-4-1BBL antibody also inhibited osteoclast formation to a lesser extent, indicating involvement of reverse signaling through 4-1BBL during inhibition of osteoclast formation. A casein kinase I (CKI) inhibitor markedly suppressed the inhibitory effect of r4-1BB on M-CSF/ RANKL-induced osteoclast formation, suggesting that CKI might be involved in 4-1BB/4-1BBL reverse signaling. r4-1BB showed no effects on M-CSF- or RANKL-induced phosphorylation of I-κB, ERK1/2, p38, or JNK, whereas RANKL-induced phosphorylation of Akt, a downstream target of phosphatidylinositol 3-kinase (PI3K), was completely abolished by r4-1BB, suggesting that 4-1BB/4-1BBL reverse signaling may interfere with PI3K/Akt pathway. r4-1BB also abolished RANKL-mediated induction of nuclear factor of activated T cells-2. This study may elucidate a novel role of 4-1BB in cell metabolism, especially osteoclastogenesis..
8. Satoshi Fukumoto, Takayoshi Kiba, Bradford Hall, Noriyuki Iehara, Takashi Nakamura, Glenn Longenecker, Paul H. Krebsbach, Antonio Nanci, Ashok B. Kulkarni, Yoshihiko Yamada, Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts, Journal of Cell Biology, 10.1083/jcb.200409077, 167, 5, 973-983, 2004.12, Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation..
9. Noriko Muraoka, Lillian Shum, Satoshi Fukumoto, Taisei Nomura, Masamichi Ohishi, Kazuaki Nonaka, Transforming growth factor-β3 promotes mesenchymal cell proliferation and angiogenesis mediated by the enhancement of cyclin D1, Flk-1, and CD31 gene expression during Cl/Fr mouse lip fusion, Birth Defects Research Part A - Clinical and Molecular Teratology, 10.1002/bdra.20191, 73, 12, 956-965, 2005.12, BACKGROUND: Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-β3 (TGF-β3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-β3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages. METHODS: To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-β3. The lips that developed in these cultures fused in 2 days. RESULTS: During normal development, we detected positive immunoreactions for TGF-β3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-β3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-β3, in comparison with controls. In tandem, TGF-β3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-β3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-β3, which suggests that TGF-β3 promoted cell proliferation. CONCLUSIONS: TGF-β3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-β3. © 2005 Wiley-Liss, Inc..
10. Muhetaer Huojia, Noriko Muraoka, Keigo Yoshizaki, Satoshi Fukumoto, Misako Nakashima, Akifumi Akamine, Kazuaki Nonaka, Masamichi Ohishi, TGF-β3 induces ectopic mineralization in fetal mouse dental pulp during tooth germ development, Development Growth and Differentiation, 10.1111/j.1440-169x.2005.00790.x, 47, 3, 141-152, 2005.04, Several members of the transforming growth factor (TGF)-β superfamily are expressed in developing teeth from the initiation stage through adulthood. Of those, TGF-β1 regulates odontoblast differentiation and dentin extracellular matrix synthesis. However, the molecular mechanism of TGF-β3 in dental pulp cells is not clearly understood. In the present study, beads soaked with human recombinant TGF-β3 induced ectopic mineralization in dental pulp from fetal mouse tooth germ samples, which increased in a dose-dependent manner. Further, TGF-β3 promoted mRNA expression, and increased protein levels of osteocalcin (OCN) and type I collagen (COL I) in dental pulp cells. We also observed that the expression of dentin sialophosphoprotein and dentin matrix protein 1 was induced by TGF-β3 in primary cultured dental pulp cells, however, not in calvaria osteoblasts, whereas OCN, osteopontin and osteonectin expression was increased after treatment with TGF-β3 in both dental pulp cells and calvaria osteoblasts. Dentin sialoprotein was also partially detected in the vicinity of TGF-β3 soaked beads in vivo. These results indicate for the first time that TGF-β3 induces ectopic mineralization through upregulation of OCN and COL I expression in dental pulp cells, and may regulate the differentiation of dental pulp stem cells to odontoblasts..
11. Satoshi Fukumoto, Yoshihiko Yamada, Review: Extracellular matrix regulates tooth morphogenesis, Connective Tissue Research, 10.1080/03008200500344017, 46, 4-5, 220-226, 2005.07, Mineralized tissues are unique in that they use proteins to attract and organize calcium and phosphate ions into a structured mineral phase, thus precise knowledge of the expression and extracellular distribution of matrix proteins is very important to understand their function. Tooth development is regulated by sequential and reciprocal interactions between neural crest-derived mesenchymal cells and the oral environment. However, the precise molecular mechanisms that mediate interactions between epithelium and mesenchymal cells are not clear, although basement membrane (BM) components have been shown to play important roles in these regulatory events. In addition, the extracellular matrix layer, whose main components are laminin, collagen IV, nidogen, and sulfated proteoglycan, and the BM layer are both considered to be involved with cell proliferation and differentiation. During tooth morphogenesis, extracellular matrices are dramatically changed. Further, the BM components, laminin and collagen IV support dental epithelium; however, in the late stage, they begin the processes of enamel matrix secretion and calcification, after which the BM structure between the dental epithelium and mesenchyme disappears. In addition, tooth abnormalities associated with several kinds of human diseases that cause mutations in the extracellular matrix, as well as the molecular mechanisms of the basement membrane and enamel matrix during tooth morphogenesis, are not clearly understood. In our review, we discuss the role of the extracellular matrix, with focus on the BM and enamel matrix during tooth morphogenesis. Copyright © Taylor & Francis Inc..
12. Aya Yamada, Emiko Fukumoto, Yoko Kamasaki, Hiroko Ida-Yonemochi, Takashi Saku, Taku Fujiwara, Satoshi Fukumoto, GD3 synthase gene found expressed in dental epithelium and shown to regulate cell proliferation, Archives of Oral Biology, 10.1016/j.archoralbio.2004.09.014, 50, 4, 393-399, 2005.04, GD3 synthase is one of the key enzymes involved with ganglioside synthesis, and its activity regulates the main profile of ganglioside expression. We analyzed the expression of the GD3 synthase gene in laser-dissected teeth germs using RT-PCR. The GD3 synthase gene was found expressed in brain, thymus, and tooth germ tissues, however, not in liver or skin specimens. Further, it was highly expressed during the early stage of tooth germ development (embryonic day 14.5), especially in dental epithelia, which gradually reduced in the molar site until postnatal day 7, whereas it was not in dental mesenchyme tissues. In addition, dental epithelial cells transiently transfected with the GD3 synthase gene showed enhanced proliferation. These results indicate that the GD3 synthase gene may be involved in early tooth development, particularly in the proliferation of dental epithelium. © 2004 Elsevier Ltd. All rights reserved..
13. Yutaka Kamimura, Keiko Furukawa, Daiji Kittaka, Masashi Nishio, Kazunori Hamamura, Satoshi Fukumoto, Koichi Furukawa, Differential enhancing effects of alpha2,8-sialyltransferase on the cell proliferation and mobility., International Journal of Oncology, 26, 2, 337-344, 2005.02, alpha2,8-Sialyltransferase (alpha2,8S-T, GD3 synthase) has been reported to be involved in the enhanced cell proliferation of malignant tumors. Using a cloned cDNA of alpha2,8S-T, transfectant cells were established and the effects of the gene expression on the cell phenotypes were analyzed. In contrast with PC12 cells, in which we reported marked growth enhancement based on the transfection of alpha2,8S-T, Swiss3T3 cells showed no enhancement in either cell proliferation or phosphorylation of MAP kinases after the transfection of alpha2,8S-T when treated with platelet-derived growth factor. Correspondingly, the receptor for platelet-derived growth factor also showed no increased phosphorylation upon the factor stimulation. However, in the wound-healing scratching assay, the Swiss3T3 transfectant cells demonstrated increased mobility as the PC12 transfectant cells. These results suggest that the enhancing effects of alpha2,8S-T on the proliferation and mobility are differential depending on the cell types, and ganglioside-associating molecules in the individual cell types need to be investigated..
14. Satoshi Fukumoto, Jeffrey H. Miner, Hiroko Ida, Emiko Fukumoto, Kenji Yuasa, Hiroshi Miyazaki, Matthew P. Hoffman, Yoshihiko Yamada, Laminin α5 is required for dental epithelium growth and polarity and the development of tooth bud and shape, Journal of Biological Chemistry, 10.1074/jbc.M509295200, 281, 8, 5008-5016, 2006.02, In tooth development, the oral ectoderm and mesenchyme coordinately and reciprocally interact through the basement membrane for their growth and differentiation to form the proper shape and size of the tooth. Laminin α5 subunit-containing laminin-10/11 (LM-511/521) is the major laminin in the tooth germ basement membrane. Here, we have examined the role of laminin α5 (Lama5) in tooth development using laminin α5-null mouse primary dental epithelium and tooth germ organ cultures. Lama5-null mice develop a small tooth germ with defective cusp formation and have reduced proliferation of dental epithelium. Also, cell polarity and formation of the monolayer of the inner dental epithelium are disturbed. The enamel knot, a signaling center for tooth germ development, is defective, and there is a significant reduction of Shh and Fgf4 expression in the dental epithelium. In the absence of laminin α5, the basement membrane in the inner dental epithelium becomes discontinuous. In normal mice, integrin α6β4, a receptor for laminin α5, is strongly localized at the basal layer of the epithelium, whereas in mutant mice, integrin α6β4 is expressed around the cell surface. In primary dental epithelium culture, laminin-10/11 promotes cell growth, spreading, and filopodia-like microspike formation. This promotion is inhibited by anti-integrin α6 and β4 antibodies and by phosphatidylinositol 3-kinase inhibitors and dominant negative Rho-GTPase family proteins Cdc42 and Rac. In organ culture, anti-integrin α6 antibody and wortmannin reduce tooth germ size and shape. Our studies demonstrate that laminin α5 is required for the proliferation and polarity of basal epithelial cells and suggest that the interaction between laminin-10/11-integrin α6β4 and the phosphatidylinositol 3-kinase-Cdc42/Rac pathways play an important role in determining the size and shape of tooth germ..
15. Satoshi Fukumoto, Aya Yamada, Kazuaki Nonaka, Yoshihiko Yamada, Essential roles of ameloblastin in maintaining ameloblast differentiation and enamel formation, Cells Tissues Organs, 10.1159/000091380, 181, 3-4, 189-195, 2006.04, During tooth development, dental epithelial cells interact with extracellular matrix components, such as the basement membrane and enamel matrix. Ameloblastin, an enamel matrix protein, plays a crucial role in maintaining the ameloblast differentiation state and is essential for enamel formation. Ameloblastin-null mice developed severe enamel hypoplasia. In mutant mice, dental epithelial cells started to differentiate into ameloblasts, but ameloblasts soon lost cell polarity, proliferated, and formed multiple cell layers, indicative of some aspects of preameloblast phenotypes. In addition, the expression of amelogenin, another component of the enamel matrix, was specifically reduced in mutant ameloblasts. More than 20% of amelobastin-null mice developed odontogenic tumors. We also found that recombinant ameloblastin specifically bound to ameloblasts and inhibited proliferation of dental epithelial cells. These results suggest that ameloblastin is an important regulator to maintain the differentiation state of ameloblasts. Copyright © 2005 S. Karger AG..
16. Susana De Vega, Tsutomu Iwamoto, Takashi Nakamura, Kentaro Hozumi, Dianalee A. McKnight, Larry W. Fisher, Satoshi Fukumoto, Yoshihiko Yamada, TM14 is a new member of the fibulin family (fibulin-7) that interacts with extracellular matrix molecules and is active for cell binding, Journal of Biological Chemistry, 10.1074/jbc.M705847200, 282, 42, 30878-30888, 2007.10, We identified a new extracellular protein, TM14, by differential hybridization using mouse tooth germ cDNA microarrays. TM14 cDNA encodes 440 amino acids containing a signal peptide. The protein contains 3 EGF modules at the center, a C-terminal domain homologous to the fibulin module, and a unique Sushi domain at the N terminus. In situ hybridization revealed that TM14 mRNA was expressed by preodontoblasts and odontoblasts in developing teeth. TM14 mRNA was also expressed in cartilage, hair follicles, and extraembryonic tissues of the placenta. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed and dentin mineralization occurred, TM14 was present in the predentin matrix and along the dentinal tubules. We found that the recombinant TM14 protein was glycosylated with N-linked oligosaccharides and interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. We also found that TM14 preferentially bound dental mesenchyme cells and odontoblasts but not dental epithelial cells or nondental cells such as HeLa, COS7, or NIH3T3 cells. Heparin, EDTA, and anti-integrin β1 antibody inhibited TM14 binding to dental mesenchyme cells, suggesting that both a heparan sulfate-containing cell surface receptor and an integrin are involved in TM14 cell binding. Our findings indicate that TM14 is a cell adhesion molecule that interacts with extracellular matrix molecules in teeth and suggest that TM14 plays important roles in both the differentiation and maintenance of odontoblasts as well as in dentin formation. Because of its protein characteristics, TM14 can be classified as a new member of the fibulin family: fibulin-7..
17. Kouji Nagata, Kouji Masumoto, Toru Uesugi, Shinya Yamamoto, Keigo Yoshizaki, Satoshi Fukumoto, Kazuaki Nonaka, Tomoaki Taguchi, Effect of insulin-like-growth factor and its receptors regarding lung development in fetal mice, Pediatric Surgery International, 10.1007/s00383-007-1977-8, 23, 10, 953-959, 2007.10, In congenital diaphragmatic hernia (CDH), both mortality and morbidity are mainly caused by pulmonary hypoplasia and persistent pulmonary hypertension. Insulin-like growth factors (IGFs) are one of the growth factors that may play an important role in the fetal lung development. Elucidating the roles of these growth factors regarding fetal lung development would thus provide new insight regarding the optimal therapy for CDH patients. The aim of this study is to investigate the role of IGFs in the fetal lung development. The mRNA expression of IGFs and its receptors was analyzed by real-time RT-PCR from embryonic day (E) 11.5 to E18.5 mice. In addition, the lungs dissected from the E17.5 mice were divided into the following three groups; lungs cultured only in the serum-free medium (group I n = 5), lungs cultured in medium containing either IGF-I (group II n = 5), or IGF-II (group III n = 5). All cultures were investigated by immunohistochemistry, using the antibodies of thyroid transcription factor (TTF)-1, prosurfactant protein (proSp)-C, alpha smooth muscle actin (α-SMA), and anti-proliferating cell nuclear antigen (PCNA). The mRNA expression level of both IGF-I and IGF-II was higher during the earlier stage than that of later stage. In contrast, the mRNA expression of both IGF-I receptor (IGF-IR) and IGF-II receptor (IGF-IIR) was higher from the E17.5 to E18.5 than that at any other stage. The number of positive cells for TTF-1, proSp-C, α-SMA and PCNA increased more in both groups II and III than in group I. Based on our findings, IGFs are suggested to induce alveolar and vascular maturation in the late stages of fetal lung development. Therefore, the administration of IGFs to the fetal CDH lung may thus be able to effectively improve the symptoms of hypoplastic lung. © 2007 Springer-Verlag..
18. Miyuki Nishiguchi, Kenji Yuasa, Kan Saito, Emiko Fukumoto, Aya Yamada, Tomokazu Hasegawa, Keigo Yoshizaki, Yoko Kamasaki, Kazuaki Nonaka, Taku Fujiwara, Satoshi Fukumoto, Amelogenin is a negative regulator of osteoclastogenesis via downregulation of RANKL, M-CSF and fibronectin expression in osteoblasts, Archives of Oral Biology, 10.1016/j.archoralbio.2006.09.016, 52, 3, 237-243, 2007.03, Amelogenin is a novel enamel matrix protein. Knockout mice showed enhanced osteoclast formation and resorption of tooth cementum. This study investigated the effects of amelogenin on osteoclastogenesis. In co-cultures with calvaria osteoblasts and purified bone marrow cells, amelogenin inhibited osteoclastogenesis dramatically. Furthermore, amelogenin inhibited the expression of receptor activator of nuclear factor κB ligand (RANKL), macrophage-colony stimulating factor (M-CSF) and fibronectin in osteoblasts, while RANKL expression was induced by fibronectin and inhibited by treatment with fibronectin small interfering RNA. These results suggest that the inhibitory effects of amelogenin on osteoclastogenesis lead to downregulation of RANKL, M-CSF and fibronectin production in osteoblasts. © 2006 Elsevier Ltd. All rights reserved..
19. 象牙芽細胞と骨芽細胞の違いを考える 象牙芽細胞特異的分子の同定と分化制御.
20. 新奇細胞-細胞間蛋白質Pannexin3(Panx3)による軟骨細胞の増殖と分化の制御機構の解明.
21. Takashi Nakamura, Susana De Vega, Satoshi Fukumoto, Lucia Jimenez, Fernando Unda, Yoshihiko Yamada, Transcription factor epiprofin is essential for tooth morphogenesis by regulating epithelial cell fate and tooth number, Journal of Biological Chemistry, 10.1074/jbc.M708388200, 283, 8, 4825-4833, 2008.02, In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation..
22. Shinya Yamamoto, Emiko Fukumoto, Keigo Yoshizaki, Tsutomu Iwamoto, Aya Yamada, Kojiro Tanaka, Hiroharu Suzuki, Shizuko Aizawa, Makiko Arakaki, Kenji Yuasa, Kyoko Oka, Yang Chai, Kazuaki Nonaka, Satoshi Fukumoto, Platelet-derived growth factor receptor regulates salivary gland morphogenesis via fibroblast growth factor expression, Journal of Biological Chemistry, 10.1074/jbc.M710308200, 283, 34, 23139-23149, 2008.08, A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is β-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor α is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRα, and PDGFRβ were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc..
23. Keigo Yoshizaki, Shinya Yamamoto, Aya Yamada, Kenji Yuasa, Tsutomu Iwamoto, Emiko Fukumoto, Hidemitsu Harada, Masahiro Saito, Akihiko Nakasima, Kazuaki Nonaka, Yoshihiko Yamada, Satoshi Fukumoto, Neurotrophic factor neurotrophin-4 regulates ameloblastin expression via full-length TrkB, Journal of Biological Chemistry, 10.1074/jbc.M704913200, 283, 6, 3385-3391, 2008.02, Neurotrophic factors play an important role in the development and maintenance of not only neural but also nonneural tissues. Several neurotrophic factors are expressed in dental tissues, but their role in tooth development is not clear. Here, we report that neurotrophic factor neurotrophin (NT)-4 promotes differentiation of dental epithelial cells and enhances the expression of enamel matrix genes. Dental epithelial cells from 3-day-old mice expressed NT-4 and three variants of TrkB receptors for neurotrophins (full-length TrkB-FL and truncated TrkB-T1 and -T2). Dental epithelial cell line HAT-7 expressed these genes, similar to those in dental epithelial cells. We found that NT-4 reduced HAT-7 cell proliferation and induced the expression of enamel matrix genes, such as ameloblastin (Ambn). Transfection of HAT-7 cells with the TrkB-FL expression construct enhanced the NT-4-mediated induction of Ambn expression. This enhancement was blocked by K252a, an inhibitor for Trk tyrosine kinases. Phosphorylation of ERK1/2, a downstream molecule of TrkB, was induced in HAT-7 cells upon NT-4 treatment. TrkB-FL but not TrkB-T1 transfection increased the phosphorylation level of ERK1/2 in NT-4-treated HAT-7 cells. These results suggest that NT-4 induced Ambn expression via the TrkB-MAPK pathway. The p75 inhibitor TAT-pep5 decreased NT-4-mediated induction of the expression of Ambn, TrkB-FL, and TrkB-T1, suggesting that both high affinity and low affinity neurotrophin receptors were required for NT-4 activity. We found that NT-4-null mice developed a thin enamel layer and had a decrease in Ambn expression. Our results suggest that NT-4 regulates proliferation and differentiation of the dental epithelium and promotes production of the enamel matrix..
24. Daiji Kittaka, Michi Ichirou Itoh, Yuhsuke Ohmi, Yuji Kondo, Satoshi Fukumoto, Takeshi Urano, Orie Tajima, Keiko Furukawa, Koichi Furukawa, Impaired hypoglossal nerve regeneration in mutant mice lacking complex gangliosides: Down-regulation of neurotrophic factors and receptors as possible mechanisms, Glycobiology, 10.1093/glycob/cwn032, 18, 7, 509-516, 2008.07, Gangliosides, sialic acid-containing glycosphingolipids, have been considered to play roles as neurotrophic factors. Exogenous gangliosides added to the culture medium of neuronal cells or injected in artificially injured sites of nerve tissues actually showed neurotrophic factor-like effects such as neurite extension and alleviation of nerve tissue deterioration. In this study, neuroregeneration in the mutant mice lacking complex gangliosides was examined. To determine whether the nervous system maintains regenerative activity in the long-term absence of complex gangliosides, we analyzed hypoglossal nerve regeneration after axotomy in the mutant mice of GM2/GD2 synthase. These mice exhibited marked impairment of regenerative activity both in the number of surviving neurons and in the number of peroxidase-positive neurons. Moreover, reduced levels of gene expression of neurotrophic factors and their receptors including CNTF, p75 NTR, TrkB, and others in hypoglossal neurons were observed in real-time reverse transcription-polymerase chain reaction combined with laser capture microdissection, suggesting that these molecules are, at least partly, involved in the regeneration of lesioned nerves and that their expression levels are precisely controlled in the presence of intact expression of complex gangliosides. The Author 2008. Published by Oxford University Press. All rights reserved..
25. Noboru Yamaguchi, Toshio Kukita, Yin Ji Li, Noriaki Kamio, Satoshi Fukumoto, Kazuaki Nonaka, Yuzo Ninomiya, Shigemasa Hanazawa, Yoshihisa Yamashita, Adiponectin inhibits induction of TNF-α/RANKL-stimulated NFATc1 via the AMPK signaling, FEBS Letters, 10.1016/j.febslet.2007.12.037, 582, 3, 451-456, 2008.02, We investigated here whether adiponectin can exhibit an inhibitory effect on tumor necrosis factor-alpha (TNF-α)- and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis by using RAW264 cell D clone with a high efficiency to form osteoclasts. Globular adiponectin (gAd) strongly inhibited TNF-α/RANKL-induced differentiation of osteoclasts by interfering with TNF receptor-associated factor 6 production and calcium signaling; consequently, the induction of nuclear factor of activated T cells c1 (NFATc1) was strongly inhibited. Moreover, we observed that inhibition of AMP-activated protein kinase abrogated gAd inhibition for TNF-α/RANKL-induced NFATc1 expression. Our data suggest that adiponectin acts as a potent regulator of bone resorption observed in diseases associated with cytokine activation. © 2008 Federation of European Biochemical Societies..
26. 象牙芽細胞の分化に関わる新奇ギャップ結合分子の同定.
27. 歯原性上皮細胞におけるエピプロフィンの機能解析.
28. 歯と歯周組織、骨の再生 エナメル質形成の分子制御機構.
29. エナメル芽細胞分化過程における細胞間結合の役割.
30. Yasunori Sasaki, Kyoko Satoh, Haruaki Hayasaki, Satoshi Fukumoto, Taku Fujiwara, Kazuaki Nonaka, The P561T polymorphism of the growth hormone receptor gene has an inhibitory effect on mandibular growth in young children, European Journal of Orthodontics, 10.1093/ejo/cjp017, 31, 5, 536-541, 2009.10, P561T heterozygous missense mutation in the growth hormone receptor (GHR) is a candidate genetic polymorphism (single-nucleotide polymorphism) for human mandibular growth. The purpose of this study was to assess whether this mutation affects mandibular growth during early childhood. The difference in mandibular growth between P561T heterozygous and wild-type individuals was analysed by cephalometric measurements during childhood. The subjects included 33 children with mandibular protrusion (aged 3-12 years, 16 males and 17 females) and 27 normal children (aged 3-13 years, 14 males and 13 females). Genomic DNA extracted from buccal epithelial cells was genotyped for the P561T heterozygous mutation with a molecular analysis (polymerase chain reaction - restriction fragment length polymorphism method). Two of the patients with normal occlusion and five with mandibular protrusion were heterozygous for the mutation. Chi-square analysis showed that the frequency of this mutation did not differ statistically between the normal and mandibular protrusion subjects. Multilevel model analysis of the 101 cephalograms showed that the mutation reduced the linear measurements of the mandible. These findings suggest that P561T heterozygous mutation affects mandibular growth during early childhood, and this mutation in the GHR gene is hypothesized to function as an inhibitory factor in the process of mandibular growth..
31. Tsutomu Iwamoto, Aya Yamada, Kenji Yuasa, Emiko Fukumoto, Takashi Nakamura, Taku Fujiwara, Satoshi Fukumoto, Influences of interferon-gamma on cell proliferation and interleukin-6 production in Down syndrome derived fibroblasts, Archives of Oral Biology, 10.1016/j.archoralbio.2009.07.009, 54, 10, 963-969, 2009.10, Objective: Down syndrome, a frequently encountered genetic disorder, is usually associated with medical problems related to infectious disease, such as periodontal diseases and prolonged wound healing. Although affected individuals are considered to have clinical problems related to high interferon (IFN) sensitivity, the molecular mechanisms of IFN activities are not completely understood. Design: Down syndrome derived fibroblasts, Detroit 539 (D1) and Hs 52.Sk (D2) cells, were used. To analyse the expressions of interferon (IFN) receptors and downstream of IFN-γ, western blotting was performed. Cell proliferation was determined by counting cells following trypan blue staining. Media levels of IL-1β, TNF-α, and IL-6 were quantified using ELISA. Results: IFN-γ receptor 2 and IFN-α receptor 1, but not IFN-γ receptor 1, were highly expressed in D1 and D2 cells, as compared to the control fibroblast cells. Cell proliferation by D1 and D2 cells was lower than that by the control fibroblasts, further, IFN-γ had a greater effect to inhibit cell proliferation by D1 and D2 cells. In addition, IFN-γ treatment increased the phosphorylation of STAT1 and MAPK in D1 cells as compared to normal fibroblasts. Also, the presence of exogenous IFN-γ in the growth medium significantly induced IL-6, but not IL-1β or TNF-α, in D1 and D2 cells. Conclusion: Taken together, our results are consistent with hypersensitive reactions to IFN-γ seen in patients with Down syndrome and may provide useful information to elucidate the mechanisms of IFN-γ activities in those individuals. © 2009 Elsevier Ltd. All rights reserved..
32. Haruaki Hayasaki, Mayuko Ishibashi, Seiji Nakamura, Satoshi Fukumoto, Kazuaki Nonaka, Dentigerous cyst in primary dentition: Case report of a 4-year-old girl, Pediatric Dentistry, 31, 4, 294-297, 2009.07, The purpose of this case report is to describe the treatment outcome of a 4-year-old girl with a dentigerous cyst on the primary mandibular right second premolar of her caries-free mouth. Her primary predecessor was extracted and the lesion decompressed by using a removable appliance with a resin projection. The succeeding premolar had not developed further 8 months later, however, and had to be extracted. Crown formation of the succeeding premolar might have been interrupted by the presence of the cyst. To our knowledge, this is the earliest reported asymptomatic dentigerous cyst in the mandibular premolar region with a vital primary second molar..
33. Satoshi Fukumoto, Aya Yamada, Tsutomu Iwamoto, Takashi Nakamura, Dental epithelium proliferation and differentiation regulated by ameloblastin., Interface Oral Health Science, 33-38, 2009.12.
34. Akira Sonoda, Tsutomu Iwamoto, Takashi Nakamura, Emiko Fukumoto, Keigo Yoshizaki, Aya Yamada, Makiko Arakaki, Hidemitsu Harada, Kazuaki Nonaka, Seiji Nakamura, Yoshihiko Yamada, Satoshi Fukumoto, Critical role of heparin binding domains of ameloblastin for dental epithelium cell adhesion and ameloblastoma proliferation, Journal of Biological Chemistry, 10.1074/jbc.M109.033464, 284, 40, 27176-27184, 2009.10, AMBN (ameloblastin) is an enamel matrix protein that regulates cell adhesion, proliferation, and differentiation of ameloblasts. In AMBN-deficient mice, ameloblasts are detached from the enamel matrix, continue to proliferate, and form a multiple cell layer; often, odontogenic tumors develop in the maxilla with age. However, the mechanism of AMBN functions in these biological processes remains unclear. By using recombinant AMBN proteins, we found that AMBN had heparin binding domains at the C-terminal half and that these domains were critical for AMBN binding to dental epithelial cells. Overexpression of full-length AMBN protein inhibited proliferation of human ameloblastoma AM-1 cells, but overexpression of heparin binding domain-deficient AMBN protein had no inhibitory effect. In full-length AMBN-overexpressing AM-1 cells, the expression of Msx2, which is involved in the dental epithelial progenitor phenotype, was decreased, whereas the expression of cell proliferation inhibitors p21 and p27 was increased. We also found that the expression of enamelin, a marker of differentiated ameloblasts, was induced, suggesting that AMBN promotes odontogenic tumor differentiation. Thus, our results suggest that AMBN promotes cell binding through the heparin binding sites and plays an important role in preventing odontogenic tumor development by suppressing cell proliferation and maintaining differentiation phenotype through Msx2, p21, and p27..
35. Kouji Nagata, Kouji Masumoto, Genshirou Esumi, Risa Teshiba, Keigo Yoshizaki, Satoshi Fukumoto, Kazuaki Nonaka, Tomoaki Taguchi, Connexin43 plays an important role in lung development, Journal of Pediatric Surgery, 10.1016/j.jpedsurg.2009.07.070, 44, 12, 2296-2301, 2009.12, Objectives: Connexin43 (Cx43) is one of the proteins associated with gap junction. Connexin43 knockout mice die after birth owing to hypoplastic lungs. The purpose of this study was to analyze the hypoplastic lung of Cx43 knockout mice to clarify the role of the Cx43 during lung development. Methods: Adult hetero Cx43 mice were mated. Newborn mice were divided into the following groups: wild, hetero, and knockout. Total RNA was extracted from the right lung, and the left lung was fixed for immunohistochemical staining. The mRNA expression of surfactant protein C, aquaporin-5, and α-smooth muscle actin were analyzed by reverse transcriptase polymerase chain reaction. H&E and immunohistochemical staining for those markers were performed. Results: The mRNA expression of aquaporin-5, surfactant protein C, and α-smooth muscle actin was significantly lower in knockout mice than that in the wild and hetero mice. H&E staining in the knockout mice showed narrow airspaces and thicker interalveolar septae. Immunohistochemical staining in all markers showed the formation of alveoli to be delayed in the knockout mice. Conclusion: Based on these findings, Cx43 is closely related to alveolar and vascular formation during lung development. © 2009 Elsevier Inc. All rights reserved..
36. 顎顔面発生研究の新規展開 若手研究者の発想とねらい 上皮器官形成における転写因子エピプロフィンの役割.
37. 象牙芽細胞分化におけるギャップ結合分子の発現および機能解析.
38. 神経栄養因子による歯原性上皮細胞の増殖、分化制御機構の解明.
39. 歯科再生医療に歯の発生生物学はどのように貢献してきたか、そして今後どのように貢献できるか 歯原性上皮の分化の分子制御メカニズム.
40. Maxillary Canine Replantation in the Patient with Eruption Disturbance due to Odontoma.
41. 唾液腺分岐におけるギャップ結合の役割解明と再生への応用.
42. エピプロフィンによる歯原性上皮細胞の増殖と分化の制御機構.
43. インテグリン-フィラミンA複合体による歯の形態形成の分子制御.
44. Tsutomu Iwamoto, Takashi Nakamura, Andrew Doyle, Masaki Ishikawa, Susana De Vega, Satoshi Fukumoto, Yoshihiko Yamada, Pannexin 3 regulates intracellular ATP/cAMP levels and promotes chondrocyte differentiation, Journal of Biological Chemistry, 10.1074/jbc.M110.127027, 285, 24, 18948-18958, 2010.06, Pannexin 3 (Panx3) is a new member of the gap junction pannexin family, but its expression profiles and physiological function are not yet clear. We demonstrate in this study that Panx3 is expressed in cartilage and regulates chondrocyte proliferation and differentiation. Panx3 mRNA was expressed in the prehypertrophic zone in the developing growth plate and was induced during the differentiation of chondrogenic ATDC5 and N1511 cells. Panx3-transfected ATDC5 and N1511 cells promoted chondrogenic differentiation, but the suppression of endogenous Panx3 inhibited differentiation of ATDC5 cells and primary chondrocytes. Panx3-transfected ATDC5 cells reduced parathyroid hormone-induced cell proliferation and promoted the release of ATP into the extracellular space, possibly by action of Panx3 as a hemichannel. Panx3 expression in ATDC5 cells reduced intracellular cAMP levels and the activation of cAMP-response element-binding, a protein kinase A downstream effector. These Panx3 activities were blocked by anti-Panx3 antibody. Our results suggest that Panx3 functions to switch the chondrocyte cell fate from proliferation to differentiation by regulating the intracellular ATP/cAMP levels..
45. Nan Wu, Tsutomu Iwamoto, Yu Sugawara, Masaharu Futaki, Keigo Yoshizaki, Shinya Yamamoto, Aya Yamada, Takashi Nakamura, Kazuaki Nonaka, Satoshi Fukumoto, PDGFs regulate tooth germ proliferation and ameloblast differentiation, Archives of Oral Biology, 10.1016/j.archoralbio.2010.03.011, 55, 6, 426-434, 2010.06, Objective: The purpose of this study was to elucidate the effects of platelet-derived growth factors (PDGFs) during tooth development, as well as the mechanisms underlying the interactions of growth factors with PDGF signalling during odontogenesis. Design: We used an ex vivo tooth germ organ culture system and two dental cell lines, SF2 cells and mDP cells, as models of odontogenesis. AG17, a tyrosine kinase inhibitor, was utilised for blocking PDGF receptor signalling. To analyse the expressions of PDGFs, reverse transcriptase (RT)-PCR and immunohistochemistry were performed. Proliferation was examined using a BrdU incorporation assay for the organ cultures and a cell counting kit for the cell lines. The expressions of Fgf2 and ameloblastin were analysed by real-time RT-PCR. Results: The PDGF ligands PDGF-A and PDGF-B, and their receptors, PDGFRα and PDGFRβ, were expressed throughout the initial stages of tooth development. In the tooth germ organ cultures, PDGF-AA, but not PDGF-BB, accelerated cusp formation. Conversely, AG17 suppressed both growth and cusp formation of tooth germs. Exogenous PDGF-BB promoted mDP cell proliferation. Furthermore, PDGF-AA decreased Fgf2 expression and increased that of ameloblastin, a marker of differentiated ameloblasts. Conclusion: Our results indicate that PDGFs are involved in initial tooth development and regulate tooth size and shape, as well as ameloblast differentiation. © 2010 Elsevier B.V. All rights reserved..
46. Masaki Ishikawa, Tsutomu Iwamoto, Takashi Nakamura, Andrew Doyle, Satoshi Fukumoto, Yoshihiko Yamada, Pannexin 3 functions as an ER Ca 2+ channel, hemichannel, and gap junction to promote osteoblast differentiation, Journal of Cell Biology, 10.1083/jcb.201101050, 193, 7, 1257-1274, 2011.06, The pannexin proteins represent a new gap junction family. However, the cellular functions of pannexins remain largely unknown. Here, we demonstrate that pannexin 3 (Panx3) promotes differentiation of osteoblasts and ex vivo growth of metatarsals. Panx3 expression was induced during osteogenic differentiation of C2C12 cells and primary calvarial cells, and suppression of this endogenous expression inhibited differentiation. Panx3 functioned as a unique Ca 2+ channel in the endoplasmic reticulum (ER), which was activated by purinergic receptor/phosphoinositide 3-kinase (PI3K)/Akt signaling, followed by activation of calmodulin signaling for differentiation. Panx3 also formed hemichannels that allowed release of ATP into the extracellular space and activation of purinergic receptors with the subsequent activation of PI3K-Akt signaling. Panx3 also formed gap junctions and propagated Ca 2+ waves between cells. Blocking the Panx3 Ca 2+ channel and gap junction activities inhibited osteoblast differentiation. Thus, Panx3 appears to be a new regulator that promotes osteoblast differentiation by functioning as an ER Ca 2+ channel and a hemichannel, and by forming gap junctions..
47. Ikarashi K, Fujiwara H, Yamazaki Y, Goto J, Kaneko K, Kato H, Fujii S, Sasaki H, Fukumoto S, Furukawa K, Waki H, Furukawa K, Impaired hippocampal long-term potentiation and failure of learning in β1,4-N-acetylgalactosaminyltransferase gene transgenic mice., Glycobiology, 10.1093/glycob/cwr090, 21, 10, 1373-1381, 2011.10.
48. Yasuko Kimura, Shigeshi Kikunaga, Ichiro Takahashi, Yuji Hatakeyama, Satoshi Fukumoto, Yasuyuki Sasano, Characterization of the calcification process modeled in rat embryonic calvarial culture, Journal of Electron Microscopy, 10.1093/jmicro/dfr068, 60, 5, 345-352, 2011.10, An organ culture system to model the physiological calcification process was designed using rat embryonic calvaria as a device for analyzing its mechanism. Standardized calvarial explants were dissected from rat embryos aged 18 and 20 days (E18 and E20) and cultured for 1, 3 and 5 days. The calcium content of the cultured explants was quantified by atomic absorption spectrophotometry. Equivalent explants were fixed, embedded in paraffin, sectioned and stained with von Kossa stain combined with hematoxylin-eosin or processed for energy-dispersive X-ray spectroscopy to determine the concentrations of calcium, phosphorus and carbon in the tissue. The total calcium content increased significantly in E18 and E20 cultured calvaria (E18cc and E20cc) over 5 days of culture. All cultured calvaria were von Kossa-positive, whereas the staining was intensified, and sound osteoblasts and osteocytes were observed in the bone matrix only in E18cc during the 5-day culture period. Concentrations of calcium and carbon increased significantly in E18cc over 5 days, whereas E20 showed little increase. Physiological calcification proceeded in E18cc, but not in E20cc. These results indicate that the organ culture system using E18 calvaria is useful for modeling the physiological calcification process in vitro. © The Author 2011. Published by Oxford University Press [on behalf of Japanese Society of Microscopy]. All rights reserved..
49. Makiko Arakaki, Masaki Ishikawa, Takashi Nakamura, Tsutomu Iwamoto, Aya Yamada, Emiko Fukumoto, Masahiro Saito, Keishi Otsu, Hidemitsu Harada, Yoshihiko Yamada, Satoshi Fukumoto, Role of epithelial-stem cell interactions during dental cell differentiation, Journal of Biological Chemistry, 10.1074/jbc.M111.285874, 287, 13, 10590-10601, 2012.03, Epithelial-mesenchymal interactions regulate the growth and morphogenesis of ectodermal organs such as teeth. Dental pulp stem cells (DPSCs) are a part of dental mesenchyme, derived from the cranial neural crest, and differentiate into dentin forming odontoblasts. However, the interactions between DPSCs and epithelium have not been clearly elucidated. In this study, we established a mouse dental pulp stem cell line (SP) comprised of enriched side population cells that displayed a multipotent capacity to differentiate into odontogenic, osteogenic, adipogenic, and neurogenic cells. We also analyzed the interactions between SP cells and cells from the rat dental epithelial SF2 line. When cultured with SF2 cells, SP cells differentiated into odontoblasts that expressed dentin sialophosphoprotein. This differentiation was regulated by BMP2 and BMP4, and inhibited by the BMP antagonist Noggin.Wealso found that mouse iPS cells cultured with mitomycin C-treated SF2-24 cells displayed an epithelial cell-like morphology. Those cells expressed the epithelial cell markers p63 and cytokeratin-14, and the ameloblast markers ameloblastin and enamelin, whereas they did not express the endodermal cell marker Gata6 or mesodermal cell marker brachyury. This is the first report of differentiation of iPS cells into ameloblasts via interactions with dental epithelium. Co-culturing with dental epithelial cells appears to induce stem cell differentiation that favors an odontogenic cell fate, which may be a useful approach for tooth bioengineering strategies..
50. Keishi Otsu, Ryota Kishigami, Ai Oikawa-Sasaki, Satoshi Fukumoto, Aya Yamada, Naoki Fujiwara, Kiyoto Ishizeki, Hidemitsu Harada, Differentiation of induced pluripotent stem cells into dental mesenchymal cells, Stem Cells and Development, 10.1089/scd.2011.0210, 21, 7, 1156-1164, 2012.05, Similar to embryonic stem cells, induced pluripotent stem (iPS) cells can differentiate into various cell types upon appropriate induction, and thus, may be valuable cell sources for regenerative medicine. However, iPS cells have not been reported to differentiate into odontogenic cells for tooth regeneration. Here we demonstrated that neural crest-like cells (NCLC) derived from mouse iPS cells have the potential to differentiate into odontogenic mesenchymal cells. We developed an efficient culture protocol to induce the differentiation of mouse iPS cells into NCLC. We confirmed that the cells exhibited neural crest (NC) cell markers as evidenced by immunocytochemistry, flow cytometry, and real-time reverse transcription-polymerase chain reaction. Further, in recombination cultures of NCLC and mouse dental epithelium, NCLC exhibited a gene expression pattern involving dental mesenchymal cells. Some NCLC also expressed dentin sialoprotein. Conditioned medium of mouse dental epithelium cultures further enhanced the differentiation of NCLC into odontoblasts. These results suggest that iPS cells are useful cell sources for tooth regeneration and tooth development studies. © Copyright 2012, Mary Ann Liebert, Inc..
51. Masaki Ishikawa, Tsutomu Iwamoto, Satoshi Fukumoto, Yoshihiko Yamada, Pannexin 3 Inhibits Proliferation of Osteoprogenitor Cells by Regulating Wnt and p21 Signaling., The Journal of Biological Chemistry, 10.1074/jbc.M113.523241, Vol.289, No.5, 2839-2851, 2013.12, Canonical Wnt signaling and BMP promote the proliferation and differentiation of osteoprogenitors, respectively. However, the regulatory mechanism involved in the transition from proliferation to differentiation is unclear. Here, we show that Panx3 (pannexin 3) plays a key role in this transition by inhibiting the proliferation and promoting the cell cycle exit. Using primary calvarial cells and explants, C3H10T1/2 cells, and C2C12 cells, we found that Panx3 expression inhibited cell growth, whereas the inhibition of endogenous Panx3 expression increased it. We also found that the Panx3 hemichannel inhibited cell growth by promoting -catenin degradation through GSK3 activation. Additionally, the Panx3 hemichannel inhibited cyclin D1 transcription and Rb phosphorylation through reduced cAMP/PKA/CREB signaling. Furthermore, the Panx3 endoplasmic reticulum Ca(2+) channel induced the transcription and phosphorylation of p21, through the calmodulin/Smad pathway, and resulted in the cell cycle exit. Our results reveal that Panx3 is a new regulator that promotes the switch from proliferation to differentiation of osteoprogenitors via multiple Panx3 signaling pathways..
52. Emiko Fukumoto, Satoshi Fukumoto, Koji Kawasaki, Reiko Furugen, Masayasu Kitamura, Yumiko Kawashita, Hideaki Hayashida, Hideki Fukuda, Youichi Iijima, Toshiyuki Saito, Cessation age of breast-feeding and pacifier use is associated with persistent finger-sucking, Pediatric Dentistry, 35, 7, 506-509, 2013.11, Although some studies have reported that breast-feeding and pacifier use influence finger-sucking, few have demonstrated whether the age at cessation of breast-feeding or pacifier use and persistent finger-sucking are related. Therefore, the purpose of this study was to examine whether the age at cessation of breast-feeding and pacifier use influenced persistent finger-sucking. Methods: A cross-sectional study of 555 36- to 47-month-olds was conducted in Nagasaki, Japan, using a questionnaire. Using the optimal cutoff point in a receiver-operating characteristic curve, the age was estimated at which cessation of pacifier use and breast-feeding had the most significant effect on persistent finger-sucking, and the estimated ages were assessed by multiple logistic regression analysis, incorporating all the questions in the questionnaire as independent variables. Results: The odds ratios for persistent finger-sucking when breast-feeding was stopped at an age younger than 12 months old or when pacifier use was stopped at an age younger than 14 months old were 3.77 (95 percent confidence interval (CI)=1.97-7.22) and 8.62 (95 percent CI=2.56-29.04), respectively. Conclusions: Cessation of breast-feeding before 12 months old or pacifier use before 14 months old was associated with persistent finger-sucking..
53. Tomoko Ikeuchi, Takashi Nakamura, Satoshi Fukumoto, Haruhiko Takada, A vitamin D3 analog augmented interleukin-8 production by human monocytic cells in response to various microbe-related synthetic ligands, especially NOD2 agonistic muramyldipeptide, International Immunopharmacology, 10.1016/j.intimp.2012.10.027, 15, 1, 15-22, 2013.01, Active metabolite vitamin D3, 1α,25-dihydroxyvitamin D3, is a pleiotropic factor and exhibits various physiological functions, including immunomodulating activities. In this study, the possible regulation of innate immune responses of human monocytic cells by a vitamin D3 analog was examined. Human monocytic THP-1 cells were pre-treated with OCT, vitamin D3 analog, 1α,25-dihydroxy-22-oxavitamin D3, followed by stimulation with various chemically synthesized Toll-like receptors (TLR) and NOD1 and NOD2 ligands. OCT-treated cells produced more IL-8 than non-treated cells upon stimulation with various chemically-synthesized ligands: TLR2-agonistic lipopeptide (FSL-1), TLR3-agonistic poly I:C, TLR4-agonistic lipid A (E. coli-type LA-15-PP), NOD1-agonistic FK565 and NOD2-agonistic muramyldipeptide (MDP). Among the ligands, MDP was the highest inducer of IL-8 production in OCT-treated THP-1 cells, and IL-8 production increased depending on the treatment time until 72 h. OCT up-regulated the expression of NOD2 in THP-1 cells, and OCT-treated cells exhibited higher activation of p38, JNK and ERK in the MAPK pathway, IκBα in the NF-κB pathway, and TAK1 upstream in response to MDP than non-treated cells. Analysis using siRNA against NOD2 and inhibitors of specific signal molecules indicated that the existence of NOD2 and activation of the above signaling molecules are required for enhanced production of IL-8 in OCT-treated THP-1 cells. These findings suggested that NOD2, NF-κB and MAPK pathways are involved in the activity of OCT to augment the response of human monocytic cells to MDP. © 2012 Elsevier B.V. All rights reserved..
54. Masaki Ishikawa, Tsutomu Iwamoto, Satoshi Fukumoto, Yoshihiko Yamada, Pannexin 3 inhibits proliferation of osteoprogenitor cells by regulating Wnt and p21 signaling, Journal of Biological Chemistry, 10.1074/jbc.M113.523241, 289, 5, 2839-2851, 2014.01, Canonical Wnt signaling and BMP promote the proliferation and differentiation of osteoprogenitors, respectively. However, the regulatory mechanism involved in the transition from proliferation to differentiation is unclear. Here, we show that Panx3 (pannexin 3) plays a key role in this transition by inhibiting the proliferation and promoting the cell cycle exit. Using primary calvarial cells and explants, C3H10T1/2 cells, and C2C12 cells, we found that Panx3 expression inhibited cell growth, whereas the inhibition of endogenous Panx3 expression increased it. We also found that the Panx3 hemichannel inhibited cell growth by promoting β-catenin degradation through GSK3β activation. Additionally, the Panx3 hemichannel inhibited cyclin D1 transcription and Rb phosphorylation through reduced cAMP/PKA/CREB signaling. Furthermore, the Panx3 endoplasmic reticulum Ca2+ channel induced the transcription and phosphorylation of p21, through the calmodulin/Smad pathway, and resulted in the cell cycle exit. Our results reveal that Panx3 is a new regulator that promotes the switch from proliferation to differentiation of osteoprogenitors via multiple Panx3 signaling pathways. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc..
55. 白血病治療を目指したヒト乳歯歯髄細胞から人工骨髄誘導法の開発.
56. Takashi Nakamura, Masahiro Naruse, Yuta Chiba, Toshihisa Komori, Keiichi Sasaki, Masahiro Iwamoto, Satoshi Fukumoto, Novel hedgehog agonists promote osteoblast differentiation in mesenchymal stem cells, Journal of Cellular Physiology, 10.1002/jcp.24823, 230, 4, 922-929, 2015.04, © 2014 Wiley Periodicals, Inc. Hedgehog (Hh) family members are involved in multiple cellular processes including proliferation, migration, differentiation, and cell fate determination. Recently, the novel Hh agonists Hh-Ag 1.3 and 1.7 were identified in a high-throughput screening of small molecule compounds that activate the expression of Gli1, a target of Hh signaling. This study demonstrates that Hh-Ag 1.3 and 1.7 strongly activate the expression of endogenous Gli1 and promote osteoblast differentiation in the mesenchymal stem cell line C3H10T1/2. Both compounds stimulated alkaline phosphatase activity in a dose-dependent manner, and induced osteoblast marker gene expression in C3H10T1/2 cells, which indicated that they had acquired an osteoblast identity. Of the markers, the expression of osterix/Sp7, a downstream target of runt-related transcription factor (Runx)2, was induced by Hh-Ag 1.7, which also rescued the osteoblast differentiation defect of RD-127, a mesenchymal cell line from Runx2-deficient mice. Hh-Ags also activated canonical Wnt signaling and synergized with low doses of BMP-2 to enhance osteoblastic potential. Thus, Hh-Ag 1.7 could be useful for bone healing in individuals with abnormalities in osteogenesis, such as osteoporosis patients and the elderly, and can contribute to the development of novel therapeutics for the treatment of bone fractures and defects..
57. Saito K, Fukumoto E, Yamada A, Yuasa K, Yoshizaki K, Iwamoto T, Saito M, Nakamura T, Fukumoto S, Interaction between fibronectin and β1 integrin is essential for tooth development., PloS one, 10.1371/journal.pone.0121667, 10, 4, e0121667, 2015.04.
58. Xuanyu Lu, Wenjin Li, Satoshi Fukumoto, Yoshihiko Yamada, Carla A. Evans, Tom Diekwisch, Xianghong Luan, The ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing, Matrix Biology, 10.1016/j.matbio.2016.02.007, 52-54, 113-126, 2016.05, © 2016 International Society of Matrix Biology. The extracellular matrix (ECM) provides structural support, cell migration anchorage, cell differentiation cues, and fine-tuned cell proliferation signals during all stages of bone fracture healing, including cartilaginous callus formation, callus remodeling, and bony bridging of the fracture gap. In the present study we have defined the role of the extracellular matrix protein ameloblastin (AMBN) in fracture resistance and fracture healing of mouse long bones. To this end, long bones from WT and AMBNδ5-6 truncation model mice were subjected to biomechanical analysis, fracture healing assays, and stem cell colony formation comparisons. The effect of exogenous AMBN addition to fracture sites was also determined. Our data indicate that lack of a functional AMBN in the bone matrix resulted in 31% decreased femur bone mass and 40% reduced energy to failure. On a cellular level, AMBN function inhibition diminished the proliferative capacity of fracture repair callus cells, as evidenced by a 58% reduction in PCNA and a 40% reduction in Cyclin D1 gene expression, as well as PCNA immunohistochemistry. In terms of fracture healing, AMBN truncation was associated with an enhanced and prolonged chondrogenic phase, resulting in delayed mineralized tissue gene expression and delayed ossification of the fracture repair callus. Underscoring a role of AMBN in fracture healing, there was a 6.9-fold increase in AMBN expression at the fracture site one week after fracture, and distinct AMBN immunolabeling in the fracture gap. Finally, application of exogenous AMBN protein to bone fracture sites accelerated callus formation and bone fracture healing (33% increase in bone volume and 19% increase in bone mineral density), validating the findings of our AMBN loss of function studies. Together, these data demonstrate the functional importance of the AMBN extracellular matrix protein in bone fracture prevention and rapid fracture healing..
59. Kanako Miyazaki, Keigo Yoshizaki, Chieko Arai, Aya Yamada, Kan Saito, Masaki Ishikawa, Han Xue, Keita Funada, Naoto Haruyama, Yoshihiko Yamada, Satoshi Fukumoto, Ichiro Takahashi, Plakophilin-1, a novel Wnt signaling regulator, is critical for tooth development and ameloblast differentiation, PLoS ONE, 10.1371/journal.pone.0152206, 11, 3, e0152206, 2016.03, Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme, and the Wnt signaling pathway is involved in this process. We found that Plakophilin (PKP)1, which is associated with diseases such as ectodermal dysplasia/skin fragility syndrome, was highly expressed in teeth and skin, and was upregulated during tooth development. We hypothesized that PKP1 regulates Wnt signaling via its armadillo repeat domain in a manner similar to β-catenin. To determine its role in tooth development, we performed Pkp1 knockdown experiments using ex vivo organ cultures and cell cultures. Loss of Pkp1 reduced the size of tooth germs and inhibited dental epithelial cell proliferation, which was stimulated by Wnt3a. Furthermore, transfected PKP1-emerald green fluorescent protein was translocated from the plasma membrane to the nucleus upon stimulation with Wnt3a and LiCl, which required the PKP1 N terminus (amino acids 161 to 270). Localization of PKP1, which is known as an adhesion-related desmosome component, shifted to the plasma membrane during ameloblast differentiation. In addition, Pkp1 knockdown disrupted the localization of Zona occludens 1 in tight junctions and inhibited ameloblast differentiation; the two proteins were shown to directly interact by immunoprecipitation. These results implicate the participation of PKP1 in early tooth morphogenesis as an effector of canonical Wnt signaling that controls ameloblast differentiation via regulation of the cell adhesion complex..
60. Jia Liu, Kan Saito, Yuriko Maruya, Takashi Nakamura, Aya Yamada, Emiko Fukumoto, Momoko Ishikawa, Tsutomu Iwamoto, Kanako Miyazaki, Keigo Yoshizaki, Lihong Ge, Satoshi Fukumoto, Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation, Scientific Reports, 10.1038/srep23670, 6, 23670, 2016.03, Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling..
61. Takashi Nakamura, Yuta Chiba, Masahiro Naruse, Kan Saito, Hidemitsu Harada, Satoshi Fukumoto, Globoside accelerates the differentiation of dental epithelial cells into ameloblasts, International Journal of Oral Science, 10.1038/ijos.2016.35, 8, 4, 205-212, 2016.12, © The Author(s) 2016. Tooth crown morphogenesis is tightly regulated by the proliferation and differentiation of dental epithelial cells. Globoside (Gb4), a globo-series glycosphingolipid, is highly expressed during embryogenesis as well as organogenesis, including tooth development. We previously reported that Gb4 is dominantly expressed in the neutral lipid fraction of dental epithelial cells. However, because its functional role in tooth development remains unknown, we investigated the involvement of Gb4 in dental epithelial cell differentiation. The expression of Gb4 was detected in ameloblasts of postnatal mouse molars and incisors. A cell culture analysis using HAT-7 cells, a rat-derived dental epithelial cell line, revealed that Gb4 did not promote dental epithelial cell proliferation. Interestingly, exogenous administration of Gb4 enhanced the gene expression of enamel extracellular matrix proteins such as ameloblastin, amelogenin, and enamelin in dental epithelial cells as well as in developing tooth germs. Gb4 also induced the expression of TrkB, one of the key receptors required for ameloblast induction in dental epithelial cells. In contrast, Gb4 downregulated the expression of p75, a receptor for neurotrophins (including neurotrophin-4) and a marker of undifferentiated dental epithelial cells. In addition, we found that exogenous administration of Gb4 to dental epithelial cells stimulated the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase signalling pathways. Furthermore, Gb4 induced the expression of epiprofin and Runx2, the positive regulators for ameloblastin gene transcription. Thus, our results suggest that Gb4 contributes to promoting the differentiation of dental epithelial cells into ameloblasts..
62. Aya Yamada, Masaharu Futagi, Emiko Fukumoto, Kan Saito, Keigo Yoshizaki, Masaki Ishikawa, Makiko Arakaki, Ryoko Hino, Yu Sugawara, Momoko Ishikawa, Masahiro Naruse, Kanako Miyazaki, Takashi Nakamura, Satoshi Fukumoto, Connexin 43 is necessary for salivary gland branching morphogenesis and FGF10-induced ERK1/2 phosphorylation, Journal of Biological Chemistry, 10.1074/jbc.M115.674663, 291, 2, 904-912, 2016.01, © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43-/- salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43-/- samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43-/- phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis..
63. Mebae Takahashi, Kazuhiro Shima, Masahiro Tsuchiya, Yoshihiro Hagiwara, Hirokazu Mizoguchi, Shinobu Sakurada, Shunji Sugawara, Takuo Fujita, Takeshi Tadano, Makoto Watanabe, Satoshi Fukumoto, Yasuo Endo, Analgesic effects of 1st generation anti-histamines in mice, Biological and Pharmaceutical Bulletin, 10.1248/bpb.b15-00755, 39, 4, 620-624, 2016.04, © 2016 The Pharmaceutical Society of Japan. Pain is sensed, transmitted, and modified by a variety of mediators and receptors. Histamine is a wellknown mediator of pain. In addition to their anti-histaminic effects, the classical, or 1st generation, anti-histamines (1st AHs) possess, to various degrees, anti-muscarinic, anti-serotonergic, anti-adrenergic, and other pharmacologic effects. Although there have been attempts to use 1st AHs as analgesics and/or analgesic adjuvants, the advent of non-steroidal anti-inflammatory drugs (NSAIDs) discouraged such trials. We previously reported that in patients with temporomandibular disorders, osteoporosis, and/or osteoarthritis, the analgesic effects of certain 1st AHs (chlorpheniramine and diphenhydramine) are superior to those of the NSAIDs flurbiprofen and indomethacin. Here, we compared analgesic effects among 1st AHs and NSAIDs against responses shown by mice to intraperitoneally injected 0.7% acetic acid. Since 1st AHs are water soluble, we selected water-soluble NSAIDs. For direct comparison, drugs were intravenously injected 30 min before the above tests. Histamine-H1-receptor-deficient (H1R-KO) mice were used for evaluating H1-receptor-independent effects. The tested 1st AHs (especially cyproheptadine) displayed or tended to display analgesic effects comparable to those of NSAIDs in normal and H1R-KO mice. Our data suggest that the anti-serotonergic and/or anti-adrenergic effects of 1st AHs make important contributions to their analgesic effects. Moreover, combination of a 1st AH with an NSAID (cyclooxygenase-1 inhibitor) produced remarkably potent analgesic effects. We propose that a 1st AH, by itself or in combination with a cyclooxygenase-1 inhibitor, should undergo testing to evaluate its usefulness in analgesia..
64. Xuanyu Lu, Satoshi Fukumoto, Yoshihiko Yamada, Carla A. Evans, Thomas G.H. Diekwisch, Xianghong Luan, Ameloblastin, an Extracellular Matrix Protein, Affects Long Bone Growth and Mineralization, Journal of Bone and Mineral Research, 10.1002/jbmr.2788, 31, 6, 1235-1246, 2016.06, © 2016 American Society for Bone and Mineral Research. Matrix molecules such as the enamel-related calcium-binding phosphoprotein ameloblastin (AMBN) are expressed in multiple tissues, including teeth, bones, and cartilage. Here we have asked whether AMBN is of functional importance for timely long bone development and, if so, how it exerts its function related to osteogenesis. Adolescent AMBN-deficient mice (AMBNΔ5-6) suffered from a 33% to 38% reduction in femur length and an 8.4% shorter trunk spinal column when compared with WT controls, whereas there was no difference between adult animals. On a cellular level, AMBN truncation resulted in a shortened growth plate and a 41% to 49% reduction in the number of proliferating tibia chondrocytes and osteoblasts. Bone marrow stromal cells (BMSCs) isolated from AMBN mutant mice displayed defects in proliferation and differentiation potential as well as cytoskeleton organization. Osteogenesis-related growth factors, such as insulin-like growth factor 1 (IGF1) and BMP7, were also significantly (46% to 73%) reduced in AMBN-deficient BMSCs. Addition of exogenous AMBN restored cytoskeleton structures in AMBN mutant BMSCs and resulted in a dramatic 400% to 600% increase in BMP2, BMP7, and Col1A expression. Block of RhoA diminished the effect of AMBN on osteogenic growth factor and matrix protein gene expression. Addition of exogenous BMP7 and IGF1 rescued the proliferation and differentiation potential of AMBN-deficient BMSCs. Confirming the effects of AMBN on long bone growth, back-crossing of mutant mice with full-length AMBN overexpressors resulted in a complete rescue of AMBNΔ5-6 bone defects. Together, these data indicate that AMBN affects extracellular matrix production and cell adhesion properties in the long bone growth plate, resulting in altered cytoskeletal dynamics, increased osteogenesis-related gene expression, as well as osteoblast and chondrocyte proliferation. We propose that AMBN facilitates rapid long bone growth and an important growth spurt during the skeletogenesis of adolescent tooth-bearing vertebrates..
65. Shimizu K, Fukushima H, Ogura K, Lien EC, Nihira NT, Zhang J, North BJ, Guo A, Nagashima K, Nakagawa T, Hoshikawa S, Watahiki A, Okabe K, Yamada A, Toker A, Asara JM, Fukumoto S, Nakayama KI, Nakayama K, Inuzuka H, Wei W, The SCFβ-TRCP E3 ubiquitin ligase complex targets Lipin1 for ubiquitination and degradation to promote hepatic lipogenesis., Science signaling, 10.1126/scisignal.aah4117, 10, 460, 2017.01.
66. Asuna Sugimoto, Aya Miyazaki, Keita Kawarabayashi, Masayuki Shono, Yuki Akazawa, Tomokazu Hasegawa, Kimiko Ueda-Yamaguchi, Takamasa Kitamura, Keigo Yoshizaki, Satoshi Fukumoto, Tsutomu Iwamoto, Piezo type mechanosensitive ion channel component 1 functions as a regulator of the cell fate determination of mesenchymal stem cells, Scientific Reports, 10.1038/s41598-017-18089-0, 7, 1, 17696, 2017.12, © 2017 The Author(s). The extracellular environment regulates the dynamic behaviors of cells. However, the effects of hydrostatic pressure (HP) on cell fate determination of mesenchymal stem cells (MSCs) are not clearly understood. Here, we established a cell culture chamber to control HP. Using this system, we found that the promotion of osteogenic differentiation by HP is depend on bone morphogenetic protein 2 (BMP2) expression regulated by Piezo type mechanosensitive ion channel component 1 (PIEZO1) in MSCs. The PIEZO1 was expressed and induced after HP loading in primary MSCs and MSC lines, UE7T-13 and SDP11. HP and Yoda1, an activator of PIEZO1, promoted BMP2 expression and osteoblast differentiation, whereas inhibits adipocyte differentiation. Conversely, PIEZO1 inhibition reduced osteoblast differentiation and BMP2 expression. Furthermore, Blocking of BMP2 function by noggin inhibits HP induced osteogenic maker genes expression. In addition, in an in vivo model of medaka with HP loading, HP promoted caudal fin ray development whereas inhibition of piezo1 using GsMTx4 suppressed its development. Thus, our results suggested that PIEZO1 is responsible for HP and could functions as a factor for cell fate determination of MSCs by regulating BMP2 expression..
67. Tsutomu Iwamoto, Takashi Nakamura, Masaki Ishikawa, Keigo Yoshizaki, Asuna Sugimoto, Hiroko Ida-Yonemochi, Hayato Ohshima, Masahiro Saito, Yoshihiko Yamada, Satoshi Fukumoto, Pannexin 3 regulates proliferation and differentiation of odontoblasts via its hemichannel activities, PLoS ONE, 10.1371/journal.pone.0177557, 12, 5, e0177557, 2017.05, © This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Highly coordinated regulation of cell proliferation and differentiation contributes to the formation of functionally shaped and sized teeth; however, the mechanism underlying the switch from cell cycle exit to cell differentiation during odontogenesis is poorly understood. Recently, we identified pannexin 3 (Panx3) as a member of the pannexin gap junction protein family from tooth germs. The expression of Panx3 was predominately localized in preodontoblasts that arise from dental papilla cells and can differentiate into dentin-secreting odontoblasts. Panx3 also co-localized with p21, a cyclin-dependent kinase inhibitor protein, in preodontoblasts. Panx3 was expressed in primary dental mesenchymal cells and in the mDP dental mesenchymal cell line. Both Panx3 and p21 were induced during the differentiation of mDP cells. Overexpression of Panx3 in mDP cells reduced cell proliferation via upregulation of p21, but not of p27, and promoted the Bone morphogenetic protein 2 (BMP2)-induced phosphorylation of Smad1/5/8 and the expression of dentin sialophosphoprotein (Dspp), a marker of differentiated odontoblasts. Furthermore, Panx3 released intracellular ATP into the extracellular space through its hemichannel and induced the phosphorylation of AMP-activated protein kinase (AMPK). 5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR), an activator of AMPK, reduced mDP cell proliferation and induced p21 expression. Conversely, knockdown of endogenous Panx3 by siRNA inhibited AMPK phosphorylation, p21 expression, and the phosphorylation of Smad1/5/8 even in the presence of BMP2. Taken together, our results suggest that Panx3 modulates intracellular ATP levels, resulting in the inhibition of odontoblast proliferation through the AMPK/p21 signaling pathway and promotion of cell differentiation by the BMP/Smad signaling pathway..
68. Chieko Arai, Keigo Yoshizaki, Kanako Miyazaki, Kan Saito, Aya Yamada, Xue Han, Keita Funada, Emiko Fukumoto, Naoto Haruyama, Tsutomu Iwamoto, Ichiro Takahashi, Satoshi Fukumoto, Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains, Scientific Reports, 10.1038/srep45181, 7, 45181, 2017.03, © 2017 The Author(s). Tooth development is initiated by epithelial-mesenchymal interactions via basement membrane (BM) and growth factors. In the present study, we found that nephronectin (Npnt), a component of the BM, is highly expressed in the developing tooth. Npnt localizes in the BM on the buccal side of the tooth germ and shows an expression pattern opposite that of the dental epithelial stem cell marker Sox2. To identify the roles of Npnt during tooth development, we performed knockdown and overexpression experiments using ex vivo organ and dental epithelial cell cultures. Our findings showed that loss of Npnt induced ectopic Sox2-positive cells and reduced tooth germ size. Over expression of Npnt showed increased proliferation, whereas the number of Sox2-positive cells was decreased in dental epithelial cells. Npnt contains 5 EGF-like repeat domains, as well as an RGD sequence and MAM domain. We found that the EGF-like repeats are critical for Sox2 expression and cell proliferation. Furthermore, Npnt activated the EGF receptor (EGFR) via the EGF-like repeat domains and induced the PI3K-Akt signaling pathway. Our results indicate that Npnt plays a critical scaffold role in dental epithelial stem cell differentiation and proliferation, and regulates Sox2 expression during tooth development..
69. Hidefumi Fukushima, Kouhei Shimizu, Asami Watahiki, Seira Hoshikawa, Tomoki Kosho, Daiju Oba, Seiji Sakano, Makiko Arakaki, Aya Yamada, Katsuyuki Nagashima, Koji Okabe, Satoshi Fukumoto, Eijiro Jimi, Anna Bigas, Keiichi I. Nakayama, Keiko Nakayama, Yoko Aoki, Wenyi Wei, Hiroyuki Inuzuka, NOTCH2 Hajdu-Cheney Mutations Escape SCFFBW7-Dependent Proteolysis to Promote Osteoporosis, Molecular Cell, 10.1016/j.molcel.2017.10.018, 68, 4, 645-658.e5, 2017.11, © 2017 Elsevier Inc. Hajdu-Cheney syndrome (HCS), a rare autosomal disorder caused by heterozygous mutations in NOTCH2, is clinically characterized by acro-osteolysis, severe osteoporosis, short stature, neurological symptoms, cardiovascular defects, and polycystic kidneys. Recent studies identified that aberrant NOTCH2 signaling and consequent osteoclast hyperactivity are closely associated with the bone-related disorder pathogenesis, but the exact molecular mechanisms remain unclear. Here, we demonstrate that sustained osteoclast activity is largely due to accumulation of NOTCH2 carrying a truncated C terminus that escapes FBW7-mediated ubiquitination and degradation. Mice with osteoclast-specific Fbw7 ablation revealed osteoporotic phenotypes reminiscent of HCS, due to elevated Notch2 signaling. Importantly, administration of Notch inhibitors in Fbw7 conditional knockout mice alleviated progressive bone resorption. These findings highlight the molecular basis of HCS pathogenesis and provide clinical insights into potential targeted therapeutic strategies for skeletal disorders associated with the aberrant FBW7/NOTCH2 pathway as observed in patients with HCS. Fukushima et al. demonstrated that the sustained osteoclast activity in Hajdu-Cheney syndrome (HCS) is largely due to elevated protein abundance of the C terminus truncating NOTCH2 mutant that escapes FBW7-mediated ubiquitination and proteolysis, suggesting that the FBW7/NOTCH2 signaling pathway is a potential therapeutic target for osteolytic bone disorders, including HCS..
70. Keigo Yoshizaki, Lizhi Hu, Thai Nguyen, Kiyoshi Sakai, Masaki Ishikawa, Ichiro Takahashi, Satoshi Fukumoto, Pamela K. DenBesten, Daniel D. Bikle, Yuko Oda, Yoshihiko Yamada, Mediator 1 contributes to enamel mineralization as a coactivator for Notch1 signaling and stimulates transcription of the alkaline phosphatase gene, Journal of Biological Chemistry, 10.1074/jbc.M117.780866, 292, 33, 13531-13540, 2017.08, Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1-deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1–RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene..
71. Takashi Nakamura, Lucia Jimenez-Rojo, Eiki Koyama, Maurizio Pacifici, Susana de Vega, Masahiro Iwamoto, Satoshi Fukumoto, Fernando Unda, Yoshihiko Yamada, Epiprofin Regulates Enamel Formation and Tooth Morphogenesis by Controlling Epithelial-Mesenchymal Interactions During Tooth Development, Journal of Bone and Mineral Research, 10.1002/jbmr.3024, 32, 3, 601-610, 2017.03, © 2016 American Society for Bone and Mineral Research The synchronization of cell proliferation and cytodifferentiation between dental epithelial and mesenchymal cells is required for the morphogenesis of teeth with the correct functional shapes and optimum sizes. Epiprofin (Epfn), a transcription factor belonging to the Sp family, regulates dental epithelial cell proliferation and is essential for ameloblast and odontoblast differentiation. Epfn deficiency results in the lack of enamel and ironically the formation of extra teeth. We investigated the mechanism underlying the functions of Epfn in tooth development through the creation of transgenic mice expressing Epfn under the control of an epithelial cell-specific K5 promoter (K5-Epfn). We found that these K5-Epfn mice developed abnormally shaped incisors and molars and formed fewer molars in the mandible. Remarkably, ameloblasts differentiated ectopically and enamel was formed on the lingual side of the K5-Epfn incisors. By contrast, ameloblasts and enamel were found only on the labial side in wild-type mice, as Follistatin (Fst) expressed in the lingual side inhibits BMP4 signaling necessary for ameloblast differentiation. We showed that Epfn transfection into the dental epithelial cell line SF2 abrogated the inhibitory activity of Fst and promoted ameloblast differentiation of SF2 cells. We found that Epfn induced FGF9 in dental epithelial cells and this dental epithelial cell-derived FGF9 promoted dental mesenchymal cell proliferation via the FGF receptor 1c (FGFR1c). Taken together, these results suggest that Epfn preserves the balance between cell proliferation and cytodifferentiation in dental epithelial and mesenchymal cells during normal tooth development and morphogenesis. © 2016 American Society for Bone and Mineral Research..
72. Han Xue, Yoshizaki Keigo, Miyazaki Kanako, Arai Chieko, Funada Keita, Yuta Tomomi, Tian Tian, Chiba Yuta, Saito Kan, Tsutomu Iwamoto, Yamada Aya, Takahashi Ichiro, Fukumoto Satoshi, The transcription factor NKX2-3 mediates p21 expression and ectodysplasin-A signaling in the enamel knot for cusp formation in tooth development, The Journal of Biological Chemistry, 10.1074/jbc.RA118.003373, Vol.293, No.38, 14572-14584, 2018.08, Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme. During tooth development, tooth cusps are regulated by precise control of proliferation of cell clusters, termed enamel knots, that are present among dental epithelial cells. The interaction of ectodysplasin-A (EDA) with its receptor, EDAR, plays a critical role in cusp formation by these enamel knots, and mutations of these genes is a cause of ectodermal dysplasia. It has also been reported that deficiency in , encoding a member of the NK2 homeobox family of transcription factors, leads to cusp absence in affected teeth. However, the molecular role of NKX2-3 in tooth morphogenesis is not clearly understood. Using gene microarray analysis in mouse embryos, we found that is highly expressed during tooth development and increased during the tooth morphogenesis, especially during cusp formation. We also demonstrate that NKX2-3 is a target molecule of EDA and critical for expression of the cell cycle regulator p21 in the enamel knot. Moreover, NKX2-3 activated the bone morphogenetic protein (BMP) signaling pathway by up-regulating expression levels of and in dental epithelium and decreased the expression of the dental epithelial stem cell marker SRY box 2 (SOX2). Together, our results indicate that EDA/NKX2-3 signaling is essential for enamel knot formation during tooth morphogenesis in mice..
73. Masato Saitoh, Yuki Nakamura, Mika Hanasaki, Issei Saitoh, Yuji Murai, Yoshihito Kurashige, Satoshi Fukumoto, Yukiko Asaka, Masaaki Yamada, Michikazu Sekine, Haruaki Hayasaki, Shigenari Kimoto, Prevalence of molar incisor hypomineralization and regional differences throughout Japan, Environmental Health and Preventive Medicine, 10.1186/s12199-018-0748-6, 23, 1, 1-6, 2018.10, © 2018 The Author(s). Background: Molar incisor hypomineralization (MIH) frequently occurs in children worldwide. However, MIH prevalence throughout Japan has not yet been investigated. The purpose of this study was to clarify MIH prevalence rates and to consider potential regional differences throughout Japan. Methods: A total of 4496 children aged 7-9 years throughout Japan were evaluated in this study. MIH prevalence rates among children were evaluated in eight regions throughout Japan. A child's residence was defined as the mother's residence during pregnancy. The localization of demarcated opacities and enamel breakdown was recorded on a standard code form using a guided record chart. Logistic regression analysis was used to evaluate whether MIH prevalence rates differed among age groups, sex, and regions. Results: The overall prevalence of MIH in Japan was 19.8%. The prevalence of MIH was 14.0% in the Hokkaido region, 11.7% in the Tohoku region, 18.5% in the Kanto Shin-Etsu region, 19.3% in the Tokai Hokuriku region, 22.3% in the Kinki region, 19.8% in the Chugoku region, 28.1% in the Shikoku region, and 25.3% in the Kyushu region. These regional differences were statistically significant. Moreover, MIH prevalence rates decreased with age. No significant sex differences in MIH prevalence rates were demonstrated. Conclusions: To our knowledge, this is the first MIH study carried out in several regions throughout Japan. Regional differences existed in MIH prevalence rates; particularly, MIH occurred more frequently in children residing in southwestern areas than those in northeastern areas of Japan..
74. Yuta Chiba, Bing He, Keigo Yoshizaki, Craig Rodes, Muneaki Ishijima, Christopher, K.E. Bleck, Erin Stempinski, Emily Y. Chu, Takashi Nakamura, Tsutomu Iwamoto, Susana de Vega, Kan Saito, Satoshi Fukumoto, Yoshihiko Yamada, The transcription factor AmeloD stimulates epithelial cell motility essential for tooth morphology, The Journal of Biological Chemistry, 10.1074/jbc.RA118.005298, Vol.294, No.10, 3406-3418, 2019.03, The development of ectodermal organs, such as teeth, requires epithelial-mesenchymal interactions. Basic-helix-loop-helix (bHLH) transcription factors regulate various aspects of tissue development, and we have previously identified a bHLH transcription factor, AmeloD, from a tooth germ cDNA library. Here, we provide both in vitro and in vivo evidence that AmeloD is important in tooth development. We created AmeloD knockout (KO) mice to identify the in vivo functions of AmeloD that are critical for tooth morphogenesis. We found that AmeloD KO mice developed enamel hypoplasia and small teeth because of increased expression of E-cadherin in inner enamel epithelial (IEE) cells and it may cause inhibition of the cellmigration. We used the CLDE dental epithelial cell line to conduct further mechanistic analyses to determine if AmeloD overexpression in CLDE cells suppresses E-cadherin expression and promotes cell migration. Knockout of epiprofin (Epfn), another transcription factor required for tooth morphogenesis and development, and analysis of AmeloD expression and deletion revealed that AmeloD also contributed to multiple tooth formation in Epfn KO mice by promoting the invasion of dental epithelial cells into the mesenchymal region. Thus, AmeloD appears to play an important role in tooth morphogenesis by modulating E-cadherin and dental epithelial-mesenchymal interactions. These findings provide detailed insights into the mechanism of ectodermal organ development..
75. 孫 梟, 加藤 大樹, 佐藤 浩, 鳥尾 倫子, 韓 旭, 張 虞, 廣藤 雄太, 加藤 隆弘, 酒井 康成, 大賀 正一, 福本 敏, 増田 啓次, 変容性骨異形成症患者由来の歯髄幹細胞から分化したドーパミン作動性ニューロンではカルシウム蓄積によりミトコンドリア障害と神経突起発達不全をきたす(Impaired neurite development and mitochondrial dysfunction associated with calcium accumulation in dopaminergic neurons differentiated from the dental pulp stem cells of a patient with metatropic dysplasia), 日本神経精神薬理学会年会・日本生物学的精神医学会年会・日本精神薬学会総会・学術集会合同年会プログラム・抄録集, 50回・42回・4回, 201-201, 2020.08.
76. Keita Funada, Keigo Yoshizaki, Kanako MIyazaki, Xue Han, Tomomi Yuta, Tian Tian, Kanji Mizuta, Yao Fu, Tsutomu Iwamoto, Aya Yamada, Ichiro Takahashi, Satoshi Fukumoto, microRNA-875-5p plays critical role for mesenchymal condensation in epithelial-mesenchymal interaction during tooth development., Scientific reports, 10.1038/s41598-020-61693-w, 10, 1, 4918-4918, 2020.03, Epithelial-mesenchymal interaction has critical roles for organ development including teeth, during which epithelial thickening and mesenchymal condensation are initiated by precise regulation of the signaling pathway. In teeth, neural crest-derived mesenchymal cells expressed PDGF receptors migrate and become condensed toward invaginated epithelium. To identify the molecular mechanism of this interaction, we explored the specific transcriptional start sites (TSSs) of tooth organs using cap analysis of gene expression (CAGE). We identified a tooth specific TSS detected in the chromosome 15qD1 region, which codes microRNA-875 (mir875). MiR875-5p is specifically expressed in dental mesenchyme during the early stage of tooth development. Furthermore, PRRX1/2 binds to the mir875 promoter region and enhances the expression of mir875. To assess the role of miR875-5p in dental mesenchyme, we transfected mimic miR875-5p into mouse dental pulp (mDP) cells, which showed that cell migration toward dental epithelial cells was significantly induced by miR875-5p via the PDGF signaling pathway. Those results also demonstrated that miR875-5p induces cell migration by inhibiting PTEN and STAT1, which are regulated by miR875-5p as part of post-transcriptional regulation. Together, our findings indicate that tooth specific miR875-5p has important roles in cell condensation of mesenchymal cells around invaginated dental epithelium and induction of epithelial-mesenchymal interaction..
77. Yuria Sato-Suzuki, Jumpei Washio, Dimas Prasetianto Wicaksono, Takuichi Sato, Satoshi Fukumoto, Nobuhiro Takahashi, Nitrite-producing oral microbiome in adults and children., Scientific reports, 10.1038/s41598-020-73479-1, 10, 1, 16652-16652, 2020.10, Recently, it was suggested that the nitrite (NO2-) produced from NO3- by oral bacteria might contribute to oral and general health. Therefore, we aimed to clarify the detailed information about the bacterial NO2-production in the oral biofilm. Dental plaque and tongue-coating samples were collected, then the NO2-producing activity was measured. Furthermore, the composition of the NO2--producing bacterial population were identified using the Griess reagent-containing agar overlay method and molecular biological method. NO2--producing activity per mg wet weight varied among individuals but was higher in dental plaque. Additionally, anaerobic bacteria exhibited higher numbers of NO2--producing bacteria, except in the adults' dental plaque. The proportion of NO2--producing bacteria also varied among individuals, but a positive correlation was found between NO2--producing activity and the number of NO2--producing bacteria, especially in dental plaque. Overall, the major NO2--producing bacteria were identified as Actinomyces, Schaalia, Veillonella and Neisseria. Furthermore, Rothia was specifically detected in the tongue coatings of children. These results suggest that dental plaque has higher NO2--producing activity and that this activity depends not on the presence of specific bacteria or the bacterial compositions, but on the number of NO2--producing bacteria, although interindividual differences were detected..
78. Yuta Chiba, Keigo Yoshizaki, Kan Saito, Tomoko Ikeuchi, Tsutomu Iwamoto, Craig Rhodes, Takashi Nakamura, Susana de Vega, Robert J Morell, Erich T Boger, Daniel Martin, Ryoko Hino, Hiroyuki Inuzuka, Christopher K E Bleck, Aya Yamada, Yoshihiko Yamada, Satoshi Fukumoto, G protein-coupled receptor Gpr115 (Adgrf4) is required for enamel mineralization mediated by ameloblasts., The Journal of Biological Chemistry, 10.1074/jbc.RA120.014281, 295, 45, 15328-15341, 2020.11, Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G protein-coupled receptors (GPCRs) function as transducers of external signals by activating associated G proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, although their activities in tooth development remain poorly understood. The present results show that the adhesion GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypomineralized enamel, with a larger acidic area because of the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line cervical loop-derived dental epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation..
79. Dong-Wook Yang, Jung-Sun Moon, Hyun-Mi Ko, Yeo-Kyeong Shin, Satoshi Fukumoto, Sun-Hun Kim, Min-Seok Kim, Direct reprogramming of fibroblasts into diverse lineage cells by DNA demethylation followed by differentiating cultures., The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology, 10.4196/kjpp.2020.24.6.463, 24, 6, 463-472, 2020.11, Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods. In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/ western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine..
80. Shahad Al Thamin, Yuta Chiba, Keigo Yoshizaki, Tian Tian, LingLing Jia, Xin Wang, Kan Saito, Jiyao Li, Aya Yamada, Satoshi Fukumoto, Transcriptional regulation of the basic helix-loop-helix factor AmeloD during tooth development., Journal of Cellular Physiology, 10.1002/jcp.30389, 236, 11, 7533-7543, 2021.04, The epithelial-mesenchymal interactions are essential for the initiation and regulation of the development of teeth. Following the initiation of tooth development, numerous growth factors are secreted by the dental epithelium and mesenchyme that play critical roles in cellular differentiation. During tooth morphogenesis, the dental epithelial stem cells differentiate into several cell types, including inner enamel epithelial cells, which then differentiate into enamel matrix-secreting ameloblasts. Recently, we reported that the novel basic-helix-loop-helix transcription factor, AmeloD, is actively engaged in the development of teeth as a regulator of dental epithelial cell motility. However, the gene regulation mechanism of AmeloD is still unknown. In this study, we aimed to uncover the mechanisms regulating AmeloD expression during tooth development. By screening growth factors that are important in the early stages of tooth formation, we found that TGF-β1 induced AmeloD expression and ameloblast differentiation in the dental epithelial cell line, SF2. TGF-β1 phosphorylated ERK1/2 and Smad2/3 to induce AmeloD expression, whereas treatment with the MEK inhibitor, U0126, inhibited AmeloD induction. Promoter analysis of AmeloD revealed that the proximal promoter of AmeloD showed high activity in dental epithelial cell lines, which was enhanced following TGF-β1 stimulation. These results suggested that TGF-β1 activates AmeloD transcription via ERK1/2 phosphorylation. Our findings provide new insights into the mechanisms that govern tooth development..
81. Keiji Masuda, Xu Han, Hiroki Kato, Hiroshi Sato, Yu Zhang, Xiao Sun, Yuta Hirofuji, Haruyoshi Yamaza, Aya Yamada, Satoshi Fukumoto, Dental Pulp-Derived Mesenchymal Stem Cells for Modeling Genetic Disorders., International Journal of Molecular Sciences, 10.3390/ijms22052269, 22, 5, 1-18, 2021.02, A subpopulation of mesenchymal stem cells, developmentally derived from multipotent neural crest cells that form multiple facial tissues, resides within the dental pulp of human teeth. These stem cells show high proliferative capacity in vitro and are multipotent, including adipogenic, myogenic, osteogenic, chondrogenic, and neurogenic potential. Teeth containing viable cells are harvested via minimally invasive procedures, based on various clinical diagnoses, but then usually discarded as medical waste, indicating the relatively low ethical considerations to reuse these cells for medical applications. Previous studies have demonstrated that stem cells derived from healthy subjects are an excellent source for cell-based medicine, tissue regeneration, and bioengineering. Furthermore, stem cells donated by patients affected by genetic disorders can serve as in vitro models of disease-specific genetic variants, indicating additional applications of these stem cells with high plasticity. This review discusses the benefits, limitations, and perspectives of patient-derived dental pulp stem cells as alternatives that may complement other excellent, yet incomplete stem cell models, such as induced pluripotent stem cells, together with our recent data..
82. Wang X, Chiba Y, Jia L, Yoshizaki K, Saito K, Yamada A, Qin M, Fukumoto S., Expression Patterns of Claudin Family Members During Tooth Development and the Role of Claudin-10 (Cldn10) in Cytodifferentiation of Stratum Intermedium, Front Cell Dev Biol, 10.3389/fcell.2020.595593, 2021.06.
83. Chiba Y, Yoshizaki K, Saito K, Ikeuchi T, Iwamoto T, Rhodes C, Nakamura T, de Vega S, Morell RJ, Boger ET, Martin D, Hino R, Inuzuka H, Bleck CKE, Yamada A, Yamada Y, Fukumoto S., G protein-coupled receptor Gpr115 (Adgrf4) is required for enamel mineralization mediated by ameloblasts, J Biol Chem, 10.1074/jbc.RA120.014281, 2020.09.
84. Hoshikawa S, Shimizu K, Watahiki A, Chiba M, Saito K, Wei W, Fukumoto S, Inuzuka H., Phosphorylation-dependent osterix degradation negatively regulates osteoblast differentiation, FASEB J, 10.1096/fj.202001340R, 2020.09.
85. Sato-Suzuki Y, Washio J, Wicaksono DP, Sato T, Fukumoto S, Takahashi N., Nitrite-producing oral microbiome in adults and children, Sci Rep, 10.1038/s41598-020-73479-1, 2020.10.
86. Yoshizaki K, Fukumoto S, Bikle DD, Oda Y., Transcriptional Regulation of Dental Epithelial Cell Fate, Int J Mol Sci. , 10.3390/ijms21238952, 2020.12.
87. Chiba Y, Saito K, Martin D, Boger ET, Rhodes C, Yoshizaki K, Nakamura T, Yamada A, Morell RJ, Yamada Y, Fukumoto S., Single-Cell RNA-Sequencing From Mouse Incisor Reveals Dental Epithelial Cell-Type Specific Genes, Front Cell Dev Biol, 10.3389/fcell.2020.00841, 2020.09.
88. Suzuki T, Uruno A, Yumoto A, Taguchi K, Suzuki M, Harada N, Ryoke R, Naganuma E, Osanai N, Goto A, Suda H, Browne R, Otsuki A, Katsuoka F, Zorzi M, Yamazaki T, Saigusa D, Koshiba S, Nakamura T, Fukumoto S, Ikehata H, Nishikawa K, Suzuki N, Hirano I, Shimizu R, Oishi T, Motohashi H, Tsubouchi H, Okada R, Kudo T, Shimomura M, Kensler TW, Mizuno H, Shirakawa M, Takahashi S, Shiba D, Yamamoto M., Nrf2 contributes to the weight gain of mice during space travel, Commun Biol, 10.1038/s42003-020-01227-2, 2020.09.
89. Kimiko Yamaguchi-Ueda 1 , Yuki Akazawa 1 , Keita Kawarabayashi 1 , Asuna Sugimoto 1 , Hiroshi Nakagawa 1 , Aya Miyazaki 1 , Rika Kurogoushi 1 , Kokoro Iwata 1 , Takamasa Kitamura 1 , Aya Yamada 2 , Tomokazu Hasegawa 1 , Satoshi Fukumoto 2 , Tsutomu Iwamoto 1, Combination of ions promotes cell migration via extracellular signal‑regulated kinase 1/2 signaling pathway in human gingival fibroblasts, Mol Med Rep, 10.3892/mmr.2019.10141, 19, 6, 5039-5045, 2019.06, Wound healing is a dynamic process that involves highly coordinated cellular events, including proliferation and migration. Oral gingival fibroblasts serve a central role in maintaining oral mucosa homeostasis, and their functions include the coordination of physiological tissue repair. Recently, surface pre‑reacted glass‑ionomer (S‑PRG) fillers have been widely applied in the field of dental materials for the prevention of dental caries, due to an excellent ability to release fluoride (F). In addition to F, S‑PRG fillers are known to release several types of ions, including aluminum (Al), boron (B), sodium (Na), silicon (Si) and strontium (Sr). However, the influence of these ions on gingival fibroblasts remains unknown. The aim of the present study was to examine the effect of various concentrations of an S‑PRG filler eluate on the growth and migration of gingival fibroblasts. The human gingival fibroblast cell line HGF‑1 was treated with various dilutions of an eluent solution of S‑PRG, which contained 32.0 ppm Al, 1,488.6 ppm B, 505.0 ppm Na, 12.9 ppm Si, 156.5 ppm Sr and 136.5 ppm F. Treatment with eluate at a dilution of 1:10,000 was observed to significantly promote the migration of HGF‑1 cells. In addition, the current study evaluated the mechanism underlying the mediated cell migration by the S‑PRG solution and revealed that it activated the phosphorylation of extracellular signal‑regulated kinase 1/2 (ERK1/2), but not of p38. Furthermore, treatment with a MEK inhibitor blocked the cell migration induced by the solution. Taken together, these results suggest that S‑PRG fillers can stimulate HGF‑1 cell migration via the ERK1/2 signaling pathway, indicating that a dental material containing this type of filler is useful for oral mucosa homeostasis and wound healing..
90. Masaki Ishikawa 1 , Geneva Williams 2 , Patricia Forcinito 2 , Momoko Ishikawa 3 , Ryan J Petrie 4 , Kan Saito 3 , Satoshi Fukumoto 3 , Yoshihiko Yamada 2, Pannexin 3 ER Ca 2+ channel gating is regulated by phosphorylation at the Serine 68 residue in osteoblast differentiation, Sci Rep, 10.1038/s41598-019-55371-9, 9, 1, 18759, 2019.12, Pannexin 3 (Panx3) is a regulator of bone formation. Panx3 forms three distinct functional channels: hemichannels, gap junctions, and endoplasmic reticulum (ER) Ca2+ channels. However, the gating mechanisms of the Panx3 channels remain unclear. Here, we show that the Panx3 ER Ca2+ channel is modulated by phosphorylation of the serine 68 residue (Ser68) to promote osteoblast differentiation. Among the 17 candidate phosphorylation sites identified, the mutation of Ser68 to Ala (Ser68Ala) was sufficient to inhibit Panx3-mediated osteoblast differentiation via reduction of Osterix and ALP expression. Using a Ser68 phospho-specific antibody (P-Panx3) revealed Panx3 was phosphorylated in prehypertrophic, hypertrophic chondrocytes, and bone areas of the newborn growth plate. In osteogenic C2C12 cells, P-Panx3 was located on the ER membranes. Importantly, the Ser68Ala mutation only affected Panx3 ER Ca2+ channel function. Ser68 on Panx3 was phosphorylated by ATP stimulation and PI3K/Akt signaling. Finally, real-time FRET imaging and ratio analysis revealed that the Panx3 channel conformation was sensitive to ATP. Together, the phosphorylation of Panx3 at Ser68 is an essential step controlling the gating of the Panx3 ER Ca2+ channel to promote osteogenesis..
91. Yukinori Tanaka 1 , Satoshi Fukumoto 2 , Shunji Sugawara 3 , Mechanisms underlying the induction of regulatory T cells by sublingual immunotherapy, J Oral Biosci, 10.1016/j.job.2019.02.001, 61, 2, 73-77, 2019.06, Background: Sublingual immunotherapy (SLIT) is used for the treatment of type 1 allergies, such as allergic rhinitis. SLIT leads to tolerance against allergens possibly via the redirection of allergen-specific T helper 2 cells to T helper 1 cells and the generation of peripheral regulatory T (Treg) cells. However, the detailed mechanisms remain unclear. Systemic tolerance to orally administered antigens (oral tolerance) has been extensively investigated. Recent studies have recognized the central role of Treg cells and classical dendritic cells (cDCs) in oral tolerance development.

Highlight: This review focuses on recent advances in the understanding of the underlying mechanisms of SLIT compared with those of oral tolerance. The sublingual administration of soluble protein antigens has been reported to induce antigen-specific Treg cells in oral mucosa-draining submandibular lymph nodes in mice. The generation of Treg cells is critical for SLIT efficacy because the transfer of SLIT-induced Treg cells confers tolerance against the antigens. A large number of oral cDCs with the CD103-CD11b+ phenotype exert retinoic acid-producing activity and convert naïve CD4+ T cells into Foxp3+ Treg cells in vitro in a transforming growth factor-β-dependent and retinoic acid-dependent manner. Oral CD103-CD11b+ cDCs transport sublingual antigens to submandibular lymph nodes and induce antigen-specific Treg cells. Sublingual antigens enter the mucosa most likely by crossing the sublingual ductal epithelium and are captured by oral antigen-presenting cells, especially macrophages..
92. Aya Miyazaki 1 , Asuna Sugimoto 1 , Keigo Yoshizaki 2 , Keita Kawarabayashi 1 , Kokoro Iwata 1 , Rika Kurogoushi 1 , Takamasa Kitamura 1 , Kunihiro Otsuka 3 , Tomokazu Hasegawa 1 , Yuki Akazawa 1 , Satoshi Fukumoto 4 , Naozumi Ishimaru 5 , Tsutomu Iwamoto 6, Coordination of WNT signaling and ciliogenesis during odontogenesis by piezo type mechanosensitive ion channel component 1, Sci Rep, 10.1038/s41598-019-51381-9, 9, 1, 14762, 2019.10, Signal transmission from the mechanical forces to the various intracellular activities is a fundamental process during tissue development. Despite their critical role, the mechanism of mechanical forces in the biological process is poorly understood. In this study, we demonstrated that in the response to hydrostatic pressure (HP), the piezo type mechanosensitive ion channel component 1 (PIEZO1) is a primary mechanosensing receptor for odontoblast differentiation through coordination of the WNT expression and ciliogenesis. In stem cells from human exfoliated deciduous teeth (SHED), HP significantly promoted calcium deposition as well as the expression of odontogenic marker genes, PANX3 and DSPP, and WNT related-genes including WNT5b and WNT16, whereas HP inhibited cell proliferation and enhanced primary cilia expression. WNT signaling inhibitor XAV939 and primary cilia inhibitor chloral hydrate blocked the HP-induced calcium deposition. The PIEZO1 activator Yoda1 inhibited cell proliferation but induced ciliogenesis and WNT16 expression. Interestingly, HP and Yoda1 promoted nuclear translocation of RUNX2, whereas siRNA-mediated silencing of PIEZO1 decreased HP-induced nuclear translocation of RUNX2. Taken together, these results suggest that PIEZO1 functions as a mechanotransducer that connects HP signal to the intracellular signalings during odontoblast differentiation..
93. Kan Saito 1 , Frederic Michon 2 , Aya Yamada 3 , Hiroyuki Inuzuka 4 , Satoko Yamaguchi 3 , Emiko Fukumoto 3 , Keigo Yoshizaki 5 , Takashi Nakamura 6 , Makiko Arakaki 3 , Yuta Chiba 3 , Masaki Ishikawa 7 , Hideyuki Okano 8 , Irma Thesleff 9 , Satoshi Fukumoto 10, Sox21 Regulates Anapc10 Expression and Determines the Fate of Ectodermal Organ, iScience, 10.1016/j.isci.2020.101329, 23, 7, 101329, 2020.07.
94. Keita Funada 1 , Keigo Yoshizaki 2 , Kanako MIyazaki 1 , Xue Han 1 , Tomomi Yuta 1 , Tian Tian 1 , Kanji Mizuta 1 , Yao Fu 1 , Tsutomu Iwamoto 3 , Aya Yamada 4 , Ichiro Takahashi 1 , Satoshi Fukumoto 5 6, microRNA-875-5p plays critical role for mesenchymal condensation in epithelial-mesenchymal interaction during tooth development, Sci Rep, 10.1038/s41598-020-61693-w, 10, 1, 4918, 2020.03, Epithelial-mesenchymal interaction has critical roles for organ development including teeth, during which epithelial thickening and mesenchymal condensation are initiated by precise regulation of the signaling pathway. In teeth, neural crest-derived mesenchymal cells expressed PDGF receptors migrate and become condensed toward invaginated epithelium. To identify the molecular mechanism of this interaction, we explored the specific transcriptional start sites (TSSs) of tooth organs using cap analysis of gene expression (CAGE). We identified a tooth specific TSS detected in the chromosome 15qD1 region, which codes microRNA-875 (mir875). MiR875-5p is specifically expressed in dental mesenchyme during the early stage of tooth development. Furthermore, PRRX1/2 binds to the mir875 promoter region and enhances the expression of mir875. To assess the role of miR875-5p in dental mesenchyme, we transfected mimic miR875-5p into mouse dental pulp (mDP) cells, which showed that cell migration toward dental epithelial cells was significantly induced by miR875-5p via the PDGF signaling pathway. Those results also demonstrated that miR875-5p induces cell migration by inhibiting PTEN and STAT1, which are regulated by miR875-5p as part of post-transcriptional regulation. Together, our findings indicate that tooth specific miR875-5p has important roles in cell condensation of mesenchymal cells around invaginated dental epithelium and induction of epithelial-mesenchymal interaction..
95. Tomoaki Nakamura 1 , Tsutomu Iwamoto 2 , Hannah M Nakamura 3 , Yuki Shindo 4 , Kan Saito 1 , Aya Yamada 1 , Yoshihiko Yamada 5 , Satoshi Fukumoto 1 , Takashi Nakamura 4, Regulation of miR-1-Mediated Connexin 43 Expression and Cell Proliferation in Dental Epithelial Cells, Front Dev Cell Biol, 10.3389/fcell.2020.00156, 8, 156, 156, 2020.03, Many genes encoding growth factors, receptors, and transcription factors are induced by the epithelial-mesenchymal interaction during tooth development. Recently, numerous functions of microRNAs (miRNAs) are reportedly involved in organogenesis and disease. miRNAs regulate gene expression by inhibiting translation and destabilizing mRNAs. However, the expression and function of miRNAs in tooth development remain poorly understood. This study aimed to analyze the expression of miRNAs produced during tooth development using a microarray system to clarify the role of miRNAs in dental development. miR-1 showed a unique expression pattern in the developing tooth. miR-1 expression in the tooth germ peaked on embryonic day 16.5, decreasing gradually on postnatal days 1 and 3. An in situ hybridization assay revealed that miR-1 is expressed at the cervical loop of the dental epithelium. The expression of miR-1 and connexin (Cx) 43, a target of miR-1, were inversely correlated both in vitro and in vivo. Knockdown of miR-1 induced the expression of Cx43 in dental epithelial cells. Interestingly, cells with miR-1 downregulation proliferated slower than the control cells. Immunocytochemistry revealed that Cx43 in cells with miR-1 knockdown formed both cell-cell gap junctions and hemichannels at the plasma membrane. Furthermore, the rate of ATP release was higher in cells with miR-1 knockdown than in control cells. Furthermore, Cx43 downregulation in developing molars was observed in Epiprofin-knockout mice, along with the induction of miR-1 expression. These results suggest that the expression pattern of Cx43 is modulated by miR-1 to control cell proliferation activity during dental epithelial cell differentiation..
96. Xu Han 1 , Hiroki Kato 1 , Hiroshi Sato 1 , Yuta Hirofuji 1 , Satoshi Fukumoto 2 , Keiji Masuda 3, Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-of-function mutation of TRPV4 associated with metatropic dysplasia, Biochem Biophys Res Commun, 10.1016/j.bbrc.2019.12.123, 523, 4, 841-846, 2020.03, Metatropic dysplasia (MD) is a congenital skeletal dysplasia characterized by severe platyspondyly and dumbbell-like long-bone deformities. These skeletal phenotypes are predominantly caused by autosomal dominant gain-of-function (GOF) mutations in transient receptor potential vanilloid 4 (TRPV4), which encodes a nonselective Ca2+-permeable cation channel. Previous studies have shown that constitutive TRPV4 channel activation leads to irregular chondrogenic proliferation and differentiation, and thus to the disorganized endochondral ossification seen in MD. Therefore, the present study investigated the role of TRPV4 in osteoblast differentiation and MD pathogenesis. Specifically, the behavior of osteoblasts differentiated from patient-derived dental pulp stem cells carrying a heterozygous single base TRPV4 mutation, c.1855C > T (p.L619F) was compared to that of osteoblasts differentiated from isogenic control cells (in which the mutation was corrected using the CRISPR/Cas9 system). The mutant osteoblasts exhibited enhanced calcification (indicated by intense Alizarin Red S staining), increased intracellular Ca2+ levels, strongly upregulated runt-related transcription factor 2 and osteocalcin expression, and increased expression and nuclear translocation of nuclear factor-activated T cell c1 (NFATc1) compared to control cells. These results suggest that the analyzed TRPV4 GOF mutation disrupts osteoblastic differentiation and induces MD-associated disorganized endochondral ossification by increasing Ca2+/NFATc1 pathway activity. Thus, inhibiting the NFATc1 pathway may be a promising potential therapeutic strategy to attenuate skeletal deformities in MD..
97. Watahiki A, Shimizu K, Hoshikawa S, Chiba M, Kitamura H, Egusa H, Fukumoto S, Inuzuka H., Lipin-2 degradation elicits a proinflammatory gene signature in macrophages, Biochem Biophys Res Commun , 10.1016/j.bbrc.2020.01.119, 524, 2, 477-483, 2020.01, Lipin-2 is a phosphatidate phosphatase with key roles in regulating lipid storage and energy homeostasis. LPIN2-genetic deficiency is associated with an autoinflammatory disorder, underscoring its critical role in innate immune signaling; however, the regulatory mechanisms underlying protein stability remain unknown. Here, we demonstrate that Lipin-2 interacts with β-TRCP, a substrate receptor subunit of the SCFβ-TRCP E3 ligase, and undergoes ubiquitination and proteasomal degradation. β-TRCP-knockout in RAW264.7 macrophages resulted in Lipin-2 accumulation, leading to the suppression of LPS-induced MAPK activation and subsequent proinflammatory gene expression. Consistent with this, treatment with MLN4924, a Cullin-neddylation inhibitor that suppresses SCF E3 activity, increased Lipin-2 protein and concomitantly decreased Il1b expression. These findings suggested that β-TRCP-mediated Lipin-2 degradation affects macrophage-elicited proinflammatory responses and could lead to new therapeutic approaches to treat inflammatory diseases..
98. Clinical Statistic Study of Surgical Procedures for Outpatient in Our Dental Hospital.
99. M.Nishiguchi,K.Yuasa,K.Saito,E.Fukumoto,A.Yamada,T.Hasegawa,K.Yoshizaki,Y.Kamasaki,K.Nonaka,T.Fujiwara,S.Fukumoto, Amelogenin is a negative regulator of osteoclastgenesis via downregulation of RANKL,M-CSF and fibronectin expression in osteoblasts, Archives of Oral Biology, 52(3)237-243, 2007.03.
100. Fukumoto S., Miner J.H., Ida H., Fukumoto E., Yuasa K., Miyazaki H., Hoffman M.P.,Yamada Y., Laminin α5 Is Required for Dental Epithelium Growth and Polarity and the Development of Tooth Bud and Shape, J Biol Chem, 281(8):5008-5016, 2006.01.
101. Muraoka N., Shum L., Fukumoto S., Nomura T., Ohishi M., Nonaka K., Transforming growth factor-b3 promotes mesenchymal cell proliferation and angiogenesis mediated by the enhancement of cyclin D1, Flk1, and CD31 gene expression during CL/Fr mouse lip fusion, Birth Defects Res A Clin Mol Teratol., 2005,73(12):956-65, 2005.07.
102. Yamada A, Fukumoto E, Kamasaki Y, Ida-Yonemochi H, Saku T, Fujiwara T, Fukumoto S., GD3 synthase gene found expressed in dental epithelium and shown to regulate cell proliferation, Arch Oral Biol, 50(4): 393-399, 2005.01.
103. Fukumoto S., Yamada Y, Review:Extracellular Matrix Regulates Tooth Morphogenesis, Connective Tissue Research, 46:220-226, 2005.01.
104. Fukumoto S., Yamada A., Nonaka K., Yamada Y, Essential Roles of Ameloblastin in Maintaining Ameloblast Differentiation and Enamel Formation, Cells Tissues Organs, 181:189-195, 2005.01.
105. Huojia M., Muraoka N., Yoshizaki K., Fukumoto S., Nakashima M., Akamine A., Nonaka K., Ohishi M., TGF-beta3 induces ectopic mineralization in fetal mouse dental pulp during tooth germ development., Dev Growth Differ., 47(3):141-52, 2005.01.
106. Satoshi Fukumoto, Takayoshi Kiba, Bradford Hall, Noriyuki Iehara, Takashi Nakamura, Glenn Longenecker, Paul Krebsbach, Antonio Nanci, Ashok B. Kulkarni, Yoshihiko Yamada, Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts, THE JOURNAL OF CELL BIOLOGY, 167(5), 973-983, 2004.12.
107. Nakamura T, Unda F, de-Vega S, Vilaxa A, Fukumoto S, Yamada KM, Yamada Y., The Kruppel-like factor epiprofin is expressed by epithelium of developing teeth, hair follicles, and limb buds and
promotes cell proliferation., J Biol Chem, 279(1):626-34, 2004.01.
108. Yuasa K*, Fukumoto S*, Kamasaki Y, Yamada A, Fukumoto E, Kanaoka K, Saito K, Harada H, Arikawa-Hirasawa E,, Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression., J Biol Chem, 279(11):10286-92, 2004.01.
109. Saito K, Ohara N, Hotokezaka H, Fukumoto S, Yuasa K, Naito M, Fujiwara T, Nakayama K., Infection-induced up-regulation of the costimulatory molecule 4-1BB in osteoblastic cells and its inhibitory effect on
M-CSF/RANKL-induced in vitro osteoclastogenesis., J Biol Chem, 279(14):13555-63, 2004.01.
110. Nishio M, Fukumoto S, Furukawa K, Ichimura A, Miyazaki H, Kusunoki S, Urano T, Furukawa K, Overexpressed GM1 suppresses nerve growth factor (NGF) signals by modulating the intracellular localization of NGF
receptors and membrane fluidity in PC12 cells., J Biol Chem, 279(32):33368-78, 2004.01.
111. Fukumoto S., Kiba T., Hall B., Iehara N., Nakamura T., Longenecker G.,Krebsbach P.H., Nanci A., Kulkarni A.B.,, Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts, Journal of Cell Biology, 167(5):973-983, 2004.01.
112. Chen HH, Fukumoto S, Furukawa K, Nakao A, Akiyama S, Urano T, Furukawa K., Suppression of lung metastasis of mouse Lewis lung cancer P29 with transfection of the ganglioside GM2/GD2 synthase
gene., Int J Cancer, 103(2):169-76, 2003.01.
113. Fukumoto E, Sakai H, Fukumoto S, Yagi T, Takagi O, Kato Y., Cadherin-related neuronal receptors in incisor development., J Dent Res, Jan;82(1):17-22, 2003.01.
114. Okada M, Itoh Mi M, Haraguchi M, Okajima T, Inoue M, Oishi H, Matsuda Y, Iwamoto T, Kawano T, Fukumoto S,, b-series Ganglioside deficiency exhibits no definite changes in the neurogenesis and the sensitivity to Fas-mediated apoptosis but impairs regeneration of the lesioned hypoglossal nerve., J Biol Chem, 277(3):1633-6, 2002.01.
115. ) Mitsuda T, Furukawa K, Fukumoto S, Miyazaki H, Urano T, Furukawa K., Overexpression of ganglioside GM1 results in the dispersion of platelet-derived growth factor receptor from glycolipid-
enriched microdomains and in the suppression of cell growth signals., J Biol Chem, 277(13):11239-46, 2002.01.
116. Yoshida S, Fukumoto S, Kawaguchi H, Sato S, Ueda R, Furukawa K., Ganglioside G(D2) in small cell lung cancer cell lines: enhancement of cell proliferation and mediation of apoptosis, Cancer Res, 61(10):4244-52, 2001.01.
117. Iwamoto T, Fukumoto S, Kanaoka K, Sakai E, Shibata M, Fukumoto E, Inokuchi Ji J, Takamiya K, Furukawa K,, Lactosylceramide is essential for the osteoclastogenesis mediated by macrophage-colony-stimulating factor and receptor
activator of nuclear factor-kappa B ligand., J Biol Chem, 276(49):46031-8, 2001.01.