九州大学 研究者情報
論文一覧
千葉 雄太(ちば ゆうた) データ更新日:2022.05.13

助教 /  歯学研究院 歯学部門 口腔保健推進学講座小児口腔医学分野


原著論文
1. Tian Tian, Kanako Miyazaki, Yuta Chiba, Keita Funada, Tomomi Yuta, Kanji Mizuta, Yao Fu, Jumpei Kawahara, Xue Han, Yuna Ando, Ami Funada, Aya Yamada, Tsutomu Iwamoto, Seiji Nakamura, Ichiro Takahashi, Satoshi Fukumoto, Keigo Yoshizaki, An ex vivo organ culture screening model revealed that low temperature conditions prevent side effects of anticancer drugs., Scientific reports, 10.1038/s41598-022-06945-7, 12, 1, 3093-3093, 2022.02, Development of chemotherapy has led to a high survival rate of cancer patients; however, the severe side effects of anticancer drugs, including organ hypoplasia, persist. To assume the side effect of anticancer drugs, we established a new ex vivo screening model and described a method for suppressing side effects. Cyclophosphamide (CPA) is a commonly used anticancer drug and causes severe side effects in developing organs with intensive proliferation, including the teeth and hair. Using the organ culture model, we found that treatment with CPA disturbed the growth of tooth germs by inducing DNA damage, apoptosis and suppressing cellular proliferation and differentiation. Furthermore, low temperature suppressed CPA-mediated inhibition of organ development. Our ex vivo and in vitro analysis revealed that low temperature impeded Rb phosphorylation and caused cell cycle arrest at the G1 phase during CPA treatment. This can prevent the CPA-mediated cell damage of DNA replication caused by the cross-linking reaction of CPA. Our findings suggest that the side effects of anticancer drugs on organ development can be avoided by maintaining the internal environment under low temperature..
2. LingLing Jia, Yuta Chiba, Kan Saito, Keigo Yoshizaki, Tian Tian, Xu Han, Kanji Mizuta, Mitsuki Chiba, Xin Wang, Shahad Al Thamin, Aya Yamada, Satoshi Fukumoto, The tooth-specific basic helix-loop-helix factor AmeloD promotes differentiation of ameloblasts., Journal of cellular physiology, 10.1002/jcp.30639, 2021.11, Tissue-specific basic helix-loop-helix (bHLH) transcription factors play an important role in cellular differentiation. We recently identified AmeloD as a tooth-specific bHLH transcription factor. However, the role of AmeloD in cellular differentiation has not been investigated. The aim of this study was to elucidate the role of AmeloD in dental epithelial cell differentiation. We found that AmeloD-knockout (AmeloD-KO) mice developed an abnormal structure and altered ion composition of enamel in molars, suggesting that AmeloD-KO mice developed enamel hypoplasia. In molars of AmeloD-KO mice, the transcription factor Sox21 encoding SRY-Box transcription factor 21 and ameloblast differentiation marker genes were significantly downregulated. Furthermore, overexpression of AmeloD in the dental epithelial cell line M3H1 upregulated Sox21 and ameloblast differentiation marker genes, indicating that AmeloD is critical for ameloblast differentiation. Our study demonstrated that AmeloD is an important transcription factor in amelogenesis for promoting ameloblast differentiation. This study provides new insights into the mechanisms of amelogenesis..
3. Y Chiba, K Yoshizaki, T Tian, K Miyazaki, D Martin, K Saito, A Yamada, S Fukumoto, Integration of Single-Cell RNA- and CAGE-seq Reveals Tooth-Enriched Genes., Journal of dental research, 10.1177/00220345211049785, 220345211049785-220345211049785, 2021.11, Organ development is dictated by the regulation of genes preferentially expressed in tissues or cell types. Gene expression profiling and identification of specific genes in organs can provide insights into organogenesis. Therefore, genome-wide analysis is a powerful tool for clarifying the mechanisms of development during organogenesis as well as tooth development. Single-cell RNA sequencing (scRNA-seq) is a suitable tool for unraveling the gene expression profile of dental cells. Using scRNA-seq, we can obtain a large pool of information on gene expression; however, identification of functional genes, which are key molecules for tooth development, via this approach remains challenging. In the present study, we performed cap analysis of gene expression sequence (CAGE-seq) using mouse tooth germ to identify the genes preferentially expressed in teeth. The CAGE-seq counts short reads at the 5'-end of transcripts; therefore, this method can quantify the amount of transcripts without bias related to the transcript length. We hypothesized that this CAGE data set would be of great help for further understanding a gene expression profile through scRNA-seq. We aimed to identify the important genes involved in tooth development via bioinformatics analyses, using a combination of scRNA-seq and CAGE-seq. We obtained the scRNA-seq data set of 12,212 cells from postnatal day 1 mouse molars and the CAGE-seq data set from postnatal day 1 molars. scRNA-seq analysis revealed the spatiotemporal expression of cell type-specific genes, and CAGE-seq helped determine whether these genes are preferentially expressed in tooth or ubiquitously. Furthermore, we identified candidate genes as novel tooth-enriched and dental cell type-specific markers. Our results show that the integration of scRNA-seq and CAGE-seq highlights the genes important for tooth development among numerous gene expression profiles. These findings should contribute to resolving the mechanism of tooth development and establishing the basis for tooth regeneration in the future..
4. Yu Sugawara, Kan Saito, Masaharu Futaki, Masahiro Naruse, Mariko Ono, Ryoko Hino, Yuta Chiba, Makiko Arakaki, Aya Yamada, Satoshi Fukumoto, Evaluation of the optimal exposure settings for occlusal photography with digital cameras, Pediatric Dental Journal, 10.1016/j.pdj.2014.04.002, 24, 2, 89-96, 2014.01, Background and objective: Recently, there have been produced several kinds of camera systems, lighting devices and image processing programs. The intraoral photographs are periodically required in pediatric dentistry, since the oral environment of children changes rapidly and dramatically because of disease, growth, and tooth eruption. However, the suitable settings for intraoral digital imaging have not yet been reported. In this study, single-lens reflex and mirrorless cameras were used with a ring flash, ring light emitting diode (LED), or standard fluorescent lamp to determine the optimal exposure parameters for occlusal photography. Materials and methods: Fifty-six dentists evaluated images of a discolored nonvital central incisor and first molar with recurrent caries and stained fissures clipped from a low-magnification photograph of the adult maxillary occlusal surface reflected in a mirror by using various F-numbers (representing aperture), shutter speeds, and International Organization for Standardization (ISO) numbers (representing light sensitivity). Results: The results showed that F-numbers between 5.6 and 10, shutter speeds faster than 1/30s, and ISO 800 produce the best occlusal images when a 60 mm f/2.8 Macro lens is used. Better images are obtained with ring LED than with ring flash. Furthermore, ISO 3200 is required for low-magnification photography in natural light. Finally, intraoral digital images should be captured under low magnification, because tilted or deflected images can be adjusted by using imaging software..
5. Bing He, Yuta Chiba, Haochuan Li, Susana de Vega, Kazuhiro Tanaka, Keigo Yoshizaki, Muneaki Ishijima, Kenji Yuasa, Masaki Ishikawa, Craig Rhodes, Kiyoshi Sakai, Peipei Zhang, Satoshi Fukumoto, Xuedong Zhou, Yoshihiko Yamada, Identification of the Novel Dental Epithelium-Specific Transcription Factor AmeloD., J Dent Res., 2018.01.
6. Aya Yamada, Keigo Yoshizaki, Masaki Ishikawa, Kan Saito, Yuta Chiba, Emiko Fukumoto, Ryoko Hino, Seira Hoshikawa, Mitsuki Chiba, Takashi Nakamura, Tsutomu Iwamoto, Satoshi Fukumoto, Connexin 43-Mediated Gap Junction Communication Regulates Ameloblast Differentiation via ERK1/2 Phosphorylation., Frontiers in physiology, 10.3389/fphys.2021.748574, 12, 748574-748574, 2021.09, Connexin 43 (Cx43) is an integral membrane protein that forms gap junction channels. These channels mediate intercellular transport and intracellular signaling to regulate organogenesis. The human disease oculodentodigital dysplasia (ODDD) is caused by mutations in Cx43 and is characterized by skeletal, ocular, and dental abnormalities including amelogenesis imperfecta. To clarify the role of Cx43 in amelogenesis, we examined the expression and function of Cx43 in tooth development. Single-cell RNA-seq analysis and immunostaining showed that Cx43 is highly expressed in pre-secretory ameloblasts, differentiated ameloblasts, and odontoblasts. Further, we investigated the pathogenic mechanisms of ODDD by analyzing Cx43-null mice. These mice developed abnormal teeth with multiple dental epithelium layers. The expression of enamel matrix proteins such as ameloblastin (Ambn), which is critical for enamel formation, was significantly reduced in Cx43-null mice. TGF-β1 induces Ambn transcription in dental epithelial cells. The induction of Ambn expression by TGF-β1 depends on the density of the cultured cells. Cell culture at low densities reduces cell-cell contact and reduces the effect of TGF-β1 on Ambn induction. When cell density was high, Ambn expression by TGF-β1 was enhanced. This induction was inhibited by the gap junction inhibitors, oleamide, and 18α-grycyrrhizic acid and was also inhibited in cells expressing Cx43 mutations (R76S and R202H). TGF-β1-mediated phosphorylation and nuclear translocation of ERK1/2, but not Smad2/3, were suppressed by gap junction inhibitors. Cx43 gap junction activity is required for TGF-β1-mediated Runx2 phosphorylation through ERK1/2, which forms complexes with Smad2/3. In addition to its gap junction activity, Cx43 may also function as a Ca2+ channel that regulates slow Ca2+ influx and ERK1/2 phosphorylation. TGF-β1 transiently increases intracellular calcium levels, and the increase in intracellular calcium over a short period was not related to the expression level of Cx43. However, long-term intracellular calcium elevation was enhanced in cells overexpressing Cx43. Our results suggest that Cx43 regulates intercellular communication through gap junction activity by modulating TGF-β1-mediated ERK signaling and enamel formation..
7. Craig S. Rhodes, Yasuo Yoshitomi, Peter D. Burbelo, Nowlan H. Freese, Takashi Nakamura, Yuta Chiba, Yoshihiko Yamada, Sp6/Epiprofin is a master regulator in the developing tooth, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2021.10.017, 2021.10.
8. 千葉 雄太, The role of the novel bHLH transcription factor AmeloD in tooth development, 東北大学 博士論文.
9. 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, 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..
10. 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, 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..
11. 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..
12. Xue Han, Keigo Yoshizaki, Kanako Miyazaki, Chieko Arai, Keita Funada, Tomomi Yuta, Tian Tian, Yuta Chiba, Kan Saito, Tsutomu Iwamoto, Aya Yamada, Ichiro Takahashi, Satoshi Fukumoto, The transcription factor NKX2-3 mediates p21 expression and ectodysplasin-A signaling in the enamel knot for cusp formation in tooth development, Journal of Biological Chemistry, 10.1074/jbc.RA118.003373, 293, 38, 14572-14584, 2018.09, 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 Nkx2-3, 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 Nkx2-3 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 Bmp2 and Bmpr2 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..
13. Koji Hirayama, Takashi Hanada, Ryoko Hino, Kan Saito, Mayu Kobayashi, Makiko Arakaki, Yuta Chiba, Norihiko Nakamura, Takeshi Sakurai, Tsutomu Iwamoto, Satoshi Fukumoto, Aya Yamada, Material properties on enamel and fissure of surface pre-reacted glass-ionomer filler-containing dental sealant, Pediatric Dental Journal, 10.1016/j.pdj.2018.05.001, 28, 2, 87-95, 2018.08, Purpose: In pediatric dentistry, sealants have been used to prevent caries. Due to its material properties such as fluoride-releasing ability and physical strength, surface pre-reacted glass (S-PRG) filler is added primarily to resin-based dental materials for clinical use. In this study, we investigated the properties of S-PRG filler containing sealant. Methods: Before using sealant, the primer was applied to extracted bovine incisors. Scanning electron microscopy (SEM) observation revealed that the primer treatment caused no structural changes of enamel surface, unlike conventional phosphoric acid etching. Further, shear bond strength test was performed to measure the initial strength and durability after thermal cycling. Bond strength of S-PRG filler containing sealant was comparable to those of other sealants even though the former does not involve phosphoric acid etching. In addition, after treating the enamel surface with the primer or phosphoric acid, it showed excellent flowability in the primer group compared to phosphoric acid treatment. Results: SEM observation showed that the sealant sealed the enamel surface as it migrated to reach the deep areas of the fissures. When the marginal sealing ability of the sealant was evaluated based on dye penetration, no dye penetration in the marginal region was observed in any specimens. In addition, measurement of the pH of an acid solution containing a cured specimen of the sealant containing S-PRG filler showed that the solution's pH became more alkaline as the immersion time increased. Conclusion: These findings suggest that the sealant is an extremely effective material for preventing caries..
14. 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..
15. Yuta Chiba, Bing He, Keigo Yoshizaki, Craig Rhodes, 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, Journal of Biological Chemistry, 10.1074/jbc.RA118.005298, 294, 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 cell migration. We used the CLDE dental epithelial cell line to conduct further mechanistic analyses to determine whether 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..
16. Kuniyuki Nakamura, Tomoko Ikeuchi, Kazuki Nara, Craig S. Rhodes, Peipei Zhang, Yuta Chiba, Saiko Kazuno, Yoshiki Miura, Tetsuro Ago, Eri Arikawa-Hirasawa, Yoh Suke Mukouyama, Yoshihiko Yamada, Perlecan regulates pericyte dynamics in the maintenance and repair of the blood-brain barrier, Journal of Cell Biology, 10.1083/JCB.201807178, 218, 10, 3506-3525, 2019.10, Ischemic stroke causes blood-brain barrier (BBB) breakdown due to significant damage to the integrity of BBB components. Recent studies have highlighted the importance of pericytes in the repair process of BBB functions triggered by PDGFRβ upregulation. Here, we show that perlecan, a major heparan sulfate proteoglycan of basement membranes, AIDS in BBB maintenance and repair through pericyte interactions. Using a transient middle cerebral artery occlusion model, we found larger infarct volumes and more BBB leakage in conditional perlecan (Hspg2)-deficient (Hspg2-/-TG) mice than in control mice. Control mice showed increased numbers of pericytes in the ischemic lesion, whereas Hspg2-/-TG mice did not. At the mechanistic level, pericytes attached to recombinant perlecan C-terminal domain V (perlecan DV, endorepellin). Perlecan DV enhanced the PDGF-BB-induced phosphorylation of PDGFRβ, SHP-2, and FAK partially through integrin α5β1 and promoted pericyte migration. Perlecan therefore appears to regulate pericyte recruitment through the cooperative functioning of PDGFRβ and integrin α5β1 to support BBB maintenance and repair following ischemic stroke..
17. Peipei Zhang, Masaki Ishikawa, Craig Rhodes, Andrew Doyle, Tomoko Ikeuchi, Kuniyuki Nakamura, Yuta Chiba, Bing He, Yoshihiko Yamada, Pannexin-3 Deficiency Delays Skin Wound Healing in Mice due to Defects in Channel Functionality, Journal of Investigative Dermatology, 10.1016/j.jid.2018.08.033, 139, 4, 909-918, 2019.04, Pannexin-3 (Panx3) is a gap junction protein that is required for regulating cell cycle exit and the differentiation of osteoblasts and chondrocytes during skeletal development. However, the role of Panx3 in skin tissue regeneration remains unclear. After dorsal skin punch biopsies, Panx3-knockout mice exhibited a significant delay in wound healing with insufficient re-epithelialization, decreased inflammatory reaction, and reduced collagen remodeling. Panx3 expression coincided with inflammatory reactions both in vivo and in vitro. By applying exogenous tumor necrosis factor-α to mimic inflammation in vitro, Panx3 expression was induced in HaCaT cells. In addition, Panx3 depletion reduced epithelial-mesenchymal transition during skin wound healing. A protein essential for signaling in epithelial-mesenchymal transition, transforming growth factor-β interacted with Panx3 by modulating intracellular adenosine triphosphate levels and thereby enhanced HaCaT cell migration ability with Panx3 overexpression. In conclusion, Panx3 plays a key role in the skin wound healing process by controlling keratinocytes and keratinocyte-mesenchyme cross-talk via hemichannel and endoplasmic reticulum Ca channel functions, which differs from another gap junction, connexin 43 (Cx43), during skin wound healing. 2+.
18. B. He, Y. Chiba, H. Li, S. de Vega, K. Tanaka, K. Yoshizaki, M. Ishijima, K. Yuasa, M. Ishikawa, C. Rhodes, K. Sakai, P. Zhang, S. Fukumoto, X. Zhou, Y. Yamada, Identification of the Novel Tooth-Specific Transcription Factor AmeloD, Journal of Dental Research, 10.1177/0022034518808254, 98, 2, 234-241, 2019.02, Basic-helix-loop-helix (bHLH) transcription factors play an important role in various organs’ development; however, a tooth-specific bHLH factor has not been reported. In this study, we identified a novel tooth-specific bHLH transcription factor, which we named AmeloD, by screening a tooth germ complementary DNA (cDNA) library using a yeast 2-hybrid system. AmeloD was mapped onto the mouse chromosome 1q32. Phylogenetic analysis showed that AmeloD belongs to the achaete-scute complex-like (ASCL) gene family and is a homologue of ASCL5. AmeloD was uniquely expressed in the inner enamel epithelium (IEE), but its expression was suppressed after IEE cell differentiation into ameloblasts. Furthermore, AmeloD expression showed an inverse expression pattern with the epithelial cell-specific cell–cell adhesion molecule E-cadherin in the dental epithelium. Overexpression of AmeloD in dental epithelial cell line CLDE cells resulted in E-cadherin suppression. We found that AmeloD bound to E-box cis-regulatory elements in the proximal promoter region of the E-cadherin gene. These results reveal that AmeloD functions as a suppressor of E-cadherin transcription in IEE cells. Our study demonstrated that AmeloD is a novel tooth-specific bHLH transcription factor that may regulate tooth development through the suppression of E-cadherin in IEE cells..
19. Kan Saito, Frederic Michon, Aya Yamada, Hiroyuki Inuzuka, Satoko Yamaguchi, Emiko Fukumoto, Keigo Yoshizaki, Takashi Nakamura, Makiko Arakaki, Yuta Chiba, Masaki Ishikawa, Hideyuki Okano, Irma Thesleff, Satoshi Fukumoto, Sox21 Regulates Anapc10 Expression and Determines the Fate of Ectodermal Organ, iScience, 10.1016/j.isci.2020.101329, 23, 7, 101329-101329, 2020.07, The transcription factor Sox21 is expressed in the epithelium of developing teeth. The present study aimed to determine the role of Sox21 in tooth development. We found that disruption of Sox21 caused severe enamel hypoplasia, regional osteoporosis, and ectopic hair formation in the gingiva in Sox21 knockout incisors. Differentiation markers were lost in ameloblasts, which formed hair follicles expressing hair keratins. Molecular analysis and chromatin immunoprecipitation sequencing indicated that Sox21 regulated Anapc10, which recognizes substrates for ubiquitination-mediated degradation, and determined dental-epithelial versus hair follicle cell fate. Disruption of either Sox21 or Anapc10 induced Smad3 expression, accelerated TGF-β1-induced promotion of epithelial-to-mesenchymal transition (EMT), and resulted in E-cadherin degradation via Skp2. We conclude that Sox21 disruption in the dental epithelium leads to the formation of a unique microenvironment promoting hair formation and that Sox21 controls dental epithelial differentiation and enamel formation by inhibiting EMT via Anapc10..
20. Yuta Chiba, Kan Saito, Daniel Martin, Erich T. Boger, Craig Rhodes, Keigo Yoshizaki, Takashi Nakamura, Aya Yamada, Robert J. Morell, Yoshihiko Yamada, Satoshi Fukumoto, Single-Cell RNA-Sequencing From Mouse Incisor Reveals Dental Epithelial Cell-Type Specific Genes, Frontiers in Cell and Developmental Biology, 10.3389/fcell.2020.00841, 8, 2020.09, Dental epithelial stem cells give rise to four types of dental epithelial cells: inner enamel epithelium (IEE), outer enamel epithelium (OEE), stratum intermedium (SI), and stellate reticulum (SR). IEE cells further differentiate into enamel-forming ameloblasts, which play distinct roles, and are essential for enamel formation. These are conventionally classified by their shape, although their transcriptome and biological roles are yet to be fully understood. Here, we aimed to use single-cell RNA sequencing to clarify the heterogeneity of dental epithelial cell types. Unbiased clustering of 6,260 single cells from incisors of postnatal day 7 mice classified them into two clusters of ameloblast, IEE/OEE, SI/SR, and two mesenchymal populations. Secretory-stage ameloblasts expressed Amel and Enam were divided into Dspp + and Ambn + ameloblasts. Pseudo-time analysis indicated Dspp + ameloblasts differentiate into Ambn + ameloblasts. Further, Dspp and Ambn could be stage-specific markers of ameloblasts. Gene ontology analysis of each cluster indicated potent roles of cell types: OEE in the regulation of tooth size and SR in the transport of nutrients. Subsequently, we identified novel dental epithelial cell marker genes, namely Pttg1, Atf3, Cldn10, and Krt15. The results not only provided a resource of transcriptome data in dental cells but also contributed to the molecular analyses of enamel formation..
21. Ryoko Hino, Aya Yamada, Yuta Chiba, Keigo Yoshizaki, Emiko Fukumoto, Tsutomu Iwamoto, Yuriko Maruya, Keishi Otsu, Hidemistu Harada, Kan Saito, Satoshi Fukumoto, Melnick-Needles syndrome associated molecule, Filamin-A regulates dental epithelial cell migration and root formation, Pediatric Dental Journal, 10.1016/j.pdj.2020.09.003, 30, 3, 208-214, 2020.12, Background: Filamin-A (FLNA) is one of the adaptor proteins binding to both integrin beta chain and actin filament. Several kind of mutation in human FLNA gene were observed in Melnick-Needles syndrome (MNS). However, the role of FLNA in tooth formation is still unknown. Matrials and methods: We analyzed the tissue expression of FLNA during tooth development using immunostaining. The role of FLNA in cell migration was analyzed using a scratch assay. In addition, we analyzed the phenotype of teeth in patients with Mernick-Needles syndrome caused by FLNA gene abnormalities. Results: FLNA expressed in inner and outer enamel epithelium in mouse tooth germ and also expressed in cervical region of dental epithelium. In postnatal stage, FLNA expressed in ameloblast and odontoblast layers. In rat dental epithelium cell line SF2, FLNA is localized at the boundary between cells and co-localized with the terminal region of F-actin filament. shRNA-FLNA transfected cells showed delayed cell migration and scratch closure compared with control cells. Analysis of actin fiber movement by time-lapse confocal microscopy showed slower fiber movement in shRNA-FLNA over expressing SF2 cells. Novel mutation in FLNA gene was observed in MNS with tooth anormaly. This patient showed tooth agenesis and short root in both primary and permanent dentition with thin enamel. Conclusion: Taken together, these results suggest that filamin-A is play a key role for dental epithelium migration and root formation..
22. 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, 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..
23. Xin Wang, Yuta Chiba, Lingling Jia, Keigo Yoshizaki, Kan Saito, Aya Yamada, Man Qin, Satoshi Fukumoto, Expression Patterns of Claudin Family Members During Tooth Development and the Role of Claudin-10 (Cldn10) in Cytodifferentiation of Stratum Intermedium, Frontiers in Cell and Developmental Biology, 10.3389/fcell.2020.595593, 8, 2020.10, There is growing evidence showing that tight junctions play an important role in developing enamel. Claudins are one of the main components of tight junctions and may have pivotal functions in modulating various cellular events, such as regulating cell differentiation and proliferation. Mutations in CLDN10 of humans are associated with HELIX syndrome and cause enamel defects. However, current knowledge regarding the expression patterns of claudins and the function of Cldn10 during tooth development remains fragmented. In this study, we aimed to analyze the expression patterns of claudin family members during tooth development and to investigate the role of Cldn10 in amelogenesis. Using cap analysis gene expression of developing mouse tooth germs compared with that of the whole body, we found that Cldn1 and Cldn10 were highly expressed in the tooth. Furthermore, single-cell RNA-sequence analysis using 7-day postnatal Krt14-RFP mouse incisors revealed Cldn1 and Cldn10 exhibited distinct expression patterns. Cldn10 has two isoforms, Cldn10a and Cldn10b, but only Cldn10b was expressed in the tooth. Immunostaining of developing tooth germs revealed claudin-10 was highly expressed in the inner enamel epithelium and stratum intermedium. We also found that overexpression of Cldn10 in the dental epithelial cell line, SF2, induced alkaline phosphatase (Alpl) expression, a marker of maturated stratum intermedium. Our findings suggest that Cldn10 may be a novel stratum intermedium marker and might play a role in cytodifferentiation of stratum intermedium..
24. 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, 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..

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