Authorship：Corresponding author   Language：English  

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Authorship：Lead author,　Corresponding author   Language：English  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.joca.2021.08.005

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Language：Japanese   Publisher：(NPO)日本口腔科学会  

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)  

© 2020 Federation of American Societies for Experimental Biology The frontal craniofacial skeleton derived from neural crest cells is vital for facial structure and masticatory functions. The exact role of Indian hedgehog (Ihh) in facial and masticatory development has not been fully explored. In this study, we generated craniofacial neural crest cells-specific Ihh deletion mice (Wnt1-Cre;Ihhfl/fl;Tomatofl/+) and found the gradual dwarfism without perinatal lethality. Morphological and histological analyses revealed unambiguous craniofacial phenotypes in mutants, where we observed skeletal malocclusion accompanied by markedly hypoplastic nasomaxillary complex and reversed incisor occlusion. Both the replacement of nasal concha cartilage by turbinate bones and the endochondral ossification of nasal septum ethmoid bone were substantially delayed. We also observed hypoplastic mandibles in mutants where the mandibular ramus was unexpectedly the most affected. Both the condylar process and mandibular angle cartilages were distorted. However, dental examination showed no significant changes in teeth and dentition. Finally, a comprehensive RNA sequence analysis utilizing condylar cartilage identified Ihh-associated gene network including several cell cycle genes and 16 genes related to the extracellular matrix, sulfate transporters, transcription factors, receptors, a ciliogenesis factor, and an adhesion molecule. Our data provide direct in vivo evidence that Ihh plays crucial roles in midface and masticatory system formation, likely by activating key genes.

DOI： 10.1096/fj.201903269R

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Campomelic dysplasia (CMPD) is a rare congenital skeletal disorder caused by SOX9 mutations. The patients are often accompanied by craniofacial and oral anomalies such as characteristic facial appearance and cleft palate. In spite of possibility of perinatal lethality, some patients could survive with milder phenotype and appropriate medical treatment. Such patients might cause critical issues in feeding and require oral management. However, there has been less information about oral surgical treatment in CMPD patients. Here, we describe our recent experience of diagnosis and cleft palate repair in a CMPD patient. A 27 month old Japanese female presenting typical CMPD phenotypes was genetically determined and a SOX9Q175Stop mutation was identified. The protein analysis demonstrated that SOX9Q175Stop mutation yields the truncated protein lacking the transactivation site. The affected infant had serious respiratory distress and always required control by an artificial ventilator. Cleft palate reconstruction under general anesthesia was performed to improve her feeding and oral functions. Modified Furlow palatoplasty was applied to minimize surgical invasion in which only a unilateral relaxation incision was designed. Artificial skin material was used to support fragile soft palate tissue. Although there was some risk to cause any serious respiratory issue, her SpO2 was maintained well through the perioperative period. Our case report might provide valuable information for syndromic cleft palate treatments.

DOI： 10.1016/j.ajoms.2018.07.007

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

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：(公社)日本口腔外科学会  

症例は生後17日の男児で、出生時に顔面異常を認めたためNICUへ搬送入院となった。両側斜顔面裂、口蓋裂および右側眼球形成不全を認めた。非定形口蓋裂を伴う両側性斜顔面裂の診断で、生後3ヵ月時に全身麻酔下にて顔面、口唇および口角形成術を行った。右側痕跡眼球に関しては、眼球様組織の摘出および角膜、結膜縫合を行い、偽眼ステントを挿入した。1歳時に非定形口蓋裂に対して、Furlow変法を用いた口蓋形成術を行った。3歳時に、創部瘢痕の除去および形態修正術を全身麻酔下にて行い、6歳時に左側の反対咬合と上顎歯列弓の狭窄に対して、緩徐拡大装置を用いた矯正治療を開始した。7歳となる現在、明らかな鼻咽腔閉鎖不全や発音障害もなく経過観察および矯正科にて矯正治療を行っている。

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Osteocytes within the mineralized bone matrix control bone remodeling by regulating osteoblast and osteoclast activity. Osteocytes express the aging suppressor Klotho, but the functional role of this protein in skeletal homeostasis is unknown. Here we identify Klotho expression in osteocytes as a potent regulator of bone formation and bone mass. Targeted deletion of Klotho from osteocytes led to a striking increase in bone formation and bone volume coupled with enhanced osteoblast activity, in sharp contrast to what is observed in Klotho hypomorphic (kl/kl) mice. Conversely, overexpression of Klotho in cultured osteoblastic cells inhibited mineralization and osteogenic activity during osteocyte differentiation. Further, the induction of chronic kidney disease with high-turnover renal osteodystrophy led to downregulation of Klotho in bone cells. This appeared to offset the skeletal impact of osteocyte-targeted Klotho deletion. Thus, our findings establish a key role of osteocyte-expressed Klotho in regulating bone metabolism and indicate a new mechanism by which osteocytes control bone formation.

DOI： 10.1016/j.kint.2017.02.014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

Odontogenic tumors and cysts, arising in the jawbones, grow by resorption and destruction of the jawbones. However, mechanisms underlying bone resorption by odontogenic tumors/cysts remain unclear. Odontogenic tumors/cysts comprise odontogenic epithelial cells and stromal fibroblasts, which originate from the developing tooth germ. It has been demonstrated that odontogenic epithelial cells of the developing tooth germ induce osteoclastogenesis to prevent the tooth germ from invading the developing bone to maintain its structure in developing bones. Thus, we hypothesized that odontogenic epithelial cells of odontogenic tumors/cysts induce osteoclast formation, which plays potential roles in tumor/cyst outgrowth into the jawbone. The purpose of this study was to examine osteoclastogenesis by cytokines, focusing on transforming growth factor-beta (TGF-beta), produced by odontogenic epithelial cells. We observed two pathways for receptor activator of NF-kappa B ligand (RANKL) induction by keratocystic odontogenic tumor fluid: the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway through interleukin-1 alpha (IL-1 alpha) signaling and non-COX-2/PGE2 pathway through TGF-beta receptor signaling. TGF-beta 1 and IL-1 alpha produced by odontogenic tumors/cysts induced osteoclastogenesis directly in the osteoclast precursor cells and indirectly via increased RANKL induction in the stroma.

DOI： 10.3892/ijo.2016.3548

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Digit formation is a process, which requires the proper segmentation, formation and growth of phalangeal bones and is precisely regulated by several important factors. One such factor is In, a gene linked to BDA1 and distal symphalangism in humans. In existing mouse models, mutations in Ihh have been shown to cause multiple synostosis in the digits but lead to perinatal lethality. To better study the exact biological and pathological events which occur in these fused digits, we used a more viable Prx1-Cre;Ihh(fl/fl) model in which Cre recombinase is expressed during mesenchymal condensation in the earliest limb buds at E9.5 dpc and found that mutant digits continuously fuse postnatally until phalanges are finally replaced by an unsegmented "one-stick bone". Mutant mice displayed osteocalcin-positive mature osteoblasts, but had reduced proliferation and abnormal osteogenesis. Because of the close interaction between Ihh and PTHrP during endochondral ossification, we also examined the digits of Prx1-Cre;PTH1R(fl/fl) mice, where the receptor for PTHrP was conditionally deleted. Surprisingly, we found PTH1R deletion caused symphalangism, demonstrating another novel function of PTH1R signaling in digit formation. We characterized the symphalangism process whereby initial cartilaginous fusion prevented epiphyseal growth plate formation, resulting in resorption and replacement of the remaining cartilage by bony tissue. Chondrocyte differentiation displayed abnormal directionality in both mutants. Lastly, Prx1-Cre;Ihh(fl/fl);Jansen Tg mice, in which a constitutively active PTH1R allele was introduced into Ihh mutants, were established to address the possible involvement of PTH1R signaling in Ihh mutant digits. These rescue mice failed to show significantly improved phenotype, suggesting that PTH1R signaling in chondrocytes is not sufficient to restore digit formation. Our results demonstrate that Ihh and PTH1R signaling in limb mesenchyme are both essential to regulate proper development of digit structures, although they appear to use different mechanisms. (c) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2015.11.017

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Indian hedgehog (Ihh) is widely recognized as an essential factor for proper skeletal development. Previous in vivo studies using mutant Ihh mouse models were limited by perinatal lethality or carried out after a growth plate formed. Thus the important role of Ihh in mesenchymal cell differentiation has not been investigated. In this study, we established Prx1-Cre;Ihh(fl/fl) mice to ablate Ihh specifically in limb mesenchyme to allow us to observe the phenotype continuously from prenatal development to 3 weeks of age. Mutant mice displayed severe limb abnormalities characterized by complete lack of secondary ossification center and growth plate, indicating an essential role for Ihh in the development of these structures. Interestingly, we discovered that osteoblast differentiation and bone formation could occur in conditions of deficient Ihh. This is a novel finding that has not been observed because of the early lethality of previous Ihh mutants. Mature osteoblasts expressing osteocalcin could be detected in the center of mutant bones at postnatal day 10 (P10). Osteoclasts and blood vessel formation were also present, suggesting active bone remodeling. Histomorphometric analyses show significant increase in osteoclast number with no major changes in bone formation rate at 3 weeks of age. Mutant long bones in the limbs were deformed, with cortices comprised of irregular woven bone. Also, there was a marked decrease in gene expression of osteoblastic and osteocytic markers. Moreover, mutant long bones displayed bone dysplasia in which we observed increased osteoclast activity and partially reduced osteoblastic and osteocytic differentiation that lead ultimately to loss of bone structures at 3 weeks of age. In summary, our data show or the first time, the presence of mature osteoblasts in long bones of the limbs despite the complete loss of growth plate formation due to Ihh deficiency. These data indicate an important function for Ihh in regulating limb mesenchymal cell differentiation. (C) 2015 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.2582

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

? 2015 Japanese Association for Oral Biology. Background Bone and cartilage are essential skeletal tissues, which not only function as structural basis of locomotive organs, but also regulate calcium homeostasis, phosphate metabolism, hematopoiesis, and glucose turnover. Several hormones and cytokines in cooperation with their downstream transcription factors regulate bone and cartilage development; therefore, it is important to understand the precise mechanisms of this regulation. Highlight Genetic studies in human and mouse have provided a wealth of information regarding the transcription factors implicated in bone and cartilage development. Moreover, innovative molecular cloning techniques identified several new transcription factors that play indispensable roles in controlling the development of bone and cartilage. The mechanisms controlling the expression of these transcription factors have been meticulously elucidated, so that the transcriptional network system, which seemed so complex and mysterious not so long ago, has become considerably clearer in recent years. Conclusion Recent advances in our knowledge about transcriptional network systems contributed to understanding the molecular underpinnings of regulation and pathological disease mechanisms in bone and cartilage.

DOI： 10.1016/j.job.2015.06.001

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Indian hedgehog (Ihh) is essential for chondrocyte differentiation and endochondral ossification and acts with parathyroid hormone-related peptide in a negative feedback loop to regulate early chondrocyte differentiation and entry to hypertrophic differentiation. Independent of this function, we and others recently reported independent Ihh functions to promote chondrocyte hypertrophy and matrix mineralization in vivo and in vitro. However, the molecular mechanisms for these actions and their functional significance are still unknown. We recently discovered that Ihh overexpression in chondrocytes stimulated the expression of late chondrocyte differentiation markers and induced matrix mineralization. Focusing on collagen type X (Col10 alpha 1) expression and transcription, we observed that hedgehog downstream transcription factors GLI-Kruppel family members (Gli) 1/2 increased COL10A1 promoter activity and identified a novel Gli1/2 response element in the 250-bp basic promoter. In addition, we found that Ihh induced Runx2 expression in chondrocytes without up-regulating other modulators of chondrocyte maturation such as Mef2c, Foxa2, and Foxa3. Runx2 promoted Col10 alpha 1 expression in cooperation with Ihh. Further analyses using promoter assays, immunofluorescence, and binding assays showed the interaction of Gli1/2 in a complex with Runx2/Smads induces chondrocyte differentiation. Finally, we could demonstrate that Ihh promotes in vitro matrix mineralization using similar molecular mechanisms. Our data provide an in vitro mechanism for Ihh signaling to positively regulate Col10 alpha 1 transcription. Thus, Ihh signaling could be an important player for not only early chondrocyte differentiation but maturation and calcification of chondrocytes.

DOI： 10.1074/jbc.M114.570507

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Histone modification, a critical step for epigenetic regulation, is an important modulator of biological events. Sox9 is a transcription factor critical for endochondral ossification
however, proof of its epigenetic regulation remains elusive. Here we identify AT-rich interactive domain 5b (Arid5b) as a transcriptional co-regulator of Sox9. Arid5b physically associates with Sox9 and synergistically induces chondrogenesis. Growth of Arid5b-/- mice is retarded with delayed endochondral ossification. Sox9-dependent chondrogenesis is attenuated in Arid5b-deficient cells. Arid5b recruits Phf2, a histone lysine demethylase, to the promoter region of Sox9 target genes and stimulates H3K9me2 demethylation of these genes. In the promoters of chondrogenic marker genes, H3K9me2 levels are increased in Arid5b-/- chondrocytes. Finally, we show that Phf2 knockdown inhibits Sox9-induced chondrocyte differentiation. Our findings establish an epigenomic mechanism of skeletal development, whereby Arid5b promotes chondrogenesis by facilitating Phf2-mediated histone demethylation of Sox9-regulated chondrogenic gene promoters.

DOI： 10.1038/ncomms3850

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS INC  

Oculodentodigital Dysplasia (ODDD) is a rare syndrome involving anomalies in eye, tooth, and digit formation, caused by mutations in CX43/GJA1. In addition to classic dental features, ODDD includes oral and craniofacial accessory symptoms such as characteristic facial appearance and cleft palate. However, there have been no reports of ODDD accompanied by cleft lip. Herein we report, for the first time, a male, sporadic, Asian proband presenting bilateral cleft lip. By direct sequence analysis, our proband was diagnosed as having ODDD with a heterozygous mutation, codon 142 G>A in GJA1 and CX43E48K. We excluded the possibility of pathogenic mutations in B3GALTL, BMP4, TFAP2A, PVRL1, IRF6, and MSX1. To address how CX43/GJA1 is related to cleft lip, we performed immunohistochemistry using mouse and human mid-facial tissue. CX43 expression was detected in the nasal compartment and nasal and maxillary processes at murine developmental stage E12.5. Furthermore, CX43 expression was found in the epithelial tissue inside the human subepithelial cleft lip that completes epithelial fusion. Therefore, we suggest that CX43/GJA1 is involved in lip formation. Our case report of ODDD with a bilateral cleft lip suggests that CX43/GJA1 might be a novel candidate gene for syndromic cleft lip.

DOI： 10.1177/0022034512447952

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS IN BIOSCIENCE INC  

Endochondral ossification is very unique and complex biological event which is associated with skeletal development and tissue partnering. Genetic studies and gene-targeting approaches identified several transcription factors that play important roles in endochondral ossification. These transcription factors sequentially and harmoniously regulate each step of endochondral ossification, and consequently maintain the spatio-temporal control of the program. Importantly, these transcription factors form large protein complex to control chromatin remodeling, histone modification, transcription and splicing steps during endochondral ossification. It is also important to understand how these transcription factors regulate expression of their target genes. Biochemical and molecular cloning techniques largely contributed to identification of the components of the transcriptional complex and the target genes. Most recently, importance of endoplasmic reticulum (ER) stress in endochondral ossification has been reported. A transcription factor, BBF2H7, functions as an ER stress sensor in chondrocytes through regulation of appropriate secretion of chondrogenic matrices. We would like to discuss how the transcription factors regulate endochondral ossification.

DOI： 10.2741/4076

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)日本口蓋裂学会  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

SRY-box-containing gene 9 (Sox9) is an essential transcription factor in chondrocyte lineage determination and differentiation. Recent studies demonstrated that Sox9 controls the transcription of chondrocyte-specific genes in association with several other transcriptional regulators. To further understand the molecular mechanisms by which Sox9 influences transcriptional events during chondrocyte differentiation, we attempted to identify transcriptional partners of Sox9 and to examine their roles in chondrocyte differentiation. We isolated AT-rich interactive domain-containing protein 5a (Arid5a; also known as Mrf1) as an activator of the Col2a1 gene promoter from an ATDC5 cDNA library. Arid5a was highly expressed in cartilage and induced during chondrocyte differentiation. Furthermore, Arid5a physically interacted with Sox9 in nuclei and up-regulated the chondrocyte-specific action of Sox9. Overexpression of Arid5a stimulated chondrocyte differentiation in vitro and in an organ culture system. In contrast, Arid5a knockdown inhibited Col2a1 expression in chondrocytes. In addition, Arid5a binds directly to the promoter region of the Col2a1 gene and stimulates acetylation of histone 3 in the region. Our results suggest that Arid5a may directly interact with Sox9 and thereby enhance its chondrocyte-specific action.

DOI： 10.1091/mbc.E10-07-0566

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Sox9 is an essential transcription factor for chondrogenesis by regulating the expression of chondrogenic genes. However, its regulatory mechanism is not fully understood. To address this, we attempted to identify the transcriptional partners of Sox9 by screening the cDNA library of the chondrogenic cell line ATDC5 using the collagen 2 alpha 1 (Col2 alpha 1) gene promoter fused to a luciferase reporter gene. One of the positive clones encoded the Znf219 gene. Whole mount in situ hybridization experiments indicated that Znf219 mRNA was specifically expressed in the developing limb buds where Col2 alpha 1 and Sox9 were strongly expressed. Znf219 markedly enhanced the transcriptional activity of Sox9 on the Col2a1 gene promoter. In addition, Znf219 is physically associated with Sox9 and is colocalized with Sox9 in the nucleus. We also found that overexpression of Znf219 profoundly increased Sox9-induced mRNA expression of Col2a1, aggrecan and Col11a2. Consistently, knockdown of Znf219 decreased the Sox9-induced mRNA expression of these genes. Furthermore, a dominant-negative mutant Znf219 inhibited Bmp2-induced chondrocyte differentiation. Our results suggest that Znf219 plays an important role in the regulation of chondrocyte differentiation as a transcriptional partner of Sox9.

DOI： 10.1242/jcs.071373

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

Sox9 is a transcription factor that plays an essential role in chondrogenesis and has been proposed to inhibit the late stages of endochondral ossification. However, the molecular mechanisms underlying the regulation of chondrocyte maturation and calcification by Sox9 remain unknown. In this study, we attempted to clarify roles of Sox9 in the late stages of chondrocyte differentiation. We found that overexpression of Sox9 alone or Sox9 together with Sox5 and Sox6 (Sox5/6/9) inhibited the maturation and calcification of murine primary chondrocytes and up-regulated parathyroid hormone-related protein (PTHrP) expression in primary chondrocytes and the mesenchymal cell line C3H10T1/2. Sox5/6/9 stimulated the early stages of chondrocyte proliferation and development. In contrast, Sox5/6/9 inhibited maturation and calcification of chondrocytes in organ culture. The inhibitory effects of Sox5/6/9 were rescued by treating with anti-PTHrP antibody. Moreover, Sox5/6/9 bound to the promoter region of the PTHrP gene and up-regulated PTHrP gene promoter activity. Interestingly, we also found that the Sox9 family members functionally collaborated with Ihh/Gli2 signaling to regulate PTHrP expression and chondrocyte differentiation. Our results provide novel evidence that Sox9 family members mediate endochondral ossification by up-regulating PTHrP expression in association with Ihh/Gli2 signaling.

DOI： 10.1091/mbc.E09-03-0227

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

AIM: The purpose of this study was to examine morphological changes of the mandibular region using frontal cephalograms following bilateral sagittal split osteotomy (BSSO) for mandibular setback. Furthermore, we compared the stability between screw and plate fixation. METHODS: Pre and postoperative frontal cephalograms were taken of 26 patients. Parameters, Angle "A" which is formed by constructed lines along the mandibular ramus, and mandibular width at the angle were calculated. The subjects were analyzed according to the types of bony fixation, whether by screws or plates. RESULTS: Angle "A" and mandibular angular width of total numbers did not widely change at early period following operation. Late postoperatively, mandibular width of total numbers decreased in conjunction with increasing Angle "A". When comparing the parameters between screw and plate groups, the plate group showed a decrease of Angle "A" as an early change. Late postoperatively, the plate group showed an increase of Angle "A" and a decrease of mandibular width, while the screw group had smaller changes. CONCLUSION: We demonstrate postoperative inclination of mandibular lower ramus towards the facial midline. We suggest that the types of rigid fixation have an influence on frontal skeletal morphology, namely that screw fixation has higher stability.

DOI： 10.1016/j.jcms.2009.04.012

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Oral and Maxillofacial Surgeons  

Since elderly patients with systemic diseases often undergo tooth extraction, it is difficult to predict the risk of and to optimally treat post-extraction hemorrhage. We report a rare case of persistent hemorrhage that occurred after tooth extraction caused by chronic disseminated intravascular coagulation (DIC) as a complication of dissecting aneurysm. A 69-year-old man who underwent extraction of a maxillary molar had persistent bleeding, which could not be stopped by continuous local treatment to promote hemostasis. After a general examination, we diagnosed chronic DIC caused by an extensive dissecting aneurysm, based on evidence of advanced fibrinogenolysis. Hemostasis was achieved by general treatment for chronic DIC. Appropriate diagnosis and treatment of both local and general conditions are necessary for post-extraction hemorrhage.

DOI： 10.5794/jjoms.54.631

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Other Link： https://jlc.jst.go.jp/DN/JALC/00322388084?from=CiNii

Language：Japanese   Publisher：(株)最新医学社  

脊椎動物の骨格の大部分は内軟骨性骨形成により形成されている.内軟骨性骨形成は,さまざまなサイトカインとその下流で機能する細胞内情報伝達経路と転写因子により,時間的・空間的に制御されている.軟骨細胞分化に重要な役割を果たすサイトカインとしては,BMP,インディアンヘッジホッグ,PTHrP,FGF,Wntの関与が明らかになっている.また転写因子としては,Sox9ファミリーとRunx2,Runx3が必須である.さらに,これら分子の作用機序の解明も飛躍的に進展している.(著者抄録)

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2008&ichushi_jid=J00516&link_issn=&doc_id=20081107140003&doc_link_id=%2Fap7saisa%2F2008%2F006311%2F004%2F2170-2175%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fap7saisa%2F2008%2F006311%2F004%2F2170-2175%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Several studies indicated that a homeobox gene, Msx2, is implicated in regulation of skeletal development by controlling enchondral ossification as well as membranous ossification. However, the molecular basis by which Msx2 conducts chondrogenesis is currently unclear. In this study, we examined the role of Msx2 in chondrocyte differentiation using mouse primary chondrocytes and embryonic metatarsal explants. Treatment with BMP2 up-regulated the expression of Msx2 mRNA along with chondrocyte differentiation in murine primary chondrocytes. Overexpression of wild-type Msx2 stimulated calcification of primary chondrocytes in the presence of BMP2. We also found that constitutively active Msx2(caMsx2) enhanced BMP2 dependent calcification more efficiently than wild- type Msx2. Consistently, caMsx2 overexpression up-regulated the expression of alkaline phosphatase and collagen type X induced by BMP2. Furthermore, organ culture experiments using mouse embryonic metatarsals indicated that caMsx2 clearly stimulated the maturation of chondrocytes into the prehypertrophic and hypertrophic stages in the presence of BMP2. In contrast, knockdown of Msx2 inhibited maturation of primary chondrocytes. The stimulatory effect of Msx2 on chondrocyte maturation was enhanced by overexpression of Smad1 and Smad4 but inhibited by Smad6, an inhibitory Smad for BMP2 signaling. These data suggest that Msx2 requires BMP2/Smad signaling for its chondrogenic action. In addition, caMsx2 overexpression induced Ihh (Indian hedgehog) expression in mouse primary chondrocytes. Importantly, treatment with cyclopamine, a specific inhibitor for hedgehogs, blocked Msx2-induced chondrogenesis. Collectively, our results indicated that Msx2 promotes the maturation of chondrocytes, at least in part, through up-regulating Ihh expression.

DOI： 10.1074/jbc.M803681200

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CLINICAL INVESTIGATION INC  

The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, alpha 1 (Col2al) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54(nrb)) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54(nrb) physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2al promoter. In ATDC5 cells, p54(nrb) colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54(nrb) suppressed Sox9-dependent Col2al expression and promoter activity. We generated a p54(nrb) mutant construct lacking RNA recognition motifs, and overexpression of mutant p54(nrb) in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2al mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54(nrb) in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54(nrb) plays.

DOI： 10.1172/JCI31373

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Language：Japanese   Publisher：(公社)日本整形外科学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

DOI： 10.1007/s00774-007-0824-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Sox9 is a transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expression. However, the precise mechanism of Sox9 activation during chondrogenesis is not fully understood. To investigate this mechanism, we performed functional gene screening to identify genes that activate SOX9-dependent transcription, using full-length cDNA libraries generated from a murine chondrogenic cell line, ATDC5. Screening revealed that TRPV4 ( (t) under bar ransient (r) under bar eceptor (p) under bar otential (v) under bar anilloid 4), a cation channel molecule, significantly elevates SOX9-dependent reporter activity. Microarray and quantitative real time PCR analyses demonstrated that during chondrogenesis in ATDC5 and C3H10T1/2 ( murine mesenchymal stem cell line), the expression pattern of TRPV4 was similar to the expression patterns of chondrogenic marker genes, such as type II collagen and aggrecan. Activation of TRPV4 by a pharmacological activator induced SOX9-dependent reporter activity, and this effect was abolished by the addition of the TRPV antagonist ruthenium red or by using a small interfering RNA for TRPV4. The SOX9-dependent reporter activity due to TRPV4 activation was abrogated by both EGTA and a calmodulin inhibitor, suggesting that the Ca2+/calmodulin signal is essential in this process. Furthermore, activation of TRPV4 in concert with insulin activity in ATDC5 cells or in concert with bone morphogenetic protein-2 in C3H10T1/2 cells promoted synthesis of sulfated glycosaminoglycan, but activation of TRPV4 had no effect alone. We showed that activation of TRPV4 increased the steady-state levels of SOX9 mRNA and protein and SOX6 mRNA. Taken together, our results suggest that TRPV4 regulates the SOX9 pathway and contributes to the process of chondrogenesis.

DOI： 10.1074/jbc.M706158200

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Doctoral thesis  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2- but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between GR2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.

DOI： 10.1091/mbc.E06-08-0743

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

In this article, a procedure involving 2-stage alveolar distraction osteogenesis using eccentric distraction devices for the augmentation of resorbed transplanted iliac bone following mandibular tumor resection is presented. A 6-month consolidation period was allowed between the first and second distractions, and endosseous implants were placed 4 months after the second distraction. Computerized tomographic images obtained before the implantation revealed that, 10 months after the first distraction, the bone generated still showed lower density compared with the basal bone, but the bone from both distractions showed enough maturity for implantation. It may be concluded that 2-stage alveolar distraction osteogenesis can be a useful and safe procedure for excessive alveolar lengthening if a sufficiently long consolidation period is allowed.

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Language：Japanese   Publisher：(NPO)日本口腔科学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

To investigate mechanisms in pathological pain conditions as the hyperalgesia and allodynia observed after dental surgery, we employed a rat dental-injury model involving the simultaneous pulpectomy to a lower incisor and extraction of an ipsilateral upper incisor. We found that hypersensitivity to tactile stimulation developed on both ipsilateral and contralateral sides in the dental-injured rats 5 days after the surgery and that this lasted for at least 30 days. Recovery from hypersensitivity to tactile stimulation was achieved by the intraperitoneal (i.p.) administration of MK-801 (0.05 mg/kg) or N-G-monomethyl-L-arginine monoacetate (L-NMMA: 10-100mg/kg), but not attained by N-G-monomethyl-D-arginine monoacetate (D-NMMA: 100 mg/kg). This recovery effect Of L-NMMA (50 mg/kg) was inhibited by pretreatment with L-arginine (600 mg/kg). In the trigeminal nucleus caudalis (SpVc), the changes in nitric oxide (NO) levels invoked by the intravenous (i.v.) administration of N-methyl-D-aspartate (NMDA; 10 mg/kg) were found to be significantly larger in the dental-injured rats than in sham-operated rats. The number of neuronal NO synthase (nNOS)-positive neurons increased in layers I-II and III-IV in the SpVc on both sides of the dental injured rats. These results suggest that hypersensitivity to tactile stimulation developed following dental injury, and that NMDA receptor/NOS/NO production pathways in the SpVc may be involved in pathological conditions.

DOI： 10.1254/jjp.90.145

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE INC  

DOI： 10.1016/j.bone.2009.01.110

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC BONE & MINERAL RES  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC BONE & MINERAL RES  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC BONE & MINERAL RES  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC BONE & MINERAL RES  

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Language：Japanese   Publisher：(NPO)日本口腔科学会  

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