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Summary Objective To observe and analyze the interaction between excessive mechanical stress (MS) and decreased sex hormones on Temporomandibular Joint Osteoarthritis (TMJ-OA), and to discover TMJ-OA disease susceptibility genes by molecular biological analysis to elucidate part of the mechanism of TMJ-OA onset. Design For experimental groups, orchiectomy (ORX) or ovariectomy (OVX) was performed on sexually mature 8-week-old mice. A metal plate was attached to the posterior surface of the maxillary incisors to apply excessive MS on mandibular condyles. Male mice were divided into control, ORX, MS, and ORX + MS groups, while female mice were divided into control, OVX, MS, and OVX + MS groups. Mandibular condyles were evaluated by histology and molecular biology. Results Histomorphometric analysis of the TMJ in ORX + MS and OVX + MS groups revealed the thinnest chondrocyte layers, highest modified Mankin scores, and significant increases in the number of osteoclasts. Gene expression analysis indicated upregulation of Angptl7 and Car1 genes in the mandibular condyles of mice subjected to the combined effects of excessive MS and reduced sex hormones. In vitro analysis suggested that cartilage-like cells overexpressing Angptl7 enhanced calcification, and osteoblast-like cells overexpression Car1 suppressed cell proliferation and calcification. Conclusions A severe TMJ-OA mouse model was successfully developed by applying excessive MS on the mandibular condyle of male and female mice with reduced sex hormones. Disease-susceptibility genes Angptl7 and Car1 were newly discovered in the experimental groups, suggesting their involvement in the onset mechanism of TMJ-OA.

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.

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.

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 a 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 for 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.

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-β (TGF-β), produced by odontogenic epithelial cells. We observed two pathways for receptor activator of NF-κB ligand (RANKL) induction by keratocystic odontogenic tumor fluid: the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway through interleukin-1α (IL-1α) signaling and non-COX-2/PGE2 pathway through TGF-β receptor signaling. TGF-β1 and IL-1α produced by odontogenic tumors/cysts induced osteoclastogenesis directly in the osteoclast precursor cells and indirectly via increased RANKL induction in the stroma.

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 2α1 (Col2α1) 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 Col2α1 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.

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.

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 (transient receptor potential vanilloid 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 (a 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.

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 Gli2 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.

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 a 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 for 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.

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 (Col10α1) 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 Col10α1 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 Col10α1 transcription. Thus, Ihh signaling could be an important player for not only early chondrocyte differentiation but maturation and calcification of chondrocytes.

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.

GTR法の5年間の臨床的評価を行うために, 成人性歯周炎を有する患者28名に対して14名14部位 (年齢21から57歳, 平均40.9歳の男性3名, 女性11名の14名) に組織再生誘導法 (GTR群), 14名14部位 (年齢23から54歳, 平均39.6歳の男性5名, 女性9名の14名) に歯肉剥離掻爬術 (FOP群) を行った。GTR群の被験部位の骨欠損形態は, 1壁性骨欠損1部位, 2壁性骨欠損11部位, 3壁性骨欠損2部位であった。FOP群の被験部位の骨欠損形態は, 1壁性骨欠損2部位, 2壁性骨欠損9部位, 3壁性骨欠損3部位であった。術前, 術後5年の臨床所見から, 歯周ボクット (PD) の減少量はGTR群3.9±1.6mm, FOP群2.9±1.5 mmであり, 臨床的アタッチメントレベル (CAL) の獲得量はGTR群3.2±1.8mm, FOP群1.1±1.4mmであり, 歯肉退縮量 (MR) は, GTR群0.7±1.3mm, FOP群1.8±1.5mmであった。GTR群の術前, 術後5年のCALに有意な改善を認めた。また, CALの獲得量は, GTR群がFOP群と比べて有意な増加を示した。さらに術後1年と5年の間の両群のPD, CALは安定していた。以上より, 5年間にわたってGTR群のPD, CALは安定しており, 有用であることが示唆された。

本研究では, 歯周ポケット底部への到達性に優れ, 塩酸ミノサイクリンを含有し, 投与直後より徐々に局所で溶解するストリップスタイプの薬剤の歯周炎の急性症状に対する局所投与の臨床的・細菌学的研究を行った。対象とした患者は, 歯周病の急性症状を呈し, 全身的疾患を有さず過去3ケ月以内に抗生物質の投与を受けていない43名とした。43名全てに歯周炎局所由来の痛みを伴う発赤・腫脹または膿瘍形成が認められた。塩酸ミノサイクリン含有のストリップスを歯周ポケット底部にまで挿入した。ストリップスを挿入後, 0, 3, 7日後の臨床症状の変化を観察し, 細菌学的変化を0, 7日後に観察した。歯肉の発赤, 腫脹, 疼痛, プロービング深さ (probing depth, PD), プロービング時出血 (bleeding on probing, BOP), 排膿 (suppuration, S), リンパ節所見および歯肉溝滲出液 (gingival crevicular fluid, GCF) 量の変化を記録した。黒色色素産生性グラム陰性嫌気性桿菌 (Black Pigmented Gram-Negative Anaerobic Rods, BPNAR) 数, 生菌数は非選択性血液平板培地で生育させて測定した。疼痛, 腫脹 (口腔内), BOPおよびSは, 薬剤投与後3日目および7日目で投与開始時に比較して有意に減少していた。さらに, PDおよびGCF量においても7日目において有意な減少が観察された。投与前に優勢であったBPNARの構成比率は投与開始直前と比較し, 7日後では明らかに減少していた。これらの結果から, 歯周ポケット内への塩酸ミノサイクリン含有ストリップスの局所投与は, 歯周炎の急性症状の改善に有効であることが示唆された。

現在, 歯肉縁下プラークのコントロール法のひとつとして, 抗生物質の歯周ポケット内投与が行われている。今回われわれは, 歯周ポケット底部への到達性に優れ, 塩酸ミノサイクワンを含有し, 投与直後より徐々に局所で溶解 (ローカルドラッグデリバリーシステム) するストリップスタイプの薬剤 (Strip type Drug for Periodontitis: SDP) の臨床効果を既存の歯科用塩酸ミノサイクリン軟膏 (Periocline ® : PER) と比較検討した。対象とした患者は, 全身疾患がなく, 4mm以上の歯周ポケットを有する歯周炎の症状を呈し, 過去3カ月以内に抗生物質の投与を受けていない68人とし, SDPを33人に, またPERを35人に局所投与した。投与は1週毎に4回連続投与した。7週後まで臨床症状の変化を観察し, 併せて細菌検査を培養法によって行った。細菌検査実施時期は薬剤投与開始時および4週後 (薬剤投与終了1週後) とした。臨床症状の観察項目は, プロービングデプス (probing depth: PD), プロービング時出血 (bleeding on probing: BOP), 排膿 (suppuration: S), 歯垢指数 (plaque index: PlI), 歯肉炎指数 (gingival index: GI) および歯肉溝滲出液 (gingival crevicular fluid: GCF) 量の変化とした。細菌はPorphyromonas gingivalis, Prevotella intermedia, Prevotella melaninogenica, Fusoacteriumz nucleatum, Eikenella corrodens, Capnocytophaga spp., Actinobacillus a ctinomycetemcomitansの7菌種を選択培地等で分離・検索し, 黒色色素産生性グラム陰性i嫌気性桿菌数, 生菌数は非選択性血液平板培地で生育させて測定した。その結果, BOP, S, GI, およびPDにおいて, 投与開始時と比較して3週後, 4週後および7週後で, 両群とも有意な減少が認められた。GCFではPER群のみ全観察期間において有意な減少が認められた。また, 臨床主要症状の改善度では両群ともに高い有効率を示したが, 両群問に有意な差は認められなかった。細菌学的検索において黒色色素産生性グラム陰性嫌気性桿菌数の変化, 生菌数の変化ともに投与の前後で有意な減少が認められ, 両群間で有意差は認められなかった。さらに上記7菌種別の消失率をみても両群問に有意な差は認められず, 両薬剤とも各菌種に対して有効であった。塩酸ミノサイクリンの局所投与における臨床的・細菌学的効果を調べたところ, 良好な結果が得られた。また, 剤型の違いによる比較を行ったところ, 臨床的・細菌学的効果に大きな差は認められず, ともに有効であることが示された。

This study shows that Arid5a interacts with Sox9 and subsequently modulates histone 3 acetylation of a chondrogenic gene, Col2a1, and stimulates chondrocyte differentiation., 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.

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.

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 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54(nrb) suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.

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 - 100 mg/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.

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.