記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Cellular communication network factor (CCN) 3 is one of the classical members of the CCN family, which are characterized by common molecular structures and multiple functionalities. Although this protein was discovered as a gene product overexpressed in a truncated form in nephroblastoma, recent studies have revealed its physiological roles in the development and homeostasis of mammalian species, in addition to its pathological association with a number of diseases. Cartilage is a tissue that creates most of the bony parts and cartilaginous tissues that constitute the human skeleton, in which CCN3 is also differentially produced to exert its molecular missions therein. In this review article, after the summary of the molecular structure and function of CCN3, recent findings on the regulation of ccn3 expression and the roles of CCN3 in endochondral ossification, cartilage development, maintenance and disorders are introduced with an emphasis on the metabolic regulation and function of this matricellular multifunctional molecule.

DOI： 10.1007/s12079-021-00629-z

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その他リンク： https://link.springer.com/article/10.1007/s12079-021-00629-z/fulltext.html

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The uncoupling protein 1 (UCP1) gene is known to be highly expressed in brown adipose tissue (BAT) that functions in thermogenesis. It has been shown that UCP1 mRNA is localized to the mouse adrenal gland, but its significance remains elusive. To explore how UCP1 expression in the adrenal gland is regulated, we generated a reporter knock-in mouse in which the GFP gene was inserted into the UCP1 locus using CRISPR-Cas9 system. Firstly, we confirmed by Western blot analysis UCP1-driven GFP protein expression in interscapular BAT of the knock-in mice kept at 4 °C. Immunohistochemistry showed that GFP protein was detected in the adrenal gland of the knock-in mice. More intense GFP expression was observed in the adrenal medulla than in the cortex of the reporter mice irrespectively of cold exposure. Immunohistochemistry using anti-UCP1 antibody, as well as Western blot analysis verified UCP1 protein expression in the wild-type adrenal medulla. These results suggest that the mouse adrenal gland is a novel organ expressing UCP1 protein and its expression is not upregulated by cold exposure.

DOI： 10.1016/j.bbrc.2021.06.013

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

Fibroblast growth factor 1 (FGF-1) is the first FGF family member, and it induces proliferation of fibroblasts and other types of the cells. However, recent studies are uncovering unexpected functions of this molecule. Our previous study redefined this growth factor as a catabolic molecule produced in cartilage upon metabolic insult. Indeed, FGF-1 was found to repress the gene expression of cellular communication network factor 2 (CCN2), which protects and regenerates cartilage, amplifying its own production through positive feedback regulation. In the present study, we investigated the molecular mechanism of this bipartite CCN2 repression and FGF1 activation by FGF-1 in chondrocytes. Repression of CCN2 and induction of FGF1 in human chondrocytic cells were both partly abolished by valproic acid, an inhibitor of histone deacetylase 1 (HDAC1), indicating the involvement of chromatin remodeling by histone acetylation in this system. In contrast, RNA degradation analysis suggested no contribution of post-transcriptional regulation of the mRNA stability to the effects conferred by FGF-1. Suspecting a regulation by a specific transcription factor, we next sought a candidate in silico from a large dataset. As a result, we found fork head box protein A1 (FOXA1) as the transcription factor that bound to both CCN2 and FGF1 loci. Functional analysis demonstrated that FOXA1 silencing significantly attenuated the CCN2 repression and FGF1 induction caused by FGF1. These findings collectively indicate that the bipartite regulation by FGF-1 is enabled by the combination of chromatin remodeling by HDACs and transcriptional modulation by FOXA1 with unknown transcriptional coactivators of opposite functionalities.

DOI： 10.1007/s12079-020-00600-4

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/ijms21207556

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.15252/embr.201948462

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Data on asthma burden in pediatric patients are limited; this real-world study investigated exacerbation frequency and health care resource utilization (HCRU) in pediatric asthma patients from the US and England. METHODS: Data from pediatric patients (aged 6-17 years) in the Optum claims database (US) or Clinical Practice Research Datalink with linkage to Hospital Episode Statistics (England) were analyzed. Patients were categorized into four hierarchical groups: treated asthma (patients with ≥1 baseline asthma medication), severe asthma (plus Global Initiative for Asthma Step 4/5), severe refractory asthma ([SRA] plus ≥2 baseline severe asthma exacerbations), and eosinophilic SRA (SRA plus blood eosinophil count ≥150 cells/µL). Exacerbation frequency and HCRU during the 12 months postindex were described. RESULTS: Of 151 549 treated asthma patients in the US, 18 086 had severe asthma, 2099 SRA, and 109 eosinophilic SRA. There were 32 893 treated asthma patients in England, of whom 2711 had severe asthma, 265 SRA, and 8 eosinophilic SRA. In the 12 months postindex, ≥1 exacerbation occurred in 12.4% and 10.8% of patients with severe asthma, and 32.6% and 42.6% with SRA in the US and England, respectively. The proportions of patients with ≥1 asthma hospitalization in the 30 days after the first asthma exacerbation were 2.7% and 4.4% (treated), 3.5% and 8.2% (severe asthma), and 6.0% and 16.8% (SRA) in the US and England, respectively. CONCLUSION: This study provides insights into current asthma management practices in the US and England and indicates that some patients with severe disease have an unmet need for effective management.

DOI： 10.1002/iid3.299

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI  

To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in RAB14 or CCN2 mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, BIP and CHOP mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes.

DOI： 10.3390/ijms21082769

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/ijms21051564

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1530/JOE-19-0022

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Fluticasone propionate 50 μg/salmeterol xinafoate 25 μg (FP/SAL) is widely used in adults and children with asthma, but there is sparse information on its use in very young children. METHODS: This was a randomized, double-blind, multicentre, controlled trial conducted in children aged 8 months to 4 years. During a 2-week run-in period, they all received FP twice daily. At randomization, they commenced FP/SAL or FP twice daily for 8 weeks. All were then given FP/SAL only, in a 16-week open-label study continuation. Medications were inhaled through an AeroChamber Plus with attached face mask. The primary end-point was mean change in total asthma symptom scores from baseline to the last 7 days of the double-blind period. Analyses were undertaken in all children randomized to treatment and who received at least one dose of study medication. RESULTS: Three hundred children were randomized 1:1 to receive FP/SAL or FP. Mean change from baseline in total asthma symptom scores was -3.97 for FP/SAL and -3.01 with FP. The between-group difference was not statistically significant (P = 0.21; 95% confidence interval: -2.47, 0.54). No new safety signals were seen with FP/SAL. CONCLUSION: This is the first randomized, double-blind study of this size to evaluate FP/SAL in very young children with asthma. FP/SAL did not show superior efficacy to FP; no clear add-on effect of SAL was demonstrated. No clinically significant differences in safety were noted with FP/SAL usage.

DOI： 10.1111/pai.13010

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担当区分：筆頭著者,　最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：AME Publishing Company  

DOI： 10.21037/biotarget.2018.08.03

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/molecules23071517

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acsbiomaterials.7b00962

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1039/C7IB00130D

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担当区分：筆頭著者,　責任著者   掲載種別：論文集(書籍)内論文   出版者・発行元：Springer New York  

DOI： 10.1007/978-1-4939-6430-7_20

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担当区分：筆頭著者,　責任著者   掲載種別：論文集(書籍)内論文   出版者・発行元：Springer New York  

DOI： 10.1007/978-1-4939-6430-7_32

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掲載種別：論文集(書籍)内論文   出版者・発行元：Springer New York  

DOI： 10.1007/978-1-4939-6430-7_15

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掲載種別：論文集(書籍)内論文   出版者・発行元：Springer New York  

DOI： 10.1007/978-1-4939-6430-7_8

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

OBJECTIVES: Platelet-rich plasma (PRP) has been widely used to enhance the regeneration of damaged joint tissues, such as osteoarthritic and rheumatoid arthritic cartilage. The aim of this study is to clarify the involvement of all of the CCN family proteins that are crucially associated with joint tissue regeneration. METHODS: Cyr61-CTGF-NOV (CCN) family proteins in human platelets and megakaryocytic cells were comprehensively analyzed by Western blotting analysis. Production of CCN family proteins in megakaryocytes in vivo was confirmed by immunofluorescence analysis of mouse bone marrow cells. Effects of CCN family proteins found in platelets on chondrocytes were evaluated by using human chondrocytic HCS-2/8 cells. RESULTS: Inclusion of CCN2, a mesenchymal tissue regenerator, was confirmed. Of note, CCN3, which counteracts CCN2, was newly found to be encapsulated in platelets. Interestingly, these two family members were not detectable in megakaryocytic cells, but their external origins were suggested. Furthermore, we found for the first time CCN5 and CCN1 that inhibits ADAMTS4 in both platelets and megakaryocytes. Finally, application of a CCN family cocktail mimicking platelets onto HCS-2/8 cells enhanced their chondrocytic phenotype. CONCLUSIONS: Multiple inclusion of CCN1, 2 and 3 in platelets was clarified, which supports the harmonized regenerative potential of PRP in joint therapeutics.

DOI： 10.3109/14397595.2016.1155255

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

CCN2/connective tissue growth factor (CTGF) is a multi-functional molecule that promotes harmonized development and regeneration of cartilage through its matricellular interaction with a variety of extracellular biomolecules. Thus, deficiency in CCN2 supply profoundly affects a variety of cellular activities including basic metabolism. A previous study showed that the expression of a number of ribosomal protein genes was markedly enhanced in Ccn2-null chondrocytes. Therefore, in this study, we analyzed the impact of CCN2 on amino acid and protein metabolism in chondrocytes. Comparative metabolome analysis of the amino acids in Ccn2-null and wild-type mouse chondrocytes revealed stable decreases in the cellular levels of all of the essential amino acids. Unexpectedly, uptake of such amino acids was rather enhanced in Ccn2-null chondrocytes, and the addition of exogenous CCN2 to human chondrocytic cells resulted in decreased amino acid uptake. However, as expected, amino acid consumption by protein synthesis was also accelerated in Ccn2-null chondrocytes. Furthermore, we newly found that expression of two genes encoding two glycolytic enzymes, as well as the previously reported Eno1 gene, was repressed in those cells. Considering the impaired glycolysis and retained mitochondrial membrane potential in Ccn2-null chondrocytes, these findings suggest that Ccn2 deficiency induces amino acid shortage in chondrocytes by accelerated amino acid consumption through protein synthesis and acquisition of aerobic energy. Interestingly, CCN2 was found to capture such free amino acids in vitro. Under physiological conditions, CCN2 may be regulating the levels of free amino acids in the extracellular matrix of cartilage.

DOI： 10.1002/jcb.25377

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jcb.25377

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JOURNAL HARD TISSUE BIOLOGY  

Sorcin is a small calcium-binding protein that is widely expressed in many tissues. Sorcin regulates cardiac myocyte apoptosis by modulating mitochondrial Ca2+ cycling and regulates fibroblast cell cycling and apoptosis via Ca2+ signaling through the endoplasmic reticulum (ER). During endochondral ossification, some chondrocytes undergo apoptosis after their terminal differentiation; however, Sorcin's expression in these cells has not been characterized. Here we examined Sorcin's gene expression in the chondrocytes derived from mouse growth plate by reverse transcription PCR (RT-PCR), and its protein localization in the chondrocytes of femoral growth plate using immunohistochemistry. Sorcin protein was detected in the chondrocytes, and was particularly abundant in the cytoplasm and nuclei of proliferative zone chondrocytes and in the nuclei of hypertrophic chondrocytes. Apoptotic analysis of the growth plate revealed that many of the hypertrophic chondrocytes undergo the DNA fragmentation. We report for the first time the localization of Sorcin in the epiphyseal growth plate in which one of the apoptotic phenomenon was detected.

DOI： 10.2485/jhtb.25.57

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その他リンク： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2016&ichushi_jid=J04713&link_issn=&doc_id=20160127380009&doc_link_id=%2Fdq4hdtib%2F2016%2F002501%2F009%2F0057-0061%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fdq4hdtib%2F2016%2F002501%2F009%2F0057-0061%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/jre.12227

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1242/bio.201411031

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MOSBY-ELSEVIER  

Background. Fibrogenic pathways in the liver are principally regulated by hepatic stellate cells (HSC), which produce and respond to fibrotic mediators such as connective tissue growth factor (CCN2). The aim of this study was to determine whether CCN2 is shuttled between HSC in membranous nanovesicles, or "exosomes."
Methods. Exosomes were incubated with HSC after isolation from conditioned medium of control or CCN2-green fluorescent protein (GFP)-transfected primary mouse HSC or human LX-2 HSC. Some exosomes were stained fluorescently with PKH26. HSC co-culture experiments were performed in the presence of GW4869 exosome inhibitor. CCN2 or CCN2-GFP were evaluated by quantitative real-time polymerase. chain reaction or Western blot.
Results. HSC-derived exosomes contained CCN2 or CCN2 mRNA, each of which increased in concentration during HSC activation or after transfection of HSC with CCN2-GFP. Exosomes, stained with either PKH26 or purified from CCN2-GFP transfected cells, were taken up by activated or quiescent HSC resulting in CCN2-GFP delivery, as shown by their direct addition to recipient cells or by the GW4869-dependency of donor HSC.
Conclusion. CCN2 is packaged into secreted, nano-sized exosomes that mediate its intercellular transfer between HSC. Exosomal CCN2 may amplify or fine tune fibrogenic signaling and, in conjunction with other exosome constituents, may have utility as a noninvasive biomarker to assess hepatic fibrosis.

DOI： 10.1016/j.surg.2014.04.014

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

Pancreatitis is an inflammatory condition of the pancreas which, in its chronic form, involves tissue destruction, exocrine and endocrine insufficiency, increased risk of pancreatic cancer, and an extensive fibrotic pathology which is due to unrelenting collagen deposition by pancreatic stellate cells (PSC). In response to noxious agents such as alcohol-excessive consumption of which is a major cause of pancreatitis in the West-normally quiescent PSC undergo a phenotypic and functional transition to activated myofibroblasts which produce and deposit collagen at high levels. This process is regulated by connective tissue growth factor (CCN2), expression of which is highly up-regulated in activated PSC. We show that CCN2 production by activated PSC is associated with enhanced expression of microRNA-21 (miR-21) which was detected at high levels in activated PSC in a murine model of alcoholic chronic pancreatitis. A positive feedback loop between CCN2 and miR-21 was identified that resulted in enhancement of their respective expression as well as that of collagen alpha 1(I). Both miR-21 and CCN2 mRNA were present in PSC-derived exosomes, which were characterized as 50-150 nm CD9-positive nanovesicles. Exosomes from CCN2-GFP- or miR-21-GFP-transfected PSC were taken up by other PSC cultures, as shown by direct fluorescence or qRT-PCR for GFP. Collectively these studies establish miR-21 and CCN2 as participants in a positive feedback loop during PSC activation and as components of the molecular payload in PSC-derived exosomes that can be delivered to other PSC. Thus interactions between cellular or exosomal miR-21 and CCN2 represent novel aspects of fibrogenic regulation in PSC. Summary Chronic injury in the pancreas is associated with fibrotic pathology which is driven in large part by CCN2-dependent collagen production in pancreatic stellate cells. This study shows that CCN2 up-regulation in PSC is associated with increased expression of miR-21 which, in turn, is able to stimulate CCN2 expression further via a positive feedback loop. Additionally miR-21 and CCN2 were identified in PSC-derived exosomes which effected their delivery to other PSC. The cellular and exosomal miR-21-CCN2 axis is a novel component in PSC fibrogenic signaling.

DOI： 10.1007/s12079-014-0220-3

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

CCN2/connective tissue growth factor (CTGF) is a unique molecule that promotes both chondrocytic differentiation and proliferation through its matricellular interaction with a number of extracellular biomolecules. This apparently contradictory functional property of CCN2 suggests its certain role in basic cellular activities such as energy metabolism, which is required for both proliferation and differentiation. Comparative metabolomic analysis of costal chondrocytes isolated from wild-type and Ccn2-null mice revealed overall impaired metabolism in the latter. Among the numerous metabolites analyzed, stable reduction in the intracellular level of ATP, GTP, CTP, or UTP was observed, indicating a profound role of CCN2 in energy metabolism. Particularly, the cellular level of ATP was decreased by more than 50% in the Ccn2-null chondrocytes. The addition of recombinant CCN2 (rCCN2) to cultured Ccn2-null chondrocytes partly redeemed the cellular ATP level attenuated by Ccn2 deletion. Next, in order to investigate the mechanistic background that mediates the reduction in ATP level in these Ccn2-null chondrocytes, we performed transcriptome analysis. As a result, several metabolism-associated genes were found to have been up-regulated or down-regulated in the mutant mice. Up-regulation of a number of ribosomal protein genes was observed upon Ccn2 deletion, whereas a few genes required for aerobic and anaerobic ATP production were down-regulated in the Ccn2-null chondrocytes. Among such genes, reduction in the expression of the enolase 1 gene was of particular note. These findings uncover a novel functional role of CCN2 as a metabolic supporter in the growth-plate chondrocytes, which is required for skeletogenesis in mammals. J. Cell. Biochem. 115: 854-865, 2014. (c) 2013 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.24728

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE INC  

The role of CCN family proteins has been proven to be of extreme importance in the process of cartilage development and endochondral ossification. The second member, CCN2, consists of 4 conserved modules that interact with a number of cofactors to display multiple functions. Although the potentially therapeutic effect of intact CCN2 on cartilage regeneration has been indicated by a number of studies, the regenerative effect of independent modules comprising CCN2 has never been evaluated before. This study aims to discover a more robust and effective CCN2 derivative to induce regeneration through assessing the effect of CCN2 independent modules on regeneration in vitro and in vivo, in comparison to the full length CCN2. In vitro evaluation using human chondrocytic cells showed a remarkable enhancing effect of several single modules on the gene expression of cartilaginous extracellular matrix components; whereas combinations of 2 or 3 modules rather diminished such effects. Interestingly, combination of all 4 modules redeemed the effect of intact CCN2 in vitro. Suspecting the re-assembly of the 4 modules, interaction among the modules was examined by surface plasmon resonance analysis. However, the results did not support the possible formation of a tetramodular complex. Next, the thrombospondin 1 type 1 repeat module (TSP1), which was found most promising in the experiments in vitro, and the combination of 4 modules were forwarded further to in vivo confirmation using 2 rat osteoarthritis (OA) models. As a result, TSP1 displayed more prominent regenerative effects than intact CCN2 on damaged cartilage. Unexpectedly, the combination of 4 modules showed limited effects in vivo. These results indicate the utility of TSP1 in the regenerative therapeutics of OA. Possible molecular mechanism that enables conditional reconstruction of CCN2 by 4 modules is discussed as well. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2013.11.010

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Background: Although the ubiquitin-proteasome pathway is thought to regulate the level of SOX9, the ubiquitin ligase involved has not yet been determined. Results: E6-AP/UBE3A was found to bind and ubiquitinate SOX9. Conclusion: E6-AP/UBE3A functions as a ubiquitin ligase toward SOX9 both in vitro and in vivo. Significance: These data provide the regulatory mechanism of the SOX9 level in chondrocytes by ubiquitin ligase.
SOX9 is a transcription factor that acts as a key regulator at various stages of cartilage differentiation. There is ample evidence that intracellular SOX9 protein levels are tightly regulated both by sumoylation and by degradation through the ubiquitin-proteasome pathway. Using a proteomics approach, here we report the identification of a SOX9-binding protein, E6-AP/UBE3A, that may act as a ubiquitin ligase toward Sox9. E6-AP bound SOX9 through the region consisting mostly of its high mobility group domain in vitro. In nuclear lysates, FLAG-tagged E6-AP coprecipitated with Sox9 and its high mobility group domain. This finding was estimated using nuclear lysates from a chondrocytic cell line that endogenously expresses E6-AP and SOX9. Accordingly, ectopically expressed E6-AP and SOX9 colocalized in the nucleus. We show that E6-AP ubiquitinates SOX9 in vitro and in vivo and that SOX9 levels are enhanced after addition of the proteasome inhibitor bortezomib. Similar, siRNA knockdown of E6-AP and the E2 ligase Ubc9 increased cellular SOX9 amounts, supporting the notion that SOX9 may be ubiquitinated in hypertrophic chondrocytes by E6-AP and degraded by proteasomes. This is in accordance with the distribution of SOX9 levels, which are high in proliferating and prehypertrophic chondrocytes but low in hypertrophic chondrocytes, whereas E6-AP levels are high in hypertrophic chondrocytes and low in prehypertrophic chondrocytes. Furthermore, E6-AP-deficient mice showed SOX9 accumulation in chondrocytes and the brain. These findings support the concept that E6-AP regulates SOX9 levels in developing cartilage by acting as a ubiquitin ligase.

DOI： 10.1074/jbc.M113.486795

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0071156

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In order to elucidate the role of β-catenin in hypertrophic cartilage zone of the growth plate, we deleted the β-catenin gene ctnnb1specifically from hypertrophic chondrocytes by mating ctnnb1(fl/fl) mice with BAC-Col10a1-Cre-deleter mice. Surprisingly, this resulted in a significant reduction of subchondral trabecular bone formation in BACCol10Cre; ctnnb1(Δ/Δ) (referred to as Cat-ko) mice, although Cre expression was restricted to hypertrophic chondrocytes. The size of the Col10a1 positive hypertrophic zone was normal, but qRT-PCR revealed reduced expression of Mmp13, and Vegfa in Cat-ko hypertrophic chondrocytes, indicating impaired terminal differentiation. Immunohistological and in situ hybridization analysis revealed the substantial deficiency of collagen I positive mature osteoblasts, but equal levels of osterix-positive cells in the subchondral bone marrow space of Cat-ko mice, indicating that the supply of osteoblast precursor cells was not reduced. The fact that in Cat-ko mice subchondral trabeculae were lacking including their calcified cartilage core indicated a strongly enhanced osteoclast activity. In fact, TRAP staining as well as in situ hybridization analysis of Mmp9 expression revealed denser occupation of the cartilage erosion zone with enlarged osteoclasts as compared to the control growth plate, suggesting increased RANKL or reduced osteoprotegerin (Opg) activity in this zone. This notion was confirmed by qRT-PCR analysis of mRNA extracted from cultured hypertrophic chondrocytes or from whole epiphyses, showing increased Rankl mRNA levels in Cat-ko as compared to control chondrocytes, whereas changes in OPG levels were not significant. These results indicate that β-catenin levels in hypertrophic chondrocytes play a key role in regulating osteoclast activity and trabecular bone formation at the cartilage-bone interface by controlling RANKL expression in hypertrophic chondrocytes.

DOI： 10.1016/j.bone.2013.03.019

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Previously we showed that CCN family member 2/connective tissue growth factor (CCN2) promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage-related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced
and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2-overexpression in transgenic mice accelerated the endochondral ossification processes, resulting in increased length of their long bones. Our results also indicate the possible involvement of locally enhanced IGF-I or IGF-II in this extended bone growth. © 2013 Tomita et al.

DOI： 10.1371/journal.pone.0059226

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

CCN family proteins 2 and 3 (CCN2 and CCN3) belong to the CCN family of proteins, all having a high level of structural similarity. It is widely known that CCN2 is a profibrotic molecule that mediates the development of fibrotic disorders in many different tissues and organs. In contrast, CCN3 has been recently suggested to act as an anti-fibrotic factor in several tissues. This CCN3 action was shown earlier to be exerted by the repression of the CCN2 gene expression in kidney tissue, whereas different findings were obtained for liver cells. Thus, the molecular action of CCN3 yielding its anti-fibrotic effect is still controversial. Here, using a general model of fibrosis, we evaluated the effect of CCN3 overexpression on the gene expression of all of the CCN family members, as well as on that of fibrotic marker genes. As a result, repression of CCN2 gene expression was modest, while type I collagen and α-smooth muscle actin gene expression was prominently repressed. Interestingly, not only CCN2, but also CCN4 gene expression showed a decrease upon CCN3 overexpression. These findings indicate that fibrotic gene induction is under the control of a complex molecular network conducted by CCN family members functioning together. © 2012 The International CCN Society.

DOI： 10.1007/s12079-012-0180-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.biochi.2012.10.016

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

To identify proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8. CCN2/CTGF and CCN3/NOV polypeptides were picked up as CCN2-binding proteins, and CCN2CCN2 and CCN2CCN3 binding domains were identified. Direct binding between CCN2 and CCN3 was confirmed by coimmunoprecipitation in vitro and in vivo and surface plasmon resonance, and the calculated dissociation constants (Kd) were 1.17 x 10-9 m for CCN2 and CCN2, and 1.95 x 10-9 m for CCN2 and CCN3. Ectopically overexpressed green fluorescent proteinCCN2 and HaloCCN3 in COS7 cells colocalized, as determined by direct fluorescence analysis. We present evidence that CCN2CCN3 interactions modulated CCN2 activity such as enhancement of ACAN and col2a1 expression. Curiously, CCN2 enhanced, whereas CCN3 inhibited, the expression of aggrecan and col2a1 mRNA in HCS-2/8 cells, and combined treatment with CCN2 and CCN3 abolished the inhibitory effect of CCN3. These effects were neutralized with an antibody against the von Willebrand factor type C domain of CCN2 (11H3). This antibody diminished the binding between CCN2 and CCN2, but enhanced that between CCN3 and CCN2. Our results suggest that CCN2 could form homotypic and heterotypic dimers with CCN2 and CCN3, respectively. Strengthening the binding between CCN2 and CCN3 with the 11H3 antibody had an enhancing effect on aggrecan expression in chondrocytes, suggesting that CCN2 had an antagonizing effect by binding to CCN3.

DOI： 10.1111/j.1742-4658.2012.08717.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

Wnt inhibitory factor 1 (Wif-1) is a secreted antagonist of Wnt signalling. We recently demonstrated that this molecule is expressed predominantly in superficial layers of epiphyseal cartilage but also in bone and tendon. Moreover, we showed that Wif-1 is capable of binding to several cartilage-related Wnt ligands and interferes with Wnt3a-dependent Wnt signalling in chondrogenic cells. Here we provide evidence that the biological function of Wif-1 may not be confined to the modulation of Wnt signalling but appears to include the regulation of other signalling pathways. Thus, we show that Wif-1 physically binds to connective tissue growth factor (CTGF/CCN2) in vitro, predominantly by interaction with the C-terminal cysteine knot domain of CTGF. In vivo such an interaction appears also likely since the expression patterns of these two secreted proteins overlap in peripheral zones of epiphyseal cartilage. In chondrocytes CTGF has been shown to induce the expression of cartilage matrix genes such as aggrecan (Acan) and collagen2a1 (Col2a1). In this study we demonstrate that Wif-1 is capable to interfere with CTGF-dependent induction of Acan and Col2a1 gene expression in primary murine chondrocytes. Conversely, CTGF does not interfere with Wif-1-dependent inhibition of Wnt signalling. These results indicate that Wif-1 may be a multifunctional modulator of signalling pathways in the cartilage compartment. J. Cell. Physiol. 227: 2207-2216, 2012. (C) 2011 Wiley Periodicals, Inc.

DOI： 10.1002/jcp.22957

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：LIPPINCOTT WILLIAMS & WILKINS  

Study Design. Kinematics of the cervical spine during head rotation was investigated using 3-dimensional (3D) magnetic resonance imaging (MRI) in patients with cervical spondylosis (CS).
Objective. To demonstrate in vivo 3D kinematics of the spondylotic cervical spine during head rotation.
Summary of Background Data. Several in vivo studies have identified kinematic differences between normal and spondylotic subjects, but only two-dimensional flexion/extension motion has been investigated. Differences of in vivo 3D cervical motion during head rotation between normal and spondylotic subjects have yet to be clarified.
Methods. Ten healthy volunteers (control group) and 15 patients with CS (CS group) underwent 3D MRI of the cervical spine with the head rotated to 5 positions (neutral, +/- 45 degrees and +/- maximal head rotation). Relative motions of the cervical spine were calculated by automatically superimposing a segmented 3D MRI of the vertebra in the neutral position over images for each position using volume registration. The 3D motions of adjacent vertebra were represented with 6 degrees of freedom by Euler angles and translations on the coordinate system.
Results. Compared with the control group, the CS group showed significantly decreased mean axial rotation and mean coupled lateral bending at C5-C6 and C6-C7 and significantly increased mean coupled lateral bending at C2-C3 and C3-C4, although both the groups showed the same pattern of coupled motions.
Conclusion. The in vivo 3D kinematics of the spondylotic cervical spine during head rotation was accurately depicted and compared with those of healthy cervical spines for the first time.

DOI： 10.1097/BRS.0b013e3181e218cb

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mutations in SOX9, a gene essential for chondrocyte differentiation cause the human disease campomelic dysplasia (CD). To understand how SOX9 activates transcription, we characterized the DNA binding and cell-free transcription ability of wild-type SOX9 and a dimerization domain SOX9 mutant. Whereas formation of monomeric mutant SOX9-DNA complex increased linearly with increasing SOX9 concentrations, formation of a wild-type SOX9-DNA dimeric complex increased more slowly suggesting a more sigmoidal-type progression. Stability of SOX9-DNA complexes, however, was unaffected by the dimerization mutation. Both wild-type and mutant SOX9 activated transcription of a naked Col2a1 DNA template. However, after nucleosomal assembly, only wild-type and not the mutant was able to remodel chromatin and activate transcription of this template. Using a cell line, in which the Col2a1 vector was stably integrated, no differences were seen in the interactions of wild-type and mutant SOX9 with the chromatin of the Col2a1 vector using ChIP. However, the mutant was unable to activate transcription in agreement with in vitro results. We hypothesize that the SOX9 dimerization domain is necessary to remodel the Col2a1 chromatin in order to allow transcription to take place. These results further clarify the mechanism that accounts for CD in patients harboring SOX9 dimerization domain mutations.

DOI： 10.1093/nar/gkq417

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COMPANY OF BIOLOGISTS LTD  

SOX9 is a transcription factor of the SRY family that regulates sex determination, cartilage development and numerous other developmental events. In the foetal growth plate, Sox9 is highly expressed in chondrocytes of the proliferating and prehypertrophic zone but declines abruptly in the hypertrophic zone, suggesting that Sox9 downregulation in hypertrophic chondrocytes might be a necessary step to initiate cartilage-bone transition in the growth plate. In order to test this hypothesis, we generated transgenic mice misexpressing Sox9 in hypertrophic chondrocytes under the control of a BAC-Col10a1 promoter. The transgenic offspring showed an almost complete lack of bone marrow in newborns, owing to strongly retarded vascular invasion into hypertrophic cartilage and impaired cartilage resorption, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed high levels of Sox9 misexpression in hypertrophic chondrocytes but deficiencies of Vegfa, Mmp13, RANKL and osteopontin expression in the non-resorbed hypertrophic cartilage, indicating that Sox9 misexpression in hypertrophic chondrocytes inhibits their terminal differentiation. Searching for the molecular mechanism of SOX9-induced inhibition of cartilage vascularization, we discovered that SOX9 is able to directly suppress Vegfa expression by binding to SRY sites in the Vegfa gene. Postnatally, bone marrow formation and cartilage resorption in transgenic offspring are resumed by massive invasion of capillaries through the cortical bone shaft, similar to secondary ossification. These findings imply that downregulation of Sox9 in the hypertrophic zone of the normal growth plate is essential for allowing vascular invasion, bone marrow formation and endochondral ossification.

DOI： 10.1242/dev.045203

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PORTLAND PRESS LTD  

CCN2/CTGF (CCN family 2/connective tissue growth factor) is a multi-cellular protein with a broad range of activities. It modulates many cellular functions, including proliferation, migration, adhesion and extracellular matrix production, and it is thus involved in many biological and pathological processes. In particular, CCN2/CTGF is essential for normal skeletal development. To identify CCN2/CTGF-interactive proteins capable of modulating its action in cartilage, we carried Out a yeast two-hybrid screening using CCN2/CTGF peptide as a bait and a cDNA library from a chondrocytic cell line, HCS-2/8. In the present paper, we report the identification of aggrecan, which is a major proteoglycan of the extracellular matrix in cartilage, its a CCN2/CTGF-binding protein. Among the four domains of CCN2/CTGF, the IGFBP [IGF (insulin-like growth factor)-binding protein-like] and/or VWC (von Willebrand factor type C) domains had a direct interaction with aggrecan in a yeast two-hybrid assay. The results of a solid-phase-binding assay using aggrecan-coated plates also showed binding to recombinant CCN2/CTGF in a dose-dependent manner. rIGFBP (recombinant IGFBP) and rVWC (recombinant VWC) module peptides had stronger binding to aggrecan compared with rTSP1 (recombinant thrombospondin type I repeat) and rCT (recombinant C-terminal cystine knot) module peptides. SPR (surface plasmon resonance) analysis showed the direct interaction between the CCN2/CTGF and aggrecan. and ectopically overexpressed CCN2/CTGF and AgG3 (G3 domain of aggrecan) confirmed their binding in vivo. Indirect immunofluorescence analysis indicated that CCN2/CTGF was extracellularly co-localized with aggrecan on HCS-2/8 cells. The rIGFBP-rVWC peptide effectively enhanced tire production and release of aggrecan compared with the rTSP-rCT peptide in chondrocytes. These results indicate that CCN2/CTGF binds to aggrecan through its N-terminal IGFBP and VWC Modules. and this binding may be related to the CCN2/CTGF-enhanced production and secretion of aggrecan by chondrocytes.

DOI： 10.1042/BJ20081991

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bone morphogenetic protein (BMP) signaling is essential for normal development of the gastrointestinal (GI) tract. BMPs also play multiple roles in vascular smooth muscle cells; however, the BMP signaling in the development of the GI musculature remains to be clarified. We investigated the expression of BMPs and their receptors in mouse embryonic GI tracts by immunohistochemistry and in situ hybridization. We demonstrated that BMP2, BMP receptor Ib and BMP receptor II were expressed in the smooth muscle progenitors from E12 to E13 for the first time. BMP signaling on smooth muscle differentiation was examined by implantation of agarose beads soaked with BMPs in the in vitro developmental model that is gut-like structures from mouse embryonic stem (ES) cells. BMP2 rather than BMP4 beads enhanced smooth muscle differentiation, and increased gut-like structures showing spontaneous contractions and expressing intensive alpha-smooth muscle actin immunoreactivity. This increase was confirmed by up-regulation of SM22 mRNA shown by real-time PCR. By addition of noggin beads or noggin to the medium at BMP2 bead implantation, the ratio of contractive gut-like structures decreased. Implantation of BMP2 beads at EB7 (EB--embryoid bodies) (corresponding to E12 or E13 of mouse embryo) showed the highest effects and up-regulation of transcription factors msx-1 after 24h. This increase was blocked by noggin, and msx-1 decreased to almost the control level after 60 h. BMP2 beads at EB7 increased platelet-derived growth factor-A (PDGF-A) in the differentiating smooth muscle cells. We have recently reported that PDGF-A is expressed in the developing inner circular smooth muscle and is crucial for the longitudinal smooth muscle differentiation. Taken together, BMP signaling was expressed for a short window in the smooth muscle progenitors and the signal, especially BMP2, plays an essential role in smooth muscle differentiation in cooperation with PDGF signaling.

DOI： 10.1016/j.diff.2008.10.003

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Previously we have shown that insertion of a LacZ reporter gene into the Col10a1 gene in the context of a bacterial artificial chromosome (BAC) drives strong and specific expression of LacZ in hypertrophic cartilage of transgenic mice [Gebhard S., Hattori T., Bauer E., Bosl M.R., Schlund B., Poschl E., Adam N., de Crombrugghe B., von der Mark K., 2007 Histochem. Cell Biol. 19 127:183-194]. BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter. Here we report on the generation of Col10a1-specific Cre deleter mice using a BAC recombineering technique based on homologous recombination in E. coli. Sixteen BAC-Col10-Cre transgenic lines were generated containing between 1 and 5 copies of the BAC-Col10-Cre gene. All lines tested so far expressed Cre specifically in hypertrophic chondrocytes of E16.5 and P1 growth plates of long bones, ribs, vertebrae and sternum as examined by crossing with ROSA26 reporter mice. Cre activity was detected as early as E13.5 when hypertrophic cartilage develops in the diaphysis of femur and humerus. The data confirm that expression of Cre under the control of the complete BAC-Col10a1 promoter occurs with high efficiency and specificity in hypertrophic chondrocytes. The BAC-Col10-Cre lines should thus provide a valuable tool to get further insight into the role of genes involved in endochondral ossification by allowing their specific deletion in the hypertrophic zone of the growth plate.

DOI： 10.1016/j.matbio.2008.07.001

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担当区分：責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.joca.2007.11.001

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER LONDON LTD  

The purpose of this study was to clarify variations in patterns of flattening in rheumatoid hindfoot. Out of 232 outpatients with rheumatoid arthritis treated at our hospital from 2001 to 2003, we studied lateral radiographs of feet of 216 patients (423 weight-bearing views). We measured the medial arch angle (MAA) and talar angle (TA) and compared the alignment of the talonavicular joint-sagittal plane of each foot. We also evaluated the relationship between the severity of flattening and inclination of the talus and performed cluster analysis. Three groups were clustered by MAA and TA. In group I, joints were normal or close to normal. In group II, both talonavicular and subtalar joints were affected. In group III, talonavicular joints were minimally affected, and the subtalar joints were primarily affected. Groups II and III were thought to be a different pattern of flattening. The present results suggest that there are at least two patterns of flattening in rheumatoid hindfoot.

DOI： 10.1007/s10067-007-0781-0

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Sox9 is a transcription factor of the SRY family required for several steps of chondrogenesis. It activates the expression of various chondrocyte-specific genes, but the mechanisms and role of cofactors involved in Sox9-regulated gene transcription are not fully understood. Here, we report on the characterization of a Tat interactive protein-60 (Tip60) as Sox9-associated protein identified in a yeast two-hybrid screen. Both in vitro and in vivo assays confirmed the specificity of interactions between Sox9 and Tip60 including the existence of an endogenous complex containing both polypeptides in chondrocytes. Gel shift assays showed the presence of a complex containing Sox9, Tip60 and the DNA of an enhancer region of the Col2a1 promoter. Reporter assays using a Col2a1 promoter with multimerized Col2a1 Sox9-binding sites indicated that Tip60 enhanced the transcriptional activity of Sox9. A larger Col2a1 promoter showed that Tip60 increased the activity of this promoter in the presence of both Sox9 and Sox5. Ectopic expression of Sox9 and transient-cotransfection with Tip60 in COS7 cells showed a more diffuse subnuclear colocalization, suggesting changes in the chromatin structure. Chromatin immunoprecipitation assays showed that Tip60, Sox9 and Sox5 associated with the same Col2a1 enhancer region. Consistent with a role of Tip60 in chondrogenesis, addition of Tip60 siRNA to limb-bud micromass cultures delayed chondrocyte differention. Tip60 enhances acetylation of Sox9 mainly through K61, 253, 398 residues; however, the K61/253/398A mutant of Sox9 still exhibited enhanced transcriptional activity by Tip60. Our results support the hypothesis that Tip60 is a coactivator of Sox9 in chondrocytes.

DOI： 10.1093/nar/gkn150

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Knowledge of the pattern of joint destruction is important for planning the therapeutic approach to rheumatoid arthritis (RA) of the elbow. Accordingly, we carried out a large-scale radiographic study with the objective of elucidating the joint destruction pattern in rheumatoid elbows. From 2001 through 2003, we examined and took plain X-rays of both elbows of 193 RA patients (i.e., 386 elbows), consisting of 18 men and 175 women, with a mean age of 57.0 years. Radiographic images of the elbow joints were used to classify the degree of bone loss in various zones on the elbow joint surface into four grades of severity, and joint destruction was compared between the left and right elbows. In addition, correlation in the extent of bone loss between each of the zones of the same elbow and differences in the extent of bone loss were analyzed statistically. The results showed direct correlations for destruction of the elbow joint surface among the zones for the left and right elbow joints and in the same elbow joint. However, more severe destruction was observed on the radial side of the humeral trochlea, and it was surmised that destruction of the elbow joint must begin at that site and gradually spread mediolaterally. In addition, in the same elbow joint, the correlation in the degree of bone loss between the trochlea of humerus and the trochlear notch was especially strong, indicating that the bone destruction at both sites represented mirror lesions. We conclude that when performing radiographic diagnosis of the joint damage in the rheumatoid elbow, knowledge of this pattern of joint destruction will be useful for assessing whether there is joint destruction in the initial stage and for deciding the therapeutic approach.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

During endochondral ossification hypertrophic chondrocytes in the growth plate of fetal long bones, ribs and vertebrae play a key role in preparing growth plate cartilage for replacement by bone. In order to establish a reporter gene mouse to facilitate functional analysis of genes expressed in hypertrophic chondrocytes in this process, Col10a1- BAC reporter gene mouse lines were established expressing LacZ specifically in hypertrophic cartilage under the control of the complete Col10a1 gene. For this purpose, a bacterial artificial chromosome (BAC RP23-192A7) containing the entire murine Col10a1 gene together with 200 kb flanking sequences was modified by inserting a LacZ-Neo cassette into the second exon of Col10a1 by homologous recombination in E. coli. Transgenic mice containing between one and seven transgene copies were generated by injection of the purified BAC-Col10a1- lLacZ DNA. X-gal staining of newborns and embryos revealed strong and robust LacZ activity exclusively in hypertrophic cartilage of the fetal and neonatal skeleton of the transgenic offspring. This indicates that expression of the reporter gene in its proper genomic context in the BAC Col10a1 environment is independent of the integration site and reflects authentic Col10a1 expression in vivo. The Col10a1 specific BAC recombination vector described here will enable the specific analysis of effector gene functions in hypertrophic cartilage during skeletal development, endochondral ossification, and fracture callus healing.

DOI： 10.1007/s00418-006-0236-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Twisted gastrulation (TSG) is an extracellular modulator of bone morphogenetic protein (BMP) activity and regulates dorsoventral axis formation in early Drosophila and Xenopus development. Studies on tsg-deficient mice also indicated a role of this protein in skeletal growth, but the mechanism of TSG activity in this process has not yet been investigated. Here we show for the first time by in situ hybridization and immunohistochemistry that TSG is strongly expressed in bovine and mouse growth plate cartilage as well as in fetal ribs, vertebral cartilage, and cartilage anlagen of the skull. Furthermore we provide evidence that TSG is directly involved in BMP-regulated chondrocyte differentiation and maturation. In vitro, TSG impaired the dose-dependent BMP-2 stimulation of collagen II and X expression in cultures of MC615 chondrocytes and primary mouse chondrocytes. In the presence of chordin, a BMP antagonist, the inhibitory effect of TSG was further enhanced. TSG also inhibited BMP-2-stimulated phosphorylation of Smad factors in chondrocytes, confirming the role of TSG as a modulator of BMP signaling. For analysis of TSG functions in cartilage development in vivo, the gene was overexpressed in transgenic mice under the control of the cartilage-specific Col2a1 promoter. As a result, Col10a1 expression was significantly reduced in the growth plates of transgenic embryos and newborns in comparison with wild type littermates as shown by in situ hybridization and by real time PCR analysis. The data suggest that TSG is an important modulator of BMP-regulated cartilage development and chondrocyte differentiation.

DOI： 10.1074/jbc.M603419200

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記述言語：日本語  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We have identified PIAS1 (protein inhibitor of activated STAT-1), -3, -x alpha, and -x beta as SOX9-associated polypeptides using the Gal4-based yeast two-hybrid system and a cDNA library derived from a chondrocytic cell line. These PIAS proteins were shown to interact directly with SOX9 in two-hybrid, co-immunoprecipitation, and electrophoretic mobility shift assays. SOX9 was sumoylated in cotransfection experiments with COS-7 cells using PIAS and SUMO-1 (small ubiquitin-like modifier-1) expression vectors. SOX9 was also sumoylated in vitro by PIAS proteins in the presence of SUMO-1, the SUMO-activating enzyme, and the SUMO-conjugating enzyme. In COS-7 cells, PIAS proteins stimulated the SOX9-dependent transcriptional activity of a Col2a1 promoter-enhancer reporter. This increase in reporter activity was paralleled by an increase in the cellular levels of SOX9. Cotransfection with a SUMO-expressing vector further enhanced the transcriptional activity of this SOX9-dependent Col2a1 reporter in COS-7 cells, and this additional activation was inhibited in the presence of either SUMO-1 mutants or PIAS RING domain mutants or by coexpression of a desumoylation enzyme. Immunofluorescence microscopy of SOX9-transfected COS-7cells showed that the subnuclear distribution of SOX9 became more diffuse in the presence of PIAS1 and SUMO-1. Our results suggest that, by controlling the cellular concentrations of SOX9, PIAS proteins and sumoylation may be part of a major regulatory system of SOX9 functions.

DOI： 10.1074/jbc.M511330200

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.febslet.2006.01.061

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Previously, we showed that gene expression of the rheumatoid arthritis-related antigen RA-A47, which is identical to human heat shock protein (HSP)47, was downregulated in chondrocytes by inflammatory cytokines such as TNFalpha. Associated with this phenomenon, RA-A47 appeared on the cell surface concomitant with upregulation of metabolic factors related to cartilage destruction. The upregulation of the metabolic factors could be achieved by downregulation of RA-A47 expression with ra-a47-specific anti-sense oligonucleotide. Here, we show that the enhanced surface expression of RA-A47 on a chondrocytic cell line, HCS-2/8 was also a direct result of RA-A47 downregulation by ra-a47 anti-sense oligonucleotide, independent of the cytokine effects. Moreover, cell-surface expression of CD9, a beta1 integrin-associated transmembrane protein that is involved in cell adhesion and cell motility events, was enhanced in the ra-a47 anti-sense oligonucleotide-treated cells. The CD9 was colocalized with RA-A47 on the cell surface, where it may have affected integrin signaling. Furthermore, Annexin-V binding to the cell surface and the level of a number of apoptosis-related genes including caspase-9 were increased after ra-a47 anti-sense oligonucleotide treatment, suggesting that enhanced surface expression of RA-A47 and CD9 may be initiating apoptosis. Differential screening using a cDNA gene array showed induction of metallothionein-III and chemokine receptor CXCR4 and of factors of the Notch signaling pathway by the anti-sense treatment, but not by TNFalpha. Thus, here we show for the first time an alternative mechanism of inducing apoptosis by downregulating molecular chaperones, independent of the action of TNFalpha. The surface-exposed RA-A47 may induce autoantibodies and inflammatory reactions in autoimmune disease situations such as rheumatoid arthritis.

DOI： 10.1002/jcp.20112

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A differentiation method of human bone marrow mesenchymal stem cells (MSCs) to chondrocytes was developed for the construction of a three-dimensional (3D) cartilage tissue. The adhesive cells, which were isolated from a human bone marrow aspirate were embedded in type I collagen in a poly-L: -lactate-glycolic acid copolymer (PLGA) mesh and cultivated for 4 week together with growth factors. The degree of cellular differentiation was estimated by quantitative RT-PCR of aggrecan and type II collagen mRNAs and by staining with Safranin O. The 3D culture showed a higher degree of differentiation even without growth factors than the conventional pellet culture with growth factors, namely, dexamethasone and transforming growth factor (TGF)-beta 3. The 3D culture for 2 week with the combined addition of dexamethasone, TGF-beta 3, and insulin-like growth factor (IGF)-I reached a 30% expression of aggrecan mRNA compared with that in primary human chondrocytes, while the aggrecan mRNA expression in the conventional pellet culture was less than 2%. The sequential two-step differentiation cultivation, during which the cells were cultivated in 3D for 1 week after the conventional two-dimensional (2D) culture for 1 week, could markedly accelerate the expression of aggrecan mRNA compared with the 3D cultivation for 2 week.

DOI： 10.1007/s10616-005-3751-x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Effects of subcultivation of human bone marrow mesenchymal stem cells on their capacities for chondrogenesis and supporting hematopoiesis, and telomea length were investigated. Mesenchymal stem cells were isolated from human bone marrow aspirates and subcultivated several times at 37 degrees C under a 5% CO(2) atmosphere employing DMEM medium containing 10% FCS up to the 20th population doubling level (PDL). The ratio of CD45(-) CD105(+) cells among these cells slightly increased as PDL increased. However, there was no marked change in the chondrogenic capacity of these cells, which was confirmed by expression assay of aggrecan mRNA and Safranin O staining after pellet cell cultivation. The change in capacity to support hematopoiesis of cord blood cells was not observed among cells with various PDLs. On the other hand, telomere length markedly decreased as PDL increased at a higher rate than that at which telomere length of primary mesenchymal stem cells decreased as the age of donor increased.

DOI： 10.1007/s10616-005-3752-9

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Previously we have identified a cis-acting regulatory domain in the human type X collagen gene upstream of the transcription start site which acts as a strong enhancer in hypertrophic, but not in resting chondrocytes. Here we show that this enhancer is highly conserved also in the murine and bovine Col10a1 genes, but not found in the known promoter sequences of chicken Col10a1. It contains a functionally active AP-1 site (TPA Responsive Element, TRE) which is essential for the high transcriptional activity of the COL10A1 enhancer in transiently transfected hypertrophic chondrocytes. Gel-shift experiments with nuclear extracts of hypertrophic chondrocytes revealed FosB and Fra-1 as candidates regulating AP-1 factors binding to the TRE site. In fact, coexpression of FosB and Fra-1 in reporter gene assays greatly stimulated transcriptional activity of enhancer bearing reporter genes. Quantitative analysis of AP-1 factor mRNA levels in distinct fractions of fetal bovine epiphyseal chondrocytes by real-time PCR confirmed significant levels of FosB and Fra-1 mRNA besides other AP-1 factors in hypertrophic chondrocytes. A key role of the enhancer element in regulating tissue-specific expression of the Col10a1 gene was shown by establishing transgenic mouse lines with a reporter gene containing a 4.6 kb murine Col10a1 promoter fragment which included the enhancer, exon 1, part of exon 2 and the first intron. Reporter gene expression was seen exclusively in hypertrophic cartilages in the growth plates of long bones, ribs, vertebrae, sternum and mandibles of 17.5-18.5 dpc embryos, confirming that the 4.6 kb promoter is able to drive specific expression of Col10a1 in hypertrophic cartilage. These established transgenic lines should facilitate the genetic analysis of regulatory pathways of chondrocyte maturation and Col10a1 gene expression in the future.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1089/104454903770238111

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Previously we have shown that the expression of RA-A47 (rheumatoid arthritis-related antigen) which is identical to HSP47, a collagen-binding chaperon, is downregulated in chondrocytes by tumor necrosis factor alpha (TNFalpha). RA-A47 was also found on the surface of chondrocytes where it is recognized as an antigen in the serum of rheumatoid arthritis (RA) patients. Its translocation to the cell surface from endoplasmic reticulum membrane where it is normally located was also enhanced by TNFalpha. To understand the significance of RA-A47 downregulation in chondrocytes independent from other effects of TNFalpha, we used an antisense oligonucleotide approach and investigated the effect of this treatment on the expression of molecules related to matrix degradation and production of growth factors for chondrocytic, endothelial, and synovial cells. Here we show that treatment of rabbit chondrocyes and human chondrosarcoma cells HCS-2/8 by ra-a47 antisense S-oligonucleotides significantly reduced the expression of ra-a47 both at mRNA and protein level. Interestingly, this TNFalpha-independent RA-A47 downregulation was associated with a strong induction of matrix metalloproteinase (MMP)-9 mRNA and inducible NO synthase (iNOS) mRNA. The induction of active-type MMP-9 was further detected by gelatin zymography. Under the same conditions, the release of basic fibroblast growth factor (bFGF) and connective tissue growth factor (CTGF) from HCS-2/8 cells into the conditioned medium (CM) was strongly enhanced. These effects were not a result of TNFalpha upregulation, since the ra-a47 antisense oligonucleotide treatment did not enhance TNFalpha synthesis. These observations indicate that downregulation of RA-A47 induces TNFalpha-independent cartilage-degrading pathways involving iNOS and MMP-9. Furthermore, the stimulation of bFGF and CTGF release from chondrocytes may stimulate the proliferation of adjacent endothelial and/or synovial cells.

DOI： 10.1002/jcp.10341

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1177/154405910308200914

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/s0006-291x(02)02739-0

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

The main functions of articular cartilages are load-bearing and reducing friction of the articular surfaces. Because the capacity of articular cartilage to repair is limited, many attempts have been made to repair articular cartilage defects, which include transplantations of various tissues or cells. Within them, autologous cultured chondrocyte or autologous bone marrow mesenchymal cell transplantations are reported to be useful methods to repair articular cartilage defects. Here, we introduce these methods of tissue engineering and gene therapy, which are expected to become new treatments of articular cartilage defects.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1046/j.0014-2956.2001.02553.x

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DOI： 10.1089/104454901317094972

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）  

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

We isolated a small segment of the 3'-untranslated region (3'-UTR) in the mouse connective tissue growth factor (ctgf/fisp12) gene and evaluated its functionality. Comparison of the nucleotide sequences of human and mouse ctgf 3'-UTRs revealed a conserved small segment of 91 bases. The corresponding segments of the 3'-UTRs shared as much as 82.4% homology, whereas the overall homology between the 3'-UTRs was 71.8%. To study the functionality of the conserved segment, the corresponding region of mouse ctgf cDNA was amplified from NIH3T3 cells. When it was fused downstream of a marker gene, it showed remarkable repressive effects on gene expression. The repressive effect of the sense form was more prominent than that of the antisense form. Computer analyses of these sequence predicted stable secondary structures, suggesting that they act at the RNA level. The predicted structures of the sense and antisense forms appeared to be slightly different, which is consistent with the difference in repressive function. These findings defined the conserved small element in the mouse ctgf gene as a potent negative regulator of gene expression, which may act at a posttranscriptional level. (C) 2000 Academic Press.

DOI： 10.1006/bbrc.2000.3780

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掲載種別：研究論文（学術雑誌）  

The purpose of the present study was to evaluate the shear bond strengths of plastic brackets and the influences of the bracket filler contents on the bonding. The shear bond strengths of 4 plastic brackets (Spirit; Spirit MB; Clear Bracket; Aesthetic-Line) bonded to enamel with 4 orthodontic adhesives (Orthomite Superbond; System 1+; Transbond XT; and Kurasper-F) were compared with the strength of a conventional metal bracket. The findings of this study indicated the following: (1) shear bond strength of the 4 plastic brackets was significantly lower than that of the conventional metal brackets (P <.05), with most of the values ranging from 3 MPa to 6 MPa; (2) when comparing the bond strengths of plastic brackets, Aesthetic-Line had the largest value followed by Spirit MB, Spirit, and Clear Bracket, and when the plastic brackets were bonded with Orthomite Superbond, they showed relatively stronger bond strengths than when bonded with the other adhesives. Clear Bracket showed relatively lower values especially when bonded with System 1+; (3) the application of primer did not increase the durability of the bond strengths when bonding Spirit and Clear brackets; and (4) fillers contained in each plastic bracket ranging from 9.18% to 19. 52% were fairly well distributed and showed the same morphology of a fiber type 10 microm in diameter with different lengths. The filler concentration tended to correlate with the bond strength. The exposed fillers on the bracket base surface may play a more important role in plastic bracket adhesion than the macro-morphology of the base surface.

DOI： 10.1016/S0889-5406(00)70163-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Aims: Imbalance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) may be involved in the breakdown of articular cartilage matrix of the temporomandibular joint (TMJ). Aims: In this study, MMPs, TIMPs, and MMP-1/TIMP-1 complex levels were examined in TMJ synovial fluid samples aspirated from TMJ osteoarthritis (OA) patients (2 males, 8 females
mean age, 29.7 years) and asymptomatic control subjects (2 males, 8 females
mean age, 23.6 years) to determine the likelihood of increased proteolytic activity in the OA joints. Methods: The various types of MMPs and TIMPs were detected by Western blotting with monoclonal antibodies and gelatin zymography. The MMP-1/ TIMP-1 complex level was measured by an enzyme-linked immunosorbent assay kit. All aspirates were first analyzed for total protein content and then individually diluted to make the total protein levels equivalent. Results: The mean MMP-1/TIMP-1 complex concentration in the synovial fluids of the OA patients was 3.92 ± 1.39 ng/mL
this value was significantly lower (P &lt
0.05) than the value from control subjects (5.46 ± 1.32 ng/mL). Matrix metalloproteinase-1 (52 kDa), MMP-3 (57 kDa), TIMP-1 (28 kDa), and TIMP-2 (26 kDa) were detected in all of the normal and the OA samples. However, MMP-1 (28 kDa), MMP-2 (72 kDa), MMP-3 (45 kDa), and MMP-9 (83 kDa) were detected in higher concentration in the OA samples. Conclusion: These findings suggest a strong association between the OA-active joints and the presence of biologically active forms of known tissue degradation enzymes (MMP-1, MMP-3, and MMP-9).

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掲載種別：研究論文（学術雑誌）  

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掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   掲載種別：学位論文（博士）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/s007740070004

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その他リンク： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2000&ichushi_jid=J04654&link_issn=&doc_id=20001102080004&doc_link_id=10.1007%2Fs007740070004&url=https%3A%2F%2Fdoi.org%2F10.1007%2Fs007740070004&type=Crossref&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00002_2.gif

担当区分：筆頭著者,　責任著者   記述言語：日本語   出版者・発行元：岡山歯学会  

ra-a47mRNAにおいてTGFβで発現量が増加し,II型コラーゲン遺伝子(COL2A1)mRNAの発現量の増加と同調していることが観察された.対称的に,腫瘍壊死因子(TNF)α,インターフェロン(IFN)β,インターロイキン(IL)-6等の炎症性サイトカイン刺激によってra-a47mRNAの発現量は減少したが,COL2A1mRNAの発現量は減少せず,むしろ増加することがHCS-2/8で観察された.更に,誘導型一酸化窒素合成酵素(iNOS)mRNA,マトリックスメタロプロテアーゼ(MMP-9)mRNAの発現がTNFα刺激で強く誘導されていることが明らかとなった

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/s0003-9969(99)00009-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/oxfordjournals.jbchem.a022376

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC  

The presence of receptors specific for connective tissue growth factor (CTGF) was demonstrated on a human chondrosarcoma-derived chondrocytic cell Line, HCS-2/8. The binding of I-125-labeIed recombinant CTGF to HCS-2/8 cells was inhibited by unlabeled CTGF but not by PDGF-BB or bFGF. Scatchard analysis revealed the presence of two classes of binding sites with Rd values of 18.6 and 259 nM on cells. A cross-linking study revealed the formation of I-12S-CTGF-receptor complex with an apparent molecular weight of 280 kDa, The I-125-CTGF-receptor complex disappeared almost completely on the addition of unlabeled CTGF but not PDGF-BB or bFGF, In addition, the I-125-CTGF-receptor complex was immunoprecipitated with anti-CTGF antiserum but not with anti-PDGF receptor antiserum. These findings suggest that CTGF directly binds to specific receptor molecules on HCS-2/8 cells. (C) 1998 Academic Press.

DOI： 10.1006/bbrc.1998.8895

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/oxfordjournals.jbchem.a021955

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1006/bbrc.1998.8505

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

An immortal human chondrocytic cell line (HCS-2/8) derived from a chondrosarcoma was used as a source of human antigens to find humoral antibodies to cell surface proteins of human chondrocytes in sera from patients with rheumatoid arthritis (RA). Membrane fractions prepared from the cell line were subjected to Western blot analysis using RA and normal sera as probes. RA sera recognized about a dozen bands, but three of these bands, with molecular weights of 105 kDa, 68 kDa, and 47 kDa, were found to be specific for the RA sera (P < 0.05). These bands disappeared following V8 protease digestion, indicating that they were proteins. Among patients with 4 years or more of RA disease activity, reactivity against 105-kDa and 68-kDa proteins was relatively high in those whose joints showed a high degree of erosion. We suspect that levels of these two antibodies are suggestive of changes associated with the natural course of RA.

DOI： 10.1007/bf02239482

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その他リンク： http://link.springer.com/article/10.1007/BF02239482/fulltext.html

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Based upon DNA sequence data and positive immunochemical reactivity of expressed protein, novel homologues of the FNR family were identified in four representative oral facultative anaerobes: Capnocytophaga ochracea, Capnocytophaga sputigena, Haemophilus aphrophilus, and Actinobacillus actinomycetemcomitans. The similarity to E. coli FNR and to HlyX (itself 71% similar to E. coli FNR, while regulating expression of hemolysin operon in Actinobacillus pleuropneumoniae) was estimated from the deduced partial amino acid sequence to be, in the above order of tested species, 98, 98, 86, and 85%, and 75, 75, 88, and 88%, respectively. The phylogenetic relatedness indicates a rather closer link of HlyX to the FNR homologues from both pathogens, H. aphrophilus and A. actinomycetemcomitans. The possibility that the A. actinomycetemcomitans FNR homologue functions as a redox-sensing transcriptional factor to regulate, in addition to anaerobic respiration, microaerobic expression of the leukotoxin operon (Itx gene) is suggested.

DOI： 10.1111/j.1574-6968.1996.tb08108.x

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掲載種別：研究論文（学術雑誌）  

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掲載種別：研究論文（学術雑誌）  

Prolonged incubation with interleukin-1β (IL-1) induced the release of large amounts of NO and subsequently inhibited DNA synthesis and the biosynthesis and accumulation of proteoglycans in cultured rabbit articular chondrocytes (RAC). IL-1 also inhibited DNA synthesis in bovine aortic endothelial cells (BAE). On the other hand, DNA synthesis in BAE cocultured with RAC was not inhibited by prolonged incubation with IL-1. Moreover, conditioned media from RAC incubated for a long period with IL-1 stimulated DNA synthesis in BAE alone. This growth stimulatory activity was mainly due to the release of basic fibroblast growth factor, a heparin-binding growth factor, into RAC culture. Gelatin zymography of the RAC culture medium revealed that IL-1 increased the production of matrix met alloproteinase-2 (MMP-2) and MMP-9. N(G)-Monomethyl-L-arginine, an inhibitor of NO synthesis, inhibited all of these actions of IL-1. These results indicate that NO from RAC treated with IL-1 stimulates MMPs, which, in turn, degrade the extracellular matrix produced by RAC, resulting in the release of large amounts of basic fibroblast growth factor stored in the matrix, which then stimulates adjacent BAE proliferation. Thus, NO produced from RAC treated with IL-1 may modulate angiogenesis in the synovium of arthritic patients.

DOI： 10.1210/endo.137.9.8756539

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掲載種別：研究論文（学術雑誌）  

With cultured osteoblastic cells, clone MC3T3-E1 derived from newborn mouse calvaria, the mRNAs encoding three representative neurotrophins, namely, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), were proved in estimation by the polymerase chain reaction (PCR) method. The increase and then subsequent decrease of all three mRNAs within the proliferation phase were followed by their gradual increase in the differentiation phase, with a tendency of enhancement by exogenous TGF-β as was particularly evident in the case of NGF. These findings were further substantiated by identification of NGF-like neurotrophins in the conditioned medium of the osteoblastic cells by an immunoblotting and neurite extension assay. © 1994 Academic Press, Inc.

DOI： 10.1006/bbrc.1994.1127

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

In facultative anaerobes, the anaerobic expression of respiratory genes is regulated by a transcriptional activator, FNR. Transcription in vitro of the E. coli fnr gene was repressed by its product, FNR. The transcription of the E. coli nar X gene encoding the nitrate sensor protein was likewise repressed. DNA truncation experiments for fnr and nar X genes indicated that multiple anaero-boxes in each promoter region are essential for repression by the FNR protein, but they also suggest that factor-independent upstream activation signals are operating with these promoters.

DOI： 10.1016/0014-5793(94)80173-8

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記述言語：日本語   出版者・発行元：歯科基礎医学会  

口腔細菌の酸素分圧の応答性を調べる目的で, E.coliの酸素センサーであると同時に嫌気的呼吸系遺伝子群の活性化因子として機能するFNR蛋白質と類似の蛋白質の口腔細菌での存在を調べた。E. coliの精製FNR蛋白質に対する家兎抗FNR血清を用いたウエスタンブロット法で, 嫌気的条件下で増殖できる代表的な5株の口腔細菌のFNR様因子の同定を行った。通性嫌気性細菌であるA. actinomycetemcomitdnsについてはFNR様因子を確認することができたが, 嫌気的呼吸系を有する微好気性細菌や偏性嫌気性細菌あるいは糖発酵やアミノ酸異化代謝をエネルギー獲得系として利用する細菌ではそのような蛋白質は確認できなかった。また, A. actinomycetemcomitansでのFNR様因子の発現量は, その増殖速度の減少に対応して増加し, 培地の酸化還元電位が-250mV付近の微好気的条件下で最大であった。

DOI： 10.2330/joralbiosci1965.34.459

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担当ページ：391-403   記述言語：英語

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記述言語：日本語 著書種別：事典・辞書

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総ページ数：1冊  

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担当区分：筆頭著者,　責任著者   記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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担当区分：最終著者,　責任著者   記述言語：日本語   出版者・発行元：日本結合組織学会  

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担当区分：最終著者,　責任著者   記述言語：日本語   出版者・発行元：日本結合組織学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

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掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

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記述言語：日本語   掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）   出版者・発行元：岡山歯学会  

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記述言語：日本語   掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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担当区分：責任著者   掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

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掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

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掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：英語   出版者・発行元：(一社)日本生理学会  

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記述言語：日本語   出版者・発行元：(NPO)日本口腔科学会  

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掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

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掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

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掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

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掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

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記述言語：日本語   出版者・発行元：岡山歯学会  

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記述言語：日本語   出版者・発行元：(NPO)日本口腔科学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

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記述言語：日本語   出版者・発行元：岡山歯学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(公社)日本矯正歯科学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：英語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：生命科学系学会合同年次大会運営事務局  

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記述言語：日本語   出版者・発行元：生命科学系学会合同年次大会運営事務局  

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：英語   出版者・発行元：Humana Press Inc.  

Recombinant proteins are important tools for understanding molecular functions in vitro. Recent progress in the generation of recombinant proteins is amazing. However, when we plan to produce them, we should choose the best method according to the nature and the use of the target recombinant protein. Degradation and mis-folding are major problems in producing active recombinant CCN2. The method shown in this chapter describes the appropriate conditions under which we can produce CCN2 and its truncated fragments in Escherichia coli.

DOI： 10.1007/978-1-4939-6430-7_8

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記述言語：英語   出版者・発行元：Humana Press Inc.  

Yeast two-hybrid screening is a powerful method to identify proteins that interact with a protein of interest. CCN2 consists of four domains, and identification of new proteins that bind to individual domains of CCN2 may reveal a variety of CCN2 functions. To identify CCN2-interactive proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8, with CCN2 cDNA used as a bait. In this chapter, we describe our methods for screening for CCN2 binding partners by this system in detail. This protocol may be applied to other CCN proteins as well.

DOI： 10.1007/978-1-4939-6430-7_15

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記述言語：英語   出版者・発行元：Humana Press Inc.  

Recent progress in gene-editing technology has provided a strong impact for improved our understanding of molecular functions in living organisms. Here we describe our method to generate transgene-overexpressing mouse models, which method involves the use of tissue-specific promoters for analyzing a certain molecule (s) in special tissues. The protocol described in this chapter uses the Col2a1 promoter-enhancer, which is known for driving specific and strong transgene expression in cartilage and is based on several of our studies showing a positive role of the connective tissue growth factor (CCN2) in cartilage-bone development and maintenance of articular cartilage. These mice show strongly accelerated endochondral ossification resulting in enhanced bone elongation, as well as resistance to age-related articular degeneration. This protocol also describes how to analyze the molecular mechanisms of these phenomena by use of chondrocytes isolated from CCN2-overexpressing cartilage.

DOI： 10.1007/978-1-4939-6430-7_32

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担当区分：筆頭著者   記述言語：英語   出版者・発行元：Humana Press Inc.  

Recent progress in molecular imaging technology has had a strong impact on improving our understanding of molecular translocation, receptor internalization, and interactions in living cells. The protocol in this chapter introduces an optimized technique for intracellular localization of CCN2 and CCN3 in live cells, one using GFP-tagged CCN2 and Halo-tagged CCN3.

DOI： 10.1007/978-1-4939-6430-7_20

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：英語   出版者・発行元：WILEY  

CCN2/connective tissue growth factor (CTGF) is a multi-functional molecule that promotes harmonized development and regeneration of cartilage through its matricellular interaction with a variety of extracellular biomolecules. Thus, deficiency in CCN2 supply profoundly affects a variety of cellular activities including basic metabolism. A previous study showed that the expression of a number of ribosomal protein genes was markedly enhanced in Ccn2-null chondrocytes. Therefore, in this study, we analyzed the impact of CCN2 on amino acid and protein metabolism in chondrocytes. Comparative metabolome analysis of the amino acids in Ccn2-null and wild-type mouse chondrocytes revealed stable decreases in the cellular levels of all of the essential amino acids. Unexpectedly, uptake of such amino acids was rather enhanced in Ccn2-null chondrocytes, and the addition of exogenous CCN2 to human chondrocytic cells resulted in decreased amino acid uptake. However, as expected, amino acid consumption by protein synthesis was also accelerated in Ccn2-null chondrocytes. Furthermore, we newly found that expression of two genes encoding two glycolytic enzymes, as well as the previously reported Eno1 gene, was repressed in those cells. Considering the impaired glycolysis and retained mitochondrial membrane potential in Ccn2-null chondrocytes, these findings suggest that Ccn2 deficiency induces amino acid shortage in chondrocytes by accelerated amino acid consumption through protein synthesis and acquisition of aerobic energy. Interestingly, CCN2 was found to capture such free amino acids in vitro. Under physiological conditions, CCN2 may be regulating the levels of free amino acids in the extracellular matrix of cartilage. J. Cell. Biochem. 117: 927-937, 2016. (c) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.25377

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記述言語：英語  

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記述言語：英語   出版者・発行元：TAYLOR & FRANCIS LTD  

Objectives: Platelet-rich plasma (PRP) has been widely used to enhance the regeneration of damaged joint tissues, such as osteoarthritic and rheumatoid arthritic cartilage. The aim of this study is to clarify the involvement of all of the CCN family proteins that are crucially associated with joint tissue regeneration.
Methods: Cyr61-CTGF-NOV (CCN) family proteins in human platelets and megakaryocytic cells were comprehensively analyzed by Western blotting analysis. Production of CCN family proteins in megakaryocytes in vivo was confirmed by immunofluorescence analysis of mouse bone marrow cells. Effects of CCN family proteins found in platelets on chondrocytes were evaluated by using human chondrocytic HCS-2/8 cells.
Results: Inclusion of CCN2, a mesenchymal tissue regenerator, was confirmed. Of note, CCN3, which counteracts CCN2, was newly found to be encapsulated in platelets. Interestingly, these two family members were not detectable in megakaryocytic cells, but their external origins were suggested. Furthermore, we found for the first time CCN5 and CCN1 that inhibits ADAMTS4 in both platelets and megakaryocytes. Finally, application of a CCN family cocktail mimicking platelets onto HCS-2/8 cells enhanced their chondrocytic phenotype.
Conclusions: Multiple inclusion of CCN1, 2 and 3 in platelets was clarified, which supports the harmonized regenerative potential of PRP in joint therapeutics.

DOI： 10.3109/14397595.2016.1155255

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：日本語   出版者・発行元：(公財)成長科学協会  

結合組織成長因子(CTGF/CCN2)を低身長治療に応用するための基礎研究として、効率良く、安全に軟骨組織でCTGF/CCN2を誘導し、骨成長を促進する方法の確立を目指した。軟骨組織特異的CCN2過剰発現マウスの培養肋軟骨細胞よりRNAの単離とマイクロアレイ遺伝子解析を行った結果、多くの遺伝子発現の増減が観察された。パスウェイ解析では内軟骨性骨形成への関与が示唆されている遺伝子の増減やIGF-I、IIの発現上昇、多くのlnc RNAの発現抑制が検出され、現在も検索、追試験を行っている。また、培養肋軟骨細胞のシグナル伝達経路を解析し、チロシンキナーゼ型受容体のリン酸化誘導を確認した結果、リン酸化が確認されたチロシンキナーゼ群は、これまで内軟骨性骨形成で活性化が確認されているものがほとんどであった。

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：英語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：英語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：岡山歯学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：日本語   出版者・発行元：岡山歯学会  

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記述言語：英語   出版者・発行元：日本再生歯科医学会  

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記述言語：英語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：英語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：英語   出版者・発行元：Informa Healthcare  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC CELL BIOLOGY  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC CELL BIOLOGY  

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記述言語：日本語   出版者・発行元：岡山歯学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：英語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：英語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：英語   出版者・発行元：日本結合組織学会・マトリックス研究会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：英語   出版者・発行元：OXFORD UNIV PRESS  

Mutations in SOX9, a gene essential for chondrocyte differentiation cause the human disease campomelic dysplasia (CD). To understand how SOX9 activates transcription, we characterized the DNA binding and cell-free transcription ability of wild-type SOX9 and a dimerization domain SOX9 mutant. Whereas formation of monomeric mutant SOX9-DNA complex increased linearly with increasing SOX9 concentrations, formation of a wild-type SOX9-DNA dimeric complex increased more slowly suggesting a more sigmoidal-type progression. Stability of SOX9-DNA complexes, however, was unaffected by the dimerization mutation. Both wild-type and mutant SOX9 activated transcription of a naked Col2a1 DNA template. However, after nucleosomal assembly, only wild-type and not the mutant was able to remodel chromatin and activate transcription of this template. Using a cell line, in which the Col2a1 vector was stably integrated, no differences were seen in the interactions of wild-type and mutant SOX9 with the chromatin of the Col2a1 vector using ChIP. However, the mutant was unable to activate transcription in agreement with in vitro results. We hypothesize that the SOX9 dimerization domain is necessary to remodel the Col2a1 chromatin in order to allow transcription to take place. These results further clarify the mechanism that accounts for CD in patients harboring SOX9 dimerization domain mutations.

DOI： 10.1093/nar/gkq417

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担当区分：筆頭著者   記述言語：英語   出版者・発行元：COMPANY OF BIOLOGISTS LTD  

SOX9 is a transcription factor of the SRY family that regulates sex determination, cartilage development and numerous other developmental events. In the foetal growth plate, Sox9 is highly expressed in chondrocytes of the proliferating and prehypertrophic zone but declines abruptly in the hypertrophic zone, suggesting that Sox9 downregulation in hypertrophic chondrocytes might be a necessary step to initiate cartilage-bone transition in the growth plate. In order to test this hypothesis, we generated transgenic mice misexpressing Sox9 in hypertrophic chondrocytes under the control of a BAC-Col10a1 promoter. The transgenic offspring showed an almost complete lack of bone marrow in newborns, owing to strongly retarded vascular invasion into hypertrophic cartilage and impaired cartilage resorption, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed high levels of Sox9 misexpression in hypertrophic chondrocytes but deficiencies of Vegfa, Mmp13, RANKL and osteopontin expression in the non-resorbed hypertrophic cartilage, indicating that Sox9 misexpression in hypertrophic chondrocytes inhibits their terminal differentiation. Searching for the molecular mechanism of SOX9-induced inhibition of cartilage vascularization, we discovered that SOX9 is able to directly suppress Vegfa expression by binding to SRY sites in the Vegfa gene. Postnatally, bone marrow formation and cartilage resorption in transgenic offspring are resumed by massive invasion of capillaries through the cortical bone shaft, similar to secondary ossification. These findings imply that downregulation of Sox9 in the hypertrophic zone of the normal growth plate is essential for allowing vascular invasion, bone marrow formation and endochondral ossification.

DOI： 10.1242/dev.045203

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：日本語   出版者・発行元：岡山歯学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：ELSEVIER SCIENCE INC  

DOI： 10.1016/j.bone.2009.01.121

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

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記述言語：英語   出版者・発行元：ELSEVIER SCIENCE BV  

Previously we have shown that insertion of a LacZ reporter gene into the Col10a1 gene in the context of a bacterial artificial chromosome (BAC) drives strong and specific expression of LacZ in hypertrophic cartilage of transgenic mice [Gebhard S., Hattori T, Bauer E., Bosl M.R., Schlund B., Poschl E., Adam N., de Crombrugghe B., von der Mark K., 2007 Histochem. Cell Biol. 19 127:183-194]. BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter. Here we report on the generation of Col10a1-specific Cre deleter mice using a BAC recombineering technique based on homologous recombination in E. coli. Sixteen BAC-Col10-Cre transgenic lines were generated containing between I and 5 copies of the BAC-Col10-Cre gene. All lines tested so far expressed Cre specifically in hypertrophic chondrocytes of E16.5 and PI growth plates of long bones, ribs, vertebrae and sternum as examined by crossing with ROSA26 reporter mice. Cre activity was detected as early as E13.5 when hypertrophic cartilage develops in the diaphysis of femur and humerus. The data confirm that expression of Cre under the control of the complete BAC-Col10a1 promoter occurs with high efficiency and specificity in hypertrophic chondrocytes. The BAC-Col10-Cre lines should thus provide a Valuable tool to get further insight into the role of genes involved in endochondral ossification by allowing their specific deletion in the hypertrophic zone of the growth plate. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.matbio.2008.07.001

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC BONE & MINERAL RES  

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担当区分：筆頭著者   記述言語：英語   出版者・発行元：OXFORD UNIV PRESS  

Sox9 is a transcription factor of the SRY family required for several steps of chondrogenesis. It activates the expression of various chondrocyte-specific genes, but the mechanisms and role of cofactors involved in Sox9-regulated gene transcription are not fully understood. Here, we report on the characterization of a Tat interactive protein-60 (Tip60) as Sox9-associated protein identified in a yeast two-hybrid screen. Both in vitro and in vivo assays confirmed the specificity of interactions between Sox9 and Tip60 including the existence of an endogenous complex containing both polypeptides in chondrocytes. Gel shift assays showed the presence of a complex containing Sox9, Tip60 and the DNA of an enhancer region of the Col2a1 promoter. Reporter assays using a Col2a1 promoter with multimerized Col2a1 Sox9-binding sites indicated that Tip60 enhanced the transcriptional activity of Sox9. A larger Col2a1 promoter showed that Tip60 increased the activity of this promoter in the presence of both Sox9 and Sox5. Ectopic expression of Sox9 and transient-cotransfection with Tip60 in COS7 cells showed a more diffuse subnuclear colocalization, suggesting changes in the chromatin structure. Chromatin immunoprecipitation assays showed that Tip60, Sox9 and Sox5 associated with the same Col2a1 enhancer region. Consistent with a role of Tip60 in chondrogenesis, addition of Tip60 siRNA to limb-bud micromass cultures delayed chondrocyte differention. Tip60 enhances acetylation of Sox9 mainly through K61, 253, 398 residues; however, the K61/253/398A mutant of Sox9 still exhibited enhanced transcriptional activity by Tip60. Our results support the hypothesis that Tip60 is a coactivator of Sox9 in chondrocytes.

DOI： 10.1093/nar/gkn150

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

J-GLOBAL

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

J-GLOBAL

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J-GLOBAL

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

J-GLOBAL

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J-GLOBAL

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

J-GLOBAL

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

J-GLOBAL

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記述言語：英語   出版者・発行元：(公社)日本生化学会  

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記述言語：英語   出版者・発行元：SPRINGER  

During endochondral ossification hypertrophic chondrocytes in the growth plate of fetal long bones, ribs and vertebrae play a key role in preparing growth plate cartilage for replacement by bone. In order to establish a reporter gene mouse to facilitate functional analysis of genes expressed in hypertrophic chondrocytes in this process, Col10a1- BAC reporter gene mouse lines were established expressing LacZ specifically in hypertrophic cartilage under the control of the complete Col10a1 gene. For this purpose, a bacterial artificial chromosome (BAC RP23-192A7) containing the entire murine Col10a1 gene together with 200 kb flanking sequences was modified by inserting a LacZ-Neo cassette into the second exon of Col10a1 by homologous recombination in E. coli. Transgenic mice containing between one and seven transgene copies were generated by injection of the purified BAC-Col10a1- lLacZ DNA. X-gal staining of newborns and embryos revealed strong and robust LacZ activity exclusively in hypertrophic cartilage of the fetal and neonatal skeleton of the transgenic offspring. This indicates that expression of the reporter gene in its proper genomic context in the BAC Col10a1 environment is independent of the integration site and reflects authentic Col10a1 expression in vivo. The Col10a1 specific BAC recombination vector described here will enable the specific analysis of effector gene functions in hypertrophic cartilage during skeletal development, endochondral ossification, and fracture callus healing.

DOI： 10.1007/s00418-006-0236-8

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記述言語：日本語   出版者・発行元：(株)メディカルレビュー社  

結合組織成長因子(connective tissue growth factor;CTGF/CCN2)は前肥大軟骨細胞層が特異的に発現する遺伝子として、また、血管内皮細胞の培養上清中に存在する線維芽細胞の増殖・遊走促進因子として単離されたが、その発現部位は軟骨組織、血管内皮細胞だけでなく、脳をはじめとする神経系にも広がり、また、血小板に大量に蓄積されて体内を循環している。生理的には、正常な内軟骨性骨形成、血管新生、胚発育に必須であることが明らかになっており、細胞外マトリックス成分の合成を強く促進し、細胞外マトリックスに富む軟骨組織で高発現していることと密接に関連している。また、肺をはじめ、心臓、肝臓、腎臓、筋、膵臓などの組織における線維芽細胞では、トランスフォーミング増殖因子(TGF)-βの誘導に伴って遺伝子発現が誘導され、各組織の線維化、線維症の形成を促進することから、そのメディエーターとしての働きが注目されている。また、乳癌細胞で高度に発現していることが観察されているが、低酸素状態によってCTGF/CCN2の発現が誘導され、これにより血管新生が惹起されることが明らかになっている。(著者抄録)

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

J-GLOBAL

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J-GLOBAL

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

J-GLOBAL

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

J-GLOBAL

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記述言語：日本語   出版者・発行元：岡山歯学会  

J-GLOBAL

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J-GLOBAL

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記述言語：日本語   出版者・発行元：(一社)歯科基礎医学会  

J-GLOBAL

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

J-GLOBAL

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

J-GLOBAL

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J-GLOBAL

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記述言語：英語  

DOI： 10.1074/jbc.M603419200

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PubMed

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記述言語：英語   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We have identified PIAS1 (protein inhibitor of activated STAT-1), -3, -x alpha, and -x beta as SOX9-associated polypeptides using the Gal4-based yeast two-hybrid system and a cDNA library derived from a chondrocytic cell line. These PIAS proteins were shown to interact directly with SOX9 in two-hybrid, co-immunoprecipitation, and electrophoretic mobility shift assays. SOX9 was sumoylated in cotransfection experiments with COS-7 cells using PIAS and SUMO-1 (small ubiquitin-like modifier-1) expression vectors. SOX9 was also sumoylated in vitro by PIAS proteins in the presence of SUMO-1, the SUMO-activating enzyme, and the SUMO-conjugating enzyme. In COS-7 cells, PIAS proteins stimulated the SOX9-dependent transcriptional activity of a Col2a1 promoter-enhancer reporter. This increase in reporter activity was paralleled by an increase in the cellular levels of SOX9. Cotransfection with a SUMO-expressing vector further enhanced the transcriptional activity of this SOX9-dependent Col2a1 reporter in COS-7 cells, and this additional activation was inhibited in the presence of either SUMO-1 mutants or PIAS RING domain mutants or by coexpression of a desumoylation enzyme. Immunofluorescence microscopy of SOX9-transfected COS-7cells showed that the subnuclear distribution of SOX9 became more diffuse in the presence of PIAS1 and SUMO-1. Our results suggest that, by controlling the cellular concentrations of SOX9, PIAS proteins and sumoylation may be part of a major regulatory system of SOX9 functions.

DOI： 10.1074/jbc.M511330200

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記述言語：日本語   出版者・発行元：(一社)日本リウマチ学会  

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記述言語：日本語   出版者・発行元：(一社)日本リウマチ学会  

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記述言語：日本語   出版者・発行元：(株)医薬ジャーナル社  

小胞体シャペロンheat shock protein/rheumatoid arthritis-related antigen(HSP47/RA-A47)は細胞内のコラーゲン成熟、分泌の機能を持ち、細胞をストレスから防御し、未成熟あるいは正常な合成に失敗したコラーゲンの細胞外への分泌を妨げる役割を担っているが、リウマチ軟骨ではその発現量が減少しており、細胞に小胞体ストレスを誘発している。また、軟骨細胞破壊につながる細胞障害性因子の放出を促し、その結果軟骨細胞はアポトーシスにより死ぬ。また、この時細胞外にHSP47/RA-A47が放出される。細胞外HSP47/RA-A47は自己抗原として認識される可能性があるが、抗原抗体反応およびそれにかかわる炎症反応を制御している可能性もある。HSP47/RA-A47の細胞内外の量のコントロールがリウマチ治療の鍵となろう。(著者抄録)

J-GLOBAL

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC BONE & MINERAL RES  

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記述言語：英語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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