Faculty Profiles - HATTORI Takako

Papers - HATTORI Takako

Division display >> ／ All the affair displays 1 - 96 of about 96

Do not overwork: cellular communication network factor 3 for life in cartilage. Reviewed International journal

Satoshi Kubota, Harumi Kawaki, Bernard Perbal, Masaharu Takigawa, Kazumi Kawata, Takako Hattori, Takashi Nishida

Journal of cell communication and signaling 17 ( 2 ) 353 - 359 2023.6

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

Cellular communication network factor (CCN) 3, which is one of the founding members of the CCN family, displays diverse functions. However, this protein generally represses the proliferation of a variety of cells. Along with skeletal development, CCN3 is produced in cartilaginous anlagen, growth plate cartilage and epiphysial cartilage. Interestingly, CCN3 is drastically induced in the growth plates of mice lacking CCN2, which promotes endochondral ossification. Notably, chondrocytes in these mutant mice with elevated CCN3 production also suffer from impaired glycolysis and energy metabolism, suggesting a critical role of CCN3 in cartilage metabolism. Recently, CCN3 was found to be strongly induced by impaired glycolysis, and in our study, we located an enhancer that mediated CCN3 regulation via starvation. Subsequent investigations specified regulatory factor binding to the X-box 1 (RFX1) as a transcription factor mediating this CCN3 regulation. Impaired glycolysis is a serious problem, resulting in an energy shortage in cartilage without vasculature. CCN3 produced under such starved conditions restricts energy consumption by repressing cell proliferation, leading chondrocytes to quiescence and survival. This CCN3 regulatory system is indicated to play an important role in articular cartilage maintenance, as well as in skeletal development. Furthermore, CCN3 continues to regulate cartilage metabolism even during the aging process, probably utilizing this regulatory system. Altogether, CCN3 seems to prevent "overwork" by chondrocytes to ensure their sustainable life in cartilage by sensing energy metabolism. Similar roles are suspected to exist in relation to systemic metabolism, since CCN3 is found in the bloodstream.

DOI： 10.1007/s12079-023-00723-4

Elevated Expression of CCN3 in Articular Cartilage Induces Osteoarthritis in Hip Joints Irrespective of Age and Weight Bearing. Reviewed International journal

Kazuki Hirose, Miho Kuwahara, Eiji Nakata, Tomonori Tetsunaga, Kazuki Yamada, Kenta Saiga, Masaharu Takigawa, Toshifumi Ozaki, Satoshi Kubota, Takako Hattori

International journal of molecular sciences 23 ( 23 ) 2022.12

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

Osteoarthritis (OA) occurs not only in the knee but also in peripheral joints throughout the whole body. Previously, we have shown that the expression of cellular communication network factor 3 (CCN3), a matricellular protein, increases with age in knee articular cartilage, and the misexpression of CCN3 in cartilage induces senescence-associated secretory phenotype (SASP) factors, indicating that CCN3 promotes cartilage senescence. Here, we investigated the correlation between CCN3 expression and OA degenerative changes, principally in human femoral head cartilage. Human femoral heads obtained from patients who received total hip arthroplasty were categorized into OA and femoral neck fracture (normal) groups without significant age differences. Gene expression analysis of RNA obtained from femoral head cartilage revealed that CCN3 and MMP-13 expression in the non-weight-bearing part was significantly higher in the OA group than in the normal group, whereas the weight-bearing OA parts and normal cartilage showed no significant differences in the expression of these genes. The expression of COL10A1, however, was significantly higher in weight-bearing OA parts compared with normal weight-bearing parts, and was also higher in weight-bearing parts compared with non-weight-bearing parts in the OA group. In contrast, OA primary chondrocytes from weight-bearing parts showed higher expression of CCN3, p16, ADAMTS4, and IL-1β than chondrocytes from the corresponding normal group, and higher ADAMTS4 and IL-1β in the non-weight-bearing part compared with the corresponding normal group. Acan expression was significantly lower in the non-weight-bearing group in OA primary chondrocytes than in the corresponding normal chondrocytes. The expression level of CCN3 did not show significant differences between the weight-bearing part and non-weight-bearing part in both OA and normal primary chondrocytes. Immunohistochemical analysis showed accumulated CCN3 and aggrecan neoepitope staining in both the weight-bearing part and non-weight-bearing part in the OA group compared with the normal group. The CCN3 expression level in cartilage had a positive correlation with the Mankin score. X-ray analysis of cartilage-specific CCN3 overexpression mice (Tg) revealed deformation of the femoral and humeral head in the early stage, and immunohistochemical analysis showed accumulated aggrecan neoepitope staining as well as CCN3 staining and the roughening of the joint surface in Tg femoral and humeral heads. Primary chondrocytes from the Tg femoral head showed enhanced expression of Ccn3, Adamts5, p16, Il-6, and Tnfα, and decreased expression of Col2a1 and -an. These findings indicate a correlation between OA degenerative changes and the expression of CCN3, irrespective of age and mechanical loading. Furthermore, the Mankin score indicates that the expression level of Ccn3 correlates with the progression of OA.

DOI： 10.3390/ijms232315311

Molecular and Genetic Interactions between CCN2 and CCN3 behind Their Yin-Yang Collaboration. Reviewed International journal

Satoshi Kubota, Kazumi Kawata, Takako Hattori, Takashi Nishida

International journal of molecular sciences 23 ( 11 ) 2022.5

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

Cellular communication network factor (CCN) 2 and 3 are the members of the CCN family that conduct the harmonized development of a variety of tissues and organs under interaction with multiple biomolecules in the microenvironment. Despite their striking structural similarities, these two members show contrastive molecular functions as well as temporospatial emergence in living tissues. Typically, CCN2 promotes cell growth, whereas CCN3 restrains it. Where CCN2 is produced, CCN3 disappears. Nevertheless, these two proteins collaborate together to execute their mission in a yin-yang fashion. The apparent functional counteractions of CCN2 and CCN3 can be ascribed to their direct molecular interaction and interference over the cofactors that are shared by the two. Recent studies have revealed the mutual negative regulation systems between CCN2 and CCN3. Moreover, the simultaneous and bidirectional regulatory system of CCN2 and CCN3 is also being clarified. It is of particular note that these regulations were found to be closely associated with glycolysis, a fundamental procedure of energy metabolism. Here, the molecular interplay and metabolic gene regulation that enable the yin-yang collaboration of CCN2 and CCN3 typically found in cartilage development/regeneration and fibrosis are described.

DOI： 10.3390/ijms23115887

Maternal Gut Microbiome Decelerates Fetal Endochondral Bone Formation by Inducing Inflammatory Reaction. Reviewed International journal

Yoko Uchida-Fukuhara, Takako Hattori, Shanqi Fu, Sei Kondo, Miho Kuwahara, Daiki Fukuhara, Md Monirul Islam, Kota Kataoka, Daisuke Ekuni, Satoshi Kubota, Manabu Morita, Mika Iikegame, Hirohiko Okamura

Microorganisms 10 ( 5 ) 2022.5

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

To investigate the effect of the maternal gut microbiome on fetal endochondral bone formation, fetuses at embryonic day 18 were obtained from germ-free (GF) and specific-pathogen-free (SPF) pregnant mothers. Skeletal preparation of the fetuses' whole bodies did not show significant morphological alterations; however, micro-CT analysis of the tibiae showed a lower bone volume fraction in the SPF tibia. Primary cultured chondrocytes from fetal SPF rib cages showed a lower cell proliferation and lower accumulation of the extracellular matrix. RNA-sequencing analysis showed the induction of inflammation-associated genes such as the interleukin (IL) 17 receptor, IL 6, and immune-response genes in SPF chondrocytes. These data indicate that the maternal gut microbiome in SPF mice affects fetal embryonic endochondral ossification, possibly by changing the expression of genes related to inflammation and the immune response in fetal cartilage. The gut microbiome may modify endochondral ossification in the fetal chondrocytes passing through the placenta.

DOI： 10.3390/microorganisms10051000

Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorption. Reviewed International journal

Hirofumi Fujita, Aoi Ando, Yohei Mizusawa, Mitsuaki Ono, Takako Hattori, Munenori Habuta, Toshitaka Oohashi, Satoshi Kubota, Hideyo Ohuchi

PloS one 17 ( 11 ) e0277307 2022

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

Cysteinyl leukotriene receptor 1 (CysLTR1) is a G protein-coupled receptor for the inflammatory lipid mediators cysteinyl leukotrienes, which are involved in smooth muscle constriction, vascular permeability, and macrophage chemokine release. The Cysltr1 gene encoding CysLTR1 is expressed in the macrophage lineage, including osteoclasts, and the CysLTR1 antagonist Montelukast has been shown to suppress the formation of osteoclasts. However, it currently remains unclear whether CysLTR1 is involved in osteoclast differentiation and bone loss. Therefore, to clarify the role of CysLTR1 in osteoclastogenesis and pathological bone loss, we herein generated CysLTR1 loss-of-function mutant mice by disrupting the cysltr1 gene using the CRISPR-Cas9 system. These mutant mice had a frameshift mutation resulting in a premature stop codon (Cysltr1 KO) or an in-frame mutation causing the deletion of the first extracellular loop (Cysltr1Δ105). Bone marrow macrophages (BMM) from these mutant mice lost the intracellular flux of calcium in response to leukotriene D4, indicating that these mutants completely lost the activity of CysLTR1 without triggering genetic compensation. However, disruption of the Cysltr1 gene did not suppress the formation of osteoclasts from BMM in vitro. We also demonstrated that the CysLTR1 antagonist Montelukast suppressed the formation of osteoclasts without functional CysLTR1. On the other hand, disruption of the Cysltr1 gene partially suppressed the formation of osteoclasts stimulated by leukotriene D4 and did not inhibit that by glutathione, functioning as a substrate in the synthesis of cysteinyl leukotrienes. Disruption of the Cysltr1 gene did not affect ovariectomy-induced osteoporosis or lipopolysaccharide-induced bone resorption. Collectively, these results suggest that the CysLT-CysLTR1 axis is dispensable for osteoclast differentiation in vitro and pathological bone loss, while the leukotriene D4-CysTR1 axis is sufficient to stimulate osteoclast formation. We concluded that the effects of glutathione and Montelukast on osteoclast formation were independent of CysLTR1.

DOI： 10.1371/journal.pone.0277307

Cellular communication network factor 3 in cartilage development and maintenance. Reviewed International journal

Satoshi Kubota, Harumi Kawaki, Bernard Perbal, Kazumi Kawata, Takako Hattori, Takashi Nishida

Journal of cell communication and signaling 15 ( 4 ) 533 - 543 2021.12

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

Other Link： https://link.springer.com/article/10.1007/s12079-021-00629-z/fulltext.html

UCP1 expression in the mouse adrenal gland is not upregulated by thermogenic conditions. Reviewed International journal

Hirofumi Fujita, Munenori Habuta, Takako Hattori, Satoshi Kubota, Hiromi Kumon, Hideyo Ohuchi

Biochemical and biophysical research communications 566 184 - 189 2021.8

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

Bipartite regulation of cellular communication network factor 2 and fibroblast growth factor 1 genes by fibroblast growth factor 1 through histone deacetylase 1 and fork head box protein A1. Reviewed International journal

Abdellatif Elseoudi, Takashi Nishida, Tomomi Mizukawa, Takako Hattori, Kazumi Kawata, Eman A Taha, Masaharu Takigawa, Satoshi Kubota

Journal of cell communication and signaling 15 ( 1 ) 81 - 91 2021.3

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

CCN3 (NOV) Drives Degradative Changes in Aging Articular Cartilage Reviewed International journal

Miho Kuwahara, Koichi Kadoya, Sei Kondo, Shanqi Fu, Yoshiko Miyake, Ayako Ogo, Mitsuaki Ono, Takayuki Furumatsu, Eiji Nakata, Takako Sasaki, Shogo Minagi, Masaharu Takigawa, Satoshi Kubota, Takako Hattori

International Journal of Molecular Sciences 21 ( 20 ) 7556 - 7556 2020.10

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI AG

Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H2O2 caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-β gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.

DOI： 10.3390/ijms21207556

CTGF/CCN2 facilitates LRP4-mediated formation of the embryonic neuromuscular junction. Reviewed International journal

Bisei Ohkawara, Akinori Kobayakawa, Shunsuke Kanbara, Takako Hattori, Satoshi Kubota, Mikako Ito, Akio Masuda, Masaharu Takigawa, Karen M Lyons, Naoki Ishiguro, Kinji Ohno

EMBO reports 21 ( 8 ) e48462 2020.6

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

At the neuromuscular junction (NMJ), lipoprotein-related receptor 4 (LRP4) mediates agrin-induced MuSK phosphorylation that leads to clustering of acetylcholine receptors (AChRs) in the postsynaptic region of the skeletal muscle. Additionally, the ectodomain of LRP4 is necessary for differentiation of the presynaptic nerve terminal. However, the molecules regulating LRP4 have not been fully elucidated yet. Here, we show that the CT domain of connective tissue growth factor (CTGF/CCN2) directly binds to the third beta-propeller domain of LRP4. CTGF/CCN2 enhances the binding of LRP4 to MuSK and facilitates the localization of LRP4 on the plasma membrane. CTGF/CCN2 enhances agrin-induced MuSK phosphorylation and AChR clustering in cultured myotubes. Ctgf-deficient mouse embryos (Ctgf-/- ) have small AChR clusters and abnormal dispersion of synaptic vesicles along the motor axon. Ultrastructurally, the presynaptic nerve terminals have reduced numbers of active zones and mitochondria. Functionally, Ctgf-/- embryos exhibit impaired NMJ signal transmission. These results indicate that CTGF/CCN2 interacts with LRP4 to facilitate clustering of AChRs at the motor endplate and the maturation of the nerve terminal.

DOI： 10.15252/embr.201948462

Burden of asthma exacerbations and health care utilization in pediatric patients with asthma in the US and England. Reviewed International journal

Mugdha Gokhale, Takako Hattori, Lee Evitt, Warren Lenney, Beth Nordstrom, Jenna Collins, Anna Schultze, Melissa K Van Dyke

Immunity, inflammation and disease 8 ( 2 ) 236 - 245 2020.6

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

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

Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes. Reviewed International journal

Mitsuhiro Hoshijima, Takako Hattori, Eriko Aoyama, Takashi Nishida, Satoshi Kubota, Hiroshi Kamioka, Masaharu Takigawa

International journal of molecular sciences 21 ( 8 ) E2769 - E2769 2020.4

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

Retrotransposons Manipulating Mammalian Skeletal Development in Chondrocytes. Invited Reviewed International journal

Satoshi Kubota, Takanori Ishikawa, Kazumi Kawata, Takako Hattori, Takashi Nishida

International journal of molecular sciences 21 ( 5 ) 1564 - 1564 2020.2

Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI AG

Retrotransposons are genetic elements that copy and paste themselves in the host genome through transcription, reverse-transcription, and integration processes. Along with their proliferation in the genome, retrotransposons inevitably modify host genes around the integration sites, and occasionally create novel genes. Even now, a number of retrotransposons are still actively editing our genomes. As such, their profound role in the evolution of mammalian genomes is obvious; thus, their contribution to mammalian skeletal evolution and development is also unquestionable. In mammals, most of the skeletal parts are formed and grown through a process entitled endochondral ossification, in which chondrocytes play central roles. In this review, current knowledge on the evolutional, physiological, and pathological roles of retrotransposons in mammalian chondrocyte differentiation and cartilage development is summarized. The possible biological impact of these mobile genetic elements in the future is also discussed.

DOI： 10.3390/ijms21051564

Circadian production of melatonin in cartilage modifies rhythmic gene expression. Reviewed International journal

Fu S, Kuwahara M, Uchida Y, Koudo S, Hayashi D, Shimomura Y, Takagaki A, Nishida T, Maruyama Y, Ikegame M, Hattori A, Kubota S, Hattori T

The Journal of endocrinology 241 ( 2 ) 161 - 173 2019.3

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Bioscientifica

Endochondral ossification, including bone growth and other metabolic events, is regulated by circadian rhythms. Herein, we provide evidence that melatonin has a direct effect on the circadian rhythm of chondrocytes. We detected mRNA expression of the genes which encode the melatonin-synthesizing enzymes AANAT (arylalkylamine N-acetyltransferase) and HIOMT (hydroxyindole O-methyltransferase), as well as the melatonin receptors MT1 and MT2 in mouse primary chondrocytes and cartilage. Production of melatonin was confirmed by mass spectrometric analysis of primary rat and chick chondrocytes. Addition of melatonin to primary BALB/c mouse chondrocytes caused enhanced cell growth and increased expression of <italic>Col2a1</italic>, <italic>Aggrecan</italic> and <italic>Sox9</italic>, but inhibited <italic>Col10a1</italic> expression. Addition of luzindole, an MT1 and MT2 antagonist, abolished these effects. These data indicate that chondrocytes produce melatonin, which regulates cartilage growth and maturation via the MT1 and MT2 receptors. Kinetic analysis showed that melatonin caused rapid upregulation of <italic>Aanat</italic>, <italic>Mt1</italic>, <italic>Mt2</italic> and <italic>Pthrp</italic> expression, followed by <italic>Sox9</italic> and <italic>Ihh</italic>. Furthermore, expression of the clock gene <italic>Bmal1</italic> was induced, while that of <italic>Per1</italic> was downregulated. Chronobiological analysis of synchronized C3H mouse chondrocytes revealed that melatonin induced the cyclic expression of <italic>Aanat</italic> and modified the cyclic rhythm of <italic>Bmal1</italic>, <italic>Mt1</italic> and <italic>Mt2</italic>. In contrast, <italic>Mt1</italic> and <italic>Mt2</italic> showed different rhythms from <italic>Bmal1</italic> and <italic>Aanat</italic>, indicating the existence of different regulatory genes. Our results indicate that exogenous and endogenous melatonin work in synergy in chondrocytes to adjust rhythmic expression to the central suprachiasmatic nucleus clock.

DOI： 10.1530/JOE-19-0022

The efficacy and safety of fluticasone/salmeterol compared to fluticasone in children younger than four years of age. Reviewed International journal

Shigemi Yoshihara, Toshikazu Tsubaki, Masanori Ikeda, Warren Lenney, Richard Tomiak, Takako Hattori, Kenichi Hashimoto, Toru Soutome, Shihona Kato

Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology 30 ( 2 ) 195 - 203 2019.3

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

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

New function of Tip60 in controlling triacylglycerol synthesis Invited Reviewed

Takako Hattori

Biotarget 2 13 - 13 2018.9

Commensal Microbiota Enhance Both Osteoclast and Osteoblast Activities. Reviewed International journal

Uchida Y, Irie K, Fukuhara D, Kataoka K, Hattori T, Ono M, Ekuni D, Kubota S, Morita M

Molecules (Basel, Switzerland) 23 ( 7 ) doi: 10, 3390/molecules2307/1517. 2018.6

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

Recent studies suggest that the commensal microbiota affects not only host energy metabolism and development of immunity but also bone remodeling by positive regulation of osteoclast activity. However, the mechanism of regulation of bone cells by the commensal microbiota has not been elucidated. In this study, 8-week-old specific pathogen-free (SPF) and germ-free (GF) mice were compared in terms of alveolar bones and primary osteoblasts isolated from calvarias. Micro-CT analysis showed that SPF mice had larger body size associated with lower bone mineral density and bone volume fraction in alveolar bones compared with GF mice. Greater numbers of osteoclasts in alveolar bone and higher serum levels of tartrate-resistant acid phosphatase 5b were observed in SPF mice. Tissue extracts from SPF alveolar bone showed higher levels of cathepsin K, indicating higher osteoclast activity. SPF alveolar extracts also showed elevated levels of γ-carboxylated glutamic acid⁻osteocalcin as a marker of mature osteoblasts compared with GF mice. Polymerase chain reaction (PCR) array analysis of RNA directly isolated from alveolar bone showed that in SPF mice, expression of mRNA of osteocalcin, which also acts as an inhibitor of bone mineralization, was strongly enhanced compared with GF mice. Cultured calvarial osteoblasts from SPF mice showed reduced mineralization but significantly enhanced expression of mRNAs of osteocalcin, alkaline phosphatase, insulin-like growth factor-I/II, and decreased ratio of osteoprotegerin/receptor activator of nuclear factor-kappa B ligand compared with GF mice. Furthermore, PCR array analyses of transcription factors in cultured calvarial osteoblasts showed strongly upregulated expression of Forkhead box g1. In contrast, Gata-binding protein 3 was strongly downregulated in SPF osteoblasts. These results suggest that the commensal microbiota prevents excessive mineralization possibly by stimulating osteocalcin expression in osteoblasts, and enhances both osteoblast and osteoclast activity by regulating specific transcription factors.

DOI： 10.3390/molecules23071517

Bioinspired Mineralization Using Chondrocyte Membrane Nanofragments Reviewed International journal

Emilio Satoshi Hara, Masahiro Okada, Noriyuki Nagaoka, Takako Hattori, Takuo Kuboki, Takayoshi Nakano, Takuya Matsumoto

ACS Biomaterials Science & Engineering 4 ( 2 ) 617 - 625 2018.2

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society ({ACS})

Biomineralization involves complex processes and interactions between organic and inorganic matters, which are controlled in part by the cells. The objectives of this study were, first, to perform a systematic and ultrastructural investigation of the initial mineral formation during secondary ossification center of mouse femur based on material science and biology viewpoint, and then develop novel biomaterials for mineralization based on the in vivo findings. First, we identified the very initial mineral deposition at postnatal day 5 (P5) at the medial side of femur epiphysis by nanocomputed tomography. Initial minerals were found in the surroundings of hypertrophic chondrocytes. Interestingly, histological and immunohistochemical analyses showed that initial mineralization until P6 was based on chondrocyte activity only, i.e., it occurred in the absence of osteoblasts. Moreover, electron microscopy-based ultrastructural analysis showed that cell-secreted matrix vesicles were absent in the early steps of osteoblast-independent endochondral ossification. Instead, chondrocyte membrane nanofragments were found in the fibrous matrix surrounding the hypertrophic chondrocytes. EDS analysis and electron diffraction study indicated that cell membrane nanofragments were not mineralized material, and could be the nucleation site for the newly formed calcospherites. The phospholipids in the cell membrane nanofragments could be a source of phosphate for subsequent calcium phosphate formation, which initially was amorphous, and later transformed into apatite crystals. Finally, artificial cell nanofragments were synthesized from ATDC5 chondrogenic cells, and in vitro assays showed that these nanofragments could promote mineral formation. Taken together, these results indicated that cell membrane nanofragments were the nucleation site for mineral formation, and could potentially be used as material for manipulation of biomineralization.

DOI： 10.1021/acsbiomaterials.7b00962

Chondrocyte burst promotes space for mineral expansion Reviewed International journal

Emilio Satoshi Hara, Masahiro Okada, Noriyuki Nagaoka, Takako Hattori, Letycia Mary Iida, Takuo Kuboki, Takayoshi Nakano, Takuya Matsumoto

Integrative Biology 10 ( 1 ) 57 - 66 2018

Language：English Publishing type：Research paper (scientific journal) Publisher：Royal Society of Chemistry ({RSC})

Analysis of tissue development from multidisciplinary approaches can result in more integrative biological findings, and can eventually allow the development of more effective bioengineering methods. In this study, we analyzed the initial steps of mineral formation during secondary ossification of mouse femur based on biological and bioengineering approaches. We first found that some chondrocytes burst near the mineralized area. External factors that could trigger chondrocyte burst were then investigated. Chondrocyte burst was shown to be modulated by mechanical and osmotic pressure. A hypotonic solution, as well as mechanical stress, significantly induced chondrocyte burst. We further hypothesized that chondrocyte burst could be associated with space-making for mineral expansion. In fact, ex vivo culture of femur epiphysis in hypotonic conditions, or under mechanical pressure, enhanced mineral formation, compared to normal culture conditions. Additionally, the effect of mechanical pressure on bone formation in vivo was investigated by immobilization of mouse lower limbs to decrease the body pressure onto the joints. The results showed that limb immobilization suppressed bone formation. Together, these results suggest chondrocyte burst as a novel fate of chondrocytes, and that manipulation of chondrocyte burst with external mechano-chemical stimuli could be an additional approach for cartilage and bone tissue engineering.

DOI： 10.1039/C7IB00130D

Generation and Analysis of Cartilage-Specific CCN2 Overexpression in Transgenic Mice

Takako Hattori, Shinsuke Itoh, Masaharu Takigawa

Methods in Molecular Biology 391 - 403 2017

Protein Imaging of CCN2 and CCN3 in Living Cells

Takako Hattori, Mitsuhiro Hoshijima, Masaharu Takigawa

Methods in Molecular Biology 211 - 215 2017

Production of Recombinant CCN2 Protein in Escherichia coli

Eriko Aoyama, Takako Hattori, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 77 - 84 2017

Protocols for Screening for Binding Partners of CCN Proteins: Yeast Two-Hybrid System

Mitsuhiro Hoshijima, Takako Hattori, Masaharu Takigawa

Methods in Molecular Biology 145 - 154 2017

Involvement of multiple CCN family members in platelets that support regeneration of joint tissues. Reviewed International journal

Chikako Hara, Satoshi Kubota, Takashi Nishida, Miki Hiasa, Takako Hattori, Eriko Aoyama, Yoshinori Moriyama, Hiroshi Kamioka, Masaharu Takigawa

Modern rheumatology 26 ( 6 ) 940 - 949 2016.11

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

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

Assessment of CCN2 Independent Modules Regenerative Capacity on Osteoarthritis and Further Selecting the Most Suitable Among them as a Potential Therapeutic Drug Reviewed

Abdelkader Tarek, Aoyama Eriko, Nishida Takashi, Hattori Takako, Janune Danilo, Hara Emilio S, Ono Mitsuaki, Tabata Yasuhiko, Kuboki Takuo, Kubota Satoshi, Takigawa Masaharu

FASEB JOURNAL 30 2016.4

Role of CCN2 in Amino Acid Metabolism of Chondrocytes. Reviewed International journal

Yurika Murase, Takako Hattori, Eriko Aoyama, Takashi Nishida, Aya Maeda-Uematsu, Harumi Kawaki, Karen M Lyons, Akira Sasaki, Masaharu Takigawa, Satoshi Kubota

Journal of cellular biochemistry 117 ( 4 ) 927 - 37 2016.4

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

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jcb.25377

Sorcin Expression in the Epiphyseal Growth Plates of Mice Reviewed

Mariko Kawai, Ning Liu, Takako Hattori, Yo-Hei Kataoka, Masaharu Takigawa, Satoshi Kubota, Toshio Yamamoto, Kiyoshi Ohura

JOURNAL OF HARD TISSUE BIOLOGY 25 ( 1 ) 57 - 61 2016.1

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

Other Link： 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

Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-alpha expression upon lipopolysaccharide stimulation in human monocytes Reviewed

H. Murata, T. Hattori, H. Maeda, S. Takashiba, M. Takigawa, J. Kido, T. Nagata

JOURNAL OF PERIODONTAL RESEARCH 50 ( 4 ) 452 - 460 2015.8

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

Background and ObjectiveTumor necrosis factor alpha (TNF-) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF- gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF- promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-B) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF- expression.
Material and MethodsTo identify DNA-binding proteins that bound to the target region of TNF- promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF- expression.
ResultsSeveral candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF- induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF- promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF- expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF- expression.
ConclusionWe identified TDP-43 as one of the novel TNF- factors and found that it bound to the LPS-responsive element in the TNF- promoter to increase TNF- expression.

DOI： 10.1111/jre.12227

Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage Reviewed International journal

Jung Park, Matthias Gebhardt, Svitlana Golovchenko, Francesc Perez-Branguli, Takako Hattori, Christine Hartmann, Xin Zhou, Benoit deCrombrugghe, Michael Stock, Holm Schneider, Klaus von der Mark

BIOLOGY OPEN 4 ( 5 ) 608 - 621 2015.5

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

According to the general understanding, the chondrocyte lineage terminates with the elimination of late hypertrophic cells by apoptosis in the growth plate. However, recent cell tracking studies have shown that murine hypertrophic chondrocytes can survive beyond "terminal'' differentiation and give rise to a progeny of osteoblasts participating in endochondral bone formation. The question how chondrocytes convert into osteoblasts, however, remained open. Following the cell fate of hypertrophic chondrocytes by genetic lineage tracing using BACCol10;Cre induced YFP-reporter gene expression we show that a progeny of Col10Cre-reporter labelled osteoprogenitor cells and osteoblasts appears in the primary spongiosa and participates - depending on the developmental stage - substantially in trabecular, endosteal, and cortical bone formation. YFP+ trabecular and endosteal cells isolated by FACS expressed Col1a1, osteocalcin and runx2, thus confirming their osteogenic phenotype. In searching for transitory cells between hypertrophic chondrocytes and trabecular osteoblasts we identified by confocal microscopy a novel, small YFP(+)Osx(+) cell type with mitotic activity in the lower hypertrophic zone at the chondro-osseous junction. When isolated from growth plates by fractional enzymatic digestion, these cells termed CDOP (chondrocyte-derived osteoprogenitor) cells expressed bone typical genes and differentiated into osteoblasts in vitro. We propose the Col10Cre-labeled CDOP cells mark the initiation point of a second pathway giving rise to endochondral osteoblasts, alternative to perichondrium derived osteoprogenitor cells. These findings add to current concepts of chondrocyte-osteocyte lineages and give new insight into the complex cartilage-bone transition process in the growth plate.

DOI： 10.1242/bio.201411031

Exosomes mediate intercellular transfer of pro-fibrogenic connective tissue growth factor (CCN2) between hepatic stellate cells, the principal fibrotic cells in the liver Reviewed International journal

Alyssa Charrier, Ruju Chen, Li Chen, Sherri Kemper, Takako Hattori, Masaharu Takigawa, David R. Brigstock

SURGERY 156 ( 3 ) 548 - 555 2014.9

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

Connective tissue growth factor (CCN2) and microRNA-21 are components of a positive feedback loop in pancreatic stellate cells (PSC) during chronic pancreatitis and are exported in PSC-derived exosomes Reviewed International journal

Alyssa Charrier, Ruju Chen, Li Chen, Sherri Kemper, Takako Hattori, Masaharu Takigawa, David R. Brigstock

JOURNAL OF CELL COMMUNICATION AND SIGNALING 8 ( 2 ) 147 - 156 2014.6

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

CCN2 as a Novel Molecule Supporting Energy Metabolism of Chondrocytes Reviewed International journal

Aya Maeda-Uematsu, Satoshi Kubota, Harumi Kawaki, Kazumi Kawata, Yoshiaki Miyake, Takako Hattori, Takashi Nishida, Norifumi Moritani, Karen M. Lyons, Seiji Iida, Masaharu Takigawa

JOURNAL OF CELLULAR BIOCHEMISTRY 115 ( 5 ) 854 - 865 2014.5

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

The regenerative effects of CCN2 independent modules on chondrocytes in vitro and osteoarthritis models in vivo Reviewed International journal

El Kader, Tarek Abd, Kubota Satoshi, Nishida Takashi, Hattori Takako, Aoyama Eriko, Janune Danilo, Hara Emilio S, Ono Mitsuaki, Tabata Yasuhiko, Kuboki Takuo, Takigawa Masaharu

Bone 59 180 - 188 2014

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

E6-AP/UBE3A Protein Acts as a Ubiquitin Ligase toward SOX9 Protein Reviewed International journal

Takako Hattori, Tetsuya Kishino, Shelley Stephen, Heidi Eberspaecher, Sayumi Maki, Masaharu Takigawa, Benoit de Crombrugghe, Hideyo Yasuda

JOURNAL OF BIOLOGICAL CHEMISTRY 288 ( 49 ) 35138 - 35148 2013.12

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

CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes Reviewed International journal

Shinsuke Itoh, Takako Hattori, Nao Tomita, Eriko Aoyama, Yasutaka Yutani, Takashi Yamashiro, Masaharu Takigawa

PLOS ONE 8 ( 8 ) e71156 2013.8

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

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage.

DOI： 10.1371/journal.pone.0071156

Deletion of beta catenin in hypertrophic growth plate chondrocytes impairs trabecular bone formation. Reviewed International journal

Svitlana Golovchenko, Takako Hattori, Christine Hartmann, Matthias Gebhardt, Sonja Gebhard, Andreas Hess, Friederike Pausch, Britta Schlund, Klaus von der Mark

Bone 55 ( 1 ) 102 - 12 2013.7

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

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

Cartilage-Specific Over-Expression of CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Stimulates Insulin-Like Growth Factor Expression and Bone Growth Reviewed International journal

Nao Tomita, Takako Hattori, Shinsuke Itoh, Eriko Aoyama, Mayumi Yao, Takashi Yamashiro, Masaharu Takigawa

PLoS ONE 8 ( 3 ) e59226 2013.3

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

Anti-fibrotic effect of CCN3 accompanied by altered gene expression profile of the CCN family Reviewed International journal

Tarek Abd El Kader, Satoshi Kubota, Danilo Janune, Takashi Nishida, Takako Hattori, Eriko Aoyama, Bernard Perbal, Takuo Kuboki, Masaharu Takigawa

Journal of Cell Communication and Signaling 7 ( 1 ) 11 - 18 2013.3

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

Novel chondrogenic and chondroprotective effects of the natural compound harmine Reviewed International journal

Emilio Satoshi Hara, Mitsuaki Ono, Satoshi Kubota, Wataru Sonoyama, Yasutaka Oida, Takako Hattori, Takashi Nishida, Takayuki Furumatsu, Toshifumi Ozaki, Masaharu Takigawa, Takuo Kuboki

Biochimie 95 ( 2 ) 374 - 381 2013.2

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

A significant number of natural compounds have been shown to regulate the behavior of the cells, in collaboration with cellular proteins. CCN2/connective tissue growth factor (CTGF) has been reported to have essential roles in cartilage development, chondrocyte proliferation and differentiation as well as regulation of the extracellular matrix metabolism. Previous studies demonstrated the capability of CCN2 to regenerate surgical defects in articular cartilage of rat knee. Also, transgenic mice over-expressing cartilage-specific CCN2 were shown to be more resistant to aging-related cartilage degradation. We hypothesized that small molecules that induce CCN2 in chondrocytes could be novel candidates to increase the resistance to aging-related cartilage degradation, or even to correct cartilage degenerative changes incurred in OA. Therefore, this study screened a compound library and identified the β-carboline alkaloid harmine as a novel inducer of CCN2 in human chondrocytic HCS-2/8 cells and osteoarthritic articular chondrocytes. Harmine increased the expression of the cartilage markers aggrecan and COL2α1, as well as that of the master regulator of chondrogenesis, SOX-9. Moreover, harmine notably induced chondrogenesis of prechondrocytic ATDC5 cells in micromass cultures. The chondroprotective effect of harmine was investigated under inflammatory condition by stimulation with TNFα, and harmine was shown to ameliorate TNFα-induced decrease in expression of CCN2 and cartilage markers. These findings uncover novel chondrogenic effects of harmine and indicate harmine as a potential drug for prevention and/or repair of cartilage degradation.

DOI： 10.1016/j.biochi.2012.10.016

The CCN2-inducer harmine promotes chondrogenesis and protects against TNFα-induced ablation of chondrocytic phenotype Reviewed

Hara, E.S, M. Ono, S. Kubota, W. Sonoyama, Y. Oida, T. Hattori, T. Nishida, T. Furumatsu, T. Ozaki, M. Takigawa, T. Kuboki

Biochimie 95 374 - 381 2013

Roles of heterotypic CCN2/CTGF-CCN3/NOV and homotypic CCN2-CCN2 interactions in expression of the differentiated phenotype of chondrocytes Reviewed International journal

Mitsuhiro Hoshijima, Takako Hattori, Eriko Aoyama, Takashi Nishida, Takashi Yamashiro, Masaharu Takigawa

FEBS JOURNAL 279 ( 19 ) 3584 - 3597 2012.10

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

The Wnt antagonist Wif-1 interacts with CTGF and inhibits CTGF activity Reviewed International journal

Cordula Surmann-Schmitt, Takako Sasaki, Takako Hattori, Nicole Eitzinger, Georg Schett, Klaus Von der Mark, Michael Stock

JOURNAL OF CELLULAR PHYSIOLOGY 227 ( 5 ) 2207 - 2216 2012.5

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

In Vivo Three-Dimensional Kinematics of the Cervical Spine During Head Rotation in Patients With Cervical Spondylosis Reviewed International journal

Yukitaka Nagamoto, Takahiro Ishii, Hironobu Sakaura, Motoki Iwasaki, Hisao Moritomo, Masafumi Kashii, Takako Hattori, Hideki Yoshikawa, Kazuomi Sugamoto

SPINE 36 ( 10 ) 778 - 783 2011.5

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

The dimerization domain of SOX9 is required for transcription activation of a chondrocyte-specific chromatin DNA template. Reviewed International journal

Françoise Coustry, Chun-do Oh, Takako Hattori, Sankar N Maity, Benoit de Crombrugghe, Hideyo Yasuda

Nucleic acids research 38 ( 18 ) 6018 - 28 2010.10

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

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

SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification Reviewed International journal

Takako Hattori, Catharina Mueller, Sonja Gebhard, Eva Bauer, Friederike Pausch, Britta Schlund, Michael R. Boesl, Andreas Hess, Cordula Surmann-Schmitt, Helga von der Mark, Benoit de Crombrugghe, Klaus von der Mark

DEVELOPMENT 137 ( 6 ) 901 - 911 2010.3

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

N-terminal domains of CCN family 2/connective tissue growth factor bind to aggrecan Reviewed International journal

Eriko Aoyama, Takako Hattori, Mitsuhiro Hoshijima, Daisuke Araki, Takashi Nishida, Satoshi Kubota, Masaharu Takigawa

BIOCHEMICAL JOURNAL 420 ( 3 ) 413 - 420 2009.6

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

The expression and crucial roles of BMP signaling in development of smooth muscle progenitor cells in the mouse embryonic gut. Reviewed International journal

Shigeko Torihashi, Takako Hattori, Hirotaka Hasegawa, Masaaki Kurahashi, Takunori Ogaeri, Toyoshi Fujimoto

Differentiation; research in biological diversity 77 ( 3 ) 277 - 89 2009.3

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

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

Specific expression of Cre recombinase in hypertrophic cartilage under the control of a BAC-Col10a1 promoter. Reviewed International journal

Sonja Gebhard, Takako Hattori, Eva Bauer, Britta Schlund, Michael R Bösl, Benoit de Crombrugghe, Klaus von der Mark

Matrix biology : journal of the International Society for Matrix Biology 27 ( 8 ) 693 - 9 2008.10

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

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

CCN family 2/connective tissue growth factor (CCN2/CTGF) stimulates proliferation and differentiation of auricular chondrocytes Reviewed

T. Fujisawa, T. Hattori, M. Ono, J. Uehara, S. Kubota, T. Kuboki, M. Takigawa

Osteoarthritis and Cartilage 16 ( 7 ) 787 - 795 2008.7

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

Objectives: CCN family 2/connective tissue growth factor (CCN2/CTGF) is an atypical growth factor for growth plate chondrocytes. It plays an important role in their proliferation and differentiation in vitro, but does not stimulate hypertrophy or calcification of articular chondrocytes. We herein report for the first time that CCN2/CTGF promotes growth and differentiation of auricular chondrocytes and maintains their molecular phenotype in vitro and in vivo. Methods: Auricular chondrocytes were isolated from rabbit auricular cartilage by trypsin-collagenase treatment, and treated with human recombinant CCN2/CTGF or infected with adenovirus harboring the ccn2/ctgf gene. Cell proliferation was measured by [3H] thymidine incorporation and MTS assay, and changes in gene expression of auricular chondrocyte markers were monitored by real-time polymerase chain reaction, Northern hybridization, and histological analysis. For in vivo studies, auricular chondrocytes were cultured as pellets and implanted subcutaneously after treatment of recombinant human CCN2/CTGF. Ectopically formed cartilage was subjected to histological analysis. Cell death was monitored by in situ TUNEL analysis. Results: CCN2/CTGF stimulated proliferation, differentiation and synthesis of elastin and proteoglycans of rabbit primary auricular chondrocytes in a dose-dependent manner. CCN2/CTGF caused a 2.5-fold increase in the expression of elastin in comparison to the control, resulting in enhanced deposition of elastin fibers in a monolayer culture of auricular chondrocytes. Mineralization was not induced; in contrast, CCN2/CTGF stimulated expression of matrix gla protein which is known to impair mineralization. Furthermore, pretreatment of pellets of auricular chondrocytes with CCN2/CTGF and subcutaneous implantation significantly enhanced the growth of ectopic auricular cartilage pieces expressing phenotypic markers of auricular chondrocytes including type II and X collagen. Notably, chondrocyte apoptosis was impaired by CCN2/CTGF. Conclusions: These findings show that CCN2/CTGF may be a suitable agent for promoting differentiation and growth of auricular chondrocytes, while preventing mineralization and apoptosis, and suggests that CCN2/CTGF may be useful for the repair or reconstruction of elastic cartilage. © 2007 Osteoarthritis Research Society International.

DOI： 10.1016/j.joca.2007.11.001

Radiological study of joint destruction patterns in rheumatoid flatfoot Reviewed International journal

Takako Hattori, Jun Hashimoto, Tetsuya Tomita, Takashi Kitamura, Hideki Yoshikawa, Kazuomi Sugamoto

CLINICAL RHEUMATOLOGY 27 ( 6 ) 733 - 737 2008.6

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

Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5. Reviewed International journal

Takako Hattori, Francoise Coustry, Shelley Stephens, Heidi Eberspaecher, Masaharu Takigawa, Hideyo Yasuda, Benoit de Crombrugghe

Nucleic acids research 36 ( 9 ) 3011 - 24 2008.5

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

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

Radiographic study of joint destruction patterns in the rheumatoid elbow. Reviewed International journal

Takashi Kitamura, Jun Hashimoto, Tsuyoshi Murase, Tetsuya Tomita, Takako Hattori, Hideki Yoshikawa, Kazuomi Sugamoto

Clinical rheumatology 26 ( 4 ) 515 - 9 2007.4

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

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.

BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter. Reviewed International journal

Sonja Gebhard, Takako Hattori, Eva Bauer, Michael R Bösl, Britta Schlund, Ernst Pöschl, Nadia Adam, Benoit de Crombrugghe, Klaus von der Mark

Histochemistry and cell biology 127 ( 2 ) 183 - 94 2007.2

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

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

Twisted gastrulation modulates bone morphogenetic protein-induced collagen II and X expression in chondrocytes in vitro and in vivo. Reviewed International journal

Martina Schmidl, Nadia Adam, Cordula Surmann-Schmitt, Takako Hattori, Michael Stock, Uwe Dietz, Benoit de Crombrugghe, Ernst Pöschl, Klaus von der Mark

The Journal of biological chemistry 281 ( 42 ) 31790 - 800 2006.10

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

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

[Autoantigen and molecular chaperone in rheumatoid arthritis--their roles in metabolism of chondrocytes]. Reviewed

Takako Hattori, Masaharu Takigawa

Clinical calcium 16 ( 9 ) 1553 - 56 2006.9

Language：Japanese

The endoplasmic reticulum chaperone; heat shock protein/rheumatoid arthritis-related antigen (HSP47/RA-A47), in addition to its important intercellular functions for collagen maturation and secretion, has chondrocytes-destructive roles such as induction of endoplasmic reticulum (ER)-stress and metabolic gene expressions result in apoptosis when HSP47/RA-A47 is downregulated. Extracellular HSP47/RA-A47 may act as an autoantigen, but also regulate autoimmune inflammation. It is, therefore, a potential new biologic therapy for rheumatoid arthritis.

Interactions between PIAS proteins and SOX9 result in an increase in the cellular concentrations of SOX9 Reviewed International journal

T Hattori, H Eberspaecher, JF Lu, R Zhang, T Nishida, T Kahyo, H Yasuda, B de Crombrugghe

JOURNAL OF BIOLOGICAL CHEMISTRY 281 ( 20 ) 14417 - 14428 2006.5

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

CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha 5 beta 1 Reviewed International journal

M Hoshijima, T Hattori, M Inoue, D Araki, H Hanagata, A Miyauchi, M Takigawa

FEBS LETTERS 580 ( 5 ) 1376 - 1382 2006.2

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

Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast-two-hybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcoma-derived chondrocytic cell line HCS-2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/CTGF and its CT domain enhanced the adhesion of HCS-2/8 cells to FN in a dose-dependent manner. The CCN2/CTGF-enhancing effect on cell adhesion to FN was abolished by a blocking antibody against alpha 5 beta 1 integrin (alpha 5 beta 1), but not by one against anti-alpha v beta 3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C-terminal CT domain with FN, and that alpha 5 beta 1 is involved in this adhesion. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2006.01.061

Downregulation of rheumatoid arthritis-related antigen RA-A47 (HSP47/colligin-2) in chondrocytic cell lines induces apoptosis and cell-surface expression of RA-A47 in association with CD9. Reviewed International journal

Takako Hattori, Klaus von der Mark, Harumi Kawaki, Yasutaka Yutani, Satoshi Kubota, Tohru Nakanishi, Heidi Eberspaecher, Benoit de Crombrugghe, Masaharu Takigawa

Journal of cellular physiology 202 ( 1 ) 191 - 204 2005.1

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

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

Differentiation of Human Bone Marrow Mesenchymal Stem Cells to Chondrocytes for Construction of Three-dimensional Cartilage Tissue. International journal

Chikayoshi Matsuda, Mutsumi Takagi, Takako Hattori, Shigeyuki Wakitani, Toshiomi Yoshida

Cytotechnology 47 ( 1-3 ) 11 - 7 2005.1

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

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

Effect of Subcultivation of Human Bone Marrow Mesenchymal Stem on their Capacities for Chondrogenesis, Supporting Hematopoiesis, and Telomea Length. International journal

Masaki Nakahara, Mutsumi Takagi, Takako Hattori, Shigeyuki Wakitani, Toshiomi Yoshida

Cytotechnology 47 ( 1-3 ) 19 - 27 2005.1

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

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

A highly conserved enhancer in mammalian type X collagen genes drives high levels of tissue-specific expression in hypertrophic cartilage in vitro and in vivo. Reviewed International journal

Sonja Gebhard, Ernst Pöschl, Silvia Riemer, Eva Bauer, Takako Hattori, Heidi Eberspaecher, Zhaoping Zhang, Veronique Lefebvre, Benoit de Crombrugghe, Klaus von der Mark

Matrix biology : journal of the International Society for Matrix Biology 23 ( 5 ) 309 - 22 2004.8

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

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.

Novel enzyme-linked immunosorbent assay systems for the quantitative analysis of connective tissue growth factor (CTGF/Hcs24/CCN2): Detection of HTLV-I tax-induced CTGF from a human carcinoma cell line Reviewed International journal

H Kawaki, S Kubota, M Minato, NH Moritani, T Hattori, H Hanagata, M Kubota, A Miyauchi, T Nakanishi, M Takigawa

DNA AND CELL BIOLOGY 22 ( 10 ) 641 - 648 2003.10

Language：English Publishing type：Research paper (scientific journal) Publisher：MARY ANN LIEBERT INC PUBL

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24/CCN2) is known as a multifunctional growth factor. It stimulates proliferation, migration, and extracellular matrix production of mesenchymal cells, and is highly expressed in hypertrophic chondrocytes. In this study, we constructed useful ELISA systems for the analysis of CTGF and its modular fragments. For this objective we prepared four different antihuman CTGF monoclonal antibodies. One, specific for the VWC module, was utilized as the detecting antibody, and the other three, recognizing CT, IGFBP, and VWC modules, respectively, were employed as capture antibodies. Then we established three novel quantitative analysis systems for CTGF. The first system recognizing CT and VWC modules was useful to measure full-length CTGF with improved sensitivity. Utilizing this system, we found significant enhancement of CTGF production from a human carcinoma cell line transduced by HTLV-I tax gene, where the finding indicates the possible involvement of Tax in carcinogenesis. The second system, seeing IGFBP and VWC modules, could quantify not only CTGF, but also may be useful to analyze processed N-terminal fragments. The third system, utilizing capture and detection antibodies against the VWC module, was able to quantify the VWC module only, while it did not recognize full-length CTGF. Since CTGF is actually processed into subfragments, and functional assignment of each module is of interest, these systems are expected to contribute to the progress of CTGF investigations.

DOI： 10.1089/104454903770238111

Downregulation of a rheumatoid arthritis-related antigen (RA-A47) by ra-a47 antisense oligonucleotides induces inflammatory factors in chondrocytes. Reviewed International journal

Takako Hattori, Harumi Kawaki, Satoshi Kubota, Yasutaka Yutani, Benoit de Crombrugghe, Klaus von der Mark, Masaharu Takigawa

Journal of cellular physiology 197 ( 1 ) 94 - 102 2003.10

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

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

A repetitive, steady mouth opening induced an osteoarthritis-like lesion in the rabbit temporomandibular joint Reviewed

T Fujisawa, T Kuboki, T Kasai, W Sonoyama, S Kojima, J Uehara, C Komori, H Yatani, Hattori, I, M Takigawa

JOURNAL OF DENTAL RESEARCH 82 ( 9 ) 731 - 735 2003.9

Language：English Publishing type：Research paper (scientific journal) Publisher：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R

Although excessive mechanical stress is assumed to be one of the factors contributing to pathogenesis of temporomandibular joint (TMJ) osteoarthritis (OA), no pure mechanical-stress-induced OA model has been developed without surgical manipulation or puncture of the joint cavity. The purpose of this study was to establish a genuine mechanical-stress-induced OA model of the rabbit TMJ. In the experimental rabbits, repetitive, forced jaw-opening, 3 hrs/day for 5 days, was applied with the use of a general anesthesia protocol. By histological assessment of the TMJ articular tissues, partial eburnation of the articular cartilage, reactive marginal proliferation of the articular cartilage chondrocytes, and nested proliferation of chondrocytes in the subchondral bone area were observed at 7 days after the repetitive, forced-jaw-opening period. These results suggest that the repetitive, forced-jaw-opening protocol without surgical intervention can induce evident OA-like lesions in the rabbit TMJ, and this OA model may greatly contribute to the elucidation of the cartilage degradation mechanism in TMJ OA.

DOI： 10.1177/154405910308200914

CTGF/Hcs24 interacts with the cytoskeletal protein actin in chondrocytes Reviewed International journal

G Yosimichi, S Kubota, T Hattori, T Nishida, K Nawachi, T Nakanishi, M Kamada, T Takano-Yamamoto, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 299 ( 5 ) 755 - 761 2002.12

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

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) displays multiple functions in several types of mesenchymal cells, including the promotion of proliferation and differentiation of chondrocytes. Recently, the internalization and intracellular function of CTGF/Hcs24 were indicated as well. In this study, a binding protein for this factor was purified from the cytosolic fraction of human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by CTGF/Hcs24-affinity chromatography. The apparent molecular weight of the protein was 42 kDa and determination of the internal amino acid sequence revealed this protein to be beta- or gamma-actin. An in vitro competitive binding assay of I-125-labeled recombinant CTGF/Hcs24 with cold-rCTGF/Hcs24 showed that the binding between actin and I-125-CTGF/Hcs24 was specific. Immunoprecipitation analysis also showed that CTGF/Hcs24 bound to actin in HCS-2/8 cells. However, rCTGF/Hcs24 had no effects on the expression level of gamma-actin mRNA or total actin protein. These findings suggest that a significant portion of intracellular CTGF/Hcs24 may regulate certain cell biological events in chondrocytes through the interaction with this particular cytoskeletal protein. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/s0006-291x(02)02739-0

[Articular cartilage regeneration].

Takako Hattori, Shigeyuki Wakitani

Clinical calcium 12 ( 2 ) 217 - 21 2002.2

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

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.

CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/42 MAPK/extracellular-signal regulated kinase (ERK) Reviewed

G Yosimichi, T Nakanishi, T Nishida, T Hattori, T Takano-Yamamoto, M Takigawa

EUROPEAN JOURNAL OF BIOCHEMISTRY 268 ( 23 ) 6058 - 6065 2001.12

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL SCIENCE LTD

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24) promotes proliferation and differentiation of chondrocytes in culture. We investigated the roles of two major types of mitogen activated protein kinase (MAPK) in the promotion of proliferation and differentiation by CTGF/Hcs24. Here we report the effects of the MAPKK/MEK 1/2 inhibitor, PD098059, and p38 MAPK inhibitor, SB203580, in a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) and rabbit growth cartilage (RGC) cells treated with CTGF/Hcs24. In the proliferation phase, CTGF/Hcs24 induced a approximate to fivefold increase in the phosphorylation of p44/42 MAPK/ERK and a approximate to twofold increase in that of p38 MAPK in an in vivo kinase assay. These inhibitors of MAPKK and MAPK suppressed phosphorylation of ets-like gene-1 (Elk-1) and nuclear activating transcription factor-2 (Atf-2) induced by CTGF/Hcs24 in a dose-dependent manner, respectively. Western blot analysis showed that phosphorylation of ERK was induced from 30 to 60 min and phosphorylation of p38 MAPK from 10 to 15 min after the addition of CTGF/Hcs24 in confluence HCS-2/8 cells. PD098059 suppressed the DNA synthesis of HCS-2/8 cells and RGC cells, while SB203580 did not. On the other hand, the p38 MAPK inhibitor, SB203580, completely inhibited the CTGF/Hcs24-induced synthesis of proteoglycans in HCS-2/8 cells and RGC cells but the MEK1/2 inhibitor, PD098059, did not. These results suggest that ERK mediates the CTGF/Hcs24-induced proliferation of chondrocytes, and that p38 MAPK mediates the CTGF/Hcs24-induced differentiation of chondrocytes.

DOI： 10.1046/j.0014-2956.2001.02553.x

Cell-type-specific trans-activation of herpes simplex virus thymidine kinase promoter by the human T-cell leukemia virus type I Tax protein. Reviewed

Kubota S, Mukudai Y, Hattori T, Eguchi T, Kondo S, Takigawa M

DNA and cell biology 20 ( 9 ) 563 - 568 2001.9

Change in cellular localization of a rheumatoid arthritis-related antigen (RA-A47) with downregulation upon stimulation by inflammatory cytokines in chondrocytes. Reviewed

Hattori, T, Kubota, S, Yutani Y, Fujisawa T, Nakanishi, T, Takahashi, K, Takigawa M

J. Cell. Physiol. 186 268 - 281 2001

Regulatory mechanism of human connective tissue growth factor (CTGF/Hcs24) gene expression in a human chondrocytic cell line, HCS-2/8. Reviewed

Eguchi, T, Kubota, S, Kondo, S, Shimo, T, Hattori, T, Nakanishi, T, Kuboki, T, Yatani, H, Takigawa, M

J. Biochem. 130 79 - 87 2001

Novel mode of processing and secretion of connective tissue growth factor/ecogenin (CTGF/Hcs24) in chondrocytic HCS-2/8 cells. Reviewed

Kubota, S, Eguchi, T, Shimo, T, Nishida T, Hattori, T, Kondo, S, Nakanishi, T, Takigawa, M

Bone 29 155 - 161 2001

Characterization of a Mouse ctgf 3′-UTR Segment That Mediates Repressive Regulation of Gene Expression Reviewed

Seiji Kondo, Satoshi Kubota, Takanori Eguchi, Takako Hattori, Tohru Nakanishi, Toshio Sugahara, Masaharu Takigawa

Biochemical and Biophysical Research Communications 278 ( 1 ) 119 - 124 2000.11

Publishing type：Research paper (scientific journal) Publisher：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

Shear bond strengths of orthodontic plastic brackets

G. Guan, T. Takano-Yamamoto, M. Miyamoto, T. Hattori, K. Ishikawa, K. Suzuki

American journal of orthodontics and dentofacial orthopedics : official publication of the American Association of Orthodontists, its constituent societies, and the American Board of Orthodontics 117 ( 4 ) 438 - 443 2000.4

Publishing type：Research paper (scientific journal)

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

Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Synovial Fluids of Patients with Temporomandibular Joint Osteoarthritis Reviewed

Manabu Kanyama, Takuo Kuboki, Shunji Kojima, Takuo Fujisawa, Takako Hattori, Masaharu Takigawa, Atsushi Yamashita

Journal of Orofacial Pain 14 ( 1 ) 20 - 30 2000

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

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).

Novel intracellular effects of human connective tissue growth factor expressed in Cos-7 cells.

Kubota, S, Hattori, T, Shimo, T, Nakanishi, T, Takigawa, M

FEBS Letters 474 58 - 62 2000

Identification of an RNA element that confers post-transcriptional repression of connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/hcs24) gene: Similarities to retroviral RNA-protein interactions. Reviewed

Kubota, S, Kondo, S, Eguchi1, T, Hattori, T, Nakanishi, T, Pomerantz, R.J, Takigawa, M

Oncogene 19 4773 - 4786 2000

軟骨由来の慢性関節リウマチ関連抗原RA-A47に関する研究（Study on a rheumatoid arthritis-related antigen (RA-A47) isolated from chondrocytes）. Reviewed

服部高子

2000

Rheumatoid arthritis-related antigen 47kDa (RA-A47) is a product of colligin-2 and acts as a human HSP47 Reviewed

T Hattori, K Takahashi, Y Yutani, T Fujisawa, T Nakanishi, M Takigawa

JOURNAL OF BONE AND MINERAL METABOLISM 18 ( 印刷中 ) 323 - 334 2000

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER-VERLAG TOKYO

We previously isolated RA-A47, which is recognized as an antigen of rheumatoid arthritis (RA), from a human chondrosarcoma-derived cell line (HCS-2/8). The N-terminal 21-amino-acid sequence of RA-A47 had 81% homology to the deduced amino acid sequence of the human heat-shock protein (HSP) 47 gene, the colligin gene, and 100% homology to that of the colligin-2 gene. Moreover, as is HSP47, RA-A47 was a heat-inducible collagen-binding protein. To further characterize RA-A47, we isolated ra-a47 cDNA from HCS-2/8 cells and human periodontal ligament fibroblast (HPLF) cells. The isolated ra-a47 cDNAs from both cells were almost the same as that of collipin-2. C-504 and G(505) in the cDNA sequences of both cells and C-598 in the cDNA of HCS-2/8 were different from the corresponding bases of colligin-2 cDNA. These differences were also observed in genomic DNA. colligin cDNA was not isolated. To show that the isolated cDNA encodes RA-A47 protein, it was expressed in Cos-7 cells. The produced protein was 47 kDa and was recognized both with RA sera and antirat HSP47 antibody, indicating that it is RA-A47 and has structural similarity to HSP47. These results taken together with our previous finding show that RA-A47 is the putative colligin-2 gene product and behaves as a human HSP47. Although colligin has been considered the human HSP47 gene, failure to detect the colligin gene and its mRNA suggests that colligin does not exist in human cells and that the HSP47 gene is identical with colligin-2, which encodes RA-A47.

DOI： 10.1007/s007740070004

Other Link： 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

軟骨細胞様培養細胞株HCS-2/8における慢性関節リウマチ関連抗原RA-A47の炎症性サイトカインによる発現抑制と細胞内局在変化. (Downregulation of a rheumatoid arthritis-related antigen (RA-A47) by stimulation of inflammatory cytokines in chondrocytic cell line, HCS-2/8). Reviewed

服部高子

岡山歯学会雑誌 19 ( 2 ) 241 - 254 2000

Authorship：Lead author,　Corresponding author Language：Japanese Publisher：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

Detection of specific antibodies against human cultured chondrosarcoma (HCS-2/8) and osteosarcoma (Saos-2) cells in the serum of patients with osteoarthritis of the temporomandibular joint Reviewed

T Kuboki, T Hattori, T Mizushima, M Kanyama, T Fujisawa, A Yamashita, M Takigawa

ARCHIVES OF ORAL BIOLOGY 44 ( 5 ) 403 - 414 1999.5

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

To find specific humoral antibodies in sera from patients with temporomandibular joint (TMJ) osteoarthritis (OA), an immortal human chondrocyte (HCS-2/8) and osteoblast (Saos-2) cell line derived from a chondrosarcoma and an osteosarcoma, respectively, were used as source proteins of human antigens. Patients with chronically painful TMJ OA (n = 18) but no other joints symptoms were selected from a consecutive series of patients with temperomandibular disorders and sex-matched asymptomatic controls (n = 8) were also recruited. Cellular proteins of the HCS-2/8 and Saos-2 cells were subjected to Western blotting with the OA and control sera as probes. Band-recognition frequency and the peak optical density of the band were compared between groups by chi(2) and t-tests. OA sera recognized various bands for the chondrocytes, and one of these (47-kDa) was specific for the OA sera. In two OA patients whose treatment outcome was less favorable, the,reactivity against the 47-kDa protein was relatively high. In addition, the OA sera clearly cross-reacted with recombinant HSP47. Based on these findings, an autoimmune reaction against chondrocytes could be one of the exaggerating and/or perpetuating mechanisms in the pathophysiology of osteoarthritic TMJs, and the humoral antibody titre against the HSP47-like protein derived from the chondrocytes could be one of the possible markers for the prognosis of the joint pathology. (C) 1999 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/s0003-9969(99)00009-6

Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1 Reviewed

T Fujisawa, T Hattori, K Takahashi, T Kuboki, A Yamashita, M Takigawa

JOURNAL OF BIOCHEMISTRY 125 ( 5 ) 966 - 975 1999.5

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

To clarify the mechanism of cartilage degradation induced by mechanical stress, we investigated the influence of cyclic tension force (CTF) on the metabolism of cultured chondrocytes. The chondrocytes were exposed to CTF using a Flexercell strain unit. Five or 15 kPa of high frequency CTF significantly inhibited the syntheses of DNA, proteoglycan, collagen, and protein, Fifteen kPa of high frequency CTF induced the expression of interleukin-1 (IL-1), matrix metalloproteinase (MMP)-2 and -9 mRNA, and increased the production of pro- and active-MMP-9. The degradation of proteoglycan was inhibited by and MMP inhibitor, indicating that MMPs are involved in the degradation of proteoglycans induced by high frequency CTF, Moreover, reducing the frequency of CTF from high to low decreased the inhibition of proteoglycan synthesis, These findings suggest that the CTF frequency is one of the key determinants of chondrocyte metabolism. Low magnitude CTF, whether high or low frequency, did not cause the gene expression of cartilage degradation factors, suggesting that this CTF magnitude causes only minor changes in the cartilage matrix, High magnitude and frequency CTF caused the gene expression of IL-1 and MMP-9, followed by increases in the production of MMP-2 and -9 proteins, suggesting that excessive and continuous cyclic mechanical stress induces the production of IL-1 and MMP-9, resulting in cartilage degradation.

DOI： 10.1093/oxfordjournals.jbchem.a022376

Involvement of cis-acting repressive element(s) in the 3'-untranslated region of human connective tissue growth factor gene. Reviewed

Kubota, S, Hattori, T, Nakanishi, T, Takigawa, M

FEBS Letters 450 84 - 88 1999

Demonstration of receptors specific for connective tissue growth factor on a human chondrocytic cell line (HCS-2/8) Reviewed

T Nishida, T Nakanishi, T Shimo, M Asano, T Hattori, T Tamatani, K Tezuka, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 247 ( 3 ) 905 - 909 1998.6

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

Nitric oxide mediates interleukin-1-induced gene expression of matrix metalloproteinases and basic fibroblast growth factor in cultured rabbit articular chondrocytes Reviewed

K Sasaki, T Hattori, T Fujisawa, K Takahashi, H Inoue, M Takigawa

JOURNAL OF BIOCHEMISTRY 123 ( 3 ) 431 - 439 1998.3

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

We recently reported that nitric oxide (NO), which is produced by chondrocytes treated with interleukin-1 beta (IL-1), releases basic fibroblast growth factor (bFGF) stored in the matrix of articular chondrocytes, To clarify the mechanism of the IL-l-induced bFGF release, we investigated the production and gene expression of bFGF, matrix metalloproteinases (MMPs), syndecan 3, and inducible NO synthase (iNOS) by IL-l-treated rabbit articular chondrocytes. IL-1 stimulated not only the release of bFGF but also the production of it, Gelatin and casein zymography revealed that IL-1 stimulated the production of not only MMP-9 but also MMP-3, The increase in the production of these MMPs preceded the IL-1-stimulated bFGF release, An MMP inhibitor partially suppressed the release of bFGF, indicating that matrix degradation is at least partially involved in the IL-l-stimulated bFGF release even if increased production of bFGF is related to the release, IL-1 sequentially stimulated mRNA expression of iNOS, membrane type 1-MMP, MMP-9 and -3, and bFGF, in that order, N-G-Monomethyl-L-arginine, an inhibitor of NO production, inhibited gene expression of MMP-9 and bFGF. These findings suggest that elevation of the NO level via iNOS mRNA expression stimulated by IL-1 mediates gene expression and production of MMPs and bFGF, resulting in the release of bFGF, and also reveal molecular mechanisms implicating the degradation of articular cartilage followed by angiogenesis in the synovium in arthritic joints.

DOI： 10.1093/oxfordjournals.jbchem.a021955

Demonstration of receptors for epidermal growth factor on cultured rabbit chondrocytes and regulation of their expression by various growth and differentiation factors. Reviewed

Nishida,T, Nakanishi,T, Shimo,T, Asano,M, Hattori,T, Tamatani,T, Tezuka,K, Takigawa,M

Biochem.Biophys.Res.Commun. 183 14 - 20 1998

Isolation and characterization of a rheumatoid arthritis-specific antigen(RA-A47) from a human chondrocytic cell line(HCS-2/8) Reviewed

Takako Hattori, Takuo Fujisawa, Kazuhiro Sasaki, Yasutaka Yutani, Tohru Nakanishi, Kojiro Takahashi, Masaharu Takigawa

Biochem. Biophys. Res. Commun. 245 ( 3 ) 679 - 683 1998

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

Two types of 47 kDa antigen specifically recognized by sera from rheumatoid arthritis (RA) patients were isolated from the membrane fraction of a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by a 2-step procedure: preparative SDS-PAGE and reverse-phase HPLC. An N-terminal amino acid sequence in one of the 47 kDa antigens, named RA-A47, had 81% homology to that deduced from the DNA sequence of the colligin gene which is reported as human hsp47 gene, and 100% homology to that deduced from the DNA sequence of colligin-2 gene, a homologue of colligin. The RA-A47 cross-reacted with a monoclonal antibody raised against chick heat shock protein (Hsp) 47 and bound to gelatin. The expression of the ra-a47 gene was enhanced by heat shock treatment and TGF-β stimulation. These findings suggest that RA-A47 is a Hsp47-like protein, presumably the product of the colligin-2 gene, and that a collagen-specific molecular chaperone(s) such as Hsp47 and/or RA-A47 is involved in cartilage destruction in RA.

DOI： 10.1006/bbrc.1998.8505

Specific serum antibodies against membranous proteins of a human immortal chondrocytic cell line (HCS-2/8) in rheumatoid arthritis and their relationship to the natural history of this disease Reviewed

Akira Sakawa, Yasutaka Yutani, Kentaro Inui, Akira Shimazu, Yoshiki Yamano, Akira Kinosita, Fujio Suzuki, Takako Hattori, Masaharu Takigawa

Journal of Bone and Mineral Metabolism 14 ( 3 ) 146 - 152 1996.9

Publishing type：Research paper (scientific journal) Publisher：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

Other Link： http://link.springer.com/article/10.1007/BF02239482/fulltext.html

Novel FNR homologues identified in four representative oral facultative anaerobes: Capnocytophaga ochracea, Capnocytophaga sputigena, Haemophilus aphrophilus, and Actinobacillus actinomycetemcomitans Reviewed

T Hattori, K Takahashi, T Nakanishi, H Ohta, K Fukui, S Taniguchi, M Takigawa

FEMS MICROBIOLOGY LETTERS 137 ( 2-3 ) 213 - 220 1996.4

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

Nitric oxide mediates interleukin-1-induced matrix degradation and basic fibroblast groth factor release in cultured rabbit articular chondrocytes: A possible mechanism of pathological neovascularization in arthritis. Reviewed

Tamura, T, Nakanishi, T, Kimura, Y, Hattori, T, Sasaki, K, Norimatsu, H, Takahashi, K, Takigawa, M

Endocrinology 137 ( 9 ) 3729 - 3737 1996

Nitric oxide mediates interleukin-1-induced matrix degradation and basic fibroblast growth factor release in cultured rabbit articular chondrocytes: A possible mechanism of pathological neovascularization in arthritis

Tomoo Tamura, Tohru Nakanishi, Yusuke Kimura, Takako Hattori, Kazuhiro Sasaki, Hiromichi Norimatsu, Kojiro Takahashi, Masaharu Takigawa

Endocrinology 137 ( 9 ) 3729 - 3737 1996

Publishing type：Research paper (scientific journal)

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

Expression of nerve growth factor family neurotrophins in a mouse osteoblastic cell line

Tohru Nakanishi, Kojiro Takahashi, Chiharu Aoki, Kiyoshi Nishikawa, Takako Hattori, Shigehiko Taniguchi

Biochemical and Biophysical Research Communications 198 ( 3 ) 891 - 897 1994.2

Publishing type：Research paper (scientific journal)

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

REPRESSION OF IN-VITRO TRANSCRIPTION OF THE ESCHERICHIA-COLI FNR AND NARX GENES BY FNR PROTEIN Reviewed

K TAKAHASHI, T HATTORI, T NAKANISHI, T NOHNO, N FUJITA, A ISHIHAMA, S TANIGUCHI

FEBS LETTERS 340 ( 1-2 ) 59 - 64 1994.2

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

Expression of nerve growth factor family neurotrophins in a mouse osteoblastic cell line. Reviewed

Nakanishi, T, Takahashi, K, Aoki, C, Nishikawa, K, Hattori, T, Taniguchi, S

Biochem. Biophys. Res. Commun. 198 ( 3 ) 891 - 897 1994

Differential expression of homeobox-containing genes Msx-1 and Msx-2 and homeoprotein Msx-2 expression during chick craniofacial development. Reviewed

Nishikawa, K, Nakanishi, T, Aoki, C, Hattori, T, Takahashi, K, Taniguchi, S

Biochem. Mol. Biol. Inter. 32 ( 4 ) 763 - 771 1994

Studies on phosphorylated transcriptional regulator(NarL)for DBE. coli nar(/)/DB operon by ³¹P-NMR spectroscopy.

K. Takahashi, T. Hattori, H. Shindo, S. Noji, T. Nohno, S. Taniguchi

Biochemistry and Molecular Biology International 31 ( 1 ) 161 - 168 1993

Publishing type：Research paper (scientific journal)

The sequential transphosphorylation from autophosphorylated nitrate-sensing protein (NarX) to the transcriptional regulator protein (NarL), both operating in signal transduction to control the expression of the respiratory nitrate reductase (nar) operon in E. coli, was demonstrated with an in vitro reconstructed system to function similarly to other bacterial two-component regulatory systems. Over-expression system established by means of the pT7 promoter/polymerase provided both NarX and NarL proteins to reconstruct the in vitro transphosphorylation system. The phosphorylated NarL was detected, and the unstable phosphorylated group was directly assigned to acyl phosphate in the in vitro system by 31P-NMR spectroscopy.

FNR-like protein as a regulator for the gene expression of anaerobic respiratory system in oral bacteria. Reviewed

Takahashi Kojiro, Hattori Takako, Ohta Hiroyuki, Noji Sumihare, Kato Keijiro, Taniguchi Shigehiko

Japanese Journal of Oral Biology 34 ( 5 ) 459 - 466 1992

Language：Japanese Publisher：Japanese Association for Oral Biology

TheE. coliFNR protein is known as a molecular oxygene sensor and a specific activator for the gene expression of anaerobic respiratory system. In order to survey metabolic response toward molecular oxygen in oral bacteria from viewpoint of FNR-like protein participation, the expression of FNR-like protein in five representative strains of oral bacteria (Actinobacillus actinomycetemcomitansand Streptococcus sanguis as facultative anaerobe, Wolinella recta as microaerophile, and Prevotella intermedia and Fusobacterium nucleatumas strict anaerobe) was investigated with the Western blot method using an anti-FNR rabbit serum prepared against the purifiedE. coliFNR. For only A. actinomycetemcomitansas a facultative anaerobe, the Western blot test was positive, whereas negative for other four strains. This implies the possibility that in A. actinomycetemcomitans, a regulatory mechanism similar to that in E. coli functions for the expression of anaerobic respiratory genes.<BR>The expression level of FNR-like protein in A. actinomycetemcomitans was found to be enhanced with decreasing rate of the fructose-limited growth at the redox potential fixed around-400 mV, andbe maximum at the redox potential around-250 mV with the growth rate fixed at 0.11 h-1 under the anaerobic conditions.

DOI： 10.2330/joralbiosci1965.34.459

　 PREV - NEXT 　

1