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Noriyuki Tsumaki

Publications and source records attributed to Noriyuki Tsumaki.

16 recordsLinked to original sources
PubMed2007

Hyaline cartilage formation and enchondral ossification modeled with KUM5 and OP9 chondroblasts.

What is it that defines a bone marrow-derived chondrocyte? We attempted to identify marrow-derived cells with chondrogenic nature and immortality without transformation, defining "immortality" simply as indefinite cell division. KUM5 mesenchymal cells, a marrow stromal cell line, generated hyaline cartilage in vivo and exhibited enchondral ossification at a later stage after implantation. Selection of KUM5 chondroblasts based on the activity of the chondrocyte-specific cis-regulatory element of the collagen alpha2(XI) gene resulted in enhancement of their chondrogenic nature. Gene chip analysis revealed that OP9 cells, another marrow stromal cell line, derived from macrophage colony-stimulating factor-deficient osteopetrotic mice and also known to be niche-constituting cells for hematopoietic stem cells expressed chondrocyte-specific or -associated genes such as type II collagen alpha1, Sox9, and cartilage oligomeric matrix protein at an extremely high level, as did KUM5 cells. After cultured OP9 micromasses exposed to TGF-beta3 and BMP2 were implanted in mice, they produced abundant metachromatic matrix with the toluidine blue stain and formed type II collagen-positive hyaline cartilage within 2 weeks in vivo. Hierarchical clustering and principal component analysis based on microarray data of the expression of cell surface markers and cell-type-specific genes resulted in grouping of KUM5 and OP9 cells into the same subcategory of "chondroblast," that is, a distinct cell type group. We here show that these two cell lines exhibit the unique characteristics of hyaline cartilage formation and enchondral ossification in vitro and in vivo.

Animals↗
PubMed2006

Oxygen tension regulates chondrocyte differentiation and function during endochondral ossification.

Cartilage functions at a lower oxygen tension than most other tissues. To determine the role of oxygen tension in chondrocyte differentiation and function, we investigated the influence of oxygen tension in the pluripotent mesenchymal cell line C3H10T1/2 and 14.5E mice embryo forelimb organ culture. 10T1/2 cells and embryo forelimbs were cultured under normoxia (20% O2) or hypoxia (5% O2) in the presence of recombinant human bone morphogenetic protein 2. To elucidate the mechanism by which oxygen tension influences chondrocyte differentiation, the Smad pathway was examined using Smad6 overexpression adenovirus and Smad6 transgenic mice embryo forelimbs. The p38 MAPK pathway was examined using dominant-negative MKK3 and FR167653, a specific p38 MAPK inhibitor. The transcriptional activities of Sox9 and Runx2 were also investigated. Hypoxia promoted bone morphogenetic protein 2-induced glycosaminoglycan production and suppressed alkaline phosphatase activity and mineralization of C3H10T1/2. Thus, hypoxia promoted chondrocytic commitment rather than osteoblastic differentiation. In the mice embryo forelimb organ culture, hypoxia increased cartilaginous matrix synthesis. These effects were primarily mediated by p38 MAPK activation, independent of Sox9. Hypoxia inhibited Col10a1 (type X collagen alpha1) expression via down-regulation of Runx2 activity by Smad suppression and histone deacetylase 4 activation. In conclusion, hypoxia promotes chondrocytic differentiation and cartilage matrix synthesis and suppresses terminal chondrocyte differentiation. These hypoxia-induced phenomena may act on chondrocytes to enhance and preserve their phenotype and function during chondrocyte differentiation and endochondral ossification.

Animals↗
PubMed2006

Bone morphogenetic proteins in bone stimulate osteoclasts and osteoblasts during bone development.

UNLABELLED: In this study, overexpression of noggin, a BMP antagonist, in developing bone caused significantly decreased osteoclast number as well as bone formation rate, resulting in increased bone mass with immature bone quality. BMP signaling plays important roles in normal bone development and regulation of bone resorption. INTRODUCTION: Bone morphogenetic proteins (BMPs) act on various types of cells. Although involvement of BMP signals in osteoblast differentiation has been studied extensively, the effects of BMPs on osteoclasts have not been widely researched. Consequently, the net effects of BMPs on bone remain unclear. The purpose of this study was to delineate more fully the role of BMPs in skeletal biology. MATERIALS AND METHODS: We generated transgenic mice that express BMP4 or noggin in bone under the control of the 2.3-kb alpha1(I) collagen chain gene (Col1a1) promoter, and analyzed their bone phenotype. We also analyzed bone of transgenic mice expressing BMP4 specifically in cartilage. RESULTS: Mice overexpressing BMP4 in bone developed severe osteopenia with increased osteoclast number. Mice overexpressing noggin, a BMP antagonist, in bone showed increased bone volume associated with decreased bone formation rate and decreased osteoclast number. The noggin-transgenic tibias exhibited reduced periosteal bone formation and reduced resorption of immature bone in marrow spaces, associated with frequent fractures at the diaphysis. Co-culture of primary osteoblasts prepared from noggin-transgenic calvariae and wildtype spleen cells resulted in poor osteoclast formation, which was rescued by addition of recombinant BMP2, suggesting that noggin inhibits osteoclast formation by attenuating BMP activities in noggin-transgenic mice. The expression levels of Rankl were not decreased in primary osteoblasts from noggin transgenic mice. Immunoblot analysis showed increased phosphorylation of Smad1/5/8 in osteoclast precursor cells after 20-minute treatment with BMPs, suggesting that these cells are stimulated by BMPs. Mice overexpressing BMP4 in cartilage had enlarged bones containing thick trabeculae, possibly because of expansion of cartilage anlagen. CONCLUSIONS: Overexpression of noggin in bone revealed that BMP signals regulate bone development through stimulation of osteoblasts and osteoclasts.

Animals↗
PubMed2006

[Effect of BMP signaling in cartilage and bone formation].

To investigate roles of bone morphogenic protein (BMP) signaling in cartilage and bone formation, tissue-specific transgenic and conditional knockout mice have been generated. BMP ligands, antagonists, receptors, Smads, and Smurfs were overexpressed or deleted. Comparison of phenotypes between these mice have elucidated general roles of BMP signals as well as individual effect of each molecule during endochondral bone formation.

Animals↗
PubMed2006

[Bone morphogenetic proteins and their related molecules in the skeletal tissue].

Biochemical studies revealed bone morphogenetic protein (BMP) receptors and intracelluar Smad proteins that mediate BMP signals. These pathways are regulated extracelluarly by antagonists such as noggin and intracellularly by Smads 6 and 7. Transgenic and conditional knockout mice have been generated and analyzed to clarify function of these molecules in skeletal tissues by using bone- or cartilage-specific promoter sequences.

Animals↗
PubMed2005

The role of bone morphogenetic proteins in endochondral bone formation.

Bone morphogenetic proteins (BMPs) were originally identified as proteins capable of inducing endochondral bone formation when implanted at extraskeletal sites. BMPs have diverse biological activities during early embryogenesis and various aspects of organogenesis. BMPs bind to BMP receptors on the cell surface, and these signals are transduced intracellularly by Smad proteins. BMP signal pathways can be inhibited by both extra- and intracellular mechanisms. As for skeletal development, genetic studies suggest that BMPs are skeletal mesoderm inducers. Recent studies of tissue-specific activation and inactivation of BMP signals have revealed that BMP signals control proliferation and differentiation of chondrocytes, differentiation of osteoblasts and bone quality. These findings may contribute not only to understanding of bone biology and pathology, but also to improvement of the clinical efficacy of BMPs.

Animals↗
PubMed2004

Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia.

Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo.

Animals↗
PubMed2004

Low-intensity pulsed ultrasound accelerates maturation of callus in patients treated with opening-wedge high tibial osteotomy by hemicallotasis.

BACKGROUND: Opening-wedge high tibial osteotomy by hemicallotasis for osteoarthritis in the medial compartment of the knee requires external fixation for a long time, until callus maturation is complete. The aim of this study was to determine if low-intensity pulsed ultrasound would accelerate callus maturation when applied after distraction to limbs treated with opening-wedge high tibial osteotomy by hemicallotasis. METHODS: Twenty-one patients with symmetric grades of osteoarthritis and similar degrees of varus deformity in the two knees underwent bilateral one-stage opening-wedge high tibial osteotomy by hemicallotasis. After completion of distraction, the bone mineral density of the distraction callus was measured. Then, one randomly selected limb was subjected to ultrasound treatment for twenty minutes daily until removal of the external fixator. The contralateral limb was left untreated to serve as the control. After four weeks of treatment, bone mineral density was measured again. RESULTS: During the four-week treatment period, the mean increase in callus bone mineral density was significantly greater in the ultrasound-treated tibiae (0.20 +/- 0.12 g/cm(2)) than in the control tibiae (0.13 +/- 0.10 g/cm(2)) (p = 0.02, unpaired t test). In eighteen patients the increase in the bone mineral density was greater in the ultrasound-treated limb than in the control limb, whereas in three patients the increase was greater in the control limb. CONCLUSIONS: We found that low-intensity pulsed ultrasound applied during the consolidation phase of distraction osteogenesis accelerates callus maturation after opening-wedge high tibial osteotomy by hemicallotasis in elderly patients.

Absorptiometry, Photon↗
PubMed2004

[Role of BMPs and Smads during endochondral bone formation].

Biochemical research has revealed that BMP signals are mainly mediated through ligand binding to the receptors followed by activation of Smad proteins. BMP signaling is subjected to regulation at extracellular level by noggin and at intracellular level by inhibitory-Smads. Analysis of transgenic mice in which BMP signals are activated or inactivated has been clarifying a role of BMP signaling during endochondral bone formation. BMPs promote chondrocyte proliferation and hypertrophy, whereas Smad signaling regulates chondrocyte hypertrophy. Proper regulation of these signals is necessary for normal bone formation.

Animals↗
PubMed2003

COL11A2 collagen gene transcription is differentially regulated by EWS/ERG sarcoma fusion protein and wild-type ERG.

A specific t(21;22) chromosomal translocation creates the chimeric EWS/ERG gene in some cases of Ewing's sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part of the ETS family protein ERG is replaced by the N terminus of the RNA-binding protein EWS. We found that both the EWS/ERG and COL11A2 genes are expressed in the Ewing's sarcoma cell line, CADO-ES1. To investigate a potential role for EWS/ERG in COL11A2 gene expression, we characterized the COL11A2 promoter and tested the ability of wild-type ERG and EWS/ERG sarcoma fusion protein to transactivate COL11A2 promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed involvement of EWS/ERG in the formation of DNA-protein complexes, and chromatin immunoprecipitation assay revealed direct interaction between COL11A2 promoter and EWS/ERG fusion protein in vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II. Treatment of cells with the histone deacetylase inhibitor trichostatin A enabled ERG to transactivate the COL11A2 promoter, therefore abolishing the differential effects of EWS/ERG and ERG. Taken together, these findings indicate that the COL11A2 gene is regulated both by potential ERG association with a histone deacetylase complex and by direct EWS/ERG recruitment of RNA polymerase II.

Animals↗
PubMed2002

Activation of cartilage-derived morphogenetic protein-1 in torn rotator cuff.

Localization and expression of cartilage-derived morphogenetic protein-1 in tissues of torn rotator cuff tendons were examined by in situ hybridization and immunohistochemical analysis. Histologic findings of torn rotator cuff tendons showed that active cells synthesizing the alpha-1 chain of collagen Type I messenger ribonucleic acid were localized predominantly in the torn edge and in the bursa side rather than in the joint side, and scarcely localized in a site distant from the torn edge. Cartilage-derived morphogenetic protein-1 had a similar distribution as the alpha-1 chain of collagen Type I. The current findings provide the first observational evidence that cartilage-derived morphogenetic protein-1 was activated specifically at the site of the torn rotator cuff tendon. The current findings suggest that the cells in the torn rotator cuffs are capable of synthesizing cartilage-derived morphogenetic protein-1, one of the known essential factors for tendon formation.

Aged↗
PubMed2002

A Krüppel-associated box-zinc finger protein, NT2, represses cell-type-specific promoter activity of the alpha 2(XI) collagen gene.

Type XI collagen is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the -530-bp promoter segment of the alpha 2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Krüppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRAB-zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.

Amino Acid Sequence↗
PubMed2002

Bone morphogenetic protein signals are required for cartilage formation and differently regulate joint development during skeletogenesis.

The bone morphogenetic protein (BMP) family consists of a large number of members and has diverse biological activities during development. Various tissues express pleural BMP family members, which seem to cooperatively regulate developmental events. Here, multiple BMP signals were inactivated in chondrocytes to clarify the function of BMPs during skeletogenesis. To obtain tissue-specific inactivation, Noggin gene (Nog) was overexpressed in cartilage under the control of a2(XI) collagen gene (Collla2) promoter/enhancer sequences. The resultant transgenic mice lacked most of their cartilaginous components, suggesting that cartilage does not develop without BMP signals. These effects seem to be mediated through down-regulation of Sox9 expression. Conversely, specific BMP signals were activated in the skeleton by targeted expression of Bmp4 in cartilage and the resultant phenotype was compared with that of transgenic mice expressing growth and differentiation factor-5 (GDF-5), another BMP family member. Overactivity of Bmp4 in the skeleton caused an increase of cartilage production and enhanced chondrocyte differentiation, as GDF5 expression did, but it did not disturb joint formation as GDF5 did. During skeletogenesis, unique roles of each BMP may reside in the regulation of joint development. Together with the common effect on the cartilage overproduction by Bmp4 and GDF5 overactivation, loss of cartilage by inactivation of multiple BMPs in Noggin transgenic mice indicates that signals for cartilage production are reinforced by multiple BMPs exclusively. These conclusions may account for the reason why multiple BMPs are coexpressed in cartilage.

Animals↗
PubMedDate not supplied

Differential expression of aggrecan mRNA isoforms by chondrosarcoma cells.

BACKGROUND: Aggrecan, the large aggregating proteoglycan, is a major component of the cartilage extracellular matrix. The C-terminal globular domain (G3) of the aggrecan molecule exhibits a pattern of alternative splicing that involves three exons. Each corresponds to a subdomain, epidermal growth factor 1(EGF1), EGF2, and a short complement repeat (SCR). MATERIALS AND METHODS: In the present study, we compared aggrecan mRNA splicing variants in osteochondrogenic tumor cells and normal cartilage using RT-PCR. RESULTS: Normal cartilage expressed two mRNA isoforms, both containing the SCR exon, but with or without the EGF1 exon. Benign cartilaginous tumors showed the same splicing pattern as normal cartilage. However, malignant chondrosarcomas expressed additional splicing isoforms, one of which lacked all three alternative exons and another with EGF2 and SCR exons. The human chondrosarcoma cell line (Ch-1 cells) expressed further isoforms, one containing either EGF1 or EGF2 exon only, and another with all the variant exons. CONCLUSION: These results imply that a change in the pattern of alternative aggrecan mRNA splicing is associated with malignant transformation of chondrocytes.

Aggrecans↗