研究者詳細 - 久保田　聡

論文 - 久保田　聡

分割表示 229 件中 181 - 200 件目／全件表示 >>

成長因子と軟骨細胞

久保田聡, 滝川正春

腎と骨代謝 16巻:103-110 2003年

CTGF/Hcs24 interacts with the cytoskeletal protein actin in chondrocytes

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年12月

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

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.

Web of Science

researchmap

Tyrosine kinase-type receptor ErbB4 in chondrocytes: interaction with connective tissue growth factor and distribution in cartilage

K Nawachi, M Inoue, S Kubota, T Nishida, G Yosimichi, T Nakanishi, M Kanyama, T Kuboki, H Yatani, T Yamaai, M Takigawa

FEBS LETTERS 528 ( 1-3 ) 109 - 113 2002年9月

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

In order to identify receptor molecules that participate in the growth and differentiation of cliondrocytes, we cloned a number of cDNA fragments from HCS-2/8 chondrocytic cells, by using tyrosine kinase-specific primers for amplification. The mRNA expression of one such receptor, ErbB4, was increased by connective tissue growth factorthypertrophic chondrocyte-specific gene product (CTGF/Hes24), which promotes all stages of the endochondral ossification in vitro. ErbB4 expression was observed through all stages of chondrocytic differentiation in vitro, corresponding to the wide distribution of CTGF/Hcs24 target cells. Furthermore, positive signals for erbB4 mRNA were detectable throughout most populations of chondrocytes, in growth and articular cartilage in vivo. These results demonstrate for the first time that ErbB4 is expressed in chondrocytes and may play some roles in chondrocytic growth and differentiation along with CTGF/Hcs24. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Web of Science

researchmap

CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, stimulates proliferation and differentiation, but not hypertrophy of cultured articular chondrocytes 査読

T Nishida, S Kubota, T Nakanishi, T Kuboki, G Yosimichi, S Kondo, M Takigawa

JOURNAL OF CELLULAR PHYSIOLOGY 192 ( 1 ) 55 - 63 2002年7月

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

We previously reported that connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTCF/Hcs24) stimulated the proliferation and differentiation of rabbit growth cartilage (RGC) cells in vitro. In this study, we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of rabbit articular cartilage (RAC) cells in vitro. RAC cells transduced by recombinant adenoviruses generating mRNA for CTGF/Hcs24 synthesized more proteoglycan than the control cells. Also, treatment of RAC cells with recombinant CTGF/Hcs24 (rCTGF/Hcs24) increased DNA and proteoglycan syntheses in a dose-dependent manner. Northern blot analysis revealed that the rCTGF/Hcs24 stimulated the gene expression of type II collagen and aggrecan core protein, which are markers of chondrocyte maturation, in both RGC and RAC cells. However, the gene expression of type X collagen, a marker of hypertrophic chondrocytes, was stimulated by rCTGF/Hcs24 only in RGC cells, but not in RAC cells. Oppositely, gene expression of tenascin-C, a marker of articular chondrocytes, was stimulated by rCTGF/Hcs24 in RAC cells, but not in RGC cells. Moreover, rCTGF/Hcs24 effectively increased both alkaline phosphatase (ALPase) activity and matrix calcification of RGC cells, but not of RAC cells. These results indicate that CTGF/Hcs24 promotes the proliferation and differentiation of articular chondrocytes, but does not promote their hypertrophy or calcification. Taken together, the data show that CTCF/Hcs24 is a direct growth and differentiation factor for articular cartilage, and suggest that it may be useful for the repair of articular cartilage. (C) 2002 Wiley-Liss, Inc.

DOI： 10.1002/jcp.10113

Web of Science

researchmap

A novel cis-element that enhances connective tissue growth factor gene expression in chondrocytic cells

T Eguchi, S Kubota, S Kondo, T Kuboki, H Yatani, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 295 ( 2 ) 445 - 451 2002年7月

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

To clarify the chondrocyte-specific regulatory mechanism of connective tissue growth factor (ctgf) gene expression, we analyzed the functionality and DNA-protein interaction of the CTGF promoter. Comparative luciferase assay of the CTGF promoter deletion mutants among HCS-2/8 chondrocytic cells and fibroblastic cells revealed that a 110-bp region in the promoter was crucial for the HCS-2/8-specific transcriptional enhancement. Subsequent competitive gel shift assay revealed that transcription factors in HCS-2/8 nuclei bound to a 60-bp portion in the corresponding region. Relative luciferase activity from a CTGF promoter with mutant TGF-beta response element (TbRE) was 16.9% lower than that from an intact promoter. On the other hand, relative luciferase activity from a CTGF promoter with 4 bp point mutations at 30 bp upstream of the TbRE was 47.7% lower than that from the intact one. The binding activity of HCS-2/8 nuclear factor(s) to the sequence over the 4-bp was remarkably higher than that of any nuclear extract from other types of cells. Therefore, we entitled the sequence 'TRENDIC', a transcription enhancer dominant in chondrocytes, which stands for a novel enhancer for chondrocyte-specific CTGF gene expression. (C) 2002 Elsevier Science (USA). All rights reserved.

Web of Science

researchmap

A trans-dominant negative HIV type 1 Rev with intact domains of NLS/NOS and NES

JH Fang, S Kubota, RJ Pomerantz

AIDS RESEARCH AND HUMAN RETROVIRUSES 18 ( 10 ) 705 - 709 2002年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MARY ANN LIEBERT INC PUBL

The nuclear diffusion inhibitory signal (NIS) is a 15-amino acid peptide motif (10-24; EDLLKAVRLIKFLYQ) in the N-terminus of the HIV-1 Rev protein. NIS appears to be involved in maintaining the proper nucleocytoplasmic trafficking and intracellular stability of HIV-1 Rev. Deletion in NIS leads to Rev functional inactivity, and these data led to further investigation of its possible inhibitory effects on Rev function. An HIV-1 proviral rescue assay was utilized to evaluate Rev function. The association between wild-type Rev molecules, or wild-type Rev with Revd23, an NIS mutant, plus Rev-responsive element (RRE) interactions in cultured cells were also evaluated. Revd23 showed a potent trans-dominant negative phenotype, while multimerization with wild-type Rev and Revd23-RRE binding in cells were found to reveal no significant changes from wildtype. These results suggest that the potential trans-dominance mechanism of Revd23 may differ from that of a Rev construct, RevM10, with mutations in the C-terminus nuclear export signal (NES). As such, these data will be useful in the rational design of novel antiretroviral approaches targeting HIV-1 Rev.

Web of Science

researchmap

Connective tissue growth factor increased by hypoxia may initiate angiogenesis in collaboration with matrix metalloproteinases

S Kondo, S Kubota, T Shimo, T Nishida, G Yosimichi, T Eguchi, T Sugahara, M Takigawa

CARCINOGENESIS 23 ( 5 ) 769 - 776 2002年5月

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

Connective tissue growth factor (CTGF) is known to be a potent angiogenic factor. Here we investigated how CTGF and matrix metalloproteinases (MMPs) are involved in the early stage of hypoxia-induced angiogenesis using human breast cancer cell line, MDA231, and vascular endothelial cells. Hypoxic stimulation (5% O-2) of MDA231 cells increased their steady-state level of ctgf mRNA by similar to2-fold within 1.5 h, and the levels remained at a plateau up to 6 h, and then decreased by 12 h as compared with the cells cultured under the normoxic condition. Membrane-type 1 MMP (MT1-MMP) mRNA levels was also increased within a few hours of the exposure to hypoxia. Indeed, ELISA revealed that the CTGF protein/cell in medium conditioned by MDA231 cells exposed to hypoxia was maximally greater at 24 h than in the medium from normoxic cultures and that the secretion rate (supernatant CTGF/cell layer CTGF) increased in a time-dependent manner from 24 to 72 h of hypoxic exposure. Hypoxic induction of CTGF was also confirmed by immunohistochemical analyses. Furthermore, zymogram analysis revealed that the production of active MMP-9 was also induced in MDA231 cells incubated under hypoxic conditions. Finally, we found that recombinant CTGF also increased the expression of a number of metalloproteinases that play a role in the vascular invasive processes and decreased the expression of tissue inhibitors of metalloproteinases by vascular endothelial cells. These findings suggest that hypoxia stimulates MDA231 cells to release CTGF as an angiogenic modulator, which initiates the invasive angiogenesis cascade by modulating the balance of extracellular matrix synthesis and degradation via MMPs secreted by endothelial cells in response to CTGF. This cascade may play critical roles in the hypoxia-induced neovascularization that accompanies tumor invasion in vivo.

Web of Science

researchmap

CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, stimulates the proliferation and differentiation but not hypertrophy of cultured articular cartilage cells.

Nishida, T, Kubota, S, Nakanishi, T, Kuboki, T, Yosimichi, G, Kondo S, Takigawa, M

J. Cell. Physiol. 2002年

The role of redox signaling in the processing of Gag polyprotein in immature retrovirus 査読

S Kubota, S Oroszlan

XI BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL 193 - 197 2002年

記述言語：英語掲載種別：研究論文（国際会議プロシーディングス）出版者・発行元：MEDIMOND S R L

Incubation of radiolabeled immature Moloney murine leukemia virus (Mo-MLV) in buffers containing the reducing agent dithiothreitol (DTT) or adsorption to NIH 3T3 cells resulted in similar time-dependent cleavage of the Pr65 Gag poly-protein by the endogenous viral protease (PR). The combined results suggest that cell membrane-associated thioreductases may initiate and accelerate retroviral polyprotein processing and that redox regulation may be involved in virus maturation, which is essential for replication.

Web of Science

researchmap

CTGF/Hcs24, a hypertrophic chondrocyte specific gene product, stimulates the proliferation and expression of the cartilage phenotype but not hypertrophy or calcification or articular cartilage in culture.

Nishida T, Kubota S, Nakanishi T, Kuboki T, Yosimichi G, Kondo S, Takigawa M

J Cell Physiol 192, 55 - 63 2002年

Connective tissue growth factor as a major angiogenic agent that is induced by hypoxia in a human breast cancer cell line

T Shimo, S Kubota, S Kondo, T Nakanishi, A Sasaki, H Mese, T Matsumura, M Takigawa

CANCER LETTERS 174 ( 1 ) 57 - 64 2001年12月

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

Connective tissue growth factor (CTGF) is known to be a potent angiogenic factor. Here, we present the evidence that the hypoxic induction of angiogenesis by human breast cancer cells (MDA-231) can be ascribed at least in part to CTGF, Our results indicate that (i) CTGF is abundantly present in MDA-231 cells in vitro and in vivo, (ii) its secretion is up-regulated by hypoxia, and (iii) its gene expression is enhanced in MDA-231 cells cultured under hypoxic conditions. These data suggest CTGF may stimulate angiogenesis by paracrine mechanisms, thereby contributing to the invasion of breast cancer cells. This is the first evidence that human cancer cells differentially express CTGF protein and mRNA under the control of hypoxic conditions. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Web of Science

researchmap

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

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

DNA AND CELL BIOLOGY 20 ( 9 ) 563 - 568 2001年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MARY ANN LIEBERT INC PUBL

The human T-cell leukemia virus type I Tax protein (HTLV-I Tax) is known as a trans-activating factor for a variety of genes, including those of cytokines. Here, we show that Tax is capable of activating the herpes simplex virus thymidine kinase (HSV-TK) promoter in certain mammalian cell lines. In murine NIH 3T3 fibroblasts and human HeLa cells, trans-activation by Tax was remarkably strong, whereas in human chondrocytic HCS-2/8 and monkey kidney Cos-7 cells, the responsiveness of the TK promoter to Tax was poor. Deletion analysis revealed that one of the two previously described Sp1 sites is required for the Tax responsiveness, whereas the CTF binding site is not. The results suggest possible interactions between the oncogenic Tax protein and the viral TK in coinfected cells in vivo. Care should be taken in the context of HTLV-I research, as the HSV-TK promoter has been widely used in molecular biology and gene therapeutics.

Web of Science

researchmap

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

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

BONE 29 ( 2 ) 155 - 161 2001年8月

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

The synthesis, processing, and secretion of human connective tissue growth factor (CTGF/Hcs24) in a human chondrocytic cell line, HCS-2/8, were analyzed immunochemically. By metabolic pulse-labeling, chasing, and subsequent immunoprecipitation analyses, active synthesis of CTGF was observed not only in growing HCS-2/8 cells, but also in confluent cells. However, secretion and processing of CTGF were found to be regulated differentially, depending upon the growth status. During phases of growth, HCS-2/8 cells released CTGF molecules immediately without sequestering them within the cell layer. In contrast, after the cells reached confluence, the secretion slowed, resulting in an accumulation of CTGF in the cells or extracellular matrices (ECMs). Also, in confluent cell layers, a 10 kDa protein that was reactive to an anti-CTGF serum was observed. This CTGF-related small protein was not detected immediately after labeling, but gradually appeared within 6 h after chase, which suggests its entity as a processed subfragment of CTGF. Surprisingly, the 10 kDa protein was stable even 48 h after synthesis, and was not released by ECM digestion, suggesting an intracellular maintenance and function. Taken together, the behavior of CTGF in HCS-2/8 cells is remarkably different from that reported in fibroblasts, which may represent unique roles for CTGF in the growth and differentiation of chondrocytes. (C) 2001 by Elsevier Science Inc. All rights reserved.

Web of Science

researchmap

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

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

JOURNAL OF BIOCHEMISTRY 130 ( 1 ) 79 - 87 2001年7月

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

CTGF/Hcs24 is a multi-functional growth factor that potentiates either the growth or differentiation of mesenchymal cells, according to the biological conditions. Among various functional aspects of CTGF/Hcs24, it is especially notable that CTGF/Hcs24 may promote endochondral ossification in growth cartilage through all stages, and it is highly expressed in a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8). In this study, to clarify the regulatory mechanism of CTGF/Hcs24 gene expression in chondrocytes, we analyzed the transcriptional activity of the CTGF/Hcs24 promoter and the effect of the CTGF/Hcs24 S'-untranslated region (3'-UTR) on gene expression in HCS-2/8 by means of an established DNA transfection and luciferase reporter gene assay system. As a result, the luciferase activity of the CTGF/Hcs24 promoter was found to be remarkably high in HCS-2/8. The 3'-UTR of the CTGF/Hcs24 gene strongly repressed the luciferase activity in HCS-2/8, when it was linked to the downstream of the luciferase reporter gene, suggesting its functionality also in chondrocytic cells. Deletion analysis of the CTGF/Hcs24 promoter clarified a major segment responsible for the enhanced CTGF/Hcs24 promoter activity in HCS-2/8, The TGF-beta response element in the DNA segment was active in HCS-2/8, and point mutations in the element moderately decreased the highly maintained promoter activity with total loss of TGF-beta responsiveness. These results indicate that the strong expression of the CTGF/Hcs24 gene in HCS-2/8 was mainly caused by high transcriptional activity of the CTGF/Hcs24 promoter, and that the TGF-beta response element is one of the critical elements that support the high transcription activity.

Web of Science

researchmap

The extinction program for Homo sapiens and cloning humans: trinucleotide expansion as a one-way track to extinction

S Kubota

MEDICAL HYPOTHESES 56 ( 3 ) 296 - 301 2001年3月

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

It has been uncovered that expansion of trinucleotide repeats in the human genome are causing a variety of genetic diseases. They are also identified in other loci that are not clearly related to particular diseases, which indicates such repeat expansion is one of the general forms of evolution occurring throughout the human genome. Trinucleotide repeat expansion has been shown to occur during meiosis, and is generally irreversible. In consequence, every human chromosome suffers from the burden of accumulating trinucleotide repeats along with the passage of generation, which eventually ends up in a deficiency of replication. Thus, all the human chromosomes are predicted to be 'mortal', so far as they replicate through meiosis. Naturally, the mortality of human chromosomes leads to the mortality of Home sapiens as a species on earth. Although it is quite controversial, a radical breakthrough for humans to survive might be found through cloning reproduction without meiosis. (C) 2001 Harcourt Publishers Ltd.

DOI： 10.1054/mehy.2000.1140

Web of Science

researchmap

Change in cellular localization of a rheumatoid arthritis-related antigen (RA-A47) with downregulation upon stimulation by inflammatory cytokines in chondrocytes: Implications for a recognition mechanism of RA-A47 as autoantigen during rheumatoid arthr・・・

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

Journal of Cellular Physiology 186 ( 2 ) 268 - 281 2001年2月

軟骨細胞とMMPs-生理学的役割を中心に

久保田聡, 滝川正春

The Bone 15巻:39-43 2001年

Characterization of a mouse ctgf 3ﾕ-UTR segment that mediates repressive regulation of gene expression.

Kondo, S, S. Kubota, T. Eguchi, T. Hattori, T. Nakanishi, T. Sugahara, M. Takigawa

Biochemical and Biophysical Research Communications 278 ( 1 ) 119 - 124 2000年11月

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

S Kubota, S Kondo, T Eguchi, T Hattori, T Nakanishi, RJ Pomerantz, M Takigawa

ONCOGENE 19 ( 41 ) 4773 - 4786 2000年9月

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

The repressive effect of the 3'-untranslated region (3'-UTR) in human connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/hcs24) mRNA on gene expression had been demonstrated in our previous study. Here, we identified a minimal RNA element in the 3'-UTR, which acts as a cis-acting element of structure-anchored repression (CAESAR). Deletion analyses of the 3'-UTR led us to minimize the element of 84 bases at the junction of the coding region and the 3'-UTR. The minimized RNA segment is predicted, and actually capable of forming a stable secondary structure in vitro. Mutational analyses disclosed a significant relationship between the predicted structure and repressive effect. The utility of CAESAR as a post-transcriptional regulatory element was represented by the fact that steady-state mRNA levels were not affected by CAESAR linked in cis, while protein levels from such a chimeric gene were markedly reduced. Of note, the CAESAR sequence exerted no effect, when it was placed upstream of the promoter. Finally, RNA gel electromobility-shift analyses demonstrated a nuclear factor that interacts with the folded CAESAR. Taken together, it was uncovered that CAESAR of ctgf is a novel post-transcriptional structured RNA regulatory element, probably acting through direct interactions with a nuclear factor as observed in retroviral RNA elements with certain proteins.

Web of Science

researchmap

The nuclear function of the nuclear diffusion inhibitory signal of human immunodeficiency virus type 1 - Critical roles in dominant nuclear localization and intracellular stability

S Kubota, RJ Pomerantz

JOURNAL OF HUMAN VIROLOGY 3 ( 4 ) 173 - 181 2000年7月

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

Objectives: Human immunodeficiency virus type 1 (HIV-1) Rev is a nucleocytoplasmic shuttling protein with dominant localization in the cell nucleus/nucleolus. To clarify the mechanism(s) that enables such a biased subcellular localization under the co-presence of nuclear/nucleolar targeting (NOS) and nuclear export signals (NES), the properties of another functional domain, a nuclear diffusion inhibitory signal (NIS), was investigated,
Study Design: The NIS was previously shown to interfere with passive nuclear entry of small proteins. Intracellular distribution and degradation profiles of Rev mutants chat harbor mutations in the NIS were analyzed biochemically and cell-biologically.
Results: A NIS-deficient Rev mutant, which was no longer functional as Rev, displayed a significantly more rapid degradation profile as a consequence of its dramatic leakage into the cytoplasm. Additionally, disabling the NOS/nuclear localization signal (NLS), as well as the NIS, resulted in further rapid degradation, which also supports the hypothetical role of the nucleolus as a Rev storage site.
Conclusions: It was uncovered that the NIS is playing a key role in HIV-1 Rev function by maintaining the nuclear-dominant localization and the intracellular stability of Rev.

Web of Science

researchmap

　前のページ - 次のページ　

<久保田 聡>

論文 - 久保田 聡

<久保田　聡>

論文 - 久保田　聡