Faculty Profiles - TAKIGAWA Masaharu

Papers - TAKIGAWA Masaharu

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Mitogenic effects of neurotrophins on a periodontal ligament cell line Reviewed

Y Tsuboi, T Nakanishi, T Takano-Yamamoto, M Miyamoto, T Yamashiro, M Takigawa

JOURNAL OF DENTAL RESEARCH 80 ( 3 ) 881 - 886 2001.3

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC DENTAL RESEARCH

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are three representative neurotrophins responsible for the differentiation and survival of neurons, and their high-affinity receptors are tropomyosin-receptor-kinase (TRK)A, TRKB, and TRKC, respectively. In this study, we investigated the expression of neurotrophins in a mouse periodontal ligament cell line (MPL), by reverse transcription-polymerase chain-reaction (RT-PCR) and enzyme-linked immunoabsorbent assay (ELISA). We also studied the expression of TRK receptors on MPL by immunostaining and the effects of neurotrophins on the proliferation of MPL, with a hypothesis of autocrine mechanism of neurotrophins. Each neurotrophin and TRK receptor was expressed, and neurotrophins enhanced the proliferation of MPL. These findings suggest that the MPL has functional neurotrophin receptors involved in an autocrine function of neurotrophins. The expression level of neurotrophins and TRKs showed the reverse pattern, and we propose an auto-regulatory mechanism of ligands and receptors in accordance with the level of synthesized neurotrophins.

DOI： 10.1177/00220345010800030701

Other Link： http://orcid.org/0000-0002-4419-9643

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

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

JOURNAL OF CELLULAR PHYSIOLOGY 186 ( 2 ) 268 - 281 2001.2

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

We previously isolated a rheumatoid arthritis-related antigen (RA-A47) protein that had reactivity with RA sera from a human chondrosarcoma-derived chondrocytic cell Line, HCS-2/8. Sequencing analysis of ra-a47 cDNA revealed RA-A47 as a product of the colligin-2 gene, which is also known as the human heat shock protein (HSP) 47 gene. Expression of hsp47 has been shown to be cooperatively altered with that of collagen genes upon stimulation. in this study, it was confirmed that the mRNA expression of ra-a47 and COL2A1, a type II collagen gene, was upregulated on stimulation with transforming growth factor (TGF) beta in chondrocytes. However, in contrast, inflammatory cytokines such as tumor necrosis factor (TNF) alpha, interferon (IFN) beta, and interleukin (IL)-6 downregulated the expression of ra-a47 mRNA, whereas the expression of COL2A1 mRNA was not repressed, or even upregulated, in HCS-2/8 cells. Of note, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP)-9 mRNAs were strongly stimulated by TNF alpha. We also found that cell-surface type II collagen disappeared upon such a stimulation, suggesting that decrement of RA-A47 may inhibit the secretion of type II collagen and lead to its accumulation inside the cells. RA-A47 was detected in the cultured medium of TNF alpha -treated HCS-2/8 cells and of IL-1-treated rabbit chondrocytes by Western blot analysis. Under the same conditions, RA-A47 was detected on the cell surface by immunofluorescence staining. These findings demonstrate that the RA-A47 chaperone protein is specifically downregulated, causing the intracellular accumulation of unsecretable type II collagen, while the extracellular matrix (ECM) is degraded by MM Ps and iNOS th rough the stimulation of chondrocytes by TNF alpha. The altered localization of RA-A47 to the surface or outside of cells may represent the mechanism for the recognition of RA-A47 as an autoantigen during rheumatoid arthritis. J. Cell. Physiol. 186:268-281, 2001. (C) 2001 Wiley-Liss, Inc.

Expression of transduced HSP70 gene protects chondrocytes from stress

T Kubo, Y Arai, K Takahashi, T Ikeda, S Ohashi, Kitajima, I, O Mazda, M Takigawa, J Imanishi, Y Hirasawa

JOURNAL OF RHEUMATOLOGY 28 ( 2 ) 330 - 335 2001.2

Language：English Publishing type：Research paper (scientific journal) Publisher：J RHEUMATOL PUBL CO

Objective. To investigate thr efficacy of adenovirus vector mediated transduction of heat shock protein 70 (HSP70) gene to human chondrocyte-like cell (HCS-2/8) against heat stress.
Methods. Two adenovirus vectors that contain wild-type (AxSHEwt) or mutant-type (AxSHEmt) HSP70 gene, and that are regulated by SR alpha promoter, were constructed. The mutant-type lacks the area that expresses stress durability. One of the 2 adenovirus vectors was added to the cultures of human chondrocyte-like cells (HCS-2/8). Heat stress (48 degreesC) was applied to the transduced cells for 2 h. and the efficacy of adenovirus vector mediated transduction of HSP70 gene against heat stress in the chondrocytes was investigated using alamar blue assay and MTT assay.
Results. Absorbance levels at 48 degreesC were 300.3 +/- 51.9 and 1.173 +/- 0.011 in the controls, 278.5 +/- 33.8 and 1.217 +/- 0.018 in the AxSHEmt transduced cells, and 349 +/- 14.7 and 1.371 +/- 0.033 in the AxSHEwt transduced cells. The level in the AxSHEwt transduced cells was significantly higher than in the other 2 groups (p < 0.05). With 37<degrees>C treatment, no significant difference was observed.
Conclusion. Chondrocytes to which HSP70 gene was transduced had a significantly higher metabolic activity and viability under heat stress.

Mechanical stimulation induces CTGF expression in rat osteocytes

T Yamashiro, T Fukunaga, N Kobashi, H Kamioka, T Nakanishi, M Takigawa, T Takano-Yamamoto

JOURNAL OF DENTAL RESEARCH 80 ( 2 ) 461 - 465 2001.2

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC DENTAL RESEARCH

Connective tissue growth factor (CTGF), which is encoded by an immediate early gene and a member of the CCN family, has been shown to be expressed in osteoblasts, fibroblasts, and chondrocytes. Although CTGF is expressed in bone and cartilage tissues, we tested the hypothesis that CTGF is regulated in mechanotransduction. In the alveolar bone during experimental tooth movement, CTGF mRNA was expressed in osteoblasts and in osteocytes localized around the periodontal ligament under control conditions. Interestingly, 12 hrs after the start of experimental tooth movement, the expression of CTGF mRNA in osteocytes and osteoblasts became more intense around the periodontal ligament, and the intense expression of CTGF extended to osteocytes situated deep in alveolar bone matrix apart from periodontal ligament in both tension and compression sides. Our present findings indicate that CTGF could play a role in regulation of osteocyte function during the mechanical stimulation of bone.

Recognition of YKL-39, a human cartilage related protein, as a target antigen in patients with rheumatoid arthritis

T Sekine, K Masuko-Hongo, T Matsui, H Asahara, M Takigawa, K Nishioka, T Kato

ANNALS OF THE RHEUMATIC DISEASES 60 ( 1 ) 49 - 54 2001.1

Language：English Publishing type：Research paper (scientific journal) Publisher：BRITISH MED JOURNAL PUBL GROUP

Objective-To investigate whether autoimmunity to YKL-39, a recently cloned cartilage protein, occurs in patients with rheumatoid arthritis (RA).
Methods-Autoantibody to YKL-39 was assayed by enzyme Linked immunosorbent assay (ELISA) and western blotting in serum samples from patients with RA, systemic lupus erythematosus (SLE), and healthy donors, using recombinant YKL-39 protein. This reactivity was compared with that against a YKL-39 homologue, YKL-40 (human cartilage gp-39/ chondrex), which has been reported to be an autoantigen in RA.
Results-Autoantibody to YKL-39 was detected in seven of 87 patients with RA (8%), but not in serum samples from patients with SLE or healthy donors. YKL-40 reactivity was found in only one of 87 RA serum samples (1%), with no cross reactivity to YKL-39.
Conclusion-The existence of anti-YKL-39 antibody in a subset of patients with RA is reported here for the first time. Further, it was shown that the immune response to YKL-39 was independent of that to YKL-40. Clarification of the antibody and T cell responses to autoantigens derived from chondrocyte, cartilage, or other joint components may lead to a better understanding of the pathophysiology of joint destruction in patients with RA.

Cationic polymer-mediated genetic transduction into cultured human chondrosarcoma-derived HCS-2/8 cells

Suzuyo Ohashi, Toshikazu Kubo, Takumi Ikeda, Yuji Arai, Kenji Takahashi, Yasusuke Hirasawa, Masaharu Takigawa, Etsuko Satoh, Jiro Imanishi, Osam Mazda

Journal of Orthopaedic Science 6 ( 1 ) 75 - 81 2001

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

The usefulness of three types of cationic polymer, i.e., degraded polyamidoamine (PAMAM) dendrimer (SuperFect Transfection Reagent
Qiagen), linear polyethylenimine (PEI: ExGen 500: Euromedex), and branched PEI in gene delivery into chondrocytes was examined comparatively. A plasmid vector containing the Escherichia coli LacZ (pSES.β) was combined with one of the three cationic polymers at various molar ratios and the resultant complex (polyplex) was used to transduce a human chondrocyte-like cell line, HCS-2/8. Gene expression was evaluated by an O-nitrophenyl β-D-galactopyranoside (ONPG) assay and by staining with 0.05% 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal: Nacalai Tesque). The ONPG assay showed that the highest delivery rate was achieved when 2μg of pSES.β was combined with either 21 μg of dendrimer, 1.7 μg of linear PEI, or 2.0 μg of branched PEI. At the same DNA/polymer ratios, the proportions of X-galstained cells were also the highest (31.3 ± 7.5%, 30.3 ± 9.0%, and 8.3 ± 3.1%, respectively). LacZ expression reached the highest level 3 days after the dendrimer-mediated transduction, and gradually declined, returning to the background level on day 14. Possible cytotoxicity was examined by trypan blue staining and phase contrast microscopic observations. Neither cytotoxicity nor morphological change was induced at the optimal dose of each polymer. The cationic polymers, particularly the degraded dendrimer and linear PEI, would be a useful nonviral vector for gene delivery to cells of chondrocytes.

DOI： 10.1007/s007760170028

軟骨細胞とMMPs-生理的役割を中心に Invited

久保田聡, 滝川正春

The Bone 5, 39-43 2001

Involvement of CTGF, a hypertrophic chondrocyte-specific gene product, in tumor angiogenesis

T Shimo, T Nakanishi, T Nishida, M Asano, A Sasaki, M Kanyama, T Kuboki, T Matsumura, M Takigawa

ONCOLOGY 61 ( 4 ) 315 - 322 2001

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

Connective tissue growth factor (CTGF) is a potent secreted signaling factor which functions in multiple stages of angiogenesis. In the present study, we examined the role of CTGF in tumor angiogenesis and made the following observations: (1) Histological analysis of human breast cancer (MDA231) cell and human fibrosarcoma (HT1080) cell xenografts in BALB/c nude mice showed a high level of neovascularization. Human squamous cell carcinoma (A431) xenografts induced only a low level of neovascularization. (2) CTGF mRNA was strongly expressed in MDA231 and in HT1080 cells in vivo and in vitro, but not in A431 cells. (3) CTGF protein was markedly produced in MDA231 cells and HT1080 cells and secreted into culture medium, and its production was greater during phases of growth rather than confluency. (4) Production of CTGF in bovine aorta endothelial cells was induced by CTGF, VEGF, bFGF and TGF-beta. (5) Neovascularization induced by HT1080 cells or MDA231 cells on chicken chorioallantoic membrane was suppressed in the presence of neutralizing CTGF-specific polyclonal antibody. These results suggest that CTGF regulates progression in tumor angiogenesis and the release or secretion of CTGF from tumor cells is essential for the angiogenesis. Copyright (C) 2001 S. Karger AG, Basel.

CTGF/Hcs24 induces chondrocyte differentiation through p44/42 MAPK/exracellular-signal regulated kinase (ERK).

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

Eur. J. Biochem. 268, 1 - 9, 2001

Cell density-dependent proliferative effects of transforming growth factor (TGF)-beta 1, beta 2, and beta 3 in human chondrosarcoma cells HCS-2/8 are associated with changes in the expression of TGF-beta receptor type I

K Boumediene, M Takigawa, JP Pujol

CANCER INVESTIGATION 19 ( 5 ) 475 - 486 2001

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

In this study, the growth properties of the human chondrosarcoma cell line HCS-2/8, its response to transforming growth factor (TGF)-beta isoforms 1, 2, and 3, and its expression of TGF-beta receptors I and II were examined. We demonstrated that these tumor cells are not contact-inhibited and that they can proliferate in the absence of additional serum growth factors. In sparse cultures, all TGF-beta forms inhibited the growth of HCS-2/8 cells, whereas they induced a 2-fold increase of DNA synthesis in serum-fed confluent cultures. In serum-free confluent conditions only TGF-beta1 stimulated the proliferation rate, whereas TGF-beta2 was without effect and TGF-beta3 was rather inhibitory. The bimodal effect of TGF-beta forms was associated with a greater level of TGF-beta receptor I mRNA in confluent HCS-2/8 than in sparse cultures suggesting that the growth response to TGF-beta forms is dependent on the receptor profile expressed.

Mitogenetic effect of neurotrophins on periodonatal ligament cell line.

Tsuboi, Y, Nakanishi, T, Takano-Yamamoto, T, Miyamoto, M, Yamashiro, T, Takigawa, M

J. Dent. Res. 80(3), 881 - 886, 2001

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

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

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 278 ( 1 ) 119 - 124 2000.11

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

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.

Expression of connective tissue growth factor in cartilaginous tumors

T Shakunaga, T Ozaki, N Ohara, K Asaumi, T Doi, K Nishida, A Kawai, T Nakanishi, M Takigawa, H Inoue

CANCER 89 ( 7 ) 1466 - 1473 2000.10

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

BACKGROUND. Connective tissue growth factor (CTGF) predominantly is expressed in hypertrophic chondrocytes and its specific receptors are demonstrated on chondrocytic cells. Therefore, CTGF may be involved in the proliferation and/or differentiation of cartilage cells. In the current study, CTGF expression was examined both in chondrosarcoma and enchondroma to clarify the relation between the expression of CTGF and the grade of malignancy.
METHODS. The expression of CTGF and proliferating cell nuclear antigen (PCNA] were analyzed immunohistochemically in 34 cartilaginous tumor specimens. Eighteen tumors were determined to be chondrosarcoma including 8 Grade 1 tumors, 6 Grade 2 tumors, and 4 Grade 3 tumors. The percentage of CTGF positive and PCNA positive cells was quantified using at least 500 cells.
RESULTS, CTGF was expressed in 70.1% of enchondroma cells, 84.0% of Grade 1 chondrosarcoma cells, 53.7% of Grade 2 tumor cells, and 26.8% of Grade 3 tumor cells (rho = -0.501; P = 0.0053). In chondrosarcoma cases, CTGF expression was correlated closely with tumor grade (rho = -0.920; P = 0.0001). There was a strong correlation between PCNA expression and tumor grade (rho = 0.907; P < 0.0001) and a strong negative correlation between CTGF and PCNA expression (rho = -0.493; P = 0.0061]. In chondrosarcoma cases, patients with high expression of CTGF (greater than or equal to 30%) showed higher overall survival compared with those with low expression (< 30%) (P = 0.004).
CONCLUSIONS. The current study revealed a correlation between the histologic grade of chondrosarcoma and prognosis, and the concomitant association between CTGF immunostaining and tumor grade and prognosis. Therefore, immunohistochemical staining with CTGF is a useful procedure for assessing the tumor grade and clinical course in patients with chondrosarcoma. Cancer 2000;89: 1466-73. (C) 2000 American Cancer Society.

Regulation of human COL2A1 gene expression in chondrocytes - Identification of C-Krox-responsive elements and modulation by phenotype alteration

C Ghayor, JF Herrouin, C Chadjichristos, L Ala-Kokko, M Takigawa, JP Pujol, P Galera

JOURNAL OF BIOLOGICAL CHEMISTRY 275 ( 35 ) 27421 - 27438 2000.9

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

To identify control motifs involved in human type II collagen gene transcription in both differentiated and dedifferentiated rabbit articular chondrocytes, transient transfection experiments were performed, A 715-base pair (bp) region of the first intron (+2127/+2842), including a 153-bp sequence so far uncharacterized (+2689/+2842), was found to mediate enhancer activity. In dedifferentiated chondrocytes, this enhancer activity was shown to be less effective than in primary cultures but still present. We then demonstrated that a zinc finger protein, C-Krox, activates COL2A1 gene transcription in differentiated chondrocytes through the enhancer region, whereas in subcultured cells, it inhibited the gene activity via a 266-bp promoter. Multicopies of the C-Krox binding site were found to mediate transactivation in both primary cultures and passaged cells, whereas C-Krox overexpression inhibited transcription in dedifferentiated chondrocytes. Additionally, we showed that C-Krox binds to several cis sequences that mediate its transcriptional effects. During chondrocyte dedifferentiation, the protein levels and binding activity of C-Krox were reduced, whereas those of NF-KB were increased. This was not associated with variations of mRNA levels, suggesting that post-transcriptional regulatory mechanisms could be involved in C-Krox expression. These results suggest that C-Krox plays a major role in type II collagen expression and the chondrocyte phenotype modulation.

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 2000.9

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

Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro

T Nishida, T Nakanishi, M Asano, T Shimo, M Takigawa

JOURNAL OF CELLULAR PHYSIOLOGY 184 ( 2 ) 197 - 206 2000.8

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

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product Hcs24 (CTGF/Hcs24) promotes the proliferation and differentiation of chondrocytes and endothelial cells which are involved in endochondral ossification (Shimo et al., 1998, J Biochem 124:130-140; Shimo el al., 1999, J Biochem 126:137-145; Nakanishi et at., 2000, Endocrinology 141:264-273). To further clarify the role of CTGF/Hcs24 in endochondral ossification, here we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of osteoblastic cell lines in vitro. A binding study using I-125-labeled recombinant CTGF/Hcs24 (rCTGF/Hcs24) disclosed two classes of specific binding sites on a human osteosarcoma cell line, Saos-2. The apparent dissociation constant (Kd) value of each binding sire was 17.2 and 391 nM, respectively. A cross-linking study revealed the formation of I-125-rCTGF/Hcs24-receptor complex with an apparent molecular weight of 280 kDa. The intensity of I-125-rCTGF/Hcs24-receptor complex decreased on the addition of increasing concentrations of unlabeled rCTGF/Hcs24, but not platelet-derived growth factor-BB homodimer or basic fibroblast growth factor. These findings suggest that osteoblastic cells have specific receptor molecules for CTGF/Hcs24. rCTGF/Hcs24 promoted the proliferation of Saos-2 cells and a mouse osteoblast cell line MC3T3-E1 in a dose- and time-dependent manner. rCTGF/Hcs24 also increased mRNA expression of type I collagen, alkaline phosphatase, osteopontin, and osteocalcin in both Saos-2 cells and MC3T3-E1 cells. Moreover, rCTGF/Hcs24 increased alkaline phosphatase activity in both cells, it also stimulated collagen synthesis in MC3T3-E1 cells. Furthermore, rCTGF/Hcs24 stimulated the matrix mineralization on MC3T3-E1 cells and its stimulatory effect was comparable to that of bone morphogenetic protein-2. These findings indicate that CTGF/Hcs24 is a novel, potent stimulator for the proliferation and differentiation of osteoblasts in addition to chondrocytes and endothelial cells. Because of these functions, we are re-defining CTGF/Hcs24 as a major factor to promote endochondral ossification to be called "ecogenin: endochondral ossification genetic factor." J. Cell. Physiol. 184:197-206, 2000. (C) 2000 Wiley-Liss, Inc.

軟骨由来の成長因子CTGF/Hcs24遺伝子の転写後制御エレメント,CAESARの構造機能連関

久保田聡, 近藤誠二, 江口傑徳, 服部高子, 中西徹, 滝川正春

生化学 72 ( 8 ) 972 - 972 2000.8

Identity of urinary trypsin inhibitor-binding protein to link protein

H Kobayashi, Y Hirashima, GW Sun, M Fujie, T Nishida, M Takigawa, T Terao

JOURNAL OF BIOLOGICAL CHEMISTRY 275 ( 28 ) 21185 - 21191 2000.7

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, directly binds to some types of cells via cell-associated UTI-binding proteins (UTI-BPs). Here we report that the 40-kDa protein (UTI-BP(40)) was purified from the cultured human chondrosarcoma cell line HCS-2/8 by UTI affinity chromatography. Purified UTI-BP(40) was digested with trypsin, and the amino acid sequences of the peptide fragments were determined. The sequences of six tryptic fragments of UTI-BP(40) were identical to subsequences present in human link protein (LP). Authentic bovine LP and UTI-BP(40) displayed identical electrophoretic and chromatographic behavior. The UTI-binding properties of UTI-BP(40) and LP were indistinguishable. Direct binding and competition studies strongly demonstrated that the NH(2)-terminal fragment is the UTI-binding part of the LP molecule, that the COOH-terminal UTI fragment (HI-8) failed to bind the NH(2)-terminal subdomain of the LP molecule, and that LP and UTI-BP(40) exhibited significant hyaluronic acid binding. These results demonstrate that UTI-BP(40) is identical to LP and that the NH(2)-terminal domain of UTI is involved in the interaction with the NH(2)-terminal fragment of LP, which is bound to hyaluronic acid in the extracellular matrix.

Expression of neurotrophins and their receptors (TRK) during fracture healing

K Asaumi, T Nakanishi, H Asahara, H Inoue, M Takigawa

BONE 26 ( 6 ) 625 - 633 2000.6

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

To clarify the roles of neurotrophins and their receptors in bone formation, expression of neurotrophins and their receptors (TRK) in a model of mouse fracture healing was investigated, A total of 120 male ICR mice were studied. The right eighth rib of 70 mice was fractured. For sham operation as a control, the right eighth rib of 50 mice was similarly exposed but not fractured, Localization of TRKA, TRKB, and TRKC in a rectangular region of the rib together with surrounding soft tissues was investigated by immunostaining. Localizations of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) at the fracture callus were also investigated by immunostaining, and their mitochondrial RNA (mRNA) expressions were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR), As a result, we observed two types of neurotrophin receptors in the bone forming al ea: immunostaining by anti-TRK was observed in almost all bone forming cells, and staining with anti-TRKC was observed in osteoblast-like cells and hypertrophic chondrocytes, but no staining was observed with anti-TRKB. On the other hand, localization of NGF was observed in almost all bone forming cells, localization of BDNF was observed in osteoblast-like cells, and localization of NT-3 was observed in osteoblast-like cells and hypertrophic chondrocytes at the fracture callus. Expression levels of the mRNA of three neurotrophins in the fractured rib were increased during the process of healing, especially those of NGF and NT-3, which peaked at 2 days after the fracture. The level of BDNF mRNA increased gradually over 8 days, These findings show that neurotrophins and their receptors were expressed in bone forming tells, and suggest that they are involved in the regulation of bone formation as an autocrine and paracrine factor in vivo. (Bone 26:625-633; 2000) (C) 2000 by Elsevier Science Inc. All rights reserved.

軟骨細胞および線維芽細胞株におけるCTGF/Ecogenin遺伝子発現の3'-UTRによる調節

近藤誠二, 久保田聡, 江口傑徳, 服部高子, 中西徹, 菅原利夫, 滝川正春

岡山歯学会雑誌 19 ( 1 ) 205 - 206 2000.6

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