Faculty Profiles - TAKIGAWA Masaharu

Papers - TAKIGAWA Masaharu

Division display 41 - 60 of about 473 ／ All the affair displays >>

The BMP-2 mutant L51P: a BMP receptor IA binding-deficient inhibitor of noggin Reviewed

Hany Mohamed Khattab, Satoshi Kubota, Masaharu Takigawa, Takuo Kuboki, Walter Sebald

Journal of Bone and Mineral Metabolism 128 ( 3 ) 1 - 7 2018.4

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

The antagonist-specific regulation in tissue engineering constitutes important attempts to achieve an improved and rapid bone regeneration by controlling the natural biological response of the natural body growth factors. L51P is molecularly engineered bone morphogentic protein-2 (BMP-2) variant with a substitution of the 51st leucine with a proline residue. L51P is deficient in BMP receptor binding, but maintains its structure and affinity for inhibitory proteins such as noggin, chordin, and gremlin. These modifications convert the BMP-2 variant L51P into a receptor-inactive inhibitor of BMP antagonists. This current approach may prevent the uncontrolled bone overgrowth using high concentration of BMPs and thus regulates the possible growth factor’s high-dose side effects. Exploring of L51P biological functions is required to broad our understanding of BMP mutant biological functions and their potential clinical applications. The progress of L51P researches would hopefully lead to the development of multiple applications for using the L51P in bone and fracture healing disorders.

DOI： 10.1007/s00774-018-0925-0

Metabolic regulation of the CCN family genes by glycolysis in chondrocytes Reviewed

Sho Akashi, Takashi Nishida, Abdellatif El-Seoudi, Masaharu Takigawa, Seiji Iida, Satoshi Kubota

Journal of Cell Communication and Signaling 12 ( 1 ) 245 - 252 2018.3

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

The CCN family consists of 6 genes in the mammalian genome and produces multifunctional proteins involved in a variety of biological processes. Recent reports indicate the profound roles of CCN2 in energy metabolism in chondrocytes, and Ccn2 deficiency is known to alter the expression of 2 other family members including Ccn3. However, almost nothing is known concerning the regulation of the CCN family genes by energy metabolism. In order to gain insight into this critical issue, we initially and comprehensively evaluated the effect of inhibition of glycolysis on the expression of all of the CCN family genes in chondrocytic cells. Upon the inhibition of a glycolytic enzyme, repression of CCN2 expression was observed, whereas CCN3 expression was conversely induced. Similar repression of CCN2 was conferred by the inhibition of aerobic ATP production, which, however, did not induce CCN3 expression. In contrast, glucose starvation significantly enhanced the expression of CCN3 in those cells. The results of a reporter gene assay using a molecular construct containing a CCN3 proximal promoter revealed a dose-dependent induction of the CCN3 promoter activity by the glycolytic inhibitor in chondrocytic cells. These results unveiled a critical role of glycolytic activity in the regulation of CCN2 and CCN3, which activity mediated the mutual regulation of these 2 major CCN family members in chondrocytes.

DOI： 10.1007/s12079-017-0420-8

An early history of CCN2/CTGF research: the road to CCN2 via hcs24, ctgf, ecogenin, and regenerin Reviewed

Masaharu Takigawa

Journal of Cell Communication and Signaling 12 ( 1 ) 253 - 264 2018.3

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

The principal aim of this historical review is to present the processes by which the different aspects of CCN2/CTGF/Hcs24 were discovered by different groups and how much CCN2/CTGF, by being integrated into CCN family, has contributed to the establishment of the basic concepts regarding the role and functions of this new class of proteins. This review should be particularly useful to new investigators who have recently entered this exciting field of study and also provides a good opportunity to acknowledge the input of those individuals who participated in the development of this scientific field.

DOI： 10.1007/s12079-017-0414-6

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment Reviewed

Takanori Eguchi, Chiharu Sogawa, Yuka Okusha, Kenta Uchibe, Ryosuke Iinuma, Kisho Ono, Keisuke Nakano, Jun Murakami, Manabu Itoh, Kazuya Arai, Toshifumi Fujiwara, Yuri Namba, Yoshiki Murata, Kazumi Ohyama, Manami Shimomura, Hirohiko Okamura, Masaharu Takigawa, Tetsuya Nakatsura, Ken-ichi Kozaki, Kuniaki Okamoto, Stuart K. Calderwood

PLoS ONE 13 ( 2 ) e0191109 2018.2

Language：English Publishing type：Research paper (scientific journal) Publisher：Public Library of Science

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

DOI： 10.1371/journal.pone.0191109

A Tumor Suppressor Gene Product, Platelet-Derived Growth Factor Receptor-Like Protein Controls Chondrocyte Proliferation and Differentiation Reviewed

Kazumi Kawata, Satoshi Kubota, Takanori Eguchi, Eriko Aoyama, Norifumi H. Moritani, Morihiko Oka, Harumi Kawaki, Masaharu Takigawa

JOURNAL OF CELLULAR BIOCHEMISTRY 118 ( 11 ) 4033 - 4044 2017.11

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

The platelet-derived growth factor receptor-like (PDGFRL) gene is regarded as a tumor suppressor gene. However, nothing is known about the molecular function of PDGFRL. In this study, we initially clarified its function in chondrocytes. Among all cell lines examined, the PDGFRL mRNA level was the highest in chondrocytic HCS-2/8 cells. Interestingly, the proliferation of chondrocytic HCS-2/8 cells was promoted by PDGFRL overexpression, whereas that of the breast cancer-derived MDA-MB-231 cells was inhibited. Of note, in PDGFRL-overexpressing HCS-2/8 cells, the expression of chondrocyte differentiation marker genes, SOX9, ACAN, COL2A1, COL10A1, and ALP, was decreased. Moreover, we confirmed the expression of PDGFRL mRNA in normal cartilage tissue and chondrocytes. Eventually, the expression of PDGFRL mRNA in condrocytes except in the case of hypertrophic chondrocytes was demonstrated in vivo and in vitro. These findings suggest that PDGFRL plays the different roles, depending upon cell types. Particularly, in chondrocytes, PDGFRL may play a new and important role which is distinct from the function previously reported. J. Cell. Biochem. 118: 4033-4044, 2017. (c) 2017 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.26059

Regulatory mechanism of CCN2 production by serotonin (5-HT) via 5-HT2A and 5-HT2B receptors in chondrocytes Reviewed

Ayaka Hori, Takashi Nishida, Shogo Takashiba, Satoshi Kubota, Masaharu Takigawa

PLOS ONE 12 ( 11 ) 8630 - 8641 2017.11

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

Serotonin (5-hydroxytryptamine: 5-HT) is recognized as a neurotransmitter in the central nerve system and as a regulator of systemic blood pressure in the peripheral tissues. Recently, it was reported that 5-HT2 receptors (5-HT(2)Rs) were expressed in cartilage tissues lacking both vessels and neurons, suggesting possible novel functions of 5-HT during cartilage development and regeneration. Our previous data indicated that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) plays a central role in cartilage development and regeneration. Therefore, the aim of this study was to investigate the effect of 5-HT on the production of CCN2 in chondrocytes. Firstly, we showed that the mRNAs of 5-HT2R subtypes 5-HT2AR and 5-HT2BR, were expressed in a human chondrocytic cell line, HCS-2/8; however, 5-HT2CR mRNA was not detected. In addition, exogenously added 5-HT did not affect the 5-HT2AR and 5-HT2BR expressions. Next, we demonstrated that CCN2 production was increased by treatment with a 5-HT2AR agonist and the combination of 5-HT and 5-HT2BR antagonist. In contrast, treatment with a 5-HT2BR agonist and the combination of 5-HT and 5-HT2AR antagonist decreased CCN2 production. Furthermore, we showed that phosphorylation of Akt and p38 MAPK were increased by treatment with 5-HT2AR agonist, and that phosphorylation of PKC epsilon, PKC zeta, ERK1/2 and JNK were increased by treatment with 5-HT2BR agonist. Finally, we found that 5-HT2AR was localized in the growth plate, whereas 5-HT2BR was localized in the articular cartilage. These findings suggest that 5-HT promotes CCN2 production through the 5-HT2AR in growth plates, and that it represses CCN2 production through the 5-HT2BR in articular cartilage for harmonized development of long bones.

DOI： 10.1371/journal.pone.0188014

Novel role of CCN3 that maintains the differentiated phenotype of articular cartilage Reviewed

Danilo Janune, Tarek Abd El Kader, Eriko Aoyama, Takashi Nishida, Yasuhiko Tabata, Satoshi Kubota, Masaharu Takigawa

JOURNAL OF BONE AND MINERAL METABOLISM 35 ( 6 ) 582 - 597 2017.11

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

Knowledge of the microenvironment of articular cartilage in health and disease is the key to accomplishing fundamental disease-modifying treatments for osteoarthritis. The proteins comprising the CCN Family are matricellular proteins with a remarkable relevance within the context of cartilage metabolism. CCN2 displays a great capability for regenerating articular cartilage, and CCN3 has been shown to activate the expression of genes related to articular chondrocytes and to repress genes related to endochondral ossification in epiphyseal chondrocytes. Moreover, mice lacking CCN3 protein have been shown to display ostearthritic changes in their knee articular cartilage. In this study, we employed a monoiodoacetic acid (MIA)-induced osteoarthritic model to investigate whether osteoarthritic changes in the cartilage are reciprocally accompanied by CCN3 down-regulation and an inducible overexpression system to evaluate the effects of CCN3 on articular chondrocytes in vitro. Finally, we also investigated the effects of exogenous CCN3 in vivo during the early stages of MIA-induced osteoarthritis. We discovered that CCN3 is expressed by articular chondrocytes in normal rat knees, whereas it is rapidly down-regulated in osteoarthritic knees. In vitro, we also discovered that CCN3 increases the proteoglycan accumulation, the gene expression of type II collagen, tenascin-C and lubricin, as well as the protein production of tenascin-C and lubricin in articular chondrocytes. In vivo, it was discovered that exogenous CCN3 increased tidemark integrity and produced an increased production of lubricin protein. The potential utility of CCN3 as a future therapeutic agent and possible strategies to improve its therapeutic functions are also discussed.

DOI： 10.1007/s00774-016-0793-4

Catabolic effects of FGF-1 on chondrocytes and its possible role in osteoarthritis Reviewed

Abdellatif El-Seoudi, Tarek Abd El Kader, Takashi Nishida, Takanori Eguchi, Eriko Aoyama, Masaharu Takigawa, Satoshi Kubota

JOURNAL OF CELL COMMUNICATION AND SIGNALING 11 ( 3 ) 255 - 263 2017.9

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

Fibroblast growth factor 1 (FGF-1) is a classical member of the FGF family and is produced by chondrocytes cultured from osteoarthritic patients. Also, this growth factor was shown to bind to CCN family protein 2 (CCN2), which regenerates damaged articular cartilage and counteracts osteoarthritis (OA) in an animal model. However, the pathophysiological role of FGF-1 in cartilage has not been well investigated. In this study, we evaluated the effects of FGF-1 in vitro and its production in vivo by use of an OA model. Treatment of human chondrocytic cells with FGF-1 resulted in marked repression of genes for cartilaginous extracellular matrix components, whereas it strongly induced matrix metalloproteinase 13 (MMP-13), representing its catabolic effects on cartilage. Interestingly, expression of the CCN2 gene was dramatically repressed by FGF-1, which repression eventually caused the reduced production of CCN2 protein from the chondrocytic cells. The results of a reporter gene assay revealed that this repression could be ascribed, at least in part, to transcriptional regulation. In contrast, the gene expression of FGF-1 was enhanced by exogenous FGF-1, indicating a positive feedback system in these cells. Of note, induction of FGF-1 was observed in the articular cartilage of a rat OA model. These results collectively indicate a pathological role of FGF-1 in OA development, which includes an insufficient cartilage regeneration response caused by CCN2 down regulation.

DOI： 10.1007/s12079-017-0384-8

CATABOLIC EFFECTS OF FGF-1 ON CHONDROCYTES WITH REDUCED CCN2 PRODUCTION THAT PROMOTES CARTILAGE REGENERATION: POSSIBLE ROLE IN OSTEOARTHRITIS

A. Elseoudi, T. Abd El Kader, T. Nishida, E. Aoyama, T. Eguchi, M. Takigawa, S. Kubota

OSTEOPOROSIS INTERNATIONAL 28 S225 - S225 2017.7

UPR transducer BBF2H7 allows export of type II collagen in a cargo- and developmental stage-specific manner Reviewed

Tokiro Ishikawa, Takuya Toyama, Yuki Nakamura, Kentaro Tamada, Hitomi Shimizu, Satoshi Ninagawa, Tetsuya Okada, Yasuhiro Kamei, Tomoko Ishikawa-Fujiwara, Takeshi Todo, Eriko Aoyama, Masaharu Takigawa, Akihiro Harada, Kazutoshi Mori

JOURNAL OF CELL BIOLOGY 216 ( 6 ) 1761 - 1774 2017.6

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

The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (Sec23/24/13/31, Tango1, Sedlin, and KLHL12) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage.

DOI： 10.1083/jcb.201609100

Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation Reviewed

T. Nishida, S. Kubota, E. Aoyama, N. Yamanaka, K. M. Lyons, M. Takigawa

OSTEOARTHRITIS AND CARTILAGE 25 ( 5 ) 759 - 769 2017.5

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

Objective: CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes cartilage regeneration in experimental osteoarthritis (OA) models. However, CCN2 production is very low in articular cartilage. The aim of this study was to investigate whether or not CCN2 was promoted by cultured chondrocytes treated with low-intensity pulsed ultrasound (LIPUS) and to clarify its mechanism.
Methods: Human chondrocytic cell line (HCS)-2/8, rat primary epiphyseal and articular cartilage cells, and Ccn2-deficient chondrocytes that impaired chondrocyte differentiation, were treated with LIPUS for 20 min at 3.0 MHz frequency and 60 mW/cm(2) power. Expressions of chondrocyte differentiation marker mRNAs were examined by real-time PCR (RT-PCR) analysis from HCS-2/8 cells and Ccn2-deficient chondrocytes at 30 min and 1 h after LIPUS treatment, respectively. CCN2 production was examined by Western blotting after 5 h of LIPUS treatment. Moreover, Ca2+ influx was measured by using a Fluo-4 probe.
Results: The gene expression of chondrocyte differentiation markers and CCN2 production were increased in cultured chondrocytes treated with LIPUS. In addition, Ca2+ influx and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2 were increased by LIPUS treatment, and the stability of TRPV4 and BKca channel mRNAs was decreased by siRNA against CCN2. Consistent with those findings, the LIPUS-induced the gene expressions of type II collagen (COL2a1) and Aggrecan (ACAN) observed in wild-type cells were not observed in the Ccn2-deficient chondrocytes.
Conclusion: These data indicate that chondrocyte differentiation represented by CCN2 production was mediated via MAPK pathways activated by LIPUS-stimulated Ca2+ influx, which in turn was supported by the induced CCN2 molecules in articular chondrocytes. (C) 2016 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.

DOI： 10.1016/j.joca.2016.10.003

Erratum: Proinsulin C-peptide regulates ribosomal RNA expression (The Journal of Biological Chemistry (2010) 285 (3462-3469) DOI: 10.1074/jbc.M109.053587) Reviewed

Emma Lindahl, Ulrika Nyman, Farasat Zaman, Carina Palmberg, Anna Cascante, Jawed Shafqat, Masaharu Takigawa, Lars Sävendahl, Hans Jörnvall, Bertrand Joseph

Journal of Biological Chemistry 285 ( 5 ) 3462 - 3469 2017.3

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Biochemistry and Molecular Biology Inc.

Proinsulin C-peptide is internalized into cells, but a function of its intracellular localization has not been established. We now demonstrate that, upon cellular entry, C-peptide is localized to the nucleoli, where it promotes transcription of genes encoding for ribosomal RNA. We find that C-peptide binds to histones and enhances acetylation of lysine residue 16 of histone H4 at the promoter region of genes for ribosomal RNA. In agreement with synchrony of ribosomal RNA synthesis and cell proliferation, we show that C-peptide stimulates proliferation in chondrocytes and HEK-293 cells. This regulation of ribosomal RNA provides a mechanism by which C-peptide can exert transcriptional effects and implies that the peptide has growth factor activity. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M109.053587

Proinsulin C-peptide regulates ribosomal RNA expression (vol 292, pg 3467, 2017)

Emma Lindahl, Ulrika Nyman, Farasat Zaman, Carina Palmberg, Anna Cascante, Jawed Shafqat, Masaharu Takigawa, Lars Saevendahl, Hans Joernvall, Bertrand Joseph

JOURNAL OF BIOLOGICAL CHEMISTRY 292 ( 10 ) 4382 - 4382 2017.3

Intracellular MMP3 Promotes HSP Gene Expression in Collaboration With Chromobox Proteins Reviewed

Takanori Eguchi, Stuart K. Calderwood, Masaharu Takigawa, Satoshi Kubota, Ken-ichi Kozaki

JOURNAL OF CELLULAR BIOCHEMISTRY 118 ( 1 ) 43 - 51 2017.1

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

Matrix metalloproteinases (MMPs) are crucial factors in tumor progression, inflammatory/immune responses and tissue development/regeneration. Of note, it has been known that MMPs promote genome instability, epithelial-mesenchymal transition, invasion, and metastasis in tumor progression. We previously reported that human MMP3 could translocate into cellular nuclei and control transcription in human chondrosarcoma-derived cells and in articular cartilage (Eguchi et al. [2008] Mol Cell Biol 28(7):2391-2413); however, further transcriptional target genes and cofactors of intranuclear MMP3 have not been uncovered. In this paper, we used transcriptomics analysis in order to examine novel transcriptional target genes regulated by intracellular MMP3. We found that mRNA levels of HSP family members (HSP70B, HSP72, HSP40/DNAJ, and HSP20/CRYAB) are upregulated by the intracellular MMP3 overload. Bioinformatic analysis predicted several transcription factors that possibly interact with MMP3. Among these factors, heat shock factor 1 (HSF1) cooperated with the MMP3 to activate the HSP70B gene promoter in reporter gene assays, while a dominant negative HSF1 blocked the role for MMP3 in the trans-activation. The hemopexin-like repeat (PEX) domain of the human MMP3 was essential for transcriptional induction of the HSP70B gene. In addition, chromobox proteins CBX5/HP1 and CBX3/HP1 cooperated with the PEX domain in induction of HSP70B mRNA. Taken together, this study newly clarified that intracellular MMP3 cooperate with CBXs/HP1s in transcriptional promotion of HSP genes. J. Cell. Biochem. 118: 43-51, 2017. (c) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.25607

Immunohistochemical analysis of CCN proteins in calcified tissues

Harumi Kawaki, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 53 - 62 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Immunohistochemistry is a major technique to determine the distribution and localization of differentially produced proteins in the context of an intact tissue. It exploits one of the properties of antibodies, specific binding to an antigen, i.e., to the epitope of its target protein, in combination with a color-developing enzymatic reaction or tagged fluorophore. We have clarified the spatial and temporal expression patterns of CCN family proteins in several different types of animal tissues by using this immunohistochemical technique to support our corresponding data obtained in vitro. In this chapter, we provide our protocol for immunohistochemistry optimized for paraffin-embedded sections after having determined the optimal conditions for the use of antibodies against each member of the CCN family.

DOI： 10.1007/978-1-4939-6430-7_6

Gene expression analysis of CCN proteins: Whole-mount in situ hybridization of Ccn2 in developing calcified tissues

Tomoichiro Yamaai, Masaharu Takigawa

Methods in Molecular Biology 1489 11 - 19 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

A procedure for whole-mount in situ hybridization developed for detecting gene expression of Ccn2 in developing calcified tissues of mouse embryos is presented. In this method, embryos are hybridized with Dig-labeled riboprobes, and the riboprobes are detected by use of the alkaline-phosphatase reaction in the presence of a 4-nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (NBT + BCIP) mixture. Obvious detection of positive signals for Ccn2 in the cartilage of developing phalanges indicates that this method can be applied to gene expression analysis of other Ccn genes in developing calcified tissues.

DOI： 10.1007/978-1-4939-6430-7_2

Western blotting analysis of CCN proteins in calcified tissues

Harumi Kawaki, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 43 - 51 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Western blotting is widely used for protein analysis. We routinely perform such analysis for evaluating the production levels of CCN family proteins in a variety of cells under various conditions. In this chapter, we describe our Western blotting protocol to estimate protein production profiles of CCN family members after having assessed the specificity of the antibodies against each CCN member protein to ensure no crossreaction with other CCN member proteins.

DOI： 10.1007/978-1-4939-6430-7_5

The ccn proteins: An overview Invited

Masaharu Takigawa

Methods in Molecular Biology 1489 1 - 8 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

I introduce the general structures and functions of CCN proteins and possible molecular mechanisms regarding the unique biological actions of this new family of signaling regulators, which may be referred to as “signal conductors.” Relevance to pathology is also briefly introduced. The information provided in this overview should be useful for readers of the following chapters.

DOI： 10.1007/978-1-4939-6430-7_1

In vitro transfection with and expression of CCN family of genes

Danilo Janune, Masaharu Takigawa

Methods in Molecular Biology 1489 107 - 113 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

The ability to engineer cells to express a protein of interest in an inducible manner and stably for a long period is a valuable tool in molecular biology and also one that holds promise for regenerative medicine in the future. CCN proteins have been suggested to be involved in tissue regeneration. In this chapter, we describe an in vitro method for stable and inducible expression of CCN protein in a chondroprogenitor cell line and in chondrocytes in primary culture that does not involve the use of any viral vector.

DOI： 10.1007/978-1-4939-6430-7_11

Production of recombinant CCN2 protein by mammalian cells

Takashi Nishida, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 95 - 105 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Recombinant CCN2 protein (rCCN2) is available from many companies
however, most of them are produced in E. coli. To investigate true functions of rCCN2, glycosylated protein with proper folding needs to be used. Therefore, we use rCCN2 produced by mammalian cells. Conditioned medium (CM) of HeLa cells stably transfected with a CCN2 expression vector are collected, and the recombinant CCN2 protein produced and secreted into the CM is purified by two-step chromatography, first with a heparin affinity column and then with an anti-CCN2 affinity column prepared with a monoclonal antibody against CCN2. The purified rCCN2 shows the bands of 36–38 kDa with sliver staining after gel electrophoresis, which can be confirmed by Western blotting. This chapter describes these methods in detail.

DOI： 10.1007/978-1-4939-6430-7_10

　 PREV - NEXT 　

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24