Faculty Profiles - TAKIGAWA Masaharu

Papers - TAKIGAWA Masaharu

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Production of recombinant CCN2 protein in Escherichia coli

Eriko Aoyama, Takako Hattori, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 77 - 84 2017

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

Recombinant proteins are important tools for understanding molecular functions in vitro. Recent progress in the generation of recombinant proteins is amazing. However, when we plan to produce them, we should choose the best method according to the nature and the use of the target recombinant protein. Degradation and mis-folding are major problems in producing active recombinant CCN2. The method shown in this chapter describes the appropriate conditions under which we can produce CCN2 and its truncated fragments in Escherichia coli.

DOI： 10.1007/978-1-4939-6430-7_8

Analysis of expression of CCN family genes in skeletal tissue-derived cells

Harumi Kawaki, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 33 - 41 2017

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

The quantitative reverse transcription polymerase chain reaction or real-time PCR has become a routine technique for the detection and comparison of amounts of specific mRNA transcripts, done by measuring amplified levels of specific cDNAs. In this chapter, we provide our real-time RT-PCR experimental procedure using SYBR Green I for the quantitative analysis of CCN family gene expression. Especially, we describe the extraction and purification steps for RNA derived from mesenchymal cells, such as chondrocytes and osteoblasts that produce a large amount of extracellular matrix in detail.

DOI： 10.1007/978-1-4939-6430-7_4

In vivo evaluation of cartilage regenerative effects of CCN2 protein

Takashi Nishida, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 273 - 282 2017

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

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) is a unique growth factor that promotes the proliferation and differentiation, but not the hypertrophy of articular chondrocytes in vitro. Based on these fi ndings, we previously evaluated the cartilage-regenerative effects of recombinant CCN2 protein (rCCN2) by using both mono-iodoacetate (MIA) injection into the rat joint cavity and formation of full-thickness defects of rat articular cartilage in vivo, and our results suggested the utility of CCN2 in the regeneration of articular cartilage. This chapter entails helpful tips to apply these two animal models for the evaluation of cartilage-regenerative effects of CCN2 or its derivatives.

DOI： 10.1007/978-1-4939-6430-7_25

骨リモデリングにおける骨細胞の役割 Invited

西田崇, 久保田聡, 滝川正春

Clinical Calcium 27 ( 12 ) 23 - 29 2017

Analysis of pathological activities of CCN proteins in bone metastasis Invited

Tsuyoshi Shimo, Norie Yoshioka, Masaharu Takigawa, Akira Sasaki

Methods in Molecular Biology 1489 505 - 512 2017

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

Bone metastasis is a common occurrence in human malignancies, including breast, prostate, and lung cancer, and is associated with a high morbidity rate because of intractable bone pain, pathological fractures, hypercalcemia, and nerve compression. Animal models of bone metastasis are important tools to investigate the pathogenesis and develop treatment strategies. However, there are few models of spontaneous bone metastasis despite the fact that animals often spontaneously develop cancer. Here, we describe methods for developing a mouse model of breast cancer bone metastasis achieved by injection of MDA-MB-231 breast cancer cells into the heart. This assay can be applied to studies on roles of CCN proteins in tumor metastasis and development of treatment strategies targeting CCN proteins.

DOI： 10.1007/978-1-4939-6430-7_42

Generation and analysis of cartilage-specific CCN2 overexpression in transgenic mice

Takako Hattori, Shinsuke Itoh, Masaharu Takigawa

Methods in Molecular Biology 1489 391 - 403 2017

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

Recent progress in gene-editing technology has provided a strong impact for improved our understanding of molecular functions in living organisms. Here we describe our method to generate transgene-overexpressing mouse models, which method involves the use of tissue-specific promoters for analyzing a certain molecule (s) in special tissues. The protocol described in this chapter uses the Col2a1 promoter-enhancer, which is known for driving specific and strong transgene expression in cartilage and is based on several of our studies showing a positive role of the connective tissue growth factor (CCN2) in cartilage-bone development and maintenance of articular cartilage. These mice show strongly accelerated endochondral ossification resulting in enhanced bone elongation, as well as resistance to age-related articular degeneration. This protocol also describes how to analyze the molecular mechanisms of these phenomena by use of chondrocytes isolated from CCN2-overexpressing cartilage.

DOI： 10.1007/978-1-4939-6430-7_32

Analysis of transcytosis of CCN2 by chondrocytes

Kazumi Kawata, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 405 - 413 2017

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

Transcytosis is a mechanism for the transcellular transport of biomolecules. Analysis of transcytosis is frequently performed in cells with distinct polarity, such as brain endothelial cells. However, in cells without evident polarity, analysis of transcytosis has not been performed. Here, we describe a method for analyzing transcytosis of a CCN family protein through chondrocytic cells having no apparent polarity.

DOI： 10.1007/978-1-4939-6430-7_33

Cell biological assays for measuring angiogenic activities of CCN proteins

Tsuyoshi Shimo, Masaharu Takigawa

Methods in Molecular Biology 1489 239 - 249 2017

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

Angiogenesis, the process of generating new blood vessels from an existing vasculature, is essential in normal developmental processes such as endochondral ossification and in numerous kinds of pathogenesis including tumor growth. A part from the action of angiogenic factor or antiangiogenic factor, it is still unknown at which stage of the angiogenic cascade these agents affect angiogenesis. Here, we describe methods for the use of connective tissue growth factor (CTGF/CCN2) and CCN2 neutralizing antibody in the currently used principal angiogenesis assays, including those in vitro ones for the proliferation, migration, adhesion, and tube formation of endothelial cells and in vivo assays such as those utilizing type I collagen implantation and the chick chorioallantoic membrane (CAM).

DOI： 10.1007/978-1-4939-6430-7_22

Preparation of module-specific antibodies against CCN family members

Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 115 - 128 2017

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

Specific antibodies against biomolecules are conventional, but robust tools for the structural and functional analysis of target molecules. Since CCN family proteins are composed of four distinct modules that together determine the functionalities as full-length molecules depending upon extracellular microenvironment, specific antibody against independent modules are quite useful in CCN family research. Three distinct strategies are considerable for raising antibodies specific to four modules: IGFBP, VWC, TSP1, and CT modules. In the first strategy, full-length CCN family proteins are used to immunize mice to obtain a number of hybridoma clones producing different monoclonal antibodies, which are to be characterized to locate the epitopes in particular modules. Second methodology is a straightforward one, in which each modular protein fragment or synthetic peptide is prepared and is used for the immunization of animals independently. Finally, DNA immunization technology is recently known to be useful in developing module-specific antibodies against CCN family proteins as well. Preparation of antibodies is a quite classical and established technique, and thus nowadays is managed mostly by professional and commercial facilities. Therefore in this chapter, essentials of each strategy are introduced, rather than experimental details in each process.

DOI： 10.1007/978-1-4939-6430-7_12

Cell biological assays for measuring chondrogenic activities of CCN2 protein

Takashi Nishida, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 219 - 237 2017

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

Growth-plate chondrocytes undergo proliferation, maturation, hypertrophic differentiation, and calcification
and these processes can be reproduced in vitro in a chondrocyte culture system. Using this system, we have shown that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes all stages of proliferation, maturation, hypertrophic differentiation, and calcification, thus suggesting that CCN2 is a multifunctional growth factor for chondrocytes and plays important roles in chondrocyte proliferation and differentiation. In this chapter, we describe how to evaluate CCN2 functions in these processes occurring in cultured chondrocytes. Evaluation strategies for cell proliferation include measuring DNA synthesis by [3 H]-thymidine incorporation, cellular metabolic activity, and cell number with a hemocytometer. Next, evaluation strategies to assess maturation are analysis of the gene expression of markers of mature chondrocytes, and examination of proteoglycan and collagen synthesis by using radioactive compounds. In addition, cytohistochemical detection of glycosaminoglycans (GAGs), such as chondroitin sulfate, by use of alcian blue and toluidine blue staining is useful to evaluate chondrocyte maturation. These methods can be also used for evaluation of physiological functions of CCN2 in permanent chondrocytes such as articular and auricular chondrocytes, which do not calcify under physiological conditions. Next, evaluation of hypertrophic differentiation is performed by detecting type X collagen, which is specific marker of hypertrophic chondrocytes, and by measuring alkaline phosphatase (ALP) activity. Finally, evaluation of calcification is performed by detecting matrix calcification by use of alizarin red staining and by examining the incorporation of 45 Ca into cartilaginous matrix. These methods would be useful for the evaluation not only of CCN2 but also of its derivatives and other CCN proteins.

DOI： 10.1007/978-1-4939-6430-7_21

Protein imaging of CCN2 AND CCN3 in living cells

Takako Hattori, Mitsuhiro Hoshijima, Masaharu Takigawa

Methods in Molecular Biology 1489 211 - 215 2017

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

Recent progress in molecular imaging technology has had a strong impact on improving our understanding of molecular translocation, receptor internalization, and interactions in living cells. The protocol in this chapter introduces an optimized technique for intracellular localization of CCN2 and CCN3 in live cells, one using GFP-tagged CCN2 and Halo-tagged CCN3.

DOI： 10.1007/978-1-4939-6430-7_20

Analysis of posttranscriptional regulation of CCN genes

Seiji Kondo, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 187 - 209 2017

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

Cells generally control the concentration of mRNA by transcriptional and posttranscriptional regulation, so the separate contributions of synthesis and degradation (“decay”) cannot be discriminated by the quantification of mRNA. To elucidate the contribution of posttranscriptional regulation, all experimental procedures for the analysis of the total transcript level, transcriptional induction, and degradation of the target mRNA are performed either individually, or in combination. From our experience, measurement of the steady-state levels of the mRNA using quantitative real-time polymerase chain reaction is an essential first step in quantifying ccn2 gene expression level. Subsequently, the effect of transcription rates should be assessed by reporter assays of the ccn2 promoter and nuclear run-on assays. Finally, the stability of ccn2 mRNAs is evaluated in the presence of a metabolic inhibitor actinomycin D, followed by mRNA degradation assays in vitro. Here, we describe the strategic methods used in a series of analyses to elucidate the possible involvement of the posttranscriptional regulatory mechanism of the ccn2 gene and show how this approach can in theory be applied to elucidating the posttranscriptional regulation of other genes belonging to the CCN family.

DOI： 10.1007/978-1-4939-6430-7_19

Evaluation of molecular interaction between CCN2 protein and its binding partners by surface plasmon resonance (SPR)

Eriko Aoyama, Masaharu Takigawa

Methods in Molecular Biology 1489 169 - 176 2017

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

The surface plasmon resonance (SPR) biosensor is a useful tool to analyze numerically the interaction of certain molecules. The most important advantage of the SPR assay as compared with other protein–protein binding assays is that it can calculate the affinity between protein and its binding partner, for this affinity is necessary to determine the priority of interactions between proteins. Although CCN proteins have been shown to have various binding partners, the affinities of many of them have not yet been determined. Therefore, it is important to determine the unknown affinities of known binding partners and to find new binding partners whose affinities need to be determined. This chapter provides helpful tips to use the instrument for determination of the affinities of binding between CCN proteins and their binding partners.

DOI： 10.1007/978-1-4939-6430-7_17

Promoter analyses of CCN genes

Takanori Eguchi, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 177 - 185 2017

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

Promoter analysis is the most basics in the analysis of gene regulation. Luciferase gene is the most commonly used reporter gene in promoter analysis. Luciferase is an enzyme that is used when fi refl y and Renilla reniformis (sea pansy) emit light. The fi rst experimental step in this reporter gene assay is to connect a particular DNA segment to a luciferase gene. The second step is to transfect the reporter construct into the cells. Thereafter, stable luciferase will be produced with the help of transcriptional machinery, mRNA transporters, and translational machinery in the cells. Luciferase assay measures the quantity of light that is emitted by luciferin–luciferase reaction. Consistent with the fact that CCN2 expression has been shown to be altered by a variety of stimuli, the CCN2 promoter region also haa been shown to be bound and regulated by multiple transcription factors such as Smad, MMP3, NF-κB, AP1, TCF/LEF, and Sox9.

DOI： 10.1007/978-1-4939-6430-7_18

Analysis of signaling pathways activated by CCN proteins

Harumi Kawaki, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 139 - 143 2017

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

CCN family proteins activate multiple intracellular phosphorylated kinase cascades to yield the multiple physiological functions of a variety of target cells. In this chapter, we describe our protocol examining the effects of these proteins on signal transduction pathways, especially mitogen-activated protein kinase cascades, activated by CCN member proteins, which examinations have been carried out mainly by using Western blotting methodologies.

DOI： 10.1007/978-1-4939-6430-7_14

Protocols for screening peptide motifs binding to ccn family proteins

Satoshi Kubota, Harumi Kawaki, Masaharu Takigawa

Methods in Molecular Biology 1489 155 - 167 2017

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

Function of CCN family proteins is determined by the interactions with multiple cofactors that are present in microenvironment. Therefore, finding out these cofactors is critically important in understanding the molecular function of the CCN family members. For this objective, bacteriophage random peptide display library is a quite feasible tool. In this library, each filamentous bacteriophage is designed to display an oligopeptide of random 12–16 amino acid residues on its surface. Bacteriophage clones that possess the peptides that bind to a CCN family protein are selected through several cycles of a process designated biopanning or affinity selection. By determining the nucleotide sequence of the DNA that encodes the displayed peptide, oligopeptides that specifically bind to the CCN family member can be specified. Obtained peptide sequences can be utilized for designing peptide aptamers for the CCN family protein, or as a key sequence to find out new CCN family cofactor candidates in silico.

DOI： 10.1007/978-1-4939-6430-7_16

Elisa of CCN family proteins in body fluids including serum and plasma

Satoshi Kubota, Harumi Kawaki, Masaharu Takigawa

Methods in Molecular Biology 1489 127 - 138 2017

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

Enzyme-linked immunosorbent assay (ELISA) is the most popular methodology for absolute quantification of particular proteins in liquid samples. Especially for CCN family members that are associated with human diseases, utility of ELISA for those proteins in clinics as well as research laboratories is emphasized. However, in order to obtain accurate and stable results in ELISA, particular care should be taken in controlling the quality and quantity of standard CCN family proteins, which bind to various materials and can be unstable in a purified form. Recently, diagnostic value of the CCN family protein fragments in body fluids has been indicated in several diseases. Therefore, module-specific detection system for the CCN family members is desired as a promising tool in clinics. It should be also noted that modular fragments of CCN family members can be more stable than the full-length in purified forms, whose quality may be easier to control than that of the full-length ones.

DOI： 10.1007/978-1-4939-6430-7_13

Protocols for screening for binding partners of CCN proteins: Yeast two-hybrid system

Mitsuhiro Hoshijima, Takako Hattori, Masaharu Takigawa

Methods in Molecular Biology 1489 145 - 154 2017

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

Yeast two-hybrid screening is a powerful method to identify proteins that interact with a protein of interest. CCN2 consists of four domains, and identification of new proteins that bind to individual domains of CCN2 may reveal a variety of CCN2 functions. To identify CCN2-interactive proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8, with CCN2 cDNA used as a bait. In this chapter, we describe our methods for screening for CCN2 binding partners by this system in detail. This protocol may be applied to other CCN proteins as well.

DOI： 10.1007/978-1-4939-6430-7_15

The role of osteocytes in bone remodeling.

Nishida, T, Kubota, S, Takigawa, M

27 23 - 29 2017

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

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

FASEB JOURNAL 30 2016.4

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