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Joon-Ii Jun, Lester F Lau
The expression of Ccn2 (CTGF) has been linked to fibrosis in many tissues and pathologies, although its activities in fibroblastic cells and precise mechanism of action in fibrogenesis are still controversial. Here, we showed that CCN2 can induce cellular senescence in fibroblasts both in vitro and in vivo, whereupon senescent cells express an anti-fibrotic "senescence-associated secretory phenotype" (SASP) that includes upregulation of matrix metalloproteinases and downregulation of collagen. Mechanistically, CCN2 induces fibroblast senescence through integrin α6β1-mediated accumulation of reactive oxygen species, leading to activation of p53 and induction of p16(INK4a)...
October 18, 2016: Journal of Cell Communication and Signaling
Takayuki Furumatsu, Toshifumi Ozaki
The multifunctional growth factor CYR61/CTGF/NOV (CCN) 2, also known as connective tissue growth factor, regulates cellular proliferation, differentiation, and tissue regeneration. Recent literatures have described important roles of CCN2 in the meniscus metabolism. However, the mechanical stress-mediated transcriptional regulation of CCN2 in the meniscus remains unclear. The meniscus is a fibrocartilaginous tissue that controls complex biomechanics of the knee joint. Therefore, the injured unstable meniscus has a poor healing potential especially in the avascular inner region...
2017: Methods in Molecular Biology
Susan V McLennan, Danqing Min, Xiaoyu Wang, Stephen M Twigg
Many studies have shown effects of members of the CCN family on matrix synthesis and accumulation but few have examined degradative pathways. This scarcity of information is in part due to the lack of suitable model systems. Here we describe methods for making rhCCN2 and also for the preparation of a biosynthetically labeled matrix substrate that can be used to measure the effect of CCN on cellular or secreted degradative pathways.
2017: Methods in Molecular Biology
María José Acuña, Enrique Brandan
CCN2 or connective tissue growth factor (CTGF) is a matricellular protein that regulates several cellular processes. In skeletal muscle, CTGF is a key modulator of fibrogenesis, is increased in pathological conditions such as muscular dystrophies, and plays a major role in the pathology outcome. Overexpression of CTGF in skeletal muscle of wild-type mice results in muscle damage, fibrosis, and reduction of strength. In contrast, a decrease in CTGF in dystrophic mice increases strength and reduces damage and fibrosis...
2017: Methods in Molecular Biology
Philip C Trackman, Debashree Saxena, Manish V Bais
A simple method for the determination of relative levels of insoluble collagen accumulation in fibroblast cultures is presented. Confluent cell cultures are provided with sodium ascorbate which is then permissive for collagen deposition. At intervals, cultures are fixed and stained successively with sirius red and then crystal Violet to, respectively, assess for relative changes in collagen accumulation in response to factors such as TGF-β1 or matricellular CCN2 and changes in DNA content as an index of changes in cell density...
2017: Methods in Molecular Biology
Hideki Yokoi, Masashi Mukoyama
Renal fibrosis is characterized by glomerulosclerosis and tubulointerstitial fibrosis. Transforming growth factor-β (TGF-β) is postulated to play a central role in the development of both fibrotic processes. Extracellular matrix proteins, particularly type I collagen and fibronectin, accumulate in the tissue during renal fibrogenesis. CCN2, also known as connective tissue growth factor (CTGF), is increased in the setting of fibrosis and modulates a number of downstream signaling pathways involved in the fibrogenic properties of TGF-β...
2017: Methods in Molecular Biology
James Hutchenreuther, Andrew Leask, Katherine Thompson
CCN2 is a profibrotic matricellular protein. CCN2 directly promotes cell adhesion and indirectly promotes fibrosis by activating adhesive signaling in response to growth factors, cytokines, and extracellular matrix. The following protocols will allow the direct assessment of other CCN family members in these processes.
2017: Methods in Molecular Biology
Andrew Leask
CCN2, formerly known as connective tissue growth factor, was discovered in 1991. Soon after its discovery, CCN2 was shown to be upregulated in response to the profibrotic cytokine transforming growth factor (TGF) β and to be constitutively overexpressed in fibrotic conditions. These early observations led to the hypothesis that CCN2 was a key regulator of fibrosis. However, only recently have data been generated that directly demonstrate this hypothesis. This review summarizes these observations and suggests a mechanism of action for CCN2...
2017: Methods in Molecular Biology
Kazumi Kawata, Satoshi Kubota, Masaharu Takigawa
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.
2017: Methods in Molecular Biology
Takako Hattori, Shinsuke Itoh, Masaharu Takigawa
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...
2017: Methods in Molecular Biology
Naohiro Toda, Hideki Yokoi, Kiyoshi Mori, Masashi Mukoyama
CCN2 has been shown to be closely involved in the progression of renal fibrosis, indicating the potential of CCN2 inhibition as a therapeutic target. Although the examination of the phenotypes of adult CCN2 knockout mice with renal diseases has yielded valuable scientific insights, perinatal death has limited studies of CCN2 in vivo. Conditional knockout technology has become widely used for the deletion of genes in the desired cell populations and time points through the use of cell-specific Cre recombinase-expressing mice...
2017: Methods in Molecular Biology
Teruko Takano-Yamamoto, Tomohiro Fukunaga, Nobuo Takeshita
To investigate mechanical-dependent bone remodeling, we had previously applied various types of mechanical loading onto the teeth of rats and mice. In vitro cultured bone cells were then used to elucidate the mechanisms underlying the specific phenomenon revealed by in vivo experiments. This review describes the techniques used to upregulate CCN2 expression in bone cells produced by different types of mechanical stress, such as fluid shear stress and substrate strain in vitro, and compression or tension force in vivo...
2017: Methods in Molecular Biology
Takashi Nishida, Satoshi Kubota, Masaharu Takigawa
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 findings, 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...
2017: Methods in Molecular Biology
Koichiro Muromachi, Hiroshi Sugiya, Nobuyuki Tani-Ishii
In postnatal dentin formation, odontoblast differentiation occurs in the pulp tissue regenerative process under pathological condition. Odontoblasts and newly differentiated odontoblast-like cells beneath the caries lesion form tertiary dentin and are highly odontogenic. To observe the activity of dentinogenesis occur within the hard tissue, a combination of immunohistological analysis and immunodetection of dentinogenesis specific molecules, such as dentin sialophosphoprotein (DSPP) and/or its cleaved products dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), is a reliable approach...
2017: Methods in Molecular Biology
Tsuyoshi Shimo, Masaharu Takigawa
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)...
2017: Methods in Molecular Biology
Takashi Nishida, Satoshi Kubota, Masaharu Takigawa
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...
2017: Methods in Molecular Biology
Takako Hattori, Mitsuhiro Hoshijima, Masaharu Takigawa
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.
2017: Methods in Molecular Biology
Seiji Kondo, Satoshi Kubota, Masaharu Takigawa
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...
2017: Methods in Molecular Biology
Takanori Eguchi, Satoshi Kubota, Masaharu Takigawa
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 firefly and Renilla reniformis (sea pansy) emit light. The first 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...
2017: Methods in Molecular Biology
Eriko Aoyama, Masaharu Takigawa
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...
2017: Methods in Molecular Biology
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